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501. Characteristics of myogenic reactivity in isolated rat mesenteric veins

By Enouri Saad; Monteith Gabrielle; Johnson RonFrom American journal of physiology. Regulatory, integrative and comparative physiology (2011), 300(2), R470-8,Language: English, Database: MEDLINE

Mechanisms of mechanically induced venous tone and its interaction with the endothelium and key vasoactiveneurohormones are not well established. We investigated the contribution of the endothelium, l-type voltage-operatedcalcium channels (L-VOCCs), and PKC and Rho kinase to myogenic reactivity in mesenteric vessels exposed toincreasing transmural pressure. The interaction of myogenic reactivity with norepinephrine (NE) and endothelin-1 (ET-1) was also investigated. Pressure myography was used to study isolated, cannulated, third-order rat mesenteric small

veins and arteries. NE and ET-1 concentration response curves were constructed at low, intermediate, and hightransmural pressures. Myogenic reactivity was not altered by nitric oxide synthase inhibition with N(ω)-nitro-L-arginine(L-NNA; 100 µM) or endothelium removal in both vessels. L-VOCCs blockade (nifedipine, 1 µM) completely abolishedarterial tone, while only partially reducing venous tone. PKC (chelerythrine, 2.5 µM) and Rho kinase (Y27632, 3 µM)inhibitors largely abolished venous and arterial myogenic reactivity. There was no significant difference in thesensitivity of NE or ET-1-induced contractions within vessels. However, veins were more sensitive to NE and ET-1when compared with corresponding arteries at low, intermediate, and high transmural pressures, respectively. Theseresults suggest that 1) myogenic factors are important contributors to net venous tone in mesenteric veins; 2) PKC andRho activation are important in myogenic reactivity in both vessels, while l-VOCCs play a limited role in the veins vs.the arteries, and the endothelium does not appear to modulate myogenic reactivity in either vessel type; and 3)mesenteric veins maintain an enhanced sensitivity to NE and ET-1 compared with the arteries when studied underconditions of changing transmural distending pressure.

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502. Morphine postconditioning attenuates ICAM-1 expression on endothelial cells

By Min Too Jae; Kim Joong-il; Kim Jae-Hwan; Noh Kyung Hee; Kim Tae Woo; Kim Woon-Young; Lee Yoon-Sook;Park Young CheolFrom Journal of Korean medical science (2011), 26(2), 290-6, Language: English, Database: MEDLINE

The purpose of this study is to determine 1) whether morphine post condition (MPostC) can attenuate the intercellularadhesion molecules-1 (ICAM-1) expression after reoxygenation injury and 2) the subtype(s) of the opioid receptors(ORs) that are involved with MPostC. Human umbilical vein endothelial cells (HUVECs) were subjected to 6 hr anoxiafollowed by 12 hr reoxygenation. Three morphine concentrations (0.3, 3, 30 µM) were used to evaluate the protectiveeffect of MPostC. We also investigated blockading the OR subtypes' effects on MPostC by using three antagonists (aµ-OR antagonist naloxone, a κ -OR antagonist nor-binaltorphimine, and a δ-OR antagonist naltrindole) and the inhibitorof protein kinase C (PKC) chelerythrine. As results, the ICAM-1 expression was significantly reduced in the MPostC(3, 30 µM) groups compared to the control group at 1, 6, 9, and 12 hours reoxygenation time. As a consequence,neutrophil adhesion was also decreased after MPostC. These effects were abolished by co administeringchelerythrine, nor-binaltorphimine or naltrindole, but not with naloxone. In conclusion, it is assumed that MPostC couldattenuate the expression of ICAM-1 on endothelial cells during reoxygenation via the κ and δ-OR (opioid receptor)-specific pathway, and this also involves a PKC-dependent pathway.

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503. Effects of the PKC inhibitors chelerythrine and bisindolylmaleimide I (GF 109203X) on delayed rectifier K+ currents

By Harmati Gabor; Papp Ferenc; Szentandrassy Norbert; Barandi Laszlo; Ruzsnavszky Ferenc; Horvath Balazs;Banyasz Tamas; Magyar Janos; Panyi Gyorgy; Krasznai Zoltan; et al

From Naunyn-Schmiedeberg's archives of pharmacology (2011), 383(2), 141-8, Language: English, Database:MEDLINE

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Protein kinase C (PKC) inhibitors are useful tools for studying PKC-dependent regulation of ion channels. For thispurpose, high PKC specificity is a basic requirement excluding any direct interaction between the PKC inhibitor and theion channel. In the present study, the effects of two frequently applied PKC inhibitors, chelerythine andbisindolylmaleimide I, were studied on the rapid and slow components of the delayed rectifier K(+) current (I(Kr) andI(Ks)) in canine ventricular cardiomyocytes and on the human ether-a-go-go-related gene (hERG) channels expressedin human embryonic kidney (HEK) cells. The whole cell version of the patch clamp technique was used in allexperiments. Chelerythrine and bisindolylmaleimide I (both 1 µM) suppressed I(Kr) in canine ventricular cells. Thisinhibition developed rapidly, suggesting a direct drug-channel interaction. In HEK cells heterologously expressinghERG channels, chelerythrine and bisindolylmaleimide I blocked hERG current in a concentration-dependent manner,having EC(50) values of 0.11 ± 0.01 and 0.76 ± 0.04 µM, respectively. Both chelerythrine and bisindolylmaleimide Istrongly modified gating kinetics of hERG--voltage dependence of activation was shifted towards more negativevoltages and activation was accelerated. Deactivation was slowed by bisindolylmaleimide I but not by chelerythrine.I(Ks) was not significantly altered by bisindolylmaleimide I and chelerythrine. No significant effect of 0.1 µMbisindolylmaleimide I or 0.1 µM PMA (PKC activator) was observed on I(Kr) arguing against significant contribution ofPKC to regulation of I(Kr). It is concluded that neither chelerythrine nor bisindolylmaleimide I is suitable for selectivePKC blockade due to their direct blocking actions on the hERG channel.

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504. The actions of benzophenanthridine alkaloids, piperonyl butoxide and (S)-methoprene at the G-protein coupledcannabinoid CB receptor in vitro

By Dhopeshwarkar Amey S; Jain Saurabh; Liao Chengyong; Ghose Sudip K; Bisset Kathleen M; Nicholson Russell AFrom European journal of pharmacology (2011), 654(1), 26-32, Language: English, Database: MEDLINE

This investigation focused primarily on the interaction of two benzophenanthridine alkaloids (chelerythrine andsanguinarine), piperonyl butoxide and (S)-methoprene with G-protein-coupled cannabinoid CB(1) receptors of mousebrain in vitro. Chelerythrine and sanguinarine inhibited the binding of the CB(1) receptor agonist [(3)H]CP-55940 tomouse whole brain membranes at low micromolar concentrations (IC(50)s: chelerythrine 2.20 µM; sanguinarine 1.10µM). The structurally related isoquinoline alkaloids (berberine and papaverine) and the phthalide isoquinoline ((-)-β-hydrastine) were either inactive or considerably below IC(50) at 30 µM. Chelerythrine and sanguinarine antagonizedCP-55940-stimulated binding of [(35)S] GTPγ S to the G-protein (IC(50)s: chelerythrine 2.09 µM; sanguinarine 1.22µM). In contrast to AM251, both compounds strongly inhibited basal binding of [(35)S]GTPγ S (IC(50)s: chelerythrine10.06 µM; sanguinarine 5.19µM). Piperonyl butoxide and S-methoprene inhibited the binding of [(3)H]CP-55940(IC(50)s: piperonyl butoxide 8.2 µM; methoprene 16.4 µM), and also inhibited agonist-stimulated (but not basal)binding of [(35)S]GTPγ S to brain membranes (IC(50)s: piperonyl butoxide 22.5 µM; (S)-methoprene 19.31 µM). PMSFdid not modify the inhibitory effect of (S)-methoprene on [(3)H]CP-55940 binding. Our data suggest that chelerythrineand sanguinarine are efficacious antagonists of G-protein-coupled CB(1) receptors. They exhibit lower potenciescompared to many conventional CB(1) receptor blockers but act differently to AM251. Reverse modulation of CB(1)receptor agonist binding resulting from benzophenanthridines engaging with the G-protein component may explain thisdifference. Piperonyl butoxide and (S)-methoprene are efficacious, low potency, neutral antagonists of CB(1)receptors. Certain of the study compounds may represent useful starting structures for development of novel/morepotent G-protein-coupled CB(1) receptor blocking drugs.

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505. Protein kinase C modulates NMDA receptors in the myenteric plexus of the guinea pig ileum during in vitro ischemiaand reperfusion

By Giaroni C; Zanetti E; Giuliani D; Oldrini R; Marchet S; Moro E; Borroni P; Trinchera M; Crema F; Lecchini S; et alFrom Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society (2011),23(2), e91-103, Language: English, Database: MEDLINE

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BACKGROUND: Ischemic episodes lead to profound functional and structural alterations of the gastrointestinal tractwhich may contribute to disorders of intestinal motility. Enhancement of glutamate overflow and the consequentactivation of NMDA (N-methyl-D-aspartate) receptors may participate to such changes by modulating different entericneurotransmitter systems, including cholinergic motor pathways. METHODS: The molecular mechanism/s underlyingactivation of NMDA receptors in the guinea pig ileum were investigated after glucose/oxygen deprivation (in vitroischemia) and during reperfusion. KEY RESULTS: The number of ileal myenteric neurons positive for NR1, thefunctional subunit of NMDA receptors, and its mRNA levels were unchanged after in vitro ischemia/reperfusion. Inthese conditions, the protein levels of NR1, and of its phosphorylated form by protein kinase C (PKC), significantlyincreased in myenteric neurons, whereas, the levels of NR1 phosphorylated by protein kinase A (PKA) did not change,with respect to control values. Spontaneous glutamate overflow increased during in vitro ischemia/reperfusion. Inthese conditions, the NMDA receptor antagonists, D(-)-2-amino-5-phosphonopentanoic acid [(D)-AP5] (10 µmol L(-1))and 5,7-dichlorokynurenic acid (5,7-diClKyn acid) (10 µmol L(-1)) and the PKC antagonist, chelerythrine (1 µmol L(-1)),but not the PKA antagonist, H-89 (1 µmol L(-1)), were able to significantly depress the increased glutamate efflux.CONCLUSIONS & INFERENCES: The present data suggest that in the guinea pig ileum during in vitroischemia/reperfusion, NR1 protein levels increase. Such event may rely upon posttranscriptional events involving NR1phosphorylation by PKC. Increased NR1 levels may, at least in part, explain the ability of NMDA receptors to modulatea positive feedback on ischemia/reperfusion-induced glutamate overflow.

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506. Fos expression induced by activation of NMDA and neurokinin-1 receptors in the trigeminal subnucleus caudalis invitro: role of protein kinases

By Allen Ceri E; Worsley Matthew A; King Anne E; Boissonade Fiona MFrom Brain research (2011), 136819-27, Language: English, Database: MEDLINE

Activity-induced neuronal plasticity is partly facilitated by the expression of the immediate-early gene c-fos and theresulting transcription factor Fos. Expression of Fos is associated with nociceptive afferent activation, but a detailedstimulation-transcription pathway for Fos expression has not yet been determined in the trigeminal system. This studyutilized a novel in vitro model to determine whether Fos expression can be induced in trigeminal subnucleus caudalisby NMDA or neurokinin-1 receptor activation, and whether inhibition of intracellular kinases has any effect on Fosexpression induced by activation of these receptors. Brainstems of male Wistar rats were excised and maintained inartificial cerebrospinal fluid at 37°C. NMDA or the specific neurokinin-1 receptor agonist [Sar(9),Met(O(2))(11)]-SP wasapplied. These agonists were subsequently tested in the presence of the protein kinase A inhibitor Rp-cAMP or proteinkinase C inhibitor chelerythrine chloride. In all experiments the sodium channel blocker tetrodotoxin was used toprevent indirect neuronal activation. Brainstems were processed immunocytochemically for Fos expression, andpositive cells were counted in the trigeminal subnucleus caudalis. NMDA and [Sar(9),Met(O(2))(11)]-SP significantlyincreased Fos expression, but these increases could be prevented by chelerythrine chloride. Rp-cAMP had no effecton Fos induced by NMDA but caused a significant reduction in Fos induced by [Sar(9),Met(O(2))(11)]-SP. These datademonstrate that in trigeminal subnucleus caudalis activation of either NK1 or NMDA receptors alone induces Fosexpression; protein kinases A and C are involved in NK1R-induced Fos while protein kinase A is not required forNMDA receptor-induced Fos.

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507. Exercise preconditioning initiates late cardioprotection against isoproterenol-induced myocardial injury in ratsindependent of protein kinase C

By Shen Yu-Jun; Pan Shan-Shan; Zhuang Tao; Wang Feng-Juan

From The journal of physiological sciences : JPS (2011), 61(1), 13-21, Language: English, Database: MEDLINEThe objective of this study was to investigate the late cardioprotective effect of exercise preconditioning (EP) onisoproterenol (ISO)-induced myocardial injury in rats and the role of protein kinase C (PKC) in EP. Rats were injectedwith ISO 24 h after running on a treadmill for four periods of 10 min each at 28-30 m/min with intervening periods ofrest of 10 min. Nonselective PKC inhibitor chelerythrine (CHE) was injected before EP. The myocardial injury wasevaluated quantitatively in terms of the serum cardiac troponin I (cTnI) levels, the myocardial ischemia/hypoxia area,and the integral optical density (IOD) of haematoxylin-basic fuchsin-picric acid (HBFP) staining, and qualitatively interms of the myocardial ultrastructure. EP markedly attenuated the ISO-induced myocardial ischemia/hypoxia andultrastructural damage with lower serum cTnI levels. CHE injection before EP did not block the protective effect of EP,displaying a mild myocardial ischemia/hypoxia and well-preserved ultrastructure with even lower serum cTnI levels.The results indicate that EP can exert a late cardioprotection against ISO-induced myocardial injury, and that aninjection of the nonselective PKC inhibitor CHE before EP may have a different effect on ISO-induced myocardialinjury. Further investigation needs to be conducted to define the role of different PKC isozymes in EP by usingisozyme-selective inhibitors.

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508. Opioid peptide deltorphin II simulates the cardioprotective effect of ischemic preconditioning: role of δ -opioidreceptors, protein kinase C, and K(ATP) channels

By Maslov L N; Barzakh E I; Krylatov A V; Chernysheva G A; Krieg T; Solenkova N V; Lishmanov A Yu; Cybulnikov SYu; Zhang Y

From Bulletin of experimental biology and medicine (2010), 149(5), 591-3, Language: English, Database: MEDLINEThe cardioprotective properties of a δ -opioid receptor agonist deltorphin II were studied in rats with coronary occlusionand reperfusion. Opioid receptor ligands and inhibitors (glybenclamide, chelerythrine, and 5-hydroxydecanoate) wereinjected intravenously before ischemia and reperfusion. A δ -opioid receptor agonist deltorphin II significantlydecreased the infarction zone/risk zone index. This effect was abolished by naltrexone, naloxone methiodide, and δ -opioid receptor antagonist naltriben, but not by a δ -opioid receptor antagonist BNTX. The infarct-limiting effect ofdeltorphin II was not observed after inhibition of protein kinase C or blockade of mitochondrial K(ATP) channels.

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509. Isoflurane preconditioning increases endothelial cell tolerance to in-vitro simulated ischaemia

By Feng Jifeng; Zuo ZhiyiFrom The Journal of pharmacy and pharmacology (2011), 63(1), 106-10, Language: English, Database: MEDLINE

OBJECTIVES: Isoflurane preconditioning has been shown to protect endothelial cells against lipopolysaccharide andcytokine induced injury. This study was designed to determine whether isoflurane preconditioning increasedendothelial cell tolerance to ischaemia. METHODS: Bovine pulmonary arterial endothelial cells were exposed or notexposed to various concentrations of isoflurane for 1 h. After a 30-min isoflurane-free period, cells were subjected tooxygen-glucose deprivation (OGD) for 3 h and reoxygenation for 1 h. Lactate dehydrogenase release from cells wasused to measure cell injury. In some experiments, various protein kinase C (PKC) inhibitors and ATP-sensitivepotassium channel (K(ATP) channel) inhibitors were present from 30 min before isoflurane treatment to the end ofisoflurane treatment. KEY FINDINGS: Isoflurane preconditioning dose-dependently decreased the OGD inducedlactate dehydrogenase release. This protection was inhibited by 2 µM chelerythrine, a general PKC inhibitor, or 10 µMGo6976, an inhibitor for the conventional PKCs. This protection was also inhibited by 0.3 µM glybenclamide, a generalK(ATP) channel inhibitor, and 500 µM 5-hydroxydecanoate, a mitochondrial K(ATP) channel blocker. In addition,pretreatment with 100 µM diazoxide, a K(ATP) channel activator, for 1 h also reduced OGD induced endothelial cellinjury. This diazoxide induced protection was inhibited by chelerythrine. CONCLUSIONS: The results suggest thatisoflurane preconditioning induces endothelial protection against in-vitro simulated ischemia. This protection may bemediated at least in part by conventional PKCs and mitochondrial K(ATP) channels. The results also indicate thatPKCs may be downstream of K(ATP) channels in causing endothelial protection.

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510. Inactivation of the Ecs ABC transporter of Staphylococcus aureus attenuates virulence by altering composition andfunction of bacterial wall

By Jonsson Ing-Marie; Juuti Jarmo T; Francois Patrice; AlMajidi Rana; Pietiainen Milla; Girard Myriam; Lindholm

Catharina; Saller Manfred J; Driessen Arnold J M; Kuusela Pentti; et alFrom PloS one (2010), 5(12), e14209, Language: English, Database: MEDLINE

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BACKGROUND: Ecs is an ATP-binding cassette (ABC) transporter present in aerobic and facultative anaerobic gram-positive Firmicutes. Inactivation of Bacillus subtilis Ecs causes pleiotropic changes in the bacterial phenotype includinginhibition of intramembrane proteolysis. The molecule(s) transported by Ecs is (are) still unknown.METHODOLOGY/PRINCIPAL FINDINGS: In this study we mutated the ecsAB operon in two Staphylococcus aureusstrains, Newman and LS-1. Phenotypic and functional characterization of these Ecs deficient mutants revealed adefect in growth, increased autolysis and lysostaphin sensitivity, altered composition of cell wall proteins including theprecursor form of staphylokinase and an altered bacterial surface texture. DNA microarray analysis indicated that theEcs deficiency changed expression of the virulence factor regulator protein Rot accompanied by differential expressionof membrane transport proteins, particularly ABC transporters and phosphate-specific transport systems, protein A,adhesins and capsular polysaccharide biosynthesis proteins. Virulence of the ecs mutants was studied in a mousemodel of hematogenous S. aureus infection. Mice inoculated with the ecs mutant strains developed markedly milderinfections than those inoculated with the wild-type strains and had consequently lower mortality, less weight loss,milder arthritis and decreased persistence of staphylococci in the kidneys. The ecs mutants had higher susceptibility toribosomal antibiotics and plant alkaloids chelerythrine and sanguinarine. CONCLUSIONS/SIGNIFICANCE: Ourresults show that Ecs is essential for staphylococcal virulence and antimicrobial resistance probably since the transportfunction of Ecs is essential for the normal structure and function of the cell wall. Thus targeting Ecs may be a newapproach in combating staphylococcal infection.

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511. Protein kinase C regulates urea permeability in the rat inner medullary collecting duct

By Wang Yanhua; Klein Janet D; Liedtke Carole M; Sands Jeff MFrom American journal of physiology. Renal physiology (2010), 299(6), F1401-6, Language: English, Database:MEDLINE

Hypertonicity increases urea transport independently of, as well as synergistically with, vasopressin in the innermedullary collect duct (IMCD). We previously showed that hypertonicity does not increase the level of cAMP in theIMCD, but it does increase the level of intracellular calcium. Since we also showed that hypertonicity increases boththe phosphorylation and biotinylation of the urea transporters UT-A1 and UT-A3, this would suggest involvement of acalcium-dependent protein kinase in the regulation of urea transport in the inner medulla. In this study, we investigatedwhether protein kinase C (PKC), which is present in the IMCD, is a regulator of urea permeability. We tested the effectof PKC inhibitors and activators on urea permeability in the isolated, perfused rat terminal IMCD. Increasing osmolalityfrom 290 to 690 mosmol/kgH(2)O significantly stimulated (doubled) urea permeability; it returned to control levels oninhibition of PKC with either 10 µM chelerythrine or 50 µM rottlerin. To determine the potential synergy betweenvasopressin and PKC, phorbol dibutyrate (PDBu) was used to stimulate PKC. Vasopressin stimulated ureapermeability 247%. Although PDBu alone did not change basal urea permeability, in the presence of vasopressin, itsignificantly increased urea permeability an additional 92%. The vasopressin and PDBu-stimulated urea permeabilitywas reduced to AVP alone levels by inhibition of PKC. We conclude that hypertonicity stimulates urea transportthrough a PKC-mediated phosphorylation. Whether PKC directly phosphorylates UT-A1 and/or UT-A3 orphosphorylates it as a consequence of a cascade of activations remains to be determined.

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512. Rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase C epsilon

By Filone Claire Marie; Hanna Sheri L; Caino M Cecilia; Bambina Shelly; Doms Robert W; Cherry SaraFrom PloS one (2010), 5(11), e15483, Language: English, Database: MEDLINE

As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector andmammalian hosts. Little is known about the cellular requirements for infection in either host. Here we developed atissue culture model for RVFV infection of human and insect cells that is amenable to high-throughput screening.Using this approach we screened a library of 1280 small molecules with pharmacologically defined activities andidentified 59 drugs that inhibited RVFV infection with 15 inhibiting RVFV replication in both human and insect cells.Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C. Furtherstudies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human andinsect cells as well as in adult flies. Altogether, these data show that inhibition of cellular factors required for earlysteps in the infection cycle including PKCε can block RVFV infection, and may represent a starting point for thedevelopment of anti-RVFV therapeutics.

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513. Cardioprotective PKG-independent NO signaling at reperfusion

By Cohen Michael V; Yang Xi-Ming; Liu Yanping; Solenkova Nataliya V; Downey James MFrom American journal of physiology. Heart and circulatory physiology (2010), 299(6), H2028-36, Language: English,Database: MEDLINE

Cell models of ischemic preconditioning (IPC) indicate nitric oxide (NO) is involved in protection accruing duringreoxygenation but disagree whether it acts through PKG. Using a more relevant intact heart model, we studied

isolated rabbit hearts subjected to 30-min coronary artery occlusion/120-min reperfusion. We previously foundprotection from PKG activator 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (CPT-cGMP) atreperfusion was blocked by A(2b) adenosine receptor (A(2b)AR), ERK, or phosphatidylinositol 3-kinase (PI3-kinase)blockers. In this investigation A(2b)AR agonist BAY 60-6583 or CPT-cGMP at reperfusion reduced infarctioncomparably to IPC. Their protection was abrogated by N(ω)-nitro-l-arginine methyl ester (l-NAME), suggesting a PKG-independent NO synthase in IPC's mediator pathway downstream of PKG and A(2b)AR. NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) at reperfusion also protected. This protection was not blocked by PI3-kinase inhibitorwortmannin or ERK antagonist PD-98059, suggesting NO acted downstream of these kinases. Protection from SNAPwas not affected by mitochondrial ATP-sensitive K(+) channel closer 5-hydroxydecanoate, PKC antagonistchelerythrine, reactive oxygen species scavenger N-2-mercaptopropionylglycine, or soluble guanylyl cyclaseantagonist 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Absence of ODQ effect indicated NO was actingindependently of PKG. BAY 58-2667, a soluble guanylyl cyclase activator, was protective, and l-NAME blocked itsinfarct-sparing effect, indicating a second signaling event dependent on NO generation but independent of PKG.SB216763, a blocker of glycogen synthase kinase-3β (GSK-3β), decreased infarct size, and its infarct-sparing effectwas not affected by l-NAME, suggesting GSK-3β acted downstream or independently of NO. Hence, NO signaling

occurs in IPC's mediator pathway downstream of Akt and ERK, and its protection is independent of PKG.

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514. AT receptor and NAD(P)H oxidase mediate angiotensin II-stimulated antioxidant enzymes and mitogen-activatedprotein kinase activity in the rat hypothalamus

By Silva Jose; Pastorello Mariella; Arzola Jorge; Zavala Lida E; De Jesus Sara; Varela Maider; Matos Maria Gabriela;del Rosario Garrido Maria; Israel AnitaFrom Journal of the renin-angiotensin-aldosterone system : JRAAS (2010), 11(4), 234-42, Language: English,Database: MEDLINE

INTRODUCTION: Angiotensin II (AngII) regulates blood pressure and water and electrolyte metabolism through thestimulation of NAD(P)H oxidase and production of reactive oxygen species (ROS) such as O , which is metabolised bysuperoxide dismutase, catalase and glutathione peroxidase. We assessed the role of AT and AT receptors, NAD(P)Hoxidase and protein kinase C (PKC) in Ang II-induced sodium and water excretion and their capacity to stimulateantioxidant enzymes in the rat hypothalamus, a brain structure known to express a high density of AngII receptors.MATERIALS AND METHODS: Male Sprague-Dawley rats were intracerebroventricularly (ICV) injected with AngII andurinary sodium and water excretion was assessed. Urine sodium concentration was determined using flamephotometry. After decapitation the hypothalamus was microdissected under stereomicroscopic control. Superoxidedismutase, catalase and glutathione peroxidase activity were determined spectrophotometrically and extracellularsignal-regulated kinase (ERK1/2) activation was analysed by Western blot. RESULTS: AngII-ICV resulted inantidiuresis and natriuresis. ICV administration of losartan, PD123319, apocynin and chelerythrine blunted natriuresis.In hypothalamus, AngII increased catalase, superoxide dismutase and glutation peroxidase activity and ERK1/2phosphorylation. These actions were prevented by losartan, apocynin and chelerythrine, and increased by PD123319.CONCLUSIONS: AT and AT receptors, NAD(P)H oxidase and PKC pathway are involved in the regulation ofhydromineral metabolism and antioxidant enzyme activity induced by AngII.

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515. Mechanism of inhibition of acetylcholine secretion in newly formed mouse synapses involving Ca2+-dependentkinases and voltage-gated K+-channels

By Bogatcheva P O; Ezhova E V; Balezina O PFrom Bulletin of experimental biology and medicine (2010), 149(2), 170-3, Language: Russian, Database: MEDLINE

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Nifedipine, a blocker of L-type Ca(2+)-channels, increased quantal content of endplate potentials in newly formednerve-muscle synapses, while R 24571 (calmodulin inhibitor) and KN 62 (inhibitor of calmodulin-dependent kinase II)did not change it. KN 62 suppressed the increase in quantal content of endplate potentials evoked by nifedipine.Similar to nifedipine, chelerythrine and bisindolylmaleimide I (blockers of protein kinase C) increased quantal content ofendplate potentials. In the presence of chelerythrine, nifedipine lost its ability to facilitate secretion of neurotransmitter.4-Aminopyridine, a blocker of voltage-gated potassium channels, increased quantal content of endplate potentials. Inthe presence of chelerythrine, 4-aminopyridine induced no additional increase in the quantal content of endplatepotentials. In its turn, chelerythrine induced no extra facilitation of secretion in the presence of 4-aminopyridine. It ishypothesized that Ca(2+)-dependent inhibition of secretion results from suppression of calmodulin-dependent kinase IIand activation of protein kinase C, which potentiates the work of voltage-gated K(+)-channels.

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516. Phytochemical and antimicrobial characterization of Macleaya cordata herb

By Kosina Pavel; Gregorova Jana; Gruz Jiri; Vacek Jan; Kolar Milan; Vogel Mathias; Roos Werner; Naumann Kathrin;Simanek Vilim; Ulrichova JitkaFrom Fitoterapia (2010), 81(8), 1006-12, Language: English, Database: MEDLINE

Macleaya cordata (plume poppy) is a source of bioactive compounds, mainly isoquinoline alkaloids which are used inphytopreparations with anti-inflammatory and antimicrobial activities. In this study, the alkaloids sanguinarine,chelerythrine, their dihydro derivatives, protopine and allocryptopine and phenolics, gallic, protocatechuic, p-

hydroxybenzoic, m-hydroxybenzoic, gentisic, p-coumaric, caffeic, ferulic and sinapic acids were determined in extractsprepared from M. cordata aerial part, seeds, and seed capsules using HPLC with UV detection and/or LC/MS withelectrospray ionization. The highest content of sanguinarine and chelerythrine was found in capsules. Protopine andallocryptopine were major alkaloids in leaves including footstalks. The seed oil contained dihydrosanguinarine,dihydrochelerythrine and twelve fatty acids of which linoleic, oleic, palmitic and stearic acids predominated. In addition,sanguinarine reductase, a key enzyme in sanguinarine/dihydrosanguinarine equilibrium in plants, was found for thefirst time, in the soluble proteins of leaves. Finally, extracts were tested for antimicrobial activity using the microdilutionmethod on standard reference bacterial strains.

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517. Anti-allodynic effect of the flavonoid myricetin in a rat model of neuropathic pain: Involvement of p38 and proteinkinase C mediated modulation of Ca2+ channels

By Hagenacker Tim; Hillebrand Imke; Wissmann Andreas; Busselberg Dietrich; Schafers MariaFrom European journal of pain (London, England) (2010), 14(10), 992-8, Language: English, Database: MEDLINE

Flavonoids are increasingly ingested by the population as chemotherapeutic and anti-inflammatory agents. Myricetin isa naturally occurring flavonoid known for its anti-neoplastic and anti-inflammatory effects. Recently, behavioral studiesindicate a potential analgesic effect in animal models of pain. Pilot studies suggest a flavonoid-induced modulation ofintracellular protein kinases and interactions with voltage activated calcium channels. The aim of this study was toinvestigate the analgesic effect of myricetin in a neuropathic pain model (spinal nerve ligation, SNL) in rats. To identifypotential mechanisms of action, in vitro whole cell patch-clamp recordings of isolated rat dorsal root ganglia (DRG)neurons were performed to analyze the modulation of voltage activated calcium channel currents (I(Ca(V))) and theinfluence of intracellular kinase phosphorylation such as p38 mitogen-activated protein kinase (p38) or protein kinase C(PKC). In vivo, a single injection of myricetin (0.1-10 mg/kg i.p.) reduced SNL-induced mechanical allodynia andthermal hyperalgesia lasting for several hours. In vitro, I(Ca(V)) (depolarization from -80 to 0 mV) were reduced (10-56%) by low (0.1-5 µM) concentrations of myricetin. This decrease was abolished by blockade of PKC (20 µMchelerythrine for 30 min), but not of p38 (10 µM SB203580 for 30 min). In contrast, higher (10-100 µM) concentrationsof myricetin induced an increase of I(Ca(V)) (20-40%), which was blocked by inhibition of p38, but not of PKC. Weconclude that myricetin transiently reduces established neuropathic pain behavior. This analgesic effect may berelated to its PKC-induced decrease of I(Ca(V)) in DRG neurons.

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518. The study of effects and mechanism of U50, 488H on electrical coupling during ischemia in the perfused isolated ratheart

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By Mao Hong-Jiao; Chen Bao-Ping; Wang Hui-Ping; Gao Yun-Feng; Xia QiangFrom Zhongguo ying yong sheng li xue za zhi = Zhongguo yingyong shenglixue zazhi = Chinese journal of appliedphysiology (2010), 26(3), 261-5, Language: Chinese, Database: MEDLINE

OBJECTIVE: To determine the effect of activation of lambda-opioid receptor with U50, 488H, a selective kappa-opioidreceptor agonist, on the changes in electrical coupling during prolonged ischemia and to explore the possiblemechanism. METHODS: The isolated rat heart was perfused in a Langendorff apparatus. The effect of U50, 488H onelectrical coupling parameters including onset of uncoupling, plateau time, slope and fold increase in r(t) was observedin isolated perfused rat heart subjected to global no-flow ischemia. The effect of U50, 488H on connexin 43 (Cx43)expression of ventricular muscle during ischemia was determined by immunohistochemistry. RESULTS: In theprolonged ischemia model, U50, 488H concentration dependently delayed the onset of uncoupling, increased time to

plateau, and decreased the maximal rate of uncoupling during ischemia. The effect of U50, 488H on electricaluncoupling parameters during ischemia was abolished by a selective kappa-opioid receptor antagonist nor-BNI or aPKC inhibitor chelerythrine. The amount of Cx43 immunoreactive signal in ventricular muscle was greatly reducedafter ischemia. U50, 488H markedly increased Cx43 expression during ischemia and its effect was also attenuated bynor-BNI or chelerythrine. CONCLUSION: These results demonstrated that U50, 488H delayed the onset ofuncoupling and plateau time, decreased the maximal rate of uncoupling and increased Cx43 expression of ventricularmuscle during ischemia, and these effects of U50, 488H were mediated by kappa-opioid receptor, in which activationof PKC was involved. The effect of U50, 488H on electrical coupling during ischemia was probably correlated withpreservation of Cx43 in cardiac muscle.

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519. Protein kinase C activation modulates reversible increase in cortical blood-brain barrier permeability and tight junction protein expression during hypoxia and posthypoxic reoxygenation

By Willis Colin L; Meske Diana S; Davis Thomas PFrom Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flowand Metabolism (2010), 30(11), 1847-59, Language: English, Database: MEDLINE

Hypoxia (Hx) is a component of many disease states including stroke. Ischemic stroke occurs when there is arestriction of cerebral blood flow and oxygen to part of the brain. During the ischemic, and subsequent reperfusionphase of stroke, blood-brain barrier (BBB) integrity is lost with tight junction (TJ) protein disruption. However, themechanisms of Hx and reoxygenation (HR)-induced loss of BBB integrity are not fully understood. We examined therole of protein kinase C (PKC) isozymes in modifying TJ protein expression in a rat model of global Hx. The Hx (6%O(2)) induced increased hippocampal and cortical vascular permeability to 4 and 10 kDa dextran fluoresceinisothiocyanate (FITC) and endogenous rat-IgG. Cortical microvessels revealed morphologic changes in nPKC-θdistribution, increased nPKC-θ and aPKC-ζ protein expression, and activation by phosphorylation of nPKC-θ (Thr538)

and aPKC-ζ (Thr410) residues after Hx treatment. Claudin-5, occludin, and ZO-1 showed disrupted organization atendothelial cell margins, whereas Western blot analysis showed increased TJ protein expression after Hx. The PKCinhibition with chelerythrine chloride (5 mg/kg intraperitoneally) attenuated Hx-induced hippocampal vascularpermeability and claudin-5, PKC (θ and ζ) expression, and phosphorylation. This study supports the hypothesis thatnPKC-θ and aPKC-ζ signaling mediates TJ protein disruption resulting in increased BBB permeability.

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520. Impaired flow-induced dilation of coronary arterioles of dogs fed a low-salt diet: roles of ANG II, PKC, and NAD(P)Hoxidase

By Huang An; Yan Changdong; Suematsu Nobuhiro; Cuevas Azita; Yang Yang-Ming; Kertowidjojo Elizabeth; HintzeThomas H; Kaley Gabor; Sun DongFrom American journal of physiology. Heart and circulatory physiology (2010), 299(5), H1476-83, Language: English,Database: MEDLINE

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Low-salt (LS) diet has been considered to be beneficial in the prevention and treatment of hypertension; however, italso increases plasma angiotensin (ANG) II and may cause adverse cardiovascular effects, such as endothelialdysfunction. We assessed endothelial function of coronary arterioles and vascular superoxide production, as afunction of LS diet. Dogs were fed with LS (0.05% NaCl) or a normal-salt (NS, 0.65% NaCl) diet for 2 wk. There werethreefold increases in plasma ANG II, associated with a 60% reduction in flow-induced dilation (FID) in coronaryarterioles of LS compared with NS dogs. In vessels of NS dogs, FID was primarily mediated by nitric oxide (NO), asindicated by an eliminated FID by N(ω)-nitro-l-arginine methyl ester (l-NAME). In vessels of LS dogs, however, FIDwas eliminated. Administration of apocynin, a NAD(P)H oxidase inhibitor, partially restored FID and additional l-NAMEeliminated FID. Generation of superoxide, measured with dihydroethidium, was significantly greater in vessels of LSthan in NS dogs, which was further increased in response to ANG II or phorbol 12,13-dibutyrate, an agonist of proteinkinase C (PKC). The enhanced superoxide was normalized by apocynin, losartan (a blocker of angiotensin type 1receptor), and chelerythrine chloride (an antagonist of PKC). Western blotting indicated an upregulation of gp91(phox)and p47(phox), associated with increased expression of phosphorylated PKC in vessels of LS dogs. In separateexperiments, dogs were fed simultaneously with LS and losartan (LS + Losa) for 2 wk. There was a significantincrease in plasma ANG II in LS + Losa dogs, which, however, was associated with normal FID and gp91(phox)expression in coronary arterioles. In conclusion, LS led to endothelial dysfunction, as indicated by an impaired flow-induced dilation caused by decreasing NO bioavailibility, a response that involves angiotensin-induced activation ofPKC that, in turn, activates vascular NAD(P)H oxidase to produce superoxide.

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521. Protein kinase C pathway is involved in the inhibition by crocetin of vascular smooth muscle cells proliferation

By Zhou Cheng-Hua; Xiang Min; He Shu-Ying; Qian Zhi-YuFrom Phytotherapy research : PTR (2010), 24(11), 1680-6, Language: English, Database: MEDLINE

Crocetin is a natural carotenoid compound isolated from Gardenia jasminoids Ellis. Our previous study showed thatcrocetin inhibits angiotensin II (Ang II)-induced proliferation of vascular smooth muscle cells (VSMCs). The presentstudy investigated the involvement of the protein kinase C (PKC) pathway in the growth-inhibitory action of crocetin inVSMCs. The findings showed that PKC activity in the membrane fraction of VSMCs increased following stimulationwith Ang II, which was suppressed significantly by pretreating the cells with crocetin. Inhibition of PKC activity bycrocetin appeared to be associated with growth inhibition in VSMCs, because chelerythrine chloride, a specific PKCinhibitor, likewise decreased cell proliferation. PKC-a, a conventional PKC isoform, was detected in bovine aortaVSMCs by RT-PCR and western blotting analysis. Crocetin inhibited Ang II-induced membrane translocation of PKC-a, and the inhibition of crocetin on PKC activity in membrane fraction coincided with its suppression on membranetranslocation of PKC-a. In addition, Ang II-induced mRNA expressions of c-fos, c-jun and c-myc were also decreasedby crocetin. Taken together, the data suggest that the inhibition by crocetin of PKC activity, at least in part due toinactivation of PKC-a, and the subsequent suppression of proto-oncogene expressions might mediate its inhibitoryeffect on VSMCs proliferation.

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522. Phorbol 12-myristate 13-acetate inhibits the antilipolytic action of insulin, probably via the activity of protein kinaseCε

By Nakamura JiroFrom European journal of pharmacology (2010), 648(1-3), 188-94, Language: English, Database: MEDLINE

Protein kinase CβI (PKCβI) mediates insulin signaling and attenuates β1-adrenoceptor-stimulated lipolysis. In thiswork, the effect of the PKC activator phorbol 12-myristate 13-acetate (PMA) on the antilipolytic action of insulin wasdetermined by analyzing lipolysis induced by a β3-adrenoceptor agonist CL 316243. PMA inhibited the insulinantilipolytic action. The pan-PKC inhibitors GF 109203X and chelerythrine inhibited the PMA effect, but the PKCα / βinhibitors Go 6976 and CGP 53353 did not. Exposure of cells to PMA downregulated PKCs α, βI, and δ within 3 h andPKCε within 12 h. The effect of PMA on insulin action greatly diminished when PKCε was downregulated. Inhibitors ofphosphatidylinositol 3-kinase (PI3-K), Akt, and phosphodiesterase 3B (PDE3B) diminished the PMA effect. PMAinhibited insulin-stimulated phosphorylation of Tyr in insulin receptor β subunit and Ser/Thr in Akt. These data suggestthat PMA inhibits the antilipolytic signal mediated by the insulin receptor, PI3-K, Akt, and PDE3B. The most probabletarget of PMA is PKCε.

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523. Consecutive pharmacological activation of PKA and PKC mimics the potent cardioprotection of temperaturepreconditioning

By Khaliulin Igor; Parker Joanna E; Halestrap Andrew PFrom Cardiovascular research (2010), 88(2), 324-33, Language: English, Database: MEDLINE

AIMS: Temperature preconditioning (TP) provides very powerful protection against ischaemia/reperfusion.Understanding the signalling pathways involved may enable the development of effective pharmacologicalcardioprotection. We investigated the interrelationship between activation of protein kinase A (PKA) and protein kinaseC (PKC) in the signalling mechanisms of TP and developed a potent pharmacological intervention based on thismechanism. METHODS AND RESULTS: Isolated rat hearts were subjected to TP, 30 min global ischaemia, and 60

min reperfusion. Other control and TP hearts were perfused with either sotalol (β-adrenergic blocker) or H-89 (PKAinhibitor). Some hearts were pre-treated with either isoproterenol (β-adrenergic agonist) or adenosine (PKC activator)that were given alone, simultaneously, or sequentially. Pre-treatment with isoproterenol, adenosine, and theconsecutive isoproterenol/adenosine treatment was also combined with the PKC inhibitor chelerythrine.Cardioprotection was evaluated by haemodynamic function recovery, lactate dehydrogenase release, measurement ofmitochondrial permeability transition pore opening, and protein carbonylation during reperfusion. Cyclic AMP and PKAactivity were increased in TP hearts. H-89 and sotalol blocked the cardioprotective effect of TP and TP-induced PKCactivation. Isoproterenol, adenosine, and the consecutive treatment increased PKC activity during pre-ischaemia.Isoproterenol significantly reduced myocardial glycogen content. Isoproterenol and adenosine, alone orsimultaneously, protected hearts but the consecutive treatment gave the highest protection. Cardioprotective effects ofadenosine were completely blocked by chelerythrine but those of the consecutive treatment only attenuated.CONCLUSION: The signal transduction pathway of TP involves PKA activation that precedes PKC activation.Pharmacologically induced consecutive PKA/PKC activation mimics TP and induces extremely potent cardioprotection.

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524. Distinctions between persistent and reversible group I mGluR-induced epileptiform burst prolongation

By Fuortes Michaelangelo G; Rico Marjorie J; Merlin Lisa RFrom Epilepsia (2010), 51(8), 1633-7, Language: English, Database: MEDLINE

We have previously shown that selective activation of group I metabotropic glutamate receptors (mGluRs) results inlong-lasting enhancement of synchronized network activity in the hippocampal slice. Data herein suggest thatactivation of group I mGluRs need not result in this potentially epileptogenic effect. (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid (ACPD), a nonselective mGluR agonist, elicits ictaform bursts identical in appearance to thoseinduced by selective agonists, but ACPD-induced bursts do not persist following removal of the agent. Like the burstsinduced by selective agonist, the ACPD bursts are blocked with group I mGluR antagonists and are not dependent onactivation of either N-methyl-D-aspartate (NMDA) receptors or protein kinase C. However, they differ from thepersistent bursts in that they do not require active protein synthesis and they are not suppressed with L-cysteinesulfinic acid, an agonist at a phospholipase D-coupled metabotropic receptor. These novel findings provide evidencethat group I mGluR-induced epileptogenesis may be preventable.

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525. Mechanism of cardioprotective effect of erythropoietin-induced preconditioning in rat heart

By Garg Kavita; Yadav Harlokesh N; Singh Manjeet; Sharma P LFrom Indian journal of pharmacology (2010), 42(4), 219-23, Language: English, Database: MEDLINE

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OBJECTIVE: The cardioprotective potential of human recombinant erythropoietin (alpha) (Epo) against ischemia-reperfusion-induced injury is well known. But, the underlying mechanisms are not well elucidated. The aim of thisstudy was to characterize the mechanism involved in the cardioprotective effect of Epo-induced preconditioning inisolated rat heart. MATERIALS AND METHODS: The heart was mounted on a Langendorff apparatus. After 10 minof stabilization, four cycles of ischemic preconditioning (IPC) were given followed by 30 min of global ischemia and 120min of reperfusion. Epo preconditioning was induced by four cycles of 5-min perfusion of K-H solution containing Epo(1.0 U/ml) followed by 5 min perfusion with K-H solution. Myocardial infarct size was estimated macroscopically usingthe triphenyltetrazolium chloride staining technique. The extent of myocardial injury was measured by release oflactate dehydrogenase and creatine kinase-MB in the coronary effluent. RESULTS: The present study demonstratesthat Epo preconditioning was almost as effective as IPC. Administration of Wortmannin (100 nM), a PI-3K inhibitor, orChelerythrine (1 µM), a protein kinase-C (PKC) inhibitor, or AG490 (5 µM), a JAK-2 inhibitor, significantly attenuatedthe cardioprotective effects of Epo-induced preconditioning. CONCLUSION: Our result suggest that thecardioprotective potential of Epo-induced preconditioning in isolated rat heart was due to an interplay of the JAK-2, PI-3K and PKC pathways. Inhibition of any one of the three pathways was sufficient to block the cardioprotective effect ofEpo-induced preconditioning in isolated rat heart.

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526. Angiotensin II inhibits the electrogenic Na+/HCO3- cotransport of cat cardiac myocytes

By De Giusti Veronica C; Orlowski Alejandro; Aiello Ernesto AFrom Journal of molecular and cellular cardiology (2010), 49(5), 812-8, Language: English, Database: MEDLINE

The Na(+)/HCO(3)(-) cotransporter (NBC) plays an important role in intracellular pH (pH(i)) regulation in the heart. Inthe myocardium co-exist the electrogenic (eNBC) and electroneutral (nNBC) isoforms of NBC. We have recentlyreported that angiotensin II (Ang II) stimulated total NBC activity during the recovery from intracellular acidosis througha reactive oxygen species (ROS) and ERK-dependent pathway. In the present work we focus our attention on eNBC.In order to study the activity of the eNBC in isolation, we induced a membrane potential depolarization by increasingextracellular K(+) [K(+)](o) from 4.5 to 45 mM (K(+) pulse). This experimental protocol enhanced eNBC driving forceleading to intracellular alkalization (0.19 ± 0.008, n=6; data expressed as an increase of pH(i) units after 14 min ofapplying the K(+) pulse). This alkalization was completely abrogated by the NBC blocker S0859 (-0.004 ± 0.016*, n=5;* indicates p<0.05 vs control) but not by the Na(+)/H(+) exchanger blocker HOE642 (0.185 ± 0.04, n=4), indicating thatwe are exclusively measuring eNBC. The K(+) pulse induced alkalization was canceled by 100 nM Ang II (-0.008 ±0.018*; n=5). This inhibitory effect was prevented when the myocytes were incubated with losartan (AT(1) receptorblocker, 0.18 ± 0.02; n=4) or SB202190 (p38 MAP kinase inhibitor, 0.25 ± 0.06; n=5). Neither chelerythrine (PKCinhibitor, -0.06 ± 0.04*; n=4), nor U0126 (ERK inhibitor, -0.07 ± 0.04*; n=4) nor MPG (ROS scavenger, -0.02 ± 0.05*;n=8) affected the Ang II-induced inhibition of eNBC. The inhibitory action of Ang II on eNBC was corroborated withperforated patch-clamp experiments, since no impact of the current produced by eNBC on action potentialrepolarization was observed in the presence of Ang II. In conclusion, we propose that Ang II, binding to AT(1)receptors, exerts an inhibitory effect on eNBC activity in a p38 kinase-dependent manner.

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527. TRPC6 channels stimulated by angiotensin II are inhibited by TRPC1/C5 channel activity through a Ca2+- andPKC-dependent mechanism in native vascular myocytes

By Shi J; Ju M; Saleh S N; Albert A P; Large W AFrom The Journal of physiology (2010), 588(Pt 19), 3671-82, Language: English, Database: MEDLINE

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The present work investigated interactions between TRPC1/C5 and TRPC6 cation channel activities evoked byangiotensin II (Ang II) in native rabbit mesenteric artery vascular smooth muscle cells (VSMCs). In low intracellularCa(2+) buffering conditions (0.1 mm BAPTA), 1 nm and 10 nm Ang II activated both 2 pS TRPC1/C5 channels and 15-45 pS TRPC6 channels in the same outside-out patches. However, increasing Ang II to 100 nm abolished TRPC6activity but further increased TRPC1/C5 channel activity. Comparison of individual patches revealed an inverserelationship between TRPC1/C5 and TRPC6 channel activity suggesting that TRPC1/C5 inhibits TRPC6 channelactivity. Inclusion of anti-TRPC1 and anti-TRPC5 antibodies, raised against intracellular epitopes, in the patch pipettesolution blocked TRPC1/C5 channel currents but potentiated by about six-fold TRPC6 channel activity evoked by 1-100 nm Ang II in outside-out patches. Bath application of T1E3, an anti-TRPC1 antibody raised against anextracellular epitope, also increased Ang II-evoked TRPC6 channel activity. With high intracellular Ca(2+) bufferingconditions (10 mm BAPTA), 10 nm Ang II-induced TRPC6 channel activity was increased by about five-fold comparedto channel activity with low Ca(2+) buffering. In addition, increasing intracellular Ca(2+) levels ([Ca(2+)](i)) at thecytosolic surface inhibited 10 nm Ang II-evoked TRPC6 channel activity in inside-out patches. Moreover, in zeroexternal Ca(2+) (0 [Ca(2+)](o)) 100 nm Ang II induced TRPC6 channel activity in outside-out patches. Pre-treatmentwith the PKC inhibitor, chelerythrine, markedly increased TRPC6 channel activity evoked by 1-100 nm Ang II andblocked the inhibitory action of [Ca(2+)](i) on TRPC6 channel activity. Co-immunoprecipitation studies shows that AngII increased phosphorylation of TRPC6 proteins which was inhibited by chelerythrine, 0 [Ca(2+)](o) and the anti-TRPC1antibody T1E3. These results show that TRPC6 channels evoked by Ang II are inhibited by TRPC1/C5-mediatedCa(2+) influx and stimulation of PKC, which phosphorylates TRPC6 subunits. These conclusions represent a novelinteraction between two distinct vasoconstrictor-activated TRPC channels expressed in the same native VSMCs.

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528. Corticosterone decreases the activity of rat glutamate transporter type 3 expressed in Xenopus oocytes

By Kang Maehwa; Ryu Junghee; Kim Jin-Hee; Na Hyoseok; Zuo Zhiyi; Do Sang-HwanFrom Steroids (2010), 75(13-14), 1113-8, Language: English, Database: MEDLINE

Glucocorticoids can increase the extracellular concentrations of glutamate, the major excitatory neurotransmitter. Weinvestigated the effects of corticosterone on the activity of a glutamate transporter, excitatory amino acid carrier 1(EAAC1; also called excitatory amino acid transporter type 3 [EAAT3]), and the roles of protein kinase C (PKC) andphosphatidylinositol 3-kinase (PI3K) in regulating these effects. Rat EAAC1 was expressed in Xenopus oocytes byinjecting mRNA. L-Glutamate (30 µM)-induced membrane currents were measured using the two-electrode voltageclamp technique. Exposure of these oocytes to corticosterone (0.01-1 µM) for 72 h decreased EAAC1 activity in adose-dependent fashion, and this inhibition was incubation time-dependent. Corticosterone (0.01 µM for 72 h)significantly decreased the V(max), but not the K(m), of EAAC1 for glutamate. Furthermore, pretreatment of oocyteswith staurosporine, a PKC inhibitor, significantly decreased EAAC1 activity (1.00±0.06 to 0.70±0.05 µC; P<0.05).However, no statistical differences were observed between oocytes treated with staurosporine, corticosterone, orcorticosterone plus staurosporine. Similar patterns of responses were achieved by chelerythrine or calphostin C, otherPKC inhibitors. Phorbol-12-myristate-13-acetate (PMA), a PKC activator, inhibited corticosterone-induced reduction inEAAC1 activity. Pretreating oocytes with wortmannin or LY294002, PI3K inhibitors, also significantly reduced EAAC1activity, but no difference was observed between oocytes treated with wortmannin, corticosterone, or wortmannin pluscorticosterone. The above results suggest that corticosterone exposure reduces EAAC1 activity and this effect is PKC-and PI3K-dependent.

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529. Toll-like receptor 2 mediates inhibition of HCO(3)(-) absorption by bacterial lipoprotein in medullary thick ascendinglimb

By Good David W; George Thampi; Watts Bruns A 3rdFrom American journal of physiology. Renal physiology (2010), 299(3), F536-44, Language: English, Database:MEDLINE

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Bacterial infection and sepsis are associated with renal tubule dysfunction and dysregulation of systemic electrolytebalance but the underlying mechanisms are incompletely understood. Recently, we demonstrated that HCO(3)(-)absorption by the medullary thick ascending limb (MTAL) is inhibited by gram-negative bacterial LPS through activationof Toll-like receptor 4 (TLR4). Here, we examined whether MTAL transport is altered by activation of TLR2, thereceptor predominantly responsible for recognizing gram-positive bacteria. Confocal immunofluorescence showedexpression of TLR2 in the basolateral membrane domain of rat and mouse MTALs. The functional role of TLR2 wasexamined in perfused MTALs using Pam(3)CSK(4), a bacterial lipoprotein analog that specifically activates TLR2.Adding Pam(3)CSK(4) to the bath decreased HCO(3)(-) absorption by 25%. The inhibition by Pam(3)CSK(4) waseliminated in MTALs from TLR2(-/-) mice. HCO(3)(-) absorption was also inhibited by the TLR2 agonists lipoteichoicacid and peptidoglycan, two cell wall components of gram-positive bacteria. The MEK/ERK inhibitor U0126 eliminatedinhibition of HCO(3)(-) absorption by bath LPS but had no effect on inhibition by Pam(3)CSK(4). The inhibition byPam(3)CSK(4) was eliminated by the protein kinase C inhibitors chelerythrine Cl and bisindolylmaleimide. Moreover,the inhibition by Pam(3)CSK(4), lipoteichoic acid, and peptidoglycan was additive to inhibition by LPS. Thus, agonistsof basolateral TLR2 and TLR4 inhibit HCO(3)(-) absorption independently through distinct signaling pathways. Weconclude that bacterial components act directly through TLRs to modify the transport function of renal tubules. Duringpolymicrobial sepsis, gram-positive bacterial molecules acting through TLR2 and gram-negative LPS acting throughTLR4 can function through parallel signaling pathways to impair MTAL transport. The inhibition of luminal acidificationmay impair the ability of the kidneys to correct systemic acidosis that contributes to sepsis pathogenesis.

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530. Effects of GH secretagogues on contractility and Ca2+ homeostasis of isolated adult rat ventricular myocytes

By Sun Qiang; Ma Yi; Zhang Lin; Zhao Yu-Feng; Zang Wei-Jin; Chen ChenFrom Endocrinology (2010), 151(9), 4446-54, Language: English, Database: MEDLINE

Ghrelin and its synthetic analogue hexarelin are specific ligands of GH secretagogue receptor (GHS-R) and induce avariety of cardiovascular protective and cardiac positive inotropic effects. The signaling system underlying immediateeffects of both GHSs in cardiomyocytes remains undefined. In the present study, we investigated the immediateeffects of GHSs on isolated ventricular myocyte shortening, intracellular Ca(2+) ([Ca(2+)](i)) transients, and the L-typeCa(2+) current (I(Ca,L)). Putative intracellular signalling cascades were studied with specific receptor and signallingblockers. In fresh isolated adult Wistar rat ventricular myocytes, GHSs produced a positive inotropic effect in aconcentration-dependent manner and increased the amplitude of [Ca(2+)](i) transients and the I(Ca,L). The positiveinotropic response was abolished by the GHS-R1a antagonist [D-Lys(3)]-GH-releasing peptide-6 (10 microm). GHS-induced increase in the I(Ca,L) was abolished by [D-Lys(3)]-GH-releasing peptide-6 and protein kinase C inhibitor,chelerythrine chloride (5 microm), but not by protein kinase A inhibitor, KT 5720 (10 microm). We conclude thathexarelin and ghrelin increase the I(Ca,L), through GHS-R1a receptor and protein kinase C signalling cascade, whichcontribute to its direct positive inotropic effect on cardiomyocytes.

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531. Muscarinic modulation of TASK-like background potassium channel in rat carotid body chemoreceptor cells

By Ortiz Fernando C; Varas RodrigoFrom Brain research (2010), 132374-83, Language: English, Database: MEDLINE

The carotid body is the main peripheral arterial chemoreceptor and it is essential to initiate the cardiovascular andrespiratory compensatory reflex responses to a decrease in the arterial oxygen. The carotid body chemoreceptor(type-I) cells respond to hypoxia with membrane depolarization, voltage-gated Ca(2+) entry and secretion oftransmitters. A key step in this response is the inhibition of a TASK-like background K(+) current. It has been reportedthat TASK-K(+) channels can be modulated by G-protein coupled receptors, such as the muscarinic acetylcholinereceptor (mAChRs). Since there is a proposed role for ACh as an autocrine/paracrine modulator of the carotid bodyfunction, we have investigated the possible regulation of the background K(+) current by mAChRs. In identified type-Icells, methacholine (100microM) or muscarine (50microM) increased intracellular Ca(2+) levels. In cell-attached patchrecordings, TASK-K(+) background channel activity was reduced by approximately 50% during mAChR activation andby the diacylglycerol analogue oleoylacetylglycerol (OAG, 20microM). The co-application of both metacholine andOAG do not further inhibit K(+) channel activity. In addition, two chemically different inhibitors of protein kinase Cactivity, calphostin C (100nM) and chelerythrine (50microM) are both able to suppress the muscarinic inhibition of theTASK-like K(+) channel and to increase channel activity in the absence of mAChR agonists. Our results suggest amuscarinic regulation of the TASK-like K(+) current in rat carotid body type-I cells through a PLC/PKC-dependentpathway. Additionally, our findings are consistent with an autocrine/paracrine role for cholinergic autoreceptors presentwithin the carotid body.

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532. Role of protein kinase Czeta in thrombin-induced RhoA activation and inter-endothelial gap formation of humandermal microvessel endothelial cell monolayers

By Minshall Richard D; Vandenbroucke Emily E; Holinstat Michael; Place Aaron T; Tiruppathi Chinnaswamy; VogelStephen M; van Nieuw Amerongen Geerten P; Mehta Dolly; Malik Asrar B

From Microvascular research (2010), 80(2), 240-9, Language: English, Database: MEDLINEWe studied the potential involvement of the Ca(2+)-independent atypical protein kinase C isoform PKCzeta inmediating the thrombin-induced increase in endothelial permeability. Studies were done using human dermalmicrovessel endothelial cells (HMEC), which we showed constitutively expressed PKCzeta. We quantified the patencyof inter-endothelial junctions (IEJs) and endothelial barrier function by measuring transendothelial electrical resistance(TER) in confluent HMEC monolayers. In control monolayers, thrombin decreased TER by approximately 50%,indicating thrombin-dependent opening of IEJs. Thrombin also elicited increases in cytosolic Ca(2+) concentration[Ca(2+)](i), actin stress fiber formation, and myosin light chain (MLC) phosphorylation. Pan-PKC inhibitors, calphostinC and chelerythrine, abrogated these responses. Thrombin also decreased TER after depletion of conventional andnovel Ca(2+)-dependent PKC isoforms using phorbol 12-myristate 13-acetate (PMA). In these PMA-treated cells,thrombin induced inter-endothelial gap formation, MLC phosphorylation, and actin stress fiber formation, but failed toincrease [Ca(2+)](i). Inhibition of PKCzeta activation using the PKCzeta pseudosubstrate peptide (PSI), depletion ofPKCzeta protein with siRNA, and competitive inhibition of PKCzeta activity using dominant-negative (dn) PKCzetamutant all prevented the thrombin-induced decrease in TER and MLC phosphorylation. Expression of dn-PKCzetaalso inhibited thrombin-induced RhoA activation. These findings reveal a novel Ca(2+)-independent, PKCzeta-dependent mechanism of thrombin-induced increase in endothelial permeability. The results raise the possibility thatinhibition of PKCzeta may be a novel drug target for thrombin-induced inflammatory hyperpermeability.

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533. FMRFamide-like peptides (FLPs) enhance voltage-gated calcium currents to elicit muscle contraction in the humanparasite Schistosoma mansoni

By Novozhilova Ekaterina; Kimber Michael J; Qian Hai; McVeigh Paul; Robertson Alan P; Zamanian Mostafa; MauleAaron G; Day Tim AFrom PLoS neglected tropical diseases (2010), 4(8), e790, Language: English, Database: MEDLINE

Schistosomes are amongst the most important and neglected pathogens in the world, and schistosomiasis controlrelies almost exclusively on a single drug. The neuromuscular system of schistosomes is fertile ground for therapeuticintervention, yet the details of physiological events involved in neuromuscular function remain largely unknown. Shortamidated neuropeptides, FMRFamide-like peptides (FLPs), are distributed abundantly throughout the nervous systemof every flatworm examined and they produce potent myoexcitation. Our goal here was to determine the mechanismby which FLPs elicit contractions of schistosome muscle fibers. Contraction studies showed that the FLP Tyr-Ile-Arg-Phe-amide (YIRFamide) contracts the muscle fibers through a mechanism that requires Ca(2+) influx throughsarcolemmal voltage operated Ca(2+) channels (VOCCs), as the contractions are inhibited by classical VOCC blockersnicardipine, verapamil and methoxyverapamil. Whole-cell patch-clamp experiments revealed that inward currentsthrough VOCCs are significantly and reversibly enhanced by the application of 1 microM YIRFamide; the sustainedinward currents were increased to 190% of controls and the peak currents were increased to 180%. In order toexamine the biochemical link between the FLP receptor and the VOCCs, PKC inhibitors calphostin C, RO 31-8220 andchelerythrine were tested and all produced concentration dependent block of the contractions elicited by 1 microMYIRFamide. Taken together, the data show that FLPs elicit contractions by enhancing Ca(2+) influx through VOCCcurrents using a PKC-dependent pathway.

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534. Modulation of Ca(2+) release through ryanodine receptors in vascular smooth muscle by protein kinase Calpha

By Peng HongLi; Yaney Gordon C; Kirber Michael TFrom Pflugers Archiv : European journal of physiology (2010), 460(4), 791-802, Language: English, Database:MEDLINE

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The role of protein kinase C (PKC) in Ca(2+) release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum(SR) of vascular smooth muscle cells (SMCs) is not well understood. Caffeine was used to activate RyRs and theintracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in both freshly isolated and cultured mouse aortic SMCs(ASMCs). Pre-activation of PKC with 1,2-dioctanoyl-sn-glycerol (DOG) prevented caffeine-induced [Ca(2+)](i)transients. Application of the PKC inhibitor calphostin C caused [Ca(2+)](i) transients which were not blocked bynifedipine or by removing extracellular Ca(2+) but were abolished after inhibition of the SR Ca(2+)-ATPase withthapsigargin or after inhibition of RyRs with ryanodine. In addition, chelerythrine and GF109203X also elevated resting[Ca(2+)](i) but no further [Ca(2+)](i) increase was seen with subsequent application of caffeine. Selective inhibition ofPKCalpha with safingol blocked caffeine-induced [Ca(2+)](i) transients, but the PKCepsilon inhibitory peptide V1-2 didnot. In cells expressing a EGFP-tagged PKCalpha, caffeine-induced [Ca(2+)](i) transients were associated with a rapidfocal translocation near the cell periphery, while application of ionomycin and DOG caused translocation to the plasmamembrane. Western blot showed that caffeine increased the relative amount of PKCalpha in the particulate fraction ina time-dependent manner. Co-immunoprecipitation of RyRs and PKCalpha indicated that they interact. In conclusion,our studies suggest that PKC activation can inhibit the gating activity of RyRs in the SR of ASMCs, and this regulationis most likely mediated by the Ca(2+)-dependent PKCalpha isoform.

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535. Synergistic neuroprotective effect of combined low concentrations of galantamine and melatonin against oxidativestress in SH-SY5Y neuroblastoma cells

By Romero Alejandro; Egea Javier; Garcia Antonio G; Lopez Manuela G

From Journal of pineal research (2010), 49(2), 141-8, Language: English, Database: MEDLINEMelatonin is a potent free radical scavenger, antioxidant and neuroprotective drug. On the other hand, galantamine isa cholinergic drug with antioxidant and neuroprotective properties linked to inhibition of acetylcholinesterase andallosteric modulation of nicotinic receptors. This investigation evaluated a possible synergistic neuroprotective effect ofsubeffective concentrations of combined galantamine and melatonin. Human neuroblastoma SH-SY5Y cells weresubjected to a mitochondrial oxidative stress, by blockade of mitochondrial complexes I and V with rotenone andoligomycin-A (R/O); cells were treated for 24 hr with R/O. This caused 40% of the cell to die as measured by lactatedehydrogenase (LDH) release. Cell incubation with increasing concentrations of galantamine (10-300 nm) ormelatonin (0.3-10 nm) for 24 hr, followed by a 24-hr period with R/O, caused a concentration-dependent protection;maximum protection was achieved with 300 nm galantamine (56% protection) and 10 nm melatonin (50% protection).Combination of subeffective concentrations of melatonin (0.3 nm) and galantamine (30 nm) caused a synergistic andsignificant protection that was similar to the maximum protection afforded by effective concentrations of melatonin orgalantamine alone. This protective effect was completely reversed when nicotinic and melatonin receptors wereblocked respectively by mecamylamine and luzindole. The neuroprotective effect was prevented by chelerythrine,LY294002, and Sn (IV) protoporphyrin IX dichloride (SnPP), indicating the participation of the PKC/PI3K/Akt activationand induction of the antioxidant enzyme heme oxygenase-1. The synthesis of novel multitarget compounds having ina single molecule the combined neuroprotective properties of galantamine and melatonin could be a new strategy forpotential therapeutic agents in neurodegenerative diseases.

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536. Peripheral acid-sensing ion channels and P2X receptors contribute to mechanical allodynia in a rodent thrombus-induced ischemic pain model

By Seo Hyoung-Sig; Roh Dae-Hyun; Yoon Seo-Yeon; Kang Suk-Yun; Moon Ji-Young; Kim Hyun-Woo; Han Ho-Jae;Chung Jin Mo; Beitz Alvin J; Lee Jang-Hern

From The journal of pain : official journal of the American Pain Society (2010), 11(8), 718-27, Language: English,Database: MEDLINE

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We have previously established a thrombus-induced ischemic pain (TIIP) model in the rat, which mimics thepathophysiology of ischemic pain in patients with peripheral arterial disease. Because ischemia commonly inducesacidosis and ATP release, one of the goals of this study was to investigate the role of acid-sensing ion channels(ASICs), transient receptor potential vanilloid-1 (TRPV1) receptors, and P2X receptors in the maintenance of ischemia-induced mechanical allodynia (MA). To test this, amiloride (an ASIC blocker), AMG-9810 (a TRPV1 blocker), orPPADS (a P2Xs antagonist) was intraplantarly injected at day 3 after FeCl(2) application onto the femoral artery.Ipsilateral administration of amiloride or PPADS but not AMG-9810 dose-dependently reduced MA. However,contralateral amiloride or PPADS did not suppress contralateral MA. Interestingly, co-administration of submaximaldoses of amiloride and PPADS produced a significantly prolonged suppression of MA. Furthermore, ipsilateral EGTA(a calcium chelator) or chelerythrine (a protein kinase C inhibitor) also significantly reduced MA. Collectively, thesefindings suggest that peripheral ASICs and P2X receptors are involved in the maintenance of TIIP, which is possiblymediated by a Ca(2+)-protein kinase C signaling mechanism. These results provide mechanistic information aboutperipheral ischemic nociception that may be useful for developing better therapeutic management of ischemic pain inpatients with peripheral arterial disease. PERSPECTIVE: The results of the current study demonstrate that peripheraladministration of an ASICs blocker or P2X antagonist significantly suppress TIIP. Co-administration of submaximaldoses of ASIC and P2X antagonists produced an even greater effect. These results implicate peripheral ASICs andP2X receptors in the maintenance of thrombus-induced ischemic pain.

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537. Protein kinase A and C signaling induces bilirubin potentiation of GABA/glycinergic synaptic transmission in ratventral cochlear nucleus neurons

By Li Chun-Yan; Shi Hai-Bo; Chen Zheng-Nong; Ye Hai-Bo; Song Ning-Ying; Yin Shan-KaiFrom Brain research (2010), 134830-41, Language: English, Database: MEDLINE

Previous studies have suggested that bilirubin can potentiate GABA/glycinergic synaptic transmission in lateralsuperior olivary nucleus neurons, but the cellular mechanism has not been defined. The present study evaluated thepossible roles of protein kinase A (PKA) and C (PKC) in bilirubin potentiation of GABA/glycinergic synaptictransmission in rat ventral cochlear nucleus (VCN) neurons. VCN neurons were acutely isolated from postnatal 10-12-day-old (P10-12) rats and were voltage-clamped in whole-cell mode. Miniature inhibitory postsynaptic currents(mIPSC) frequencies, but not amplitude, were increased by bilirubin. Forskolin (PKA activator) and H-89 (PKAinhibitor) also individually increased mIPSCs frequency, with an additional increase induced by co-incubation withbilirubin and H-89. Pretreatment with forskolin blocked bilirubin potentiation. mIPSC frequency was not altered byphorbol 12,13-diacetate (PKC activator), but mIPSC frequency was increased following co-application of bilirubin. ThemIPSC frequency was increased by chelerythrine (PKC inhibitor), and then further increased after the addition ofbilirubin. Neither H-89, forskolin, nor PDA, nor their co-application with bilirubin affected mIPSC amplitudes of GABA-activated (I(GABA))/glycine-activated (I(gly)) currents, suggesting a presynaptic locus of activity. Chelerythrinedecreased the mIPSC amplitudes and I(GABA)/I(gly), suggesting a postsynaptic locus of activity. These data suggestthat both PKA and PKC can modulate GABA and glycine release in rat VCN neurons. Bilirubin facilitates transmitterrelease via presynaptic PKA activation, which might provide insight into the cellular mechanism underlying bilirubin-induced hearing dysfunction.

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538. Obestatin affords cardioprotection to the ischemic-reperfused isolated rat heart and inhibits apoptosis in cultures ofsimilarly stressed cardiomyocytes

By Alloatti Giuseppe; Arnoletti Elisa; Bassino Eleonora; Penna Claudia; Perrelli Maria Giulia; Ghe Corrado; Muccioli

GiampieroFrom American journal of physiology. Heart and circulatory physiology (2010), 299(2), H470-81, Language: English,Database: MEDLINE

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Obestatin, a newly discovered peptide encoded by the ghrelin gene, induces the expression of genes regulatingpancreatic beta-cell differentiation, insulin biosynthesis, and glucose metabolism. It also activates antiapoptoticsignaling pathways such as phosphoinositide 3-kinase (PI3K) and ERK1/2 in pancreatic beta-cells and human islets.Since these kinases have been shown to protect against myocardial injury, we sought to investigate whether obestatinwould exert cardioprotective effects. Both isolated perfused rat heart and cultured cardiomyocyte models of ischemia-reperfusion (I/R) were used to measure infarct size and cell apoptosis as end points of injury. The presence of specificobestatin receptors on cardiac cells as well as the signaling pathways underlying the obestatin effect were alsostudied. In the isolated heart, the addition of rat obestatin-(1-23) before ischemia reduced infarct size and contractiledysfunction in a concentration-dependent manner, whereas obestatin-(23-1), a synthetic analog with an inverseaminoacid sequence, was ineffective. The cardioprotective effect of obestatin-(1-23) was observed at concentrationsof 10-50 nmol/l and was abolished by inhibiting PI3K or PKC by the addition of wortmannin (100 nmol/l) orchelerythrine, (5 micromol/l), respectively. In rat H9c2 cardiac cells or isolated ventricular myocytes subjected to I/R,50 nmol/l obestatin-(1-23) reduced cardiomyocyte apoptosis and reduced caspase-3 activation; the antiapoptotic effectwas blocked by the inhibition of PKC, PI3K, or ERK1/2 pathways. In keeping with these functional findings,radioreceptor binding results revealed the presence of specific high-affinity obestatin-binding sites, mainly localized onmembranes of the ventricular myocardium and cardiomyocytes. Our data suggest that, by acting on specific receptors,obestatin-(1-23) activates PI3K, PKC-epsilon, PKC-delta, and ERK1/2 signaling and protects cardiac cells againstmyocardial injury and apoptosis induced by I/R.

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539. Extractions of isoquinoline alkaloids with butanol and octanol

By Gregorova Jana; Babica Jan; Marek Radek; Paulova Hana; Taborska Eva; Dostal JiriFrom Fitoterapia (2010), 81(6), 565-8, Language: English, Database: MEDLINE

Six different isoquinoline alkaloids (sanguinarine, chelerythrine, berberine, coptisine, allocryptopine, and protopine)were extracted by butanol and octanol from aqueous solution, pH 4.5. The samples were analyzed by HPLC. Butanolextraction was non-selective, alkaloids passed into organic phase in 83-98%. Octanol extraction provided moreselective yields: sanguinarine 99%, chelerythrine 94%, berberine 18%, coptisine 16%, allocryptopine 7.5%, protopine7%. Further, we tested octanol treatment of extract from Dicranostigma lactucoides. The octanol extraction yieldswere also selective: sanguinarine 98%, chelerythrine 92%, chelirubine 92.5%, protopine 6% and allocryptopine 3.5%.6-Butoxy-5,6-dihydrosanguinarine and 6-butoxy-5,6-dihydrochelerythrine were prepared and their NMR and MS dataare reported and discussed.

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540. Effects of a protein kinase C inhibitor combined with cisplatin on non-small cell lung cancer

By Gao Zhi-qiang; Han Bao-hui; Sha Hui-fang; Shi Zhen-yu; Yang Xiao-hua; Feng Jiu-xianFrom Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratorydiseases (2010), 33(4), 284-8, Language: Chinese, Database: MEDLINE

OBJECTIVE: To study the effects and possible mechanisms of the protein kinase C (PKC) inhibitor chelerythrinechloride (CH), combined with cisplatin (DDP) on human non-small cell lung cancer. METHODS: The effect of CH,DDP and the combination on proliferation and apoptosis of human lung cancer cell line A549 were evaluated by MTTassay and flow cytometry respectively. The inhibitory effects of CH and DDP on neoplasia were verified onsubcutaneous implanted tumor of nude mice. Implanted tumor models were constructed in nude mice using humanlung adenocarcinoma cell line A549. Twenty-four BALB/c nude mice with implanted tumors were divided into 4 groupsrandomly: group CH, group DDP, group CH + DDP, and normal saline group (group NS), each with 5 mice. CH, DDPor NS were intraperitoneally injected into nude mice for 3 weeks (DDP or NS was injected once a week, CH wasinjected twice a week). RESULTS: CH inhibited A549 cell proliferation in a concentration-dependent pattern. WhenCH and DDP were combined, the inhibitory effect was enhanced in a synergistic or additive pattern. Both CH and DDPsignificantly increased the apoptosis of A549 cells, and this action was remarkably increased when DDP was combinedwith CH. CH and DDP inhibited the growth of subcutaneous implanted tumor in nude mice and the inhibitory rate ofgroup CH + DDP (80.5%) was significantly higher than that of group CH (72.4%) or group DDP (64.3%) (t = 11.34, P <0.01). CONCLUSION: CH combined with DDP shows significantly synergistic anti-tumor effects on non-small cell lungcancer cell line A549 and subcutaneous implanted tumor in nude mice, possibly by enhancement of growth inhibitionand apoptosis induction on tumor cells.

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541. Rod photoreceptor cell death is induced by okadaic acid through activation of PKC and L-type voltage-dependentCa2+ channels and prevented by IGF-1

By Adao-Novaes Juliana; de Cassia Belem Guterrres Ceulem; Linden Rafael; Sholl-Franco AlfredFrom Neurochemistry international (2010), 57(2), 128-35, Language: English, Database: MEDLINE

Retinal dystrophies involve extensive photoreceptor apoptosis. Neuroprotective effects of insulin-like growth factor(IGF)-1 have been demonstrated in various tissues, including the retina. The aim of this study was to investigate: (i)

the action of IGF-1 upon selective photoreceptor death induced by okadaic acid (OA); and (ii) signaling pathwaysrelated to both OA-induced cell death and IGF-1 neuroprotective effect. Retinal explants were incubated with 5nM OA,a protein phosphatase type 1 and type 2A inhibitor, which induces cell death detected by the identification of pyknoticmorphology of photoreceptors immunostained for rhodopsin. OA increased both the number of pyknotic Rho 4D2(+)profiles, and Ca(2+) influx, measured through the incorporation of (45)CaCl(2), in a dose- and time-dependent way,while treatment with 10ng/mL IGF-1 abrogated both effects. Treatment with phorbol 12-myristate 13-acetate (PMA),an activator of protein kinase C, modulated OA effects, indicating the involvement of PKC. Furthermore, either10microM chelerythrine chloride, an inhibitor of PKC, or 10microM nifedipine, a L-voltage-sensitive Ca(2+) channelblocker, inhibited both Ca(2+) influx and cell death induced by OA. The data show that okadaic acid induces rodphotoreceptor cell death in retinal tissue through activation of PKC and ensuing Ca(2+) influx through L-type Ca(2+)channels, which is counteracted by a neuroprotective effect of IGF-1.

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542. In vivo anthelmintic activity of five alkaloids from Macleaya microcarpa (Maxim) Fedde against Dactylogyrusintermedius in Carassius auratus

By Wang Gao-Xue; Zhou Zhuang; Jiang Dong-Xin; Han Jing; Wang Jian-Fu; Zhao Liang-Wei; Li JunFrom Veterinary parasitology (2010), 171(3-4), 305-13, Language: English, Database: MEDLINE

The present study aims to evaluate the anthelmintic properties of aerial part of Macleaya microcarpa (Maxim) Fedde.Bioassay-guided fractionation and isolation of the compounds with anthelmintic activity were performed on theethanolic extract of M. microcarpa yielding five bioactive alkaloids namely: sanguinarine, cryptopine, beta-allocryptopine, protopine and 6-methoxyl-dihydro-chelerythrine by comparing spectral data (UV, NMR, and EI-MS) withliterature values. According to in vivo anthelmintic assays, they were found to be 100% effective at the concentrationsof 0.7, 8.0, 8.0, 16.0 and 7.0 mgl(-1), and the median effective concentration (EC(50)) values for the five compoundswere 0.37, 3.31, 4.64, 8.13 and 3.63 mgl(-1), respectively. Additionally, the acute toxicity on goldfish for the five active

compounds was also investigated with median lethal concentrations (LC(50)) values of 1.13, 16.12, 15.88, 21.69 and10.91 mgl(-1), respectively. The resulting therapeutic indices for sanguinarine, cryptopine, beta-allocryptopine,protopine and 6-methoxyl-dihydro-chelerythrine were 3.03, 4.82, 3.40, 2.66 and 2.99 correspondingly. Correlationsanalysis between the logP and EC(50), LC(50) of the five alkaloids revealed that the activity of the five alkaloids waswell correlated with their hydrophobicity and r(2)=0.45 is for anthelmintic activity while r(2)=0.47 is for acute toxicity forgoldfish, respectively. These results provided evidence that the studied plant extract, as well as the isolatedcompounds, especially sanguinarine, might be potential plant-based medicines for the treatment of D. intermediusinfection.

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543. Role of protein kinase C in protecting rats against pulmonary ischemia reperfusion injury through opening ofmitochondrial ATP sensitive potassium channel

By Lu De-Qin; Chen Ying; Li Tao; Li Bao-LuoFrom Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition (2010), 41(3), 436-40, Language: Chinese, Database: MEDLINE

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OBJECTIVE: To investigate the effect of opening of mitochondrial ATP-sensitive potassium channel on inducing lungprotection against ischemia reperfusion injury (I/R) and the role of PKC in the protective effect. METHODS: Fifty-sixrats were divided into six groups, including sham-operation group, I/R group, DE (diazoxide) +I/R group, 5-HD (5-hydroxydecanoic acid) +DE+I/R group, CHE (chelerythrine) +DE+I/R group and 5-HD +PMA (phorbol 12-myristate 13-acetate)+ I/R group. Pulmonary I/R injury rat models were established by 45 min of left hilar clipping followed by 180min of reperfusion. Morphological changes in lung tissues were detected by HE staining. The wet-to-dry weight ratioof lung tissues was evaluated. Cytochrome C expression in lung tissues was assessed by immunohistochemicalstaining. TUNEL was used to determine apoptosis. Protein kinase C (PKC) activity in lung tissues was assessed byPepTag non-radioactive assay. RESULTS: Compared with I/R group, the lung tissue morphology of the rats in theDE+I/R group was preserved well and the wet-to-dry weight ratio, expression of cytochrome C and apoptosis indexdecreased significantly (P < 0.05). The PKC activity in the lung tissues of the rats in the DE+ I/R group increaseddramatically. Both pretreatment with 5-HD and CHE blocked the protective effect induced by DE preconditioning.There were no differences between 5-HD+PMA+I/R group and I/R group in the above indicators except for PKCactivation. These results showed that blocking of mitochondrial ATP-sensitive potassium channel by 5-HD did notprotect lung from ischemia reperfusion injury even though PKC were activated. CONCLUSION: Opening ofmitochondrial ATP-sensitive potassium channel plays an essential protective role in pulmonary ischemia reperfusioninjury. The pulmonary protection appears to be dependent on PKC activation.

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544. Activation of phosphatidylinositol-linked D1-like receptors increases spontaneous glutamate release in ratsomatosensory cortical neurons in vitro

By Chu Hong-Yuan; Yang Zhi; Zhao Bin; Jin Guo-Zhang; Hu Guo-Yuan; Zhen XuechuFrom Brain research (2010), 134320-7, Language: English, Database: MEDLINE

Central dopaminergic system exerts profound modulation on spontaneous glutamate release in various brain regionsmainly through D(1) receptor/cAMP/PKA pathway. It remains unclear whether the phosphatidylinositol (PI)-linked D(1)-like receptors are also involved in such modulatory actions. The identification of substituted phenylbenzazepineSKF83959 as the selective agonist for the atypical D(1)-like receptors has given impetus to study their influence on thespontaneous glutamate release in the brain. In the present study the effects of SKF83959 on the spontaneousexcitatory postsynaptic currents (sEPSCs) were investigated through whole-cell recording from layer V-VI pyramidalneurons in rat somatosensory cortical slices. Perfusion with SKF83959 (10-100 microM) considerably increased thefrequency of sEPSCs, while had no significant effect on the amplitude of sEPSCs. The increase of sEPSC frequencyby SKF83959 was blocked by SCH23390, a D(1)-like receptor antagonist, but not by the antagonists for D(2) receptor,alpha(1)-adrenoceptor and 5-HT(2A/2C) receptor. U-73122 (PLCbeta inhibitor), 2-APB (IP(3) receptor antagonist),chelerythrine chloride (PKC inhibitor) and capsazepine (TRPV1 antagonist) could block the effects of SKF83959,whereas H-89 (PKA inhibitor) and forskolin (adenylyl cyclase activator) had no effect. Taken together, sensitization ofTRPV1 channels by PKC after activation of D(1) receptor/PLCbeta signaling pathway mediated SKF83959-inducedincrease in the sEPSC frequency. To our knowledge, this is the first pharmacological evidence that PI-linked D(1)-likedopamine receptors do exist in presynaptic terminals of cortical neurons and play an important role in controlling thespontaneous glutamate release.

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545. Mechanisms of hydrogen peroxide-induced vasoconstriction in the isolated perfused rat kidney

By Moreno J M; Rodriguez Gomez I; Wangensteen R; Perez-Abud R; Duarte J; Osuna A; Vargas FFrom Journal of physiology and pharmacology : an official journal of the Polish Physiological Society (2010), 61(3),

325-32, Language: English, Database: MEDLINE

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The vasoconstrictor effect of hydrogen peroxide (H(2)O(2)) on isolated perfused rat kidney was investigated. H(2)O(2)induced vasoconstriction in the isolated rat kidney in a concentration-dependent manner. The vasoconstrictor effectsof H(2)O(2) were completely inhibited by 1200 U/ml catalase. Endothelium-removal potentiated the renal response toH(2)O(2). The H(2)O(2) dose-response curve was not significantly modified by administration of the NO inhibitor L-NAME (10(-4) mol/l), whereas it was increased by the non-specific inhibitor of K+-channels, tetraethylammonium(3.10(-3) mol/l). Separately, removal of extracellular Ca(2+), administration of a mixture of calcium desensitizingagents (nitroprusside, papaverine, and diazoxide), and administration of a protein kinase C (PKC) inhibitor(chelerythrine, 10(-5) mol/l) each significantly attenuated the vasoconstrictor response to H(2)O(2), which was virtuallysuppressed when they were performed together. The pressor response to H(2)O(2) was not affected by: dimethylsulfoxide (7.10(-5) mol/l) plus mannitol (3.10(-5) mol/l); intracellular Ca(2+) chelation using BAPTA (10(-5) mol/l);calcium store depletion after repeated doses of phenylephrine (10(-5) g/g kidney); or the presence of indomethacin(10(-5) mol/l), ODYA (2.10(-6) mol/l) or genistein (10(-5) mol/l). We conclude that the vasoconstrictor response toH(2)O(2) in the rat renal vasculature comprises the following components: 1) extracellular calcium influx, 2) activationof PKC, and 3) stimulation of pathways leading to sensitization of contractile elements to calcium. Moreover, areduced pressor responsiveness to H(2)O(2) in female kidneys was observed.

~2 Citings

546. A study on separation and extraction of four main alkaloids in Macleaya cordata (Willd) R. Br. with strip dispersionhybrid liquid membrane

By Ouyang Li; Su Xiaoli; He Dingsheng; Chen Yuanyuan; Ma Ming; Xie Qingji; Yao Shouzhuo

From Journal of separation science (2010), 33(13), 2026-34, Language: English, Database: MEDLINEA technique based on strip dispersion hybrid liquid membrane was developed for the separation and extraction of fourmain alkaloids from fruits of Macleaya cordata (Willd) R. Br. A microporous polypropylene membrane impregnatedwith an organic membrane solution comprised the heart of the strip dispersion hybrid liquid membrane system. Themembrane solution was made by dissolving a cationic carrier, di-(2-ethylhexyl) phosphoric acid in an inexpensive, lesstoxic membrane solvent, kerosene. The transport of alkaloids from an aqueous feed solution through the membrane toa strip dispersion phase was driven by the concentration gradient of H(+) and facilitated by di-(2-ethylhexyl) phosphoricacid. The effects of the extraction time and reuse times of the membrane, the strip solution composition, the carrierconcentration, the volume ratio of the aqueous strip solution to the organic membrane solution, and the flow rates ofthe feed solution and the strip dispersion phase on the transport of alkaloids were investigated. Under the optimalconditions, the permeability coefficients obtained for the four main alkaloids allocryptopine, protopine, sanguinarine,and chelerythrine were 1.66, 1.99, 2.98, and 3.06 x 10(-4) cm/s, and the transport efficiencies were as high as 68, 77,83, and 85%, respectively.

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547. Enhancement of mesenteric artery contraction to 5-HT depends on Rho kinase and Src kinase pathways in theob/ob mouse model of type 2 diabetes

By Matsumoto Takayuki; Kobayashi Tsuneo; Ishida Keiko; Taguchi Kumiko; Kamata KatsuoFrom British journal of pharmacology (2010), 160(5), 1092-104, Language: English, Database: MEDLINE

BACKGROUND AND PURPOSE: Arteries from hypertensive subjects are reportedly hyperresponsive to 5-hydroxytryptamine (5-HT), but it remains unclear whether this is true in chronic type 2 diabetes. We have assessedresponses to 5-HT shown by mesenteric arteries from type 2 diabetic ob/ob mice (27-32 weeks old) and have identifiedthe molecular mechanisms involved. EXPERIMENTAL APPROACH: Contractions of mesenteric rings to 5-HT wereexamined in vitro. Activation of mesenteric RhoA, Rho kinase and Src was measured by Western blotting or bymodified enzyme-linked immunosorbent assay. KEY RESULTS: Concentration-dependent contractions to 5-HT weregreater in mesenteric rings from the ob/ob than in those from the age-matched control ('Lean') group. In each group,there was no significant change in the 5-HT-induced contractions after inhibition of nitric oxide synthase (with N(G)-nitro-L-arginine), of cyclooxygenase (with indomethacin) or of protein kinase C (with chelerythrine). However inhibitionof the MEK/ERK pathway (with PD98059) decreased the response to 5-HT. Although the diabetes-relatedenhancement of the 5-HT response was preserved with each of these inhibitors, enhancement was abolished by a Rhokinase inhibitor (Y27632) and by Src kinase inhibitors (PP1 analogue or Src kinase inhibitor I). 5-HT-induced activationof RhoA, Rho kinase and Src kinase in mesenteric arteries was greater in the ob/ob than in the Lean group, but theexpression of RhoA, Rho kinase isoforms and Src did not differ between these groups. CONCLUSIONS ANDIMPLICATIONS: These results suggest that the enhancement of 5-HT-induced contraction in mesenteric arteries fromob/ob mice may be attributable to increased activation of RhoA/Rho kinase and Src kinase.

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548. Rho kinase activation and ROS production contributes to the cooling enhanced contraction in cutaneous equinedigital veins

By Zerpa H; Berhane Y; Woodcock H; Elliott J; Bailey S RFrom Journal of applied physiology (Bethesda, Md. : 1985) (2010), 109(1), 11-8, Language: English, Database:MEDLINE

A decrease in environmental temperature can directly affect the contractility of cutaneous vasculature, mediated in partby alpha(2)-adrenoceptors. Most of the cellular mechanisms underlying the cooling-enhanced contractility to alpha(2)-adrenoceptor agonists have been reported in cutaneous arteries but little information is available on cutaneous veins.To investigate the cellular mechanisms associated with the cooling-enhanced contraction to UK-14304 (alpha(2)-adrenoceptor agonist), isolated equine digital veins (EDVs) were studied at 30 degrees C and 22 degrees C. Theeffects of inhibitors were studied on the contractile response to UK-14304 (0.1 microM). The cooling-enhancedresponses were inhibited by Rho kinase inhibitors [maximum response to UK-14304 95.2 +/- 8% of response todepolarizing Krebs solution (DKS) in control vessels cooled to 22 degrees C, compared with 31.4 +/- 6% in thepresence of fasudil 1 microM and 75.8 +/- 6% with Y-27632 0.1 microM] and the effects of these inhibitors wereconsiderably less at 30 degrees C (control response 56.4 +/- 5% of DKS; 34.9 +/- 6% with fasudil 1 microM and 50.6+/- 9% with Y-27632 0.1 microM). Furthermore, Western blotting showed that one of the downstream targets for Rhokinase activity, ezrin/radixin/moesin, was phosphorylated after cooling and reduced by fasudil (1 microM) only at 22degrees C. The activation of protein kinase C contributed to the contractile response, but predominantly at 30 degreesC (maximum response 82.3 +/- 9% of DKS for control; 57.7 +/- 10% in the presence of chelerythrine 10 microM) withno significant effect at 22 degrees C. The reduction of the response at 22 degrees C by antioxidants, rotenone (14%

reduction), and tempol (21% reduction) suggested the contribution of reactive oxygen species (ROS). No evidencewas obtained to support the participation of tyrosine kinase. These data demonstrate that Rho kinase activation andthe production of ROS contributes to the cooling-enhanced contraction in these cutaneous digital veins.

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549. High glucose increases periostin expression and the related signal pathway in adult rat cardiac fibroblasts

By Zou Ping; Wu Li-Ling; Wu Dan; Fan Dong; Cui Xiao-Bing; Zhou Yun; Wang Cheng; Li LiFrom Sheng li xue bao : [Acta physiologica Sinica] (2010), 62(3), 247-54, Language: Chinese, Database: MEDLINE

Cardiac fibrosis is a major mechanism contributing to myocardial systolic and diastolic dysfunction in diabetic

cardiomyopathy. Periostin is a novel extracellular matrix protein, secreted from cardiac fibroblasts, and closely relatedwith cardiac fibrosis and remodeling. The present study aimed to investigate the effect of high glucose on periostinexpression and the related signal transduction pathway in cardiac fibroblasts. Adult rat cardiac fibroblasts werecultured and stimulated with high glucose (25 mmol/L). The mRNA and protein expressions of periostin were detectedby RT-PCR and Western blot, respectively. Intracellular reactive oxygen species (ROS) production was measuredusing 2, 7-dichlorofluorescein diacetate (DCF-DA), an oxidant-sensitive fluorescent probe. Results showed that themRNA expression of periostin in adult rat cardiac fibroblasts was increased by 117.26% when treated with highglucose for 12 h. Incubation with high glucose for 24 h enhanced periostin protein expression by up to 93.12%. Highglucose induced the production of ROS in adult rat cardiac fibroblasts, which was reduced by chelerythrine (CLT), aprotein kinase C (PKC) inhibitor. High glucose-induced periostin protein expression was decreased significantly whenpretreated with CLT or N-acetylcysteine (NAC), a ROS scavenger. The phosphorylation of c-jun N-terminal proteinkinase (JNK) was increased markedly when stimulated with high glucose for 30 and 60 min, which was abolished whenpretreated with CLT or NAC. SP600125, a specific JNK inhibitor, significantly decreased periostin expression inducedby high glucose. In conclusion, high glucose stimulates periostin protein expression via a PKC/ROS/JNK-dependentpathway in adult rat cardiac fibroblasts.

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550. Regulation of (pro)renin receptor expression by glucose-induced mitogen-activated protein kinase, nuclear factor-kappaB, and activator protein-1 signaling pathways

By Huang Jiqian; Siragy Helmy MFrom Endocrinology (2010), 151(7), 3317-25, Language: English, Database: MEDLINE

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Renal (pro)renin receptor (PRR) expression is increased in diabetes. The exact mechanisms involved in this processare not well established. We hypothesized that high glucose up-regulates PRR through protein kinase C (PKC)-Raf-ERK and PKC-c-Jun N-terminal kinase (JNK)-c-Jun signaling pathways. Rat mesangial cells exposed to 30 mm d-glucose demonstrated significant increase in PRR mRNA and protein expression, intracellular phosphorylation of Raf-1(Y340/341), ERK, JNK, nuclear factor-kappaB (NF-kappaB) p65 (S536) and c-Jun (S63). By chromatinimmunoprecipitation assay and EMSA, high glucose induced more functional NF-kappaB and activator protein (AP)-1dimers bound to corresponding cis-regulatory elements in the predicted PRR promoter to up-regulate PRRtranscription. Conventional and novel PKC inhibitors Chelerythrine and Rottlerin, Raf-1 inhibitor GW5074, MEK1/2inhibitor U0126, JNK inhibitor SP600125, NF-kappaB inhibitor Quinazoline, and AP-1 inhibitor Curcumin, respectively,attenuated glucose-induced PRR up-regulation. Chelerythrine and Rottlerin also inhibited glucose-inducedphosphorylation of Raf-1 (Y340/341), ERK1/2, JNK, NF-kappaB p65 (S536), and c-Jun (S63). GW5074 and U0126inhibited the phosphorylation of ERK1/2 and NF-kappaB p65 (S536). SP600125 inhibited phosphorylation of NF-kappaB p65 (S536) and c-Jun (S63). We conclude that high glucose up-regulates the expression of PRR throughmechanisms dependent on both PKC-Raf-ERK and PKC-JNK-c-Jun signaling pathways. NF-kappaB and AP-1 areinvolved in high-glucose-induced PRR up-regulation in rat mesangial cells.

~1 Citing

551. Water permeability through aquaporin-4 is regulated by protein kinase C and becomes rate-limiting for gliomainvasion

By McCoy E S; Haas B R; Sontheimer H

From Neuroscience (2010), 168(4), 971-81, Language: English, Database: MEDLINEGlial-derived tumors, gliomas, are highly invasive cancers that invade normal brain through the extracellular space. Tonavigate the tortuous extracellular spaces, cells undergo dynamic changes in cell volume, which entails water fluxacross the membrane through aquaporins (AQPs). Two members of this family, AQP1 and AQP4 are highlyexpressed in primary brain tumor biopsies and both have a consensus phosphorylation site for protein kinase C (PKC),which is a known regulator of glioma cell invasion. AQP4 colocalizes with PKC to the leading edge of invadingprocesses and clustered with chloride channel (ClC2) and K(+)-Cl(-) cotransporter 1 (KCC1), believed to provide thepathways for Cl(-) and K(+) secretion to accomplish volume changes. Using D54MG glioma cells stably transfectedwith either AQP1 or AQP4, we show that PKC activity regulates water permeability through phosphorylation of AQP4.Activation of PKC with either phorbol 12-myristate 13-acetate or thrombin enhanced AQP4 phosphorylation, reducedwater permeability and significantly decreased cell invasion. Conversely, inhibition of PKC activity with chelerythrinereduced AQP4 phosphorylation, enhanced water permeability and significantly enhanced tumor invasion. PKCregulation of AQP4 was lost after mutational inactivation of the consensus PKC phosphorylation site S180A.Interestingly, AQP1 expressing glioma cells, by contrast, were completely unaffected by changes in PKC activity. Todemonstrate a role for AQPs in glioma invasion in vivo, cells selectively expressing AQP1, AQP4 or the mutatedS180A-AQP4 were implanted intracranially into SCID mice. AQP4 expressing glioma cells showed significantlyreduced invasion compared to AQP1 and S180 expressing tumors as determined by quantitative stereology, consistentwith a differential role for AQP1 and AQP4 in this process.

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552. Tribulosin protects rat hearts from ischemia/reperfusion injury

By Zhang Shuang; Li Hong; Yang Shi-jieFrom Acta pharmacologica Sinica (2010), 31(6), 671-8, Language: English, Database: MEDLINE

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AIM: To investigate the protective effect of tribulosin, a monomer of the gross saponins from Tribulus terrestris,against cardiac ischemia/reperfusion injury and the underlying mechanism in rats. METHODS: Isolated rat heartswere subjected to 30 min of ischemia followed by 120 min of reperfusion using Langendorff's technique. The heartswere assigned to seven groups: control, ischemia/reperfusion (I/R), treatment with gross saponins from Tribulusterrestris (GSTT) 100 mg/L, treatment with tribulosin (100, 10, and 1 nmol/L) and treatment with a PKC inhibitor(chelerythrine) (1 micromol/L). Infarct size was assessed by triphenyltetrazolium chloride staining. Malondialdehyde(MDA), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) contents as well as superoxidedismutase (SOD) and creatine kinase (CK) activities were determined after the treatment. Histopathological changesin the myocardium were observed using hematoxylin-eosin (H&E) staining. Apoptosis was detected with terminaldeoxynucleotidyl transferase nick-end labeling (TUNEL) assay. Bcl-2, Bax, caspase-3, and PKCepsilon proteinexpression were examined using Western blotting. RESULTS: Tribulosin treatment significantly reduced MDA, AST,CK and LDH contents, and increased the activity of SOD. The infarct size of I/R group was 40.21% of the total area.GSTT and various concentrations of tribulosin treatment decreased the infarct size to 24.33%, 20.24%, 23.19%, and30.32% (P<0.01). Tribulosin treatment reduced the myocardial apoptosis rate in a concentration-dependent manner.Bcl-2 and PKCepsilon protein expression was increased after tribulosin preconditioning, whereas Bax and caspase-3expression was decreased. In the chelerythrine group, Bcl-2 and PKCepsilon expression was decreased, whereasBax and caspase-3 expression was increased. CONCLUSION: Tribulosin protects myocardium againstischemia/reperfusion injury through PKCepsilon activation.

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553. The anti-apoptosis effect of erythropoietin on neonatal rat cardiocytes during hypoxia/reoxygenation injury and its

possible mechanism

By Wang Hua-jun; Jiang Hui-lin; Chen Xiao-hui; Lin Pei-yi; Zhu Yong-cheng; Tao Li-liFrom Zhongguo wei zhong bing ji jiu yi xue = Chinese critical care medicine = Zhongguo weizhongbing jijiuyixue(2010), 22(5), 302-5, Language: Chinese, Database: MEDLINE

OBJECTIVE: To investigate the anti-apoptosis effect of erythropoietin (EPO) on myocardial cells afterhypoxia/reoxygenation in vitro, and the relationship among protein kinase C (PKC), the mitochondrial ATP-sensitivepotassium (mitoKATP) channel and EPO in the anti-apoptotic signaling pathways. METHODS: Cardiocytes wereharvested from neonatal rats and cultured. Cultured myocardial cells were divided into the control group, thehypoxia/reoxygenation group, the EPO group and the chelerythrine group, and a hypoxia/reoxygenation model ofcardiocytes was reproduced. Apoptosis rate was assayed by flow cytometry. Flavoprotein fluorescence was scannedby confocal laser microscope to assess the mitoKATP channel activity. RESULTS: Apoptosis rate was significantlyhigher in hypoxia/reoxygenation group than that of control group [(42.56+/-8.00)% vs. (17.88+/-2.00)%, P<0.05]. Therewas no statistically significant difference in flavoprotein fluorescence between this group and the control group[(0.278+/-0.170)x10(-2) vs. (0.149+/-0.050)x10(-2), P>0.05]. Myocardial cell apoptosis rate in EPO group was lowerthan that in hypoxia/reoxygenation group [(22.73+/-5.00)% vs. (42.56+/-8.00)%, P<0.05], and flavoprotein fluorescenceintensity was significantly enhanced when compared with hypoxia/reoxygenation group [(2.201+/-1.090)x10(-2) vs.(0.278+/-0.170)x10(-2), P<0.01]. However, when chelerythrine was added, the anti-apoptosis effect of EPO wasblocked, and the intensity of cardiocytes flavoprotein fluorescence was decreased [the apoptosis rate was (46.72+/-17.00)% and the flavoprotein fluorescence intensity was (0.986+/-0.320)x10(-2) ]. When compared with EPO groupthere was statistically significant difference (P<0.01 and P<0.05). CONCLUSION: Myocardial cell apoptosis occurs inhypoxia/reoxygenation injury, and EPO can protect rat cardiomyocytes from hypoxia/reoxygenation induced apoptosis.The protective effect is partly associated with the PKC/mitoKATP pathway.

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554. Role of Ca(2+)/calmodulin-dependent protein kinase II in the regulation of the cardiac L-type Ca(2+) current duringendothelin-1 stimulation

By Komukai Kimiaki; O-Uchi Jin; Morimoto Satoshi; Kawai Makoto; Hongo Kenichi; Yoshimura Michihiro; KuriharaSatoshiFrom American journal of physiology. Heart and circulatory physiology (2010), 298(6), H1902-7, Language: English,Database: MEDLINE

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Endothelin-1 (ET-1) shows a positive inotropic effect on cardiac muscle. Although the L-type Ca(2+) current (I(Ca)) isone of the important determinants of cardiac excitation-contraction coupling, the effect of ET-1 on the I(Ca) is notalways clear. The controversial results appear to be due to different patch-clamp methods. The present studymeasured the effect of ET-1 on the I(Ca) of rat ventricular myocytes using the perforated patch-clamp technique. Theholding potential was set to -40 mV, and depolarization was applied every 10 s. ET-1 (10 nM) increased the I(Ca) in amonophasic manner. The current reached a steady state 15 min after the application of ET-1, when the measurementwas done. Endothelin receptor subtype expression was also investigated using Western immunoblotting. ET(A)-receptor protein was expressed, but ET(B)-receptor protein was not expressed, in the cell membranes of rat ventricularmyocytes. The effect of ET-1 on the I(Ca) was inhibited by a selective ET(A)-receptor antagonist, BQ-123, but not by aselective ET(B)-receptor antagonist, BQ-788. The effect was inhibited by protein kinase C (PKC) inhibitor chelerythrineand Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93, but not by its inactive analog KN-92. Theeffect of ET-1 was also blocked by another CaMKII inhibitor, autocamtide-2-related inhibitory peptide. These resultssuggest that ET-1 increases the I(Ca) via the ET(A)-receptor-PKC-CaMKII pathway.

~2 Citings

555. Orexins activates protein kinase C-mediated Ca(2+) signaling in isolated rat primary sensory neurons

By Ozcan M; Ayar A; Serhatlioglu I; Alcin E; Sahin Z; Kelestimur HFrom Physiological research / Academia Scientiarum Bohemoslovaca (2010), 59(2), 255-62, Language: English,Database: MEDLINE

Previous results have suggested that orexins causes a rise of intracellular free calcium ([Ca(2+)](i)) in cultured ratdorsal root ganglion (DRG) neurons, implicating a role in nociception, but the underlying mechanism is unknown.Hence, the aim of the present study was to investigate whether the orexins-mediated signaling involves the PKCpathways in these sensory neurons. Cultured DRG neurons were loaded with 1 micromol Fura-2 AM and [Ca(2+)](i)responses were quantified by the changes in 340/380 ratio using fluorescence imaging system. The orexin-1 receptorantagonist SB-334867-A (1 microM) inhibited the calcium responses to orexin-A and orexin-B (59.1+/-5.1 % vs. 200nM orexin-A, n=8, and 67+/-3.8 % vs. 200 nM orexin-B, n=12, respectively). The PKC inhibitor chelerythrine (10 and100 microM) significantly decreased the orexin-A (200 nM)-induced [Ca(2+)](i) increase (59.4+/-4.8 % P<0.01, n=10and 4.9+/-1.6 %, P<0.01, n=9) versus response to orexin-A). It was also found that chelerythrine dose-dependentlyinhibited the [Ca(2+)](i) response to 200 nM orexin-B. In conclusion, our results suggest that orexins activateintracellular calcium signaling in cultured rat sensory neurons through PKC-dependent pathway, which may haveimportant implications for nociceptive modulation and pain.

~0 Citings

556. Role of PKC in regulation of CD73 by lysophosphatidylcholine in human endothelial cells

By Zhang Qun-ying; Han Jun-yong; Zhang Hua; Tan JieFrom Zhongguo ying yong sheng li xue za zhi = Zhongguo yingyong shenglixue zazhi = Chinese journal of appliedphysiology (2010), 26(1), 102-4, Language: Chinese, Database: MEDLINE

OBJECTIVE: To discuss the effect of protein kinase C (PKC) on regulation of ecto-5'-nucleotidase activity bylysophosphatidylcholine(LPC) in human umbilical endothelial cells (HUVEC). METHODS: Experiments wereconducted in HUVEC grown on dishes which were divided into 4 groups (n=15): (1) Control group in which only eAMP(5 micromol/L) was added; (2) LPC group in which HUVEC were incubated with LPC (10 micromol/L) before eAMPwas added; (3) Chelerythrine group in which cells were pre-incubated with the PKC inhibitor chelerythrine (100micromol/L) before LPC and eAMP were added; (4) alpha, beta-Methyladenosine-5'-Diphosphate (AOPCP) group inwhich cells were incubated with AOPCP (10 micromol/L) before eAMP was added. Etheno-adenosine production wasdetected at 15th, 30th, 45th min with high performance liquid chromatography(HPLC) respectively. RESULTS:Comparing to the control group LPC significantly increased etheno-adenosine production at three time pointsrespectively (P < 0.05). Furthermore, PKC inhibitor chelerythrine abolished this effect of LPC and theethenoadenosine production at three time points were at the same level of control group (P > 0.05). CD73 inhibitorAOPCP significantly decreased the etheno-adenosine production compared to the other three groups (P < 0.01).CONCLUSION: Ecto-5'-nucleotidase can be modulated within minutes following exposure of HUVEC to LPC and thisresponse may be mediated by PKC in HUVEC.

~0 Citings

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557. Involvement of inositol-1,4,5-trisphosphate receptors in the bidirectional synaptic plasticity induced in hippocampalCA1 neurons by 1-10 Hz low-frequency stimulation

By Fujii S; Yamazaki Y; Kuroda Y; Mikoshiba KFrom Neuroscience (2010), 168(2), 346-58, Language: English, Database: MEDLINE

In the present study, both potentiation and depression of the synaptic response were induced in hippocampal CA1neurons by systematically varying the frequency of low frequency afferent stimulation (LFS) between 0.5 and 25 Hzand the pulse number between 40 and 1000. The input-response relationship for CA1 synapses showed that LFS at ahigher frequency or with a smaller pulse number increased the magnitude of potentiation of the synaptic response byincreasing the contribution of N-methyl-D-aspartate receptors (NMDARs) and metabotropic glutamate receptors

(mGluRs) to induction of potentiation. One possible mechanism for this bidirectional plasticity was that specificpatterns of LFS differentially activate a uniform receptor population in producing depression or potentiation of synapticresponses. However, a pharmacological study indicated that, despite their opposite effects, both the synapticdepression induced by LFS at 1 Hz and the synaptic potentiation induced by LFS at 10 Hz were triggered by co-activation of NMDARs and mGluRs at CA1 synapses. We suggest that activation of protein kinase C or inositol-1,4,5-trisphosphate receptors, both coupled to group 1 mGluRs, is involved in the bidirectional synaptic plasticity induced inhippocampal CA1 neurons by 1-10 Hz LFS.

~0 Citings

558. Attenuation of renal ischemia-reperfusion injury by postconditioning involves adenosine receptor and protein kinaseC activation

By Eldaif Shady M; Deneve Jeremiah A; Wang Ning-Ping; Jiang Rong; Mosunjac Mario; Mutrie Christopher J; GuytonRobert A; Zhao Zhi-Qing; Vinten-Johansen JakobFrom Transplant international : official journal of the European Society for Organ Transplantation (2010), 23(2), 217-26,Language: English, Database: MEDLINE

SUMMARY: Significant organ injury occurs after transplantation and reflow (i.e., reperfusion injury). Postconditioning(PoC), consisting of alternating periods of reperfusion and re-occlusion at onset of reperfusion, attenuates reperfusioninjury in organs including heart and brain. We tested whether PoC attenuates renal ischemia-reperfusion (I/R) injury inthe kidney by activating adenosine receptors (AR) and protein kinase C (PKC). The single kidney rat I/R model wasused. Groups: (1) sham: time-matched surgical protocol only. In all others, the left renal artery (RA) was occluded for45 min and reperfused for 24 h. (2) Control: I/R with no intervention at R. All antagonists were administered 5 minbefore reperfusion. (3) PoC: I/R + four cycles of 45 s of R and 45 s of re-occlusion before full R. (4) PoC + ARi: PoCplus the AR antagonist 8-rho-(sulfophenyl) theophylline (8-SPT). (5) PoC + PKCi: PoC plus the PKC antagonistchelerythrine (Che). In shams, plasma blood urea nitrogen (BUN mg/dl) at 24 h averaged 23.2 +/- 5.3 and creatinine(Cr mg/dl) averaged 1.28 +/- 0.2. PoC reduced BUN (87.2 +/- 10 in Control vs. 38.8 +/- 9, P = 0.001) and Cr (4.2 +/-0.6 in Control vs. 1.5 +/- 0.2, P < 0.001). 8-SPT and Che reversed renal protection indices after PoC. I/R increasedapoptosis, which was reduced by PoC, which was reversed by 8-SPT and Che. Postconditioning attenuates renal I/Rinjury by adenosine receptor activation and PKC signaling.

~1 Citing

559. Multiple kinase pathways regulate voltage-dependent Ca2+ influx and migration in oligodendrocyte precursor cells

By Paez Pablo M; Fulton Daniel J; Spreur Vilma; Handley Vance; Campagnoni Anthony TFrom The Journal of neuroscience : the official journal of the Society for Neuroscience (2010), 30(18), 6422-33,Language: English, Database: MEDLINE

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It is becoming increasingly clear that voltage-operated Ca(2+) channels (VOCCs) play a fundamental role in thedevelopment of oligodendrocyte progenitor cells (OPCs). Because direct phosphorylation by different kinases is one ofthe most important mechanisms involved in VOCC modulation, the aim of this study was to evaluate the participation ofserine-threonine kinases and tyrosine kinases (TKs) on Ca(2+) influx mediated by VOCCs in OPCs. Calcium imagingrevealed that OPCs exhibited Ca(2+) influx after plasma membrane depolarization via L-type VOCCs. Furthermore,VOCC-mediated Ca(2+) influx declined with OPC differentiation, indicating that VOCCs are developmentally regulatedin OPCs. PKC activation significantly increased VOCC activity in OPCs, whereas PKA activation produced theopposite effect. The results also indicated that OPC morphological changes induced by PKC activation were partiallymediated by VOCCs. Our data clearly suggest that TKs exert an activating influence on VOCC function in OPCs.Furthermore, using the PDGF response as a model to probe the role of TK receptors (TKr) on OPC Ca(2+) uptake, wefound that TKr activation potentiated Ca(2+) influx after membrane depolarization. Interestingly, this TKr modulation ofVOCCs appeared to be essential for the PDGF enhancement of OPC migration rate, because cell motility wascompletely blocked by TKr antagonists, as well as VOCC inhibitors, in migration assays. The present study stronglydemonstrates that PKC and TKrs enhance Ca(2+) influx induced by depolarization in OPCs, whereas PKA has aninhibitory effect. These kinases modulate voltage-operated Ca(2+) uptake in OPCs and participate in the modulation ofprocess extension and migration.

~1 Citing

560. Long-term protection and mechanism of pacing-induced postconditioning in the heart

By Babiker Fawzi A; Lorenzen-Schmidt Ilka; Mokelke Eric; Vanagt Ward Y; Delhaas Tammo; Waltenberger Johannes;

Cleutjens Jack P; Prinzen Frits WFrom Basic research in cardiology (2010), 105(4), 523-33, Language: English, Database: MEDLINE

Brief periods of ventricular pacing during the early reperfusion phase (pacing-induced postconditioning, PPC) havebeen shown to reduce infarct size as measured after 2 h of reperfusion. In this study, we investigated (1) whether PPCleads to maintained reduction in infarct size, (2) whether abnormal mechanical load due to asynchronous activation isthe trigger for PPC and (3) the signaling pathways that are involved in PPC. Rabbit hearts were subjected to 30 min ofcoronary occlusion in vivo, followed by 6 weeks of reperfusion. PPC consisted of ten 30-s intervals of left ventricular(LV) pacing, starting at reperfusion. PPC reduced infarct size (TTC staining) normalized to area at risk, from 49.0 +/-3.3% in control to 22.9 +/- 5.7% in PPC rabbits. In isolated ejecting rabbit hearts, replacing LV pacing by biventricularpacing abolished the protective effect of PPC, whereas ten 30-s periods of high preload provided a protective effectsimilar to PPC. The protective effect of PPC was neither affected by the adenosine receptor blocker 8-SPT nor by theangiotensin II receptor blocker candesartan, but was abrogated by the cytoskeletal microtubule-disrupting agentcolchicine. Blockers of the mitochondrial K(ATP) channel (5HD), PKC (chelerythrine) and PI3-kinase (wortmannin) allabrogated the protection provided by PPC. In the in situ pig heart, PPC reduced infarct size from 35 +/- 4 to 16 +/-12%, a protection which was abolished by the stretch-activated channel blocker gadolinium. No infarct size reductionwas achieved if PPC application was delayed by 5 min or if only five pacing cycles were used. The present studyindicates that (1) PPC permanently reduces myocardial injury, (2) abnormal mechanical loading is a more likely triggerfor PPC than electrical stimulation or G-coupled receptor stimulation and (3) PPC may share downstream pathwayswith other modes of cardioprotection.

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561. Berberine suppresses intestinal disaccharidases with beneficial metabolic effects in diabetic states, evidences fromin vivo and in vitro study

By Liu Li; Yu Yun-Li; Yang Jian-Song; Li Yang; Liu Yao-Wu; Liang Yan; Liu Xiao-Dong; Xie Lin; Wang Guang-Ji

From Naunyn-Schmiedeberg's archives of pharmacology (2010), 381(4), 371-81, Language: English, Database:MEDLINE

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Clinical reports have demonstrated that berberine is a potential antidiabetic agent, but the underlying mechanism isunclear. The purpose of this study was to investigate if berberine exerts its hypoglycemic action via inhibiting intestinaldisaccharidases using in vivo and in vitro experiments. Streptozotocin-induced diabetic rats received berberine (100 or200 mg/kg) orally once daily or acarbose (40 mg/kg) orally twice daily for 5 weeks. Disaccharidase activities andsucrase-isomaltase (SI) complex messenger RNA (mRNA) expression in intestinal regions were assessed. The sametreatment was operated in normal rats. Sucrose and maltose loading tests were also documented. In addition, Caco-2cells were cultured in medium containing berberine or berberine plus chelerythrine. Compound C or H-89 for 5 days,disaccharidase activities, and SI complex mRNA levels were measured. The animal experiments showed thatberberine significantly decreased the disaccharidase activities and SI complex mRNA expression both in diabetic ratsand normal rats. Berberine can also significantly lower postprandial blood glucose levels induced by sucrose ormaltose loading in normal rats. The cellular results showed that berberine may suppress disaccharidase activities anddownregulate SI complex mRNA expression in a concentration-dependent manner. Only H-89, an inhibitor of proteinkinase A (PKA), may reverse the decrease in disaccharidase activities and SI complex mRNA expression induced byberberine. In conclusion, berberine suppresses disaccharidase activities and SI complex mRNA expression withbeneficial metabolic effects in diabetic states. The inhibitory effect, at least partly, involves the PKA-dependentpathway.

~3 Citings

562. Androgens induce nongenomic stimulation of colonic contractile activity through induction of calcium sensitizationand phosphorylation of LC20 and CPI-17

By Gonzalez-Montelongo Maria C; Marin Raquel; Gomez Tomas; Marrero-Alonso Jorge; Diaz MarioFrom Molecular endocrinology (Baltimore, Md.) (2010), 24(5), 1007-23, Language: English, Database: MEDLINE

We show that androgens, testosterone and 5alpha-dihydrotestosterone (DHT), acutely (approximately 40 min) provokethe mechanical potentiation of spontaneous and agonist-induced contractile activity in mouse colonic longitudinalsmooth muscle. The results using flutamide, finasteride, cycloheximide, and actinomycin D indicate that androgen-induced potentiation is dependent on androgen receptors, requires reduction of testosterone to DHT, and occursindependently of transcriptional and translational events. Using permeabilized colonic smooth muscle preparations, wecould demonstrate that mechanical potentiation is entirely due to calcium sensitization of contractile machinery. Inaddition, DHT (10 nm) increased phosphorylation of both 20-kDa myosin light chain (LC(20)) [regulatory myosin lightchain, (MLC)] and CPI-17 (an endogenous inhibitor of MLC phosphatase). Paralleling these findings, inhibition of Rho-associated Rho kinase (ROK) and/or protein kinase C (PKC) with, respectively, Y27632 and chelerythrine, preventedLC(20) phosphorylation and abolished calcium sensitization. In addition, inhibition of ROK prevents CPI-17phosphorylation, indicating that ROK is located upstream PKC-mediated CPI-17 modulation in the signalling cascade.Additionally, androgens induce a rapid activation of RhoA and its translocation to the plasma membrane to activateROK. The results demonstrate that androgens induce sensitization of colonic smooth muscle to calcium throughactivation of ROK, which in turn, activates PKC to induce CPI-17 phosphorylation. Activation of this pathway induces apotent steady stimulation of LC(20) by inhibiting MLC phosphatase and displacing the equilibrium of the regulatorysubunit towards its phosphorylated state. This is the first demonstration that colonic smooth muscle is a physiologicaltarget for androgen hormones, and that androgens modulate force generation of smooth muscle contractile machinerythrough nongenomic calcium sensitization pathways.

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563. Simultaneous determination of seven main alkaloids of Chelidonium majus L. by ultra-performance LC withphotodiode-array detection

By Gu Yue; Qian Dawei; Duan Jin-ao; Wang Zhenzhong; Guo Jianming; Tang Yuping; Guo ShengFrom Journal of separation science (2010), 33(8), 1004-9, Language: English, Database: MEDLINE

A simple and rapid method for the simultaneous determination of seven isoquinoline alkaloids, protopine, chelidonine,coptisine, stylopine, sanguinarine, berberine, and chelerythrine, in Chelidonium majus L. (Ch. majus) samples by ultra-performance LC method with photodiode array detection is described. The baseline separation of these compoundswas performed with (A) acetonitrile-(B) ammonium acetate (10 mM, adjusted to pH 3.0 with acetic acid) as the mobilephase using a C18 RP column (2.1x100 mm, 1.7 microm). Optimized conditions resulted in excellent peak shapes.The seven alkaloids were completely separated within 20 min. Good linear behaviors (r > or = 0.9992) over theinvestigated concentration ranges were observed for all the analytes. Validation proved the repeatability of the methodwas good and recovery was satisfactory. The validated method was successfully applied for 20 batches of Ch. majus.These results demonstrated that the ultra-performance LC photodiode array method proposed was very useful in theanalysis and quality control of Ch. majus.

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~0 Citings

564. The identification of inhibitors of Schistosoma mansoni miracidial transformation by incorporating a medium-throughput small-molecule screen

By Taft Andrew S; Norante Francesca A; Yoshino Timothy PFrom Experimental parasitology (2010), 125(2), 84-94, Language: English, Database: MEDLINE

In Schistosoma mansoni, the miracidium-to-primary sporocyst transformation process is associated with manyphysiological, morphological, transcriptional and biochemical changes. In the present study, we use a medium-throughput small-molecule screen to identify chemical compounds inhibiting or delaying the in vitro transformation ofmiracidia to the sporocyst stage. The Sigma-Aldrich Library of Pharmacologically Active Compounds (LOPAC)contains 1280 well-characterized chemical compounds with various modes of action including enzyme inhibitors,antibiotics, cell-cycle regulators, apoptosis inducers and GPCR ligands. We identified 47 compounds that greatlyreduce or delay this transformation process during a primary screen of live miracidia. The majority of compoundsinhibiting larval transformation were from dopaminergic, serotonergic, ion channel and phosphorylation classes.Specifically, we found that dopamine D2-type antagonists, serotonin reuptake inhibitors, voltage-gated calcium channelantagonists and a PKC activator significantly reduced in vitro miracidial transformation rates. Many of the targets ofthese compounds regulate adenylyl cyclase activity, with the inhibition or activation of these targets resulting inincreased cAMP levels in miracidia and concomitant blocking/delaying of larval transformation.

~1 Citing

565. Urotensin II inhibits glucokinase expression and glucose-induced insulin secretion

By Liu Fang; Zhu Yi-ChunFrom Sheng li xue bao : [Acta physiologica Sinica] (2010), 62(2), 129-36, Language: Chinese, Database: MEDLINE

The purpose of the present study is to investigate the effects of urotensin II (UII) on insulin secretion in islet beta cellsand the underlying mechanism. Glucose tolerance test was performed in Wistar rats to evaluate the effect of UII onthe levels of plasma glucose and insulin. Static incubation experiment was employed to investigate the effect of UII onglucose-induced insulin secretion (GIIS) in betaTC-6 cells. After the incubation, insulin content and mRNA level inbetaTC-6 cells were analyzed. Finally, Western blot was used to find out if UII could change the expression levels ofpancreatic duodenal homeobox-1 (PDX-1) and glucokinase (GCK). It was observed that intravenous administration ofUII (30, 300 nmol/kg) resulted in a significant decrease in insulin level 15 min after glucose load, and induced anobvious increase in plasma glucose 90 min after the load. In vitro, two hours of UII incubation inhibited GIIS in betaTC-6 cells without affecting insulin content and mRNA levels. The inhibitory effect of UII was blocked by UII receptorantagonist (urantide), and partially blunted by protein kinase C (PKC) inhibitor (chelerythrine) and somatostatinreceptor antagonist (cyclosomatostatin). Moreover, we found that GCK protein level was significantly reduced by UII,while PDX-1, a key regulator of insulin gene transcription in beta cells, was not affected. These results suggest thatUII-induced inhibition of GIIS in betaTC-6 cells are mediated by UII receptor and PKC pathway, as well as somatostatinreceptor which could be activated by high dose of UII. The inhibitory effect of UII on insulin secretion is ratherassociated with a suppression of GCK expression than a regulation on PDX-1 expression.

~1 Citing

566. Rho kinase and protein kinase C involvement in vascular smooth muscle myofilament calcium sensitization inarteries from diabetic rats

By Kizub I V; Pavlova O O; Johnson C D; Soloviev A I; Zholos A VFrom British journal of pharmacology (2010), 159(8), 1724-31, Language: English, Database: MEDLINE

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BACKGROUND AND PURPOSE: Diabetes mellitus (DM) causes multiple dysfunctions including circulatory disorderssuch as cardiomyopathy, angiopathy, atherosclerosis and arterial hypertension. Rho kinase (ROCK) and proteinkinase C (PKC) regulate vascular smooth muscle (VSM) Ca(2+) sensitivity, thus enhancing VSM contraction, and up-regulation of both enzymes in DM is well known. We postulated that in DM, Ca(2+) sensitization occurs in diabeticarteries due to increased ROCK and/or PKC activity. EXPERIMENTAL APPROACH: Rats were renderedhyperglycaemic by i.p. injection of streptozotocin. Age-matched control tissues were used for comparison. Contractileresponses to phenylephrine (Phe) and different Ca(2+) concentrations were recorded, respectively, from intact andchemically permeabilized vascular rings from aorta, tail and mesenteric arteries. KEY RESULTS: Diabetic tail andmesenteric arteries demonstrated markedly enhanced sensitivity to Phe while these changes were not observed inaorta. The ROCK inhibitor HA1077, but not the PKC inhibitor chelerythrine, caused significant reduction in sensitivityto agonist in diabetic vessels. Similar changes were observed for myofilament Ca(2+) sensitivity, which was againenhanced in DM in tail and mesenteric arteries, but not in aorta, and could be reduced by both the ROCK and PKCblockers. CONCLUSIONS AND IMPLICATIONS: We conclude that in DM enhanced myofilament Ca(2+) sensitivity ismainly manifested in muscular-type blood vessels and thus likely to contribute to the development of hypertension.Both PKC and, in particular, ROCK are involved in this phenomenon. This highlights their potential usefulness as drugtargets in the pharmacological management of DM-associated vascular dysfunction.

~2 Citings

567. Inhibition of ATP-induced Ca2+ influx by corticosterone in dorsal root ganglion neurons

By Liu Xiaohong; Zeng Junwei; Zhao Yandong; Xiao Zhi; Fang Chuanqing; Ruan Huaizhen

From Neurochemical research (2010), 35(5), 804-10, Language: English, Database: MEDLINEIn addition to the classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomic effects onneurons. In the present study, the effect of corticosterone (CORT) on ATP-induced Ca(2+) mobilization in cultureddorsal root ganglion (DRG) neurons were detected with confocal laser scanning microscopy using fluo-4/AM as acalcium fluorescent indicator that could monitor real-time alterations of intracellular calcium concentration ([Ca(2+)]i).ATP, an algesic agent, caused [Ca(2+)]i increase in DRG neurons by activation of P2X receptor. Pretreatment withCORT (1 nM-1 microM for 5 min) inhibited ATP-induced [Ca(2+)]i increase in DRG neurons. The rapid inhibition ofATP-induced Ca(2+) response by CORT was concentration-dependent, reversible and could be blocked byglucocorticoid receptor antagonist RU38486 (10 microM). Furthermore, the inhibitory effect of CORT was abolished byprotein kinase A inhibitor H89 (10 microM), but was not influenced by protein kinase C inhibitor Chelerythrine chloride(10 microM). On the other hand, membrane-impermeable bovine serum albumin-conjugated corticosterone had noeffect on ATP-induced [Ca(2+)]i transients. These observations suggest that a nongenomic pathways may be involvedin the effect of CORT on ATP-induced [Ca(2+)]i transients in cultured DRG neurons.

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568. The PLC/IP 3 R/PKC pathway is required for ethanol-enhanced GABA release

By Kelm M Katherine; Weinberg Richard J; Criswell Hugh E; Breese George RFrom Neuropharmacology (2010), 58(7), 1179-86, Language: English, Database: MEDLINE

Research on the actions of ethanol at the GABAergic synapse has traditionally focused on postsynaptic mechanisms,but recent data demonstrate that ethanol also increases both evoked and spontaneous GABA release in many brainregions. Using whole-cell voltage-clamp recordings, we previously showed that ethanol increases spontaneous GABArelease at the rat interneuron-Purkinje cell synapse. This presynaptic ethanol effect is dependent on calcium releasefrom internal stores, possibly through activation of inositol 1,4,5-trisphosphate receptors (IP(3)Rs). After confirmingthat ethanol targets vesicular GABA release, in the present study we used electron microscopic immunohistochemistryto demonstrate that IP(3)Rs are located in presynaptic terminals of cerebellar interneurons. Activation of IP(3)Rsrequires binding of IP(3), generated through activation of phospholipase C (PLC). We find that the PLC antagonistedelfosine prevents ethanol from increasing spontaneous GABA release. Diacylglycerol generated by PLC andcalcium released by activation of the IP(3)R activate protein kinase C (PKC). Ethanol-enhanced GABA release wasblocked by two PKC antagonists, chelerythrine and calphostin C. When a membrane impermeable PKC antagonist,PKC (19-36), was delivered intracellularly to the postsynaptic neuron, ethanol continued to increase spontaneousGABA release. Overall, these results suggest that activation of the PLC/IP(3)R/PKC pathway is necessary for ethanolto increase spontaneous GABA release from presynaptic terminals onto Purkinje cells.

~2 Citings

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569. Hydrogen sulfide protects neurons against hypoxic injury via stimulation of ATP-sensitive potassium channel/proteinkinase C/extracellular signal-regulated kinase/heat shock protein 90 pathway

By Tay A S; Hu L F; Lu M; Wong P T H; Bian J SFrom Neuroscience (2010), 167(2), 277-86, Language: English, Database: MEDLINE

Cerebral hypoxia is one of the main causes of cerebral injury. This study was conducted to investigate the potentialprotective effect of H(2)S in in vitro hypoxic models by subjecting SH-SY5Y cells to either oxygen-glucose deprivationor Na(2)S(2)O(4) (an oxygen scavenger) treatment. We found that treatment with NaHS (an H(2)S donor, 10-100microM) 15 min prior to hypoxia increased cell viability in a concentration-dependent manner. Time-course studyshowed that NaHS was able to exert its protective effect even when added 8 h before or less than 4 h after hypoxia

induction. Interestingly, endogenous H(2)S level was markedly reduced by hypoxia induction. Over-expression ofcystathionine-beta-synthase prevented hypoxia induced cell apoptosis. Blockade of ATP-sensitive K(+) (K(ATP))channels with glibenclamide and HMR-1098, protein kinase C (PKC) with its three specific inhibitors (chelerythrine,bisindolylmaleide I and calphostin C), extracellular signal-regulated kinase 1/2 (ERK1/2) with PD98059 and heat shockprotein 90 (Hsp90) with geldanamycin and radicicol significantly attenuated the protective effects of NaHS. Westernblots showed that NaHS significantly stimulated ERK1/2 activation and Hsp90 expression. In conclusion, H(2)S exertsa protective effect against cerebral hypoxia induced neuronal cell death via K(ATP)/PKC/ERK1/2/Hsp90 pathway. Ourfindings emphasize the important neuroprotective role of H(2)S in the brain during cerebral hypoxia.

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570. Use of human cancer cell lines mitochondria to explore the mechanisms of BH3 peptides and ABT-737-inducedmitochondrial membrane permeabilization

By Buron Nelly; Porceddu Mathieu; Brabant Magali; Desgue Diana; Racoeur Cindy; Lassalle Myriam; PechouxChristine; Rustin Pierre; Jacotot Etienne; Borgne-Sanchez AnnieFrom PloS one (2010), 5(3), e9924, Language: English, Database: MEDLINE

Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. Anumber of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor celldeath. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze theeffect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effectsrelated to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated incancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptidesfrom Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found thatABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 butnot AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cellmitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane.Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xLbut not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent onisolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membranepermeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profileagainst cancer cell mitochondria but devoid of toxicity against healthy mitochondria.

~2 Citings

571. Protein kinase C-dependent inhibition of BK(Ca) current in rat aorta smooth muscle cells following gamma-irradiation

By Kizub Igor V; Pavlova Oleksandra O; Ivanova Irina V; Soloviev Anatoly IFrom International journal of radiation biology (2010), 86(4), 291-9, Language: English, Database: MEDLINE

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PURPOSE: The aim of this study was to estimate the effects of non-fatal whole-body gamma-irradiation on outwardpotassium plasma membrane conductivity in rat vascular smooth muscle cells (VSMC), and to identify underlyingmechanisms. MATERIALS AND METHODS: Rats were exposed to a 6 Gy dose irradiation from a cobalt(60) source.Whole-cell potassium current was measured in freshly isolated rat aorta smooth muscle cells using standard patch-clamp technique. RESULTS: We have determined that whole-body ionising irradiation significantly inhibits whole-celloutward K(+) current in rat aortic VSMC obtained from irradiated rats 9 and 30 days after irradiation, and this inhibitionappears to be increased throughout post-irradiation period. Using selective inhibitors of small conductance Ca(2+)-activated K(+) channels (SK(Ca)), apamin (1 microM), intermediate conductance Ca(2+)-activated K(+) channels(IK(Ca,)), charybdotoxin (1 microM) and a large conductance Ca(2+)-activated K(+) channels (BK(Ca)), paxilline (500nM), we established that the main component of whole-cell outward K(+) current in rat aortic VSMC is due to BK(Ca).It is clear that on the 9th day after irradiation paxilline had only a small effect on whole-cell outward K(+) current inVSMC, and was without effect on the 30th day post-irradiation, suggesting complete suppression of the BK(Ca)current. The PKC inhibitor, chelerythrine (100 nM), effectively reversed the suppression of whole-cell outward K(+)current induced by ionising irradiation in the post-irradiation period of 9 and 30 days. CONCLUSIONS: The resultssuggest that irradiation-evoked inhibition of the BK(Ca) current in aortic VSMC is mediated by PKC. Taken together,our data indicate that one of the mechanisms leading to elevation of vascular tone and related arterial hypertensiondevelopment under ionising irradiation impact is a PKC-mediated inhibition of BK(Ca) channels in VSMC.

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572. Activation of muscarinic M-1 cholinoceptors by curcumin to increase contractility in urinary bladder isolated fromWistar rats

By Cheng Tse-Chou; Lu Chih-Cheng; Chung Hsien-Hui; Hsu Chih-Chieh; Kakizawa Nozomi; Yamada Shizuo; ChengJuei-TangFrom Neuroscience letters (2010), 473(2), 107-9, Language: English, Database: MEDLINE

Curcumin is an active principle contained in rhizome of Curcuma longa, and it has been recently mentioned to showaffinity to muscarinic M-1 cholinoceptors (M(1)-mAChR). In the present study, we found that curcumin caused aconcentration-dependent increase of muscle tone in urinary bladder isolated from Wistar rats. This action wasinhibited by pirenzepine at concentration enough to block M(1)-mAChR. In radioligand-binding assay, specific bindingof [(3)H]-oxotremorine (OXO-M) in the rat bladder homogenates was also displaced by curcumin in a concentration-dependent manner. In the presence of inhibitors for PLC-PKC pathway, either U73122 (phospholipase C inhibitor) orchelerythrine (protein kinase C inhibitor), curcumin-stimulated contraction in urinary bladder was markedly reduced. Inconclusion, the obtained results suggest that curcumin can activate M(1)-mAChR at concentrations lower than toscavenge free radicals to increase of muscle tone in urinary bladder through PLC-PKC pathway.

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573. Role of progesterone on the regulation of vascular muscle cells proliferation, migration and apoptosis

By Cutini Pablo H; Massheimer Virginia LFrom Steroids (2010), 75(4-5), 355-61, Language: English, Database: MEDLINE

The purpose of this study was to investigate the effect of progesterone (Pg) on cellular growth, migration, apoptosis,and the molecular mechanism of action displayed by the steroid. To that end, rat aortic vascular smooth muscle cell(VSMC) cultures were employed. Pg (10nM) significantly increased [(3)H]thymidine incorporation after 24h oftreatment. The enhancement in DNA synthesis was blunted in the presence of an antagonist of Pg receptor (RU486compound). The mitogenic action of the steroid was suppressed by the presence of the compounds PD98059 (MEKinhibitor), chelerythrine (PKC inhibitor), and indomethacin (cyclooxygenase antagonist) suggesting that the stimulationof DNA synthesis involves MAPK, PKC, and cyclooxygenase transduction pathways. The proliferative effect of thehormone depends on the presence of endothelial cells (EC). When muscle cells were incubated with conditionedmedia obtained of EC treated with Pg, the mitogenic action of the steroid declined. Wounding assays shows that 10nMPg enhances VSMC migration and motility. The role of the steroid on programmed cell death was measured usingDNA fragmentation technique. Four hours of treatment with 10nM Pg enhanced DNA laddering in a similarly extent tothe apoptotic effect induced by the apoptogen hydrogen peroxide (H(2)O(2)). In summary the results presentedprovide evidence that Pg enhances cell proliferation, migration, and apoptosis of VSMC. The modulation of cell growthelicited by the steroid involves integration between genomic and signal transduction pathways activation.

~0 Citings

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574. Effect of quaternary benzo[c]phenanthridine alkaloids sanguilutine and chelilutine on normal and cancer cells

By Slunska Z; Gelnarova E; Hammerova J; Taborska E; Slaninova IFrom Toxicology in vitro : an international journal published in association with BIBRA (2010), 24(3), 697-706,Language: English, Database: MEDLINE

Sanguilutine and chelilutine, quaternary benzo[c]phenanthridine alkaloids, were studied for their antiproliferativeactivities with regard to their ability to induce oxidative stress. We observed potent antiproliferative activities for bothalkaloids against three tumour (HeLa; HL-60; A-2780) and two normal (V-79; LEP) cell lines. Both alkaloids wereefficient inductors of apoptosis. Statistical analysis revealed higher toxicity for sanguilutine compared to chelilutine andunequal sensitivity with regard to individual cell lines, although independent of the character of the cell line (i.e. tumour

vs. normal). Dihydrofluorescein diacetate staining was used to measure intracellular ROS accumulation aftertreatment with sanguilutine, chelilutine, sanguinarine, and chelerythrine. In addition, anti-oxidative effects werestudied. The effects of the alkaloids were compared with the effects of commonly used anti-oxidants, such as trolox,caffeine acid, and chlorogenic acid. None of the tested alkaloids (0.1 and 1 microg/ml) increased ROS production.Pre-incubation of sanguinarine and chelilutine (at all tested concentrations) and sanguilutine and chelerythrine (1microg/ml) decreased oxidative stress caused by H(2)O(2). These findings indicate high antiproliferative and pro-apoptotic effects of sanguilutine and chelilutine that are not accompanied by oxidative stress induction, to the contrary,both alkaloids showed anti-oxidative effects.

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575. MLCK and PKC Involvements via Gi and Rho A Protein in Contraction by the Electrical Field Stimulation in FelineEsophageal Smooth Muscle

By Park Sun Young; Shim Jae Ho; Kim Mina; Sun Yih Hsiu; Kwak Hyun Soo; Yan Xiangmei; Choi Byung-Chul; ImChaeuk; Sim Sang Soo; Jeong Ji Hoon; et alFrom The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and theKorean Society of Pharmacology (2010), 14(1), 29-35, Language: English, Database: MEDLINE

We have shown that myosin light chain kinase (MLCK) was required for the off-contraction in response to the electricalfield stimulation (EFS) of feline esophageal smooth muscle. In this study, we investigated whether protein kinase C(PKC) may require the on-contraction in response to EFS using feline esophageal smooth muscle. The contractionswere recorded using an isometric force transducer. On-contraction occurred in the presence of N(G)-nitro-L-argininemethyl ester (L-NAME), suggesting that nitric oxide acts as an inhibitory mediator in smooth muscle. The excitatorycomposition of both contractions was cholinergic dependent which was blocked by tetrodotoxin or atropine. The on-contraction was abolished in Ca(2+)-free buffer but reappeared in normal Ca(2+)-containing buffer indicating that thecontraction was Ca(2+) dependent. 4-aminopyridine (4-AP), voltage-dependent K(+) channel blocker, significantlyenhanced on-contraction. Aluminum fluoride (a G-protein activator) increased on-contraction. Pertussis toxin (a G(i)inactivator) and C3 exoenzyme (a rhoA inactivator) significantly decreased on-contraction suggesting that Gi or rhoAprotein may be related with Ca(2+) and K(+) channel. ML-9, a MLCK inhibitor, significantly inhibited on-contraction,and chelerythrine (PKC inhibitor) affected on the contraction. These results suggest that endogenous cholinergiccontractions activated directly by low-frequency EFS may be mediated by Ca(2+), and G proteins, such as Gi andrhoA, which resulted in the activation of MLCK, and PKC to produce the contraction in feline distal esophageal smoothmuscle.

~1 Citing

576. The effects of sympathetic outflow on upregulation of vanilloid receptors TRPV(1) in primary afferent neuronsevoked by intradermal capsaicin

By Xu Xijin; Wang Peng; Zou Xiaoju; Li Dingge; Fang Li; Gong Kerui; Lin QingFrom Experimental neurology (2010), 222(1), 93-107, Language: English, Database: MEDLINE

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The vanilloid receptor TRPV(1) is a key nociceptive molecule located in primary afferent nociceptive neurons in dorsalroot ganglia (DRG) for initiating neurogenic inflammation and pain. Our recent study demonstrates that up-regulationof TRPV(1) receptors by intradermal injection of capsaicin is modulated by activation of the protein kinase C (PKC)cascade. Neurogenic inflammation and pain resulting from capsaicin injection are sympathetically dependent,responding to norepinephrine, adenosine 5'-triphosphate (ATP) and/or neuropeptide Y released from sympatheticefferents. In a rat model of acute neurogenic inflammatory pain produced by capsaicin injection, we usedimmunofluorescence and Western blots combined with pharmacology and surgical sympathectomies to analyzewhether the capsaicin-evoked up-regulation of TRPV(1) in DRG neurons is affected by sympathetic outflow by way ofactivating the PKC cascade. Sympathetic denervation reduced significantly the capsaicin-evoked expressions ofTRPV(1), calcitonin gene-related peptide and/or phosphorylated PKC and their co-expression. These reductions couldbe restored by exogenous pretreatment with an analog of ATP, alpha,beta-methylene ATP. Inhibition of PKC withchelerythrine chloride prevented the ATP effect. Consistent results were obtained from experiments in whichcapsaicin-evoked changes in cutaneous inflammation (vasodilation and edema) were examined after sympatheticdenervation, and the effects of the above pharmacological manipulations were evaluated. Our findings suggest thatthe capsaicin-evoked up-regulation of TRPV(1) receptors in DRG neurons is modulated sympathetically by the actionof ATP released from sympathetic efferents to activate the PKC cascade. Thus, this study proposes a potential newmechanism of sympathetic modulation of neurogenic inflammation.

~2 Citings

577. Critical involvement of postsynaptic protein kinase activation in long-term potentiation at hippocampal mossy fibersynapses on CA3 interneurons

By Galvan Emilio J; Cosgrove Kathleen E; Mauna Jocelyn C; Card J Patrick; Thiels Edda; Meriney Stephen D;Barrionuevo GermanFrom The Journal of neuroscience : the official journal of the Society for Neuroscience (2010), 30(8), 2844-55,Language: English, Database: MEDLINE

Hippocampal mossy fiber (MF) synapses on area CA3 lacunosum-moleculare (L-M) interneurons are capable ofundergoing a Hebbian form of NMDA receptor (NMDAR)-independent long-term potentiation (LTP) induced by thesame type of high-frequency stimulation (HFS) that induces LTP at MF synapses on pyramidal cells. LTP of MF inputto L-M interneurons occurs only at synapses containing mostly calcium-impermeable (CI)-AMPA receptors (AMPARs).Here, we demonstrate that HFS-induced LTP at these MF-interneuron synapses requires postsynaptic activation ofprotein kinase A (PKA) and protein kinase C (PKC). Brief extracellular stimulation of PKA with forskolin (FSK) alone orin combination with 1-Methyl-3-isobutylxanthine (IBMX) induced a long-lasting synaptic enhancement at MF synapsespredominantly containing CI-AMPARs. However, the FSK/IBMX-induced potentiation in cells loaded with the specificPKA inhibitor peptide PKI(6-22) failed to be maintained. Consistent with these data, delivery of HFS to MFs synapsingonto L-M interneurons loaded with PKI(6-22) induced posttetanic potentiation (PTP) but not LTP. Hippocampalsections stained for the catalytic subunit of PKA revealed abundant immunoreactivity in interneurons located in strataradiatum and L-M of area CA3. We also found that extracellular activation of PKC with phorbol 12,13-diacetateinduced a pharmacological potentiation of the isolated CI-AMPAR component of the MF EPSP. However, HFSdelivered to MF synapses on cells loaded with the PKC inhibitor chelerythrine exhibited PTP followed by a significantdepression. Together, our data indicate that MF LTP in L-M interneurons at synapses containing primarily CI-AMPARsrequires some of the same signaling cascades as does LTP of glutamatergic input to CA3 or CA1 pyramidal cells.

~1 Citing

578. Regulation of K-Cl cotransport in erythrocytes of frog Rana temporaria by commonly used protein kinase andprotein phosphatase inhibitors

By Gusev Gennadii Petrovich; Agalakova Natalia IvanovnaFrom Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology (2010), 180(3), 385-91, Language: English, Database: MEDLINE

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Recently (Agalakova and Gusev in J Comp Physiol 179:443-450, 2009), we demonstrated that the activity of K-Clcotransport (KCC) in frog red blood cells is inhibited under stimulation of protein kinase C (PKC) with phorbol esterPMA (12-myristate-13-acetate). Present work was performed to uncover possible implication of protein kinases andprotein phosphatases (PPs) in the regulation of baseline and volume-dependent KCC activity in these cells. K+ influxwas estimated as 86Rb uptake by the cells in isotonic or hypotonic media in the presence of ouabain, K+ efflux wasdetermined as the difference between K+ loss by the cells incubated in parallel in isotonic or hypotonic K(+)-free Cl(-)-and NO(3)(-)-media. Swelling of the cells in hypotonic medium was accompanied by approximately 50% activation ofCl-dependent K+ influx and efflux. Protein tyrosine kinase (PTK) inhibitor genistein (0.1 mM) stably and considerably(up to 89%) suppressed both baseline and volume-dependent KCC activity in each direction. Other PTK blockers(tyrphostin 23 and quercetin) had no influence on KCC activity in frog erythrocytes. PKC inhibitor chelerythrine (20microM) and both PP inhibitors, fluoride (5 mM) and okadaic acid (1 microM), reduced KCC activity by 25-70%.Neither basal nor swelling-activated KCC in frog erythrocytes was affected by PKC inhibitor staurosporine (1 microM).Based on the previous and present results, we can suggest that the main role in the maintenance of basal and volume-dependent KCC activity in frog erythrocytes belongs to PTKs and PPs, whereas PKC is a negative regulator of this ionsystem.

~0 Citings

579. Role of Rho-kinase in mediating contraction of chicken embryo femoral arteries

By Zoer Bea; Blanco Carlos E; Villamor EduardoFrom Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology (2010), 180(3), 427-

35, Language: English, Database: MEDLINERho-kinase-dependent Ca2+ sensitization is an essential process for contraction of mammalian vascular smoothmuscle but the information about its effects in non-mammalian vessels is scarce. We aimed to investigate, using theRho-kinase inhibitor hydroxyfasudil, the potential role of the Rho-kinase pathway of Ca2+ sensitization indepolarization- and agonist-mediated contraction of chicken embryo (at day 19 of the 21 days of incubation) femoralarteries. Contraction elicited by KCl (125 mM) comprised two phases (phasic and tonic contraction), both of whichwere abolished in the absence of extracellular Ca2+. Hydroxyfasudil (10 microM) left the initial phasic componentnearly intact but abolished the tonic component. Hydroxyfasudil also induced a marked impairment of the contractionselicited by phenylephrine (PE), the thromboxane A2 mimetic U46619, and endothelin-1. In contrast, inhibition ofprotein kinase C (PKC) by chelerythrine did not affect KCl- or PE-induced contractions, indicating lack of participationof PKC-mediated Ca2+ sensitization. Incubation under chronic hypoxia (15% O2 from day 0) impaired embryonicgrowth but did not significantly affect hydroxyfasudil-mediated relaxation. In summary, our findings are indicative of arole for Rho-kinase activity in depolarization- and agonist-induced force generation in chicken embryo femoral arteries.

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580. Ecstasy and methamphetamine elicit action potential bursts via different mechanisms in a central snail neuron

By Lin Pei-Lin; Tsai Ming-Cheng; Lu Guan-Ling; Lu Dah-Yuu; Chuang Chieh-Min; Yang Han-Yin; Huang Shiang-Suo;Chen Yi-HungFrom Neurotoxicology (2010), 31(1), 26-41, Language: English, Database: MEDLINE

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This study sought to determine the effects of (+) methamphetamine (METH) and its ring-substituted analog (+/-)3,4-methylenedioxymethamphetamine (MDMA; ecstasy) on electrophysiological behavior and their relationships to secondmessenger systems in an identifiable RP4 neuron of the African snail, Achatina fulica Ferussac. Extracellularapplication of MDMA at 1mM and METH at 3mM elicited action potential bursts that were not blocked after immersingthe neurons in Ca(2+)-free solution. Notably, MDMA- (1mM) elicited action potential bursts were blocked bypretreatment with the protein kinase C (PKC) inhibitors chelerythrine (20 microM) and Ro 31-8220 (20 microM), but notby the PKA inhibitors KT-5720 (10 microM) and H89 (10 microM). The PKC activator phorbol 12,13-dibutyrate (PDBu;3 microM), but not the PKA activator forskolin (50 microM), facilitated the induction of bursts elicited by MDMA at alower concentration (0.3mM). In contrast, METH- (3mM) elicited action potential bursts were blocked by pretreatmentwith KT-5720 (10 microM) and H89 (10 microM), but not by chelerythrine (20 microM) and Ro 31-8220 (20 microM).Forskolin (50 microM), but not PDBu (3 microM) facilitated the induction of bursts elicited by METH at a lowerconcentration (1mM). Tetraethylammonium chloride (TEA), a blocker of the delayed rectifying K(+) current (I(KD)), didnot elicit bursts at a concentration of 5mM but did facilitate the induction of action potential bursts elicited by bothMETH and MDMA. Voltage clamp studies revealed that both METH and MDMA decreased the TEA-sensitive I(KD) ofthe RP4 neuron. Forskolin (50 microM) or dibutyryl cAMP (1mM), a membrane-permeable cAMP analog, alone did notelicit action potential bursts. However, co-administration with forskolin (50 microM) and TEA (5mM) or co-administration with dibutyryl cAMP (1mM) and TEA (50mM) elicited action potential bursts in the presence of the PKCinhibitor chelerythrine (20 microM). Similarly, PDBu (10 microM) or phorbol 12-myristate 13-acetate (PMA; 3 microM)alone did not elicit action potential bursts. However, co-administration with PDBu (10 microM) and TEA (5mM) or co-administration with PMA (3 microM) and TEA (5mM) elicited action potential bursts in the presence of the PKA inhibitorKT-5720 (10 microM). These data suggest that action potential bursts in the RP4 neuron were not due to Ca(2+)-dependent synaptic effects. Rather, action potential bursts may be elicited through (1) combined activation of thecAMP-PKA signaling pathway and inhibition of the I(KD) and (2) combined activation of PKC and inhibition of the I(KD).

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581. Modulation of glucagon-like peptide-1 release by berberine: in vivo and in vitro studies

By Yu Yunli; Liu Li; Wang Xinting; Liu Xiang; Liu Xiaodong; Xie Lin; Wang GuangjiFrom Biochemical pharmacology (2010), 79(7), 1000-6, Language: English, Database: MEDLINE

Glucagon-like peptide (GLP)-1 is a potent glucose-dependent insulinotropic gut hormone released from intestinal Lcells. Our previous studies showed that berberine increased GLP-1 secretion in streptozotocin-induced diabetic rats.The aim of this study was to investigate whether berberine affected GLP-1 release in normal rats and in NCI-H716cells. Proglucagon and prohormone convertase 3 genes regulating GLP-1 biosynthesis were analyzed by RT-PCR.Effects of pharmacological inhibitors on berberine-mediated GLP-1 release were studied. In vivo, 5-week treatment ofberberine enhanced GLP-1 secretion induced by glucose load and promoted proglucagon mRNA expression as well asL cell proliferation in intestine. In vitro, berberine concentration-dependently stimulated GLP-1 release in NCI-H716cells. Berberine also promoted both prohormone convertase 3 and proglucagon mRNA expression. Chelerythrine(inhibitor of PKC) concentration-dependently suppressed berberine-mediated GLP-1 secretion. Compound C (inhibitorof AMPK) also inhibited berberine-mediated GLP-1 secretion. But only low concentrations of H89 (inhibitor of PKA)showed inhibitory effects on berberine-mediated GLP-1 release. The present results demonstrated that berberineshowed its modulation on GLP-1 via promoting GLP-1 secretion and GLP-1 biosynthesis. Some signal pathwaysincluding PKC-dependent pathway were involved in this process. Elucidation of mechanisms controlling berberine-mediated GLP-1 secretion may facilitate the understanding of berberine's antidiabetic effects.

~2 Citings

582. Autophagy and protein kinase C are required for cardioprotection by sulfaphenazole

By Huang Chengqun; Liu Wayne; Perry Cynthia N; Yitzhaki Smadar; Lee Youngil; Yuan Hua; Tsukada Yayoi Tetsuo;Hamacher-Brady Anne; Mentzer Robert M Jr; Gottlieb Roberta AFrom American journal of physiology. Heart and circulatory physiology (2010), 298(2), H570-9, Language: English,Database: MEDLINE

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Previously, we showed that sulfaphenazole (SUL), an antimicrobial agent that is a potent inhibitor of cytochromeP4502C9, is protective against ischemia-reperfusion (I/R) injury (Ref. 15). The mechanism, however, underlying thiscardioprotection, is largely unknown. With evidence that activation of autophagy is protective against simulated I/R inHL-1 cells, and evidence that autophagy is upregulated in preconditioned hearts, we hypothesized that SUL-mediatedcardioprotection might resemble ischemic preconditioning with respect to activation of protein kinase C and autophagy.We used the Langendorff model of global ischemia to assess the role of autophagy and protein kinase C in myocardialprotection by SUL during I/R. We show that SUL enhanced recovery of function, reduced creatine kinase release,decreased infarct size, and induced autophagy. SUL also triggered PKC translocation, whereas inhibition of PKC withchelerythrine blocked the activation of autophagy in adult rat cardiomyocytes. In the Langendorff model, chelerythrinesuppressed autophagy and abolished the protection mediated by SUL. SUL increased autophagy in adult ratcardiomyocytes infected with GFP-LC3 adenovirus, in isolated perfused rat hearts, and in mCherry-LC3 transgenicmice. To establish the role of autophagy in cardioprotection, we used the cell-permeable dominant-negative inhibitor ofautophagy, Tat-Atg5(K130R). Autophagy and cardioprotection were abolished in rat hearts perfused with recombinantTat-Atg5(K130R). Taken together, these studies indicate that cardioprotection mediated by SUL involves a PKC-dependent induction of autophagy. The findings suggest that autophagy may be a fundamental process that enhancesthe heart's tolerance to ischemia.

~1 Citing

583. Involvement of protein kinase C-CPI-17 in androgen modulation of angiotensin II-renal vasoconstriction

By Song Jin; Eyster Kathleen M; Kost Curtis K Jr; Kjellsen Barton; Martin Douglas S

From Cardiovascular research (2010), 85(3), 614-21, Language: English, Database: MEDLINEAIMS: Previous studies suggested that androgens augmented renal vascular responses to angiotensin II (Ang II). Theprotein kinase C (PKC)-CPI-17 pathway is involved in vascular constriction. We tested the hypothesis that thispathway may contribute to androgenic amplification of Ang II-renal vasoconstriction in the New Zealand geneticallyhypertensive (NZGH) rat. METHODS AND RESULTS: NZGH underwent sham operation, castration, or castrationwith testosterone replacement at 5 weeks of age. When the rats were 16-17 weeks of age, mean arterial pressure(MAP) and renal vascular resistance (RVR) responses to intravenous Ang II infusion (20, 40, and 80 ng/kg/min) wererecorded before and after treatment with a PKC inhibitor, chelerythrine. mRNA expression of PKC isoforms and CPI-17protein expression were analysed in renal cortex. MAP and RVR responses to Ang II were enhanced in androgen-replete NZGH. The Ang II-induced increase in RVR was significantly lower in castrated NZGH (ranged from 100 +/-8% to 161 +/- 9% of baseline) than in sham-operated NZGH (ranged between 123 +/- 3% and 237 +/- 19% ofbaseline). Testosterone treatment restored RVR responses to Ang II in castrated rats. Chelerythrine treatmentmarkedly reduced the MAP and RVR responses to Ang II in each group and attenuated the differential MAP and RVRresponses to Ang II amongst the three groups. PKCdelta and PKCepsilon mRNA levels were significantly reduced bycastration and increased by testosterone treatment. In contrast, no significant differences in protein expression weredetected for these PKC isoforms. Castration decreased while testosterone treatment increased CPI-17 and phospho-CPI-17 expression. CONCLUSION: Collectively, these results suggest that androgens modulate renal vascularresponses to Ang II in part via an effect on the PKC-CPI-17 signalling pathway.

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584. High glucose increases expression of cyclooxygenase-2, increases oxidative stress and decreases the generationof nitric oxide in mouse microvessel endothelial cells

By Aljofan Mohamad; Ding HongFrom Journal of cellular physiology (2010), 222(3), 669-75, Language: English, Database: MEDLINE

Hyperglycaemia is a key factor that contributes to the development of diabetes-related microvascular disease. Bothcyclooxygenase I and cyclooxygenase II are expressed in endothelial cells and play key roles in the regulation ofcardiovascular function. In the current study we tested the hypothesis that hyperglycaemia-induced increasedexpression of cyclooxygenase II is a contributing factor both to the increased oxidative stress and to the reduction inthe generation of nitric oxide in microvessel endothelial cells following their exposure to high glucose. Wedemonstrated that the exposure of mouse microvascular endothelial cells to high glucose for 3 days decreased thegeneration of nitric oxide and enhanced production of superoxide. Western blots illustrated that exposure to highglucose also increased endothelial nitric oxide synthase and cyclooxygenase II protein expression levels anddecreased the dimer/monomer ratio of endothelial nitric oxide synthase protein. All the changes induced by the highglucose culture media could be reversed by either the cyclooxygenase II inhibitor CAY10404, the non-selectivecyclooxygenase inhibitor indomethacin or the protein kinase C inhibitor chelerythrine, but not solely by preincubationwith the antioxidant and putative NADPH oxidase inhibitor, apocynin. Our data indicate that high glucose inducedoxidative stress is linked to an increase in the expression of cyclooxygenase II and a reduced generation of nitric oxidethat is associated with an uncoupled endothelial nitric oxide synthase, possibly due to decreased dimer/monomer ratio.

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585. PTTH-stimulated ERK phosphorylation in prothoracic glands of the silkworm, Bombyx mori: role ofCa(2+)/calmodulin and receptor tyrosine kinase

By Gu Shi-Hong; Lin Ju-Ling; Lin Pei-LingFrom Journal of insect physiology (2010), 56(1), 93-101, Language: English, Database: MEDLINE

Our previous studies showed that the prothoracicotropic hormone (PTTH) stimulated extracellular signal-regulatedkinase (ERK) phosphorylation in prothoracic glands of Bombyx mori both in vitro and in vivo. In the present study, thesignaling pathway by which PTTH activates ERK phosphorylation was further investigated using PTTH, secondmessenger analogs, and various inhibitors. ERK phosphorylation induced by PTTH was partially reduced in Ca(2+)-free medium. The calmodulin antagonist, calmidazolium, partially inhibited both PTTH-stimulated ERK phosphorylationand ecdysteroidogenesis, indicating the involvement of calmodulin. When the prothoracic glands were treated withagents that directly elevate the intracellular Ca(2+) concentration [either A23187, thapsigargin, or the protein kinase C(PKC) activator, phorbol 12-myristate acetate (PMA)], a great increase in ERK phosphorylation was observed. Inaddition, it was found that PTTH-stimulated ecdysteroidogenesis was greatly attenuated by treatment with PKCinhibitors (either calphostin C or chelerythrine C). However, PTTH-stimulated ERK phosphorylation was not attenuatedby the above PKC inhibitors, indicating that PKC is not involved in PTTH-stimulated ERK phosphorylation. A potentand specific inhibitor of insulin receptor tyrosine kinase, HNMPA-(AM)(3), greatly inhibited the ability of PTTH toactivate ERK phosphorylation and stimulate ecdysteroidogenesis. However, genistein, another tyrosine kinaseinhibitor, did not inhibit PTTH-stimulated ERK phosphorylation, although it did markedly attenuate the ability of A23187to activate ERK phosphorylation. From these results, it is suggested that PTTH-stimulated ERK phosphorylation isonly partially Ca(2+)- and calmodulin-dependent and that HNMPA-(AM)(3)-sensitive receptor tyrosine kinase isinvolved in activation of ERK phosphorylation by PTTH.

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586. Differential contributions of protein kinase C isoforms in the regulation of group IIA secreted phospholipase A2expression in cytokine-stimulated rat fibroblasts

By Sugita Mizuki; Kuwata Hiroshi; Kudo Ichiro; Hara ShuntaroFrom Biochimica et biophysica acta (2010), 1801(1), 70-6, Language: English, Database: MEDLINE

Protein kinase C (PKC) is a family of serine/threonine kinases involved in various signal transduction pathways. Weinvestigated the roles of PKC in the regulation of group IIA secreted phospholipase A(2) (sPLA(2)-IIA) expression incytokine-stimulated rat fibroblastic 3Y1 cells. Here we show that the induction of sPLA(2)-IIA by proinflammatorycytokines was under the control of both classical cPKCalpha and atypical aPKClambda/iota pathways by using PKCinhibitors, a PKC activator, and PKC knockdowns. Treatment of 3Y1 cells with PKC selective inhibitors having broadspecificity, such as chelerythrine chloride and GF109203X, blocked IL-1beta/TNFalpha-dependent induction ofsPLA(2)-IIA protein in a dose-dependent manner. Treatment with the PKC activator phorbol 12-myristate 13-acetate(PMA), which activates cPKC and novel nPKC isoforms, markedly attenuated the cytokine-dependent induction ofsPLA(2)-IIA expression. In comparison, 24-h pretreatment with PMA, which down-regulates these PKC isoforms,markedly enhanced sPLA(2)-IIA expression. Results with short hairpin RNA (shRNA)-mediated knockdown of PKCisoforms revealed that the cytokine-induced sPLA(2)-IIA expression was markedly enhanced in cPKCalpha knockdowncells compared to those in replicate control cells. In contrast, knockdown of the aPKClambda/iota isoform reduced thecytokine-induced expression of sPLA(2)-IIA. These results suggest that the aPKClambda/iota pathway is required forthe induction of sPLA(2)-IIA expression and that the cPKCalpha pathway acts as a negative regulator of sPLA(2)-IIAexpression in cytokine-stimulated rat fibroblasts.

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