pkc and mapk signalling pathways regulate vascular endothelin receptor expression

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gy 580 (2008) 190–200www.elsevier.com/locate/ejphar

European Journal of Pharmacolo

PKC and MAPK signalling pathways regulate vascular endothelinreceptor expression

David Nilsson a, Angelica Wackenfors a, Lotta Gustafsson a, Martin Ugander b,Richard Ingemansson c, Lars Edvinsson a, Malin Malmsjö a,d,⁎

a Department of Medicine, Lund University Hospital, Lund, Swedenb Department of Clinical Physiology, Lund University Hospital, Lund, Sweden

c Department of Cardiothoracic Surgery, Lund University Hospital, Lund, Swedend Department of Ophthalmology, Lund University Hospital, Lund, Sweden

Received 23 May 2007; received in revised form 12 October 2007; accepted 18 October 2007Available online 1 November 2007

Abstract

Up-regulation of vascular endothelin type A (ETA) and type B (ETB) receptors are implicated in the pathogenesis of cardiovascular disease.Culture of arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for in detaildelineation of the regulation of endothelin receptors. We hypothesize that protein kinase C (PKC) and mitogen-activated kinases (MAPK) areinvolved in the regulation of endothelin receptors. Porcine coronary arteries were studied before and after 24 h of culture, using in vitropharmacology, real-time PCR and immunofluorescence techniques. Sarafotoxin 6c and endothelin ET-1 were used to examine the endothelin ETAand ETB receptor effects. The involvement of PKC and MAPK in the receptor regulation was examined by culture in the presence of antagonists.Organ culture resulted in increased sarafotoxin 6c and endothelin-1 contractions, endothelin ETA and ETB receptor immunofluorescence stainingintensities and endothelin ETB, but not ETA, receptor mRNA levels. The general PKC inhibitors, bisindolylmaleimide I (10 μM) or Ro-32-0432(10 μM), inhibited these effects. Also, the increase in sarafotoxin 6c contraction, endothelin ETB receptor and mRNA levels and endothelin ETAand ETB immunofluorescence staining intensities were inhibited by MAPK inhibitors for extracellular signal related kinases 1 and 2 (ERK1/2),PD98059 (10 μM), C-jun terminal kinase (JNK), SP600125 (10 μM), but not by p38 MAPK, SB203580 (10 μM). In conclusion, PKC and MAPKseem to be involved in the regulation of endothelin receptor expression in porcine coronary arteries. Inhibiting these intracellular signaltransduction pathways may provide a future therapeutic target for hindering the development of vascular endothelin receptor changes incardiovascular disease.© 2007 Elsevier B.V. All rights reserved.

Keywords: Endothelin; Coronary artery; Contraction; Vascular

1. Introduction

Endothelin-1 (ET-1) has been proposed to be of majorimportance in the development of cardiovascular disorders. ET-1 mediates its effect through two distinct G-protein coupled

⁎ Corresponding author. Experimental Vascular Research, BMC A13, LundUniversity Hospital, SE-221 84 Lund, Sweden. Tel.: +46 733 565 650; fax: +4646 222 0616.

E-mail address: [email protected] (M. Malmsjö).

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.10.071

receptors; the endothelin type A (ETA) and type B (ETB)receptors (Arai et al., 1990; Sakurai et al., 1990). Duringphysiologic conditions, the endothelin ETA receptor is thedominant receptor subtype on vascular smooth muscle cells andmediates contraction, while the endothelin ETB receptor isprimarily located on endothelial cells and mediates vasodilata-tion via the release of nitric oxide and prostaglandins (Maguireand Davenport, 1995; Szok et al., 2001). However endothelinETB receptors on vascular smooth muscle cells have beenobserved to be up-regulated during pathological conditions suchas atherosclerosis (Dagassan et al., 1996), congestive heart

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191D. Nilsson et al. / European Journal of Pharmacology 580 (2008) 190–200

failure (Cannan et al., 1996), and ischemic heart disease(Wackenfors et al., 2004b). Endothelin receptors on vascularsmooth muscle cells are both mitogenic, leading to atheroscle-rosis, and mediate strong vasoconstriction which may lead toelevated vascular tone frequently observed in cardiovasculardisease.

Little is known about the intracellular signal transductionpathways that are involved in endothelin receptor regulation. Inthe present study we hypothesised that protein kinase C (PKC)and mitogen-activated kinase (MAPK) signalling pathways areinvolved, which has been shown before in studies in rats(Henriksson et al., 2006; Uddman et al., 2003). PKC is a familyof serine/threonine kinases participating in signal transductionevents in response to specific hormonal, neuronal and growthfactor stimuli (Henriksson et al., 2006). MAPK representanother group of serine/threonine kinases that are thought to actdownstream from protein kinase C in the smooth muscle cellregulatory cascade (Schonwasser et al., 1998). There are threemajor groups of distinctly regulated MAPKs leading to alteredgene expression in humans. The extracellular signal relatedkinases 1 and 2 (ERK1/2), the C-jun terminal kinase (JNK) andthe p38 MAPK. MAPKs are known to play an important role inthe intracellular signalling in response to extracellular stimuli(Hazzalin and Mahadevan, 2002). Upon activation, MAPKscause phosphorylation and activation of transcription factorspresent in the cytoplasm or nucleus, thereby leading toexpression of target genes resulting in biological responses(Kaminska, 2005).

We have developed an in vitro method for the study ofendothelin receptor regulation using an organ culture model ofintact blood vessels. Endothelin receptors on smooth musclecells are up-regulated when whole blood vessels are incubatedfor 12 to 48 h. From previous in detailed studies, we knowthat the receptor regulation is dependent on the presence of37 °C and glucose (Adner et al., 1996). Endothelin receptorsare up-regulated in human coronary arteries after organculture, in a similar way as in ischemic heart disease in man(Wackenfors et al., 2004b). Endothelin receptor changes alsooccur during organ culture in rat cerebral and peripheralarteries, mimicking that observed in peripheral artery disease,stroke and subarachnoidal haemorrhage (Hansen-Schwartz,2004; Lind et al., 1999; Stenman et al., 2002) Organ culturehas, therefore, been suggested as a suitable model for studyingendothelin receptor regulation. Detailed delineation of theregulation can be performed by culture in the presence ofdifferent humoral factors or intracellular signal transductionpathway inhibitors.

In the present study, we used the organ culture model toexamine the involvement of PKC and MAPK pathways in theregulation of endothelin receptors in coronary arteries. Seg-ments of porcine coronary arteries were cultured for 24 h in theabsence or presence of PKC inhibitors (Ro-32-0432 andbisindolylmaleimide I) and inhibitors of the three majorMAPK pathways in mammals (ERK1/2, JNK and p38MAPK). The contractile effects and the levels of receptorprotein and mRNAwere evaluated using in vitro pharmacology,immunofluorescence and real-time PCR.

2. Materials and methods

2.1. Ethics

The study was approved by the Ethics Committee of LundUniversity in Sweden.

2.2. Tissue preparation

The coronary arteries were obtained from domestic landracepigs. The pigs were of both genders and had a mean body weightof 50 kg. Before surgery they were fasted overnight with freeaccess to water. Anaesthesia was induced by an intramuscularinjection of ketamine (Ketaminol vet™ 100 mg/ml; FarmaceuticiGellini S.p.A, Aprilia, Italy) 15 mg/kg body weight,in combination with midazolam (Dormicum, 1 mg/ml, Roche,Stockholm, Sweden) 0.1 mg/kg and xylazine (Rompun vet.™20 mg/ml; Bayer AG, Leverkusen, Germany) 2 mg/kg.Anaesthesia was maintained by continuous intravenous infusionof propofol (0.1–0.2 mg/kg/min, Diprivan™ 20 mg/ml; AstraZeneca, Sweden) in combination with intermittent fentanyl(0.02 μg/kg, Leptanal™; Lilly, France) and atracurium besylate(0.2–0.5 mg/kg, Tracrium™; Glaxo, Täby, Sweden). The heartswere removed through a midline sternotomy and immediately puton ice. The left anterior descending coronary artery was dissected,immersed in cold sterile Dulbeccos' modified Eagles' medium(DMEM) and transported to the laboratory on ice. In thelaboratory, the coronary arteries were dissected free from adheringtissue, and then cut into cylindrical segments (3–4 mm long).

2.3. Organ culture procedure

The artery segments were divided into two groups; one thatwas not cultured and one that was cultured for 24 h. The arterysegments for culture were placed in a 48 well plate, one seg-ments in each well, containing 1 ml DMEM (for composition,see below) and incubated for 24 h at 37 °C in humidified 5%CO2 in air. The method of blood vessel culture has beendescribed previously (Adner et al., 1996), and similar resultshave been observed when culture in DMEM and a bicarbonatebuffer solution (Krebs buffer, for composition see below). Thesegments were cultured in the absence or presence of thegeneral PKC inhibitors Ro-32-0432 (10 μM) or bisindolylma-leimide I (10 μM) or the MAPK inhibitors SP600125 (10 μM,JNK inhibitor), PD98059 (10 μM, ERK1/2 inhibitor), orSB203580 (10 μM, p38 MAPK inhibitor).

• Ro-32-0432: 2-{8-[(Dimethylamino)methyl]-6,7,8,9-tetra-hydropyrido[1,2-a]indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide

• Bisindolylamaleimide I: 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide

• SP600125: 1,9-Pyrazoloanthrone Anthrapyrazolone• PD 98059: 2-(2-Amino-3-methoxyphenyl)-4H-1-benzo-pyran-4-one

• SB 203580: 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole

192 D. Nilsson et al. / European Journal of Pharmacology 580 (2008) 190–200

2.4. In vitro pharmacology

For in vitro pharmacological experiments, the arterialsegments were mounted on two L-shaped metal prongs, oneof which was connected to a force displacement transducer forcontinuous recording of the isometric tension (Högestätt et al.,1983).The mounted segments were immersed in temperaturecontrolled (37 °C) tissue baths containing a bicarbonate basedbuffer solution (for composition, see below), which wascontinuously gassed with 5% CO2 in O2 resulting in a PH of7.4. Eight to sixteen segments were studied at the same time inseparate tissue baths. The segments had to stabilize at a restingtension of 4 mN for 1 h before the experiments were started. Aresting tension of 2 to 8 mN provides propitious conditions forstudying vascular contraction in coronary arteries (SaetrumOpgaard and Edvinsson, 1997; Wackenfors et al., 2003). Thecontractile capacity of each arterial vessel segment wasexamined by exposure to a potassium rich (63.5 mM) buffersolution.

The endothelin ETB receptor agonist, sarafotoxin 6c, wasfirst added at increasing concentrations (10−11–10−6 M). Thearteries were washed and ET-1 was added at increasingconcentrations (10−11–10−6 M). At this stage the endothelinETB receptors were desensitized (Lodge et al., 1995), allowingET-1 to act selectively on endothelin ETA receptors.

The ET-1 experiments were run in the absence (control)and presence of 0.1 μM of the selective endothelin ETAreceptor antagonist, FR139317 ((R)2-[(R)-2-[(S)-2-[[1-(hex-ahydro-1H-azepinyl)]carbonyl]amino-4-methylpentanoyl]amino-3-[3-(1-methyl-1H-indoyl)]propionyl]amino-3-(2-pyridyl)propionic acid). The sarafotoxin 6c experiments were run in theabsence (control) and presence of the selective endothelin ETBreceptor antagonist BQ788 (0.1 μM). All antagonists were added15 min prior to each agonist.

In order to check the desensitization of endothelin ETB

receptors, the effect of the selective endothelin ETB receptorantagonist, BQ788 (0.1 μM), on the ET-1 contraction aftersarafotoxin 6c desensitization, was examined. The ET-1contraction was similar both in the presence and absence ofBQ788, suggesting activation of endothelin ETA receptorssolely.

2.5. Drugs and solutions for in vitro pharmacology

The bicarbonate buffer had the following composition: NaCl(119 mM), NaHCO3 (15 mM), KCl (4.6 mM), MgCl (1.2 mM),NaH2PO4 (1.2 mM), CaCl2, (1.5 mM) and glucose (5.5 mM).The DMEM (Gibco BRL, Praisley, UK) was serum-free andcontained D-glucose (1 g/l), sodium pyruvate (100 mg/l) andwas supplemented with penicillin (100 U/ml), streptomycin(100 μg/ml) and amphotericin B (0.25 μg/ml). All drugs for thein vitro pharmacological experiments were purchased fromSigma Chemical Co (St. Louis, MO). ET-1 and sarafotoxin 6cwere dissolved in 0.9% NaCl with 10% albumin. BQ788 andFR139317 were dissolved in 0.9% saline. The PKC and MAPKinhibitors were dissolved in dimethylsulphoxide (DMSO,Calbiochem ®, Darmstadt, Germany).

2.6. Real-time PCR

The arteries for real-time PCR experiments were frozen inliquid nitrogen and stored at −80 °C until the experiments wereperformed. Endothelin ETA and ETB receptor mRNA expressionlevels were quantified by real-time PCR. Total cellular RNAwasextracted usingTRIzol®LS according to the supplier's instructions(Life Technologies, Paisley, UK). Reverse transcription of totalRNA to cDNAwas carried out using the GeneAmpRNAPCR kitin a DNA Thermal cycler (Perkin-Elmer Applied Biosystems,Foster City, CA, USA). Real-time PCR was performed with theGene-Amp SYBR Green PCR kit (PE Applied Biosystems) in aPerker-Elmer real-time PCR machine 7300.

The cDNA synthesised above served as template in a (25 μl)reaction. A non-template control was included in all experi-ments. The GeneAmp 7300 sequence detection system moni-tored the binding of a fluorescent dye to double-strand DNA byreal-time detection of the fluorescence during each cycle of PCRamplification. Specific primers were designed as follows:

Endothelin ETA receptor forward 5′-TCCTCA ATC TCTGCG CTC TCA-3′Endothelin ETA receptor reverse 5′-ATC TCA ATG GCTGTG ACC AAT G-3′Endothelin ETB receptor forward 5′-TGC GAA ACG GCCCTA ACA-3′Endothelin ETB receptor reverse 5′-TCG GCG AGC AGCTTG TAG TAG A-3′GAPDH forward 5′-GTG ATG GGC ATG AAC CAT GA-3′GAPDH reverse 5′-GCTGATGATCTTGAGGCTGTTG-3′β-actin forward 5′-CCT TCA ACT CGA TCA TGA AGTGC-3′β-actin reverse 5′-CGT AGA GGT CCT TCC TGA TGTCC-3′

The housekeeping genes, β-actin and glyceraldehyde 3-phos-phate dehydrogenase (GAPDH) were used as references due totheir continuous expression in cells. The real-time PCR reactionwas performed at a temperature of 50 °C for 2 min, 95 °C for10 min, and the following 40 PCR cycles with 95 °C for 15 s and60° for 1 min.

2.7. Drugs and solutions for real-time PCR

Oligonucleotides and reagents for the PCR assay werepurchased from Perkin-Elmer, Applied Biosystems Foster City,CA, USA.

2.8. Immunofluorescence

Endothelin ETA and ETB receptor protein expression wasvisualized using immunofluorescence. The artery segmentswere sectioned into 7 μm slices and placed on poly-L-lysincoated glass slides. The slides were fixed for 10 min in ice coldacetone and rehydrated in PBS for 10 min. The tissues werethen permeabilized and blocked in 0.2% Triton X-100/1%bovine serum albumin (BSA) in PBS for 1 h followed by


incubation overnight with mouse anti-porcine CD31 (Serotec,Raleigh, NC, USA) 1:3 and rabbit anti-human endothelin ETB

receptor (IBL-10253, Immuno-Biological Laboratories Co.,Gunma, Japan) 1:50 or goat anti-human endothelin ETAreceptor (R-19, Santa Cruz Biotechnology Inc., Santa Cruz,CA, USA) 1:50 in PBS with 1% BSA. The slides were washedtwice in 0.2% Triton X-100/1% bovine serum albumin (BSA) inPBS followed by incubation overnight with donkey anti-mouseCy™5 (Jackson Immuno Research Laboratories Inc., WestGrove, PA, USA) 1:200 and goat-anti-rabbit Alexa-633(Molecular Probes, or donkey anti-goat Cy™3 (JacksonImmuno Research Laboratories Inc., West Grove, PA, USA)1:200 or in PBS with 1% BSA. The slides were washed twice inPBS and analyzed by confocal microscopy. For illustration ofvessel morphology, a transmission image, corresponding toeach immunofluorescence image, was captured by a highresolution CCD camera. All arteries for immunofluorescencewere examined at the same time to minimize the variability.Confocal scans were recorded with identical settings in eachexperiment and optical sections were set to 1.0 μm. Primary andsecondary antibody controls were run in parallel in each ex-periment. Staining intensity is expressed as arbitrary fluores-cence units.

2.9. Calculations and statistics

Calculations and statistics were performed using MicrosoftOffice Excel 2003 and Graph Pad 4.0 software. The experi-ments were repeated six to eight times. Statistical significance

Fig. 1. (A) Endothelin-1 and (B) sarafotoxin 6c contractions and (C) endothelin ETA aarterial segments. Values are presented as mean values±S.E.M. Statistical analyses wThe values for PCR are presented as mean values±S.E.M. relative to the β-actin le

was accepted when Pb0.05, using Student's t-test. The resultsare expressed as mean±standard error of the mean (S.E.M.).

In vitro pharmacology: the maximum contraction (Emax) wascalculated as percentage of the contractile capacity of 63.5 mMpotassium. The negative logarithm of the concentration thatelicited 50% contraction (pEC50) was determined by linearregression analysis using the values immediately above andbelow half-maximum response.

Real-time PCR: the amount of endothelin ETA and ETB

receptor mRNA expression was calculated as relative to theamount GAPDH or β-actin in the same sample by the formulaX0/R0=2CtR−CtX, where X0=amount of endothelin ETA orETB mRNA, R0=original amount of GAPDH or β-actinmRNA, CtR=Ct value for GAPDH or β-actin and CtX=Ctvalue for the endothelin ETA or ETB receptor mRNA.

Immunofluorescence: the staining intensities for endothelinETA and ETB receptor protein was quantified using confocalmicroscopy Image J (http://rsb.info.nih.gov/ij/).

3. Results

3.1. Effects of organ culture on endothelin ETA and ETBreceptors

The endothelin ETB receptor mediated contraction wasstudied using the selective agonist sarafotoxin 6c. The ET-1induced vasoconstriction was studied after desensitizing theendothelin ETB receptors with sarafotoxin 6c prior to addingET-1, leaving only endothelin ETA receptors to respond.

nd (D) ETB receptor mRNA levels in cultured and non-cultured porcine coronaryere performed using Student's t-test where Pb0.05 was considered significant.vels.

http://rsb.info.nih.gov/ij/

Fig. 2. Contractile responses elicited by cumulative application of endothelin-1 or sarafotoxin 6c. The arterial segments were either not cultured or cultured in theabsence or presence of the PKC inhibitors bisindolylmaleimide I or Ro-32-0432. Values are presented as mean values±S.E.M. Statistical analyses were performedusing Student's t-test where Pb0.05 was considered significant.

Fig. 3. Endothelin ETB receptor mRNA levels assessed by real-time PCR inporcine coronary arteries. The arteries were either not cultured or cultured in theabsence or presence of the PKC inhibitors bisindolylmaleimide I or Ro-32-0432.Values are presented as mean values±S.E.M. relative to the β-actin levels.Statistical analyses were performed using Student's t-test where Pb0.05 wasconsidered significant.


In fresh porcine coronary arteries, both ET-1 and sarafotoxin6c induced potent contractions (Fig. 1). The selective endothelinETA receptor antagonist FR139317 inhibited the ET-1 response(pEC50=7.7±0.3 without and 7.1±0.2 with FR139317). Thesarafotoxin 6c contraction was inhibited by the selectiveendothelin ETB receptor antagonist, BQ788 (pEC50=8.6±0.2without and 8.1±0.3 with BQ788).

The efficacy of the ET-1- and sarafotoxin 6c-contractions weresignificantly increased after culture (Fig. 1), suggesting up-regulated endothelin ETA and ETB receptor mediated responses.After culture, the selective endothelin ETA receptor antagonistFR139317 inhibited the ET-1 response (pEC50=7.6±0.3withoutand 7.0±0.2with FR139317). The sarafotoxin 6c contractionwasinhibited by the selective endothelin ETB receptor antagonist,BQ788 (pEC50=8.1±0.2 without and 7.5±0.2 with BQ788).

The K+ contraction was not affected by culture (12.3±2.3 mN before and 14.1±2.1 mN after organ culture, P=n.s.),indicating unchanged smooth muscle cell function.

Real-time PCR experiments demonstrated elevated levels ofendothelin ETB, but not ETA, receptor mRNA, after culture(Fig. 1). Immunofluorescence experiments demonstrated in-creased staining intensities for both endothelin ETA and ETB

receptor protein in the vascular smooth muscle cells, after organculture (Figs. 4 and 7).

3.2. Inhibition of PKC

The increase in ET-1 and sarafotoxin 6c contraction andendothelin ETB receptor mRNA levels during organ culture wasinhibited by culture in the presence of either of the general PKCinhibitors, bisindolylmaleimide I or Ro-32-0432 (Figs. 2 and 3).Also, the increase in endothelin ETA and ETB receptor immuno-

fluorescence staining intensities, during organ culture, wereinhibited by bisindolylmaleimide I (Fig. 4).

3.3. Inhibition of MAPK

Culture in the presence of the ERK1/2 pathway inhibitor,PD98059 (10 μM), and the JNK pathway inhibitor, SP600125(10 μM), inhibited the increase in sarafotoxin 6c contraction,endothelin ETB receptor mRNA levels and endothelin ETA andETB receptor immunofluorescence staining intensities duringorgan culture, while there was only a tendency towards inhibitingthe up-regulated ET-1 contraction (Figs. 5, 6 and 7). The p38

Fig. 4. The staining intensities for (A) endothelin ETA and (B) ETB receptors was quantified in the smooth muscle cell layer of the porcine coronary arteries, beforeculture (0 h), after culture (24 h), and after culture in the presence of bisindolylmaleimide I (24 h, bis), using confocal microscopy. The staining intensities are expressedin arbitrary fluorescence units and are presented as mean values±S.E.M. Statistical analyses were performed using Students' t-test, where Pb0.05 was consideredsignificant. Bottom panels show confocal images with representative examples of endothelin ETB receptor staining. The images are captured to show both theendothelial cell (EC) and smooth muscle cell (SMC) layer, where the vessel lumen is on the top of the images. Below each immunofluorescence image is a transmissionimage for illustration of morphology. Note how the endothelin ETB receptor fluorescence is more intense in the smooth muscle cell layer, after culture.


MAPK pathway inhibitor, SB203580, did not have such effects(Figs. 5–7).

4. Discussion

4.1. Main findings

The present work has in part elucidated the intracellular signaltransduction pathways involved in the endothelin receptor regu-lation. Organ culture of porcine coronary arteries result in up-regulation of endothelin ETA and ETB receptor mediated contrac-

tion. Likewise, the endothelin ETA and ETB receptor immunoflu-orescence staining intensities and the endothelin ETB, but not ETA,receptor mRNA levels were increased after organ culture.Inhibition experiments revealed that PKC and MAPK signallingpathways were involved in the endothelin receptor regulation.

4.2. Organ culture and endothelin receptor up-regulation

From previous studies in other vascular beds, organ culturehas been suggested as an experimental model for in detaildelineation of the regulation of endothelin receptors, since the

Fig. 5. Contractile responses elicited by cumulative application of endothelin-1 (A) and sarafotoxin 6c (B). The arterial segments were either not cultured or cultured inthe absence or the presence of mitogen-activated kinase pathway inhibitors; the ERK1/2 inhibitor PD98059, the JNK inhibitor SP600125 or the P38 MAPK inhibitorSB203580. Values are presented as mean values±S.E.M. Statistical analyses were performed using Student's t-test where Pb0.05 was considered significant.

Fig. 6. Endothelin ETB receptor mRNA levels assessed by real-time PCR inporcine coronary arteries. The arteries were either not cultured or cultured in theabsence or the presence of mitogen-activated kinase pathway inhibitors; theERK1/2 inhibitor PD98059, the JNK inhibitor SP600125 or the P38 MAPKinhibitor SB203580. Values are presented as mean values±S.E.M. relative to theβ-actin levels. Statistical analyses were performed using Student's t-test wherePb0.05 was considered significant.


changes are similar to those frequently observed in cardiovas-cular disease. This is the first time that organ culture, as anexperimental model for endothelin receptor regulation, has beenevaluated in porcine coronary arteries. Culture of porcinecoronary artery segments for 24 h resulted in increased ET-1and sarafotoxin 6c contractions. The changes were endothelinreceptor pathway specific since the response to 63.5 mMpotassium is not altered, indicating intact smooth muscle cellfunction. Immunofluorescence experiments show increasedendothelin ETA and ETB receptor staining intensities on smoothmuscle cells. Real time PCR results suggest elevated endothelinETB, but not ETA, receptor mRNA levels. Taken together, theseresults suggest that the organ culture model is suitable forstimulating up-regulation of endothelin receptors in porcinecoronary arteries. The changes that occur during organ culturemay be comparable to those that take place in cardiovasculardisease. Endothelin ETB receptors are up-regulated in humancoronary arteries during organ culture (Wackenfors et al.,2004b) thereby mimicking the changes that can be observed incoronary arteries from patients with ischemic heart disease orcongestive heart failure (Dagassan et al., 1996; Wackenforset al., 2004b). Indeed, endothelin ETB receptors appear tocontribute to basal vascular tone, induced by endogenousendothelin release, to a higher extent in patients with coronaryheart diseases (Wenzel et al., 1996).

Similar comparisons between the endothelin receptorregulation in organ culture and that in cardio- and cerebrovas-cular disease have been made before. Organ culture of cerebraland temporal arteries result in up-regulation of endothelin ETB

receptors (Hansen-Schwartz et al., 2002; White et al., 1999),mimicking the changes that occur in the rat middle cerebralartery during experimental stroke (Stenman et al., 2002). Also,endothelin ETB receptor mRNA and protein levels in rat middlecerebral arteries and basilar arteries have been shown to beincreased in a time dependent manner after experimentalsubarachnoidal haemorrhage, thus in line with what happensin organ culture (Beg et al., 2006; Beg et al., 2007). Organculture may provide an experimental model in which thedevelopment of receptor changes on smooth muscle cells can be

Fig. 7. The staining intensities for (A) endothelin ETA and (B) ETB receptors was quantified in the smooth muscle cell layer of the porcine coronary arteries, beforeculture (0 h), after culture (24 h), and after culture in the presence the ERK1/2 inhibitor PD98059, the JNK inhibitor SP600125 or the P38 MAPK inhibitor SB203580,using confocal microscopy. The staining intensities are expressed in arbitrary fluorescence units and are presented as mean values±S.E.M. Statistical analyses wereperformed using Students' t-test, where Pb0.05 was considered significant. Bottom panels show confocal images with representative examples of endothelin ETB

receptor staining. The images are captured to show both the endothelial cell (EC) and smooth muscle cell (SMC) layer, where the vessel lumen is on the top of theimages. Below each immunofluorescence image is a transmission image for illustration of morphology.


studied in detail to further delineate the molecular mechanismsinvolved. Culture in the presence of different humoral factors orintracellular messenger inhibitors may reveal important path-ways involved in the regulation of endothelin receptors. Themethod thereby combines the advantage of cell culturingtechniques with the advantage of functional evaluation of intactblood vessels.

Both the endothelin ETA and ETB mediated contractionswere up-regulated during organ culture. Also, immunofluores-cence experiments suggest enhanced endothelin ETA and ETB

receptor protein expression. Real-time PCR experimentsshowed up-regulated endothelin ETB, but not ETA, receptormRNA levels. These results suggest that the alteration in

endothelin ETB receptor expression during culture involvesincreased transcription, while the alteration in endothelin ETAcontraction may be due to increased translation from mRNA toprotein or increased activity on the protein level. The reason forthis discrepancy cannot be deduced from the present study.

The increased endothelin contraction in organ culture hasbeen attributed to the up-regulation of contractile endothelinreceptors on smooth muscle cells. Organ culture is known to beaccompanied by decreased endothelium function, mimickingthe endothelium dysfunction that develops in cardiovasculardisease (Alm et al., 2002; de Meyer, 2000). Likewise, recentresults from our group show that endothelin ETB receptors onthe endothelium are downregulated during culture (Nilsson


et al., in press), although these effects are minor and will notsignificantly affect the conclusions drawn in the present study.

4.3. PKC in the endothelin receptor upregulation

This study was aimed to elucidate the role for the intracellularsignal transduction pathways, PKC and MAPK, in theendothelin ETA and ETB receptor regulation. The PKC familyis divided into the classic isoforms (α, βI, βII, and γ), activatedby phospatidylserine, calcium and diacylglycerol or phorbolesters, the novel PKCs (δ, ε, η, θ, and μ) activated bydiacylglycerol and phorbol esters and the atypical PKCs(ξ and λ) not activated by calcium, phorbol esters ordiacylglycerol (Murphy and Frishman, 2005). We used thegeneral PKC inhibitors Ro-32-0432 and bisindolylmaleimide I(Henriksson et al., 2007). Ro-32-0432 principally affects the α-subtype but also to a smaller extent the βI, βII, and γ-subtypes(Wilkinson et al., 1993), whereas bisindolylmaleimide I inhibitsthe four classical subtypes α, βI, βII, and γ with a similarpotency (Toullec et al., 1991). Ro-32-0432 and bisindolylma-leimide I each inhibited the increase in endothelin ETA and ETB

receptor contraction and endothelin ETB receptor mRNA levels,during organ culture. Furthermore, bisindolylmaleimide Iinhibited the increase in endothelin ETA and ETB receptorimmunofluorescence staining intensities during organ culture.

These results strongly suggest that PKC pathways areinvolved in the up-regulation of endothelin receptors duringorgan culture. This is supported by previous studies in ratswhich have demonstrated that the endothelin ETB receptor up-regulation involves PKC (Hansen-Schwartz et al., 2002;Henriksson et al., 2003; Uddman et al., 2002). Furthermore invivo rat studies have demonstrated that PKC inhibitors preventup-regulation of vascular endothelin ETB receptors and reversescerebral blood flow reduction after subarachnoidal haemorrhage(Beg et al., 2007). PKC has previously been reported tocontribute to the vascular remodelling in hypertension (Sala-manca and Khalil, 2005). In addition, PKC has been implicatedin the induction of cardiomyocyte hypertrophy, and PKCactivation has also been shown to aggravate hypoxic myocar-dial injury and has been demonstrated to be proarrhythmic(Murphy and Frishman, 2005).

4.4. MAPK in the endothelin receptor up-regulation

The MAPK pathways are thought to act downstream fromPKC in the smooth muscle cell regulatory cascade (Schonwas-ser et al., 1998) MAPKs represent a family of serine/threonineprotein kinases that mediate fundamental biological processesand cellular responses in response to external stress signals(Kaminska, 2005). In order to further elucidate the signallingpathways that are involved in the endothelin receptor up-regulation during organ culture, the role for the three MAPKkinases; ERK1/2, JNK and P38 MAPK pathways wereevaluated by culture in the presence of selective inhibitors.The results show that ERK1/2 and JNK may be involved in theup-regulation of endothelin receptors, while a role for the p38MAPK pathway is less probable.

4.4.1. ERK1/2PD98059 is a known antagonist of the ERK1/2 pathway

(Nishimoto and Nishida, 2006). In the present experiments,PD98059 inhibited the increase in sarafotoxin 6c contraction,endothelin ETB receptor mRNA levels and endothelin ETA andETB receptor immunofluorescence staining intensities, duringorgan culture, suggesting a role for the ERK1/2 pathway in theendothelin receptor regulation. ERKs mediate cellularresponses initiated by growth factors (Aoki et al., 2000), andhave been suggested to be implicated in cardio- andcerebrovascular disease. Down-regulation of ERK1/2, usingantisense oligonucleotides, inhibits intimal hyperplasia in aporcine model of coronary balloon angioplasty (Liu et al.,2002). Inhibition of ERK1/2 attenuates human vascular smoothmuscle cell growth induced by lipoprotein (a) which is anindependent risk factor for cardiovascular disease (Komai et al.,2002). ERK1/2 is involved in the up-regulation of contractileendothelin ETB receptors in rat cerebral arteries (Henrikssonet al., 2004). In vivo studies have shown that ERK 1/2 inhibitorsprevents the up-regulation of vascular endothelin ETB receptorsand reverses the cerebral blood flow reduction after subarach-noid haemorrhage in rats (Beg et al., 2006).

4.4.2. JNKThree different JNK pathways (JNK1,−2 and −3) have been

identified in humans. JNK1 and JNK2 have a broad tissuedistribution, whereas JNK3 primarily located to neuronaltissues and the cardiac myocyte. SP600125 inhibits the JNK1,−2 and −3 pathways (Bennett et al., 2001). In the presentexperiments, SP600125 inhibited the increase in sarafotoxin 6ccontraction, endothelin ETB receptor mRNA levels andendothelin ETA and ETB receptor immunofluorescence stainingintensities, during organ culture, suggesting a role for JNK inthe endothelin receptor regulation. JNK is a stress-activatedprotein kinase that can be induced by inflammatory cytokines,bacterial endotoxin, osmotic shock, UV radiation and hypoxia(Bennett et al., 2001). Previous studies have suggested that JNKis implicated in cardiovascular disease. JNK or ERK1/2 isinvolved in intimal hyperplasia induced by balloon injury in rats(Lodge et al., 1995) and cardiac hypertrophy in hypertensiverats (Vogel et al., 2001). Also, JNK is activated during ischemia/reperfusion in rats (Force et al., 2004). Inhibition of the JNKpathway during myocardial ischemia/reperfusion reducesmyocardial apoptosis and improves post ischemic cardiacfunctional in rats (Ferrandi et al., 2004).

4.5. Regulation of other receptor types in organ culture

Not much is known about the regulation of other receptortypes in culture of whole blood vessels. There have been a fewstudies on the regulation of angiotensin II and 5-HT1B receptors.Angiotensin II receptors in human coronary arteries decreaseduring culture (Wackenfors et al., 2004a), while being up-regulated in the rat middle cerebral artery during culture(Stenman et al., 2002). Furthermore, 5-HT1B receptors areupregulated in culture of whole rat basilar arteries (Hansen-Schwartz et al., 2002). The 5-HT1B receptor up-regulation was


inhibited by PKC antagonists (Hansen-Schwartz et al., 2002).We await future studies on the regulation of other receptorstypes in organ culture.

5. Conclusion

In conclusion, the present findings demonstrate that endothe-lin receptors in porcine coronary arteries are up-regulated afterorgan culture, mimicking the changes that occur in cardiovas-cular disease. Inhibition experiments revealed that PKC andMAPK signalling pathways were involved in the endothelinreceptor up-regulation. Inhibiting these intracellular signaltransduction pathways may provide a future therapeutic targetfor hindering the development of vascular endothelin receptorchanges in cardiovascular disease.

Acknowledgements

This study was supported by the Åke Wiberg Foundation, theM. Bergvall Foundation, Anna Lisa and Sven-Eric Lundgrensfoundation for medical research, the Anders Otto SwärdsFoundation/Ulrika Eklunds Foundation, the Swedish MedicalAssociation, the Royal Physiographic Society in Lund, theSwedish Medical Research Council, the Crafoord Foundation,the Swedish Heart–Lung Foundation, the Swedish GovernmentGrant for Clinical Research and theSwedishHypertensionSociety.

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