CalciumAnd

Cell Injury

by康 照 洲

Institute of Toxicology

細胞毒性之機制

1. 毒性化學物質

Toxic agents, including toxins and toxicants, can be classified by target organs, use, source, and effects. The term toxin refers to toxic agents that are produced by biological systems such as plant, animal, fungi, bacteria. Toxicant is toxic substance thatis produced by or is a by-product of anthropogenic (man-made) activities.

Paracelsus (1493-1541AD): “Alle Ding sindGift und nichts ohn Gift; alein die Dosis machtdas ein Ding kein Gift ist”

…it is the “dose” that makes the poison…

"There are no harmless substance, only harmless way of using substances.“

2. 毒化物之吸收、分布、代謝與排除 (ADME)

暴露途徑，吸收、分布、排泄、蓄積，代謝解毒、活化

3. 毒物之作用機制1)分子交互作用

a.非共價見之結合 b.共價見之結合c.半抗原之形成 d.氧化還原反應e.酵素作用

2)細胞之影響a.改變細胞之調控，含基因表現之失調（細胞分裂，程序性死亡）及細胞生理功能之失調（肌肉，神經）

b.破壞細胞之衡定，含內部衡定（ATP合成，離子濃度，訊息傳遞）及外部衡定（促進或抗凝血）

c.細胞結構（細胞膜，細胞骨架）或大分子（DNA，蛋白質）之破壞

Calcium Homeostasis in Cell

Figure 1. Spatial aspects of Ca2+ signalling. a, Elementary events (red) result from the entry of external Ca2+ across the plasma membrane or release from internal stores in the endoplasmic or sarcoplasmic reticulum (ER/SR). They generate localized concentrations of Ca2+ that can activate many processes, including export of cellular material (1), opening of K+ channels (2) and metabolism in the mitochondria (3). The Ca2+ signals can also enter the nucleus (4). All of these processes respond to the very high concentrations of Ca2+ that build up within the sub-domain of the elementary events. b, Global Ca2+ signals are produced by coordinating the activity of elementary events to produce a Ca2+ wave that spreads throughout the cell. c, The activity of neighbouring cells within a tissue can be coordinated by an intercellular wave that spreads from one cell to the next. From: Berridge: Nature, Volume 395(6703).October 15, 1998.645-648

Figure 2. Local Ca2+ signalling through elementary events such as 'sparks' and 'puffs'. Groups of either inositol-1,4,5-trisphosphate (InsP3) receptors or ryanodine receptors (RYRs) located on the endoplasmic reticulum (ER) release localized bursts of Ca2+, giving rise to elementary events such as sparks where the local concentration is very high before the Ca2+ diffuses away to contribute to the global Ca2+ signal. a, An elementary event located near the plasma membrane in smooth muscle cells opens high-affinity, Ca2+-sensitive K+ channels to give small, spontaneous, outward currents which can sum to cause the muscle to relax by hyperpolarizing the membrane [9]. It appears, therefore, that smooth muscle uses AM signalling, in that low-intensity global signals stimulate contraction whereas the high but localized concentrations within the elementary events near the membrane cause relaxation. Depending on how it is presented, Ca2+ can stimulate either contraction or relaxation. b, Release of granules from pituitary cells appears to be triggered by InsP3-induced release of stored Ca2+ [10]; release might be triggered by the high concentrations associated with the elementary events. c, Energy metabolism by mitochondria can be activated by the high concentrations of Ca2+associated with the elementary events that give rise to typical Ca2+ oscillations

Figure 1. Differential gene activation in B cells through amplitude modulation of the Ca2+ signalling pathway responsible for transmitting information to the nucleus. As Dolmetsch and colleagues [2] show, depending on the cell (naive or self-tolerant), antigen acting on the B-cell receptor (BCR) can stimulate either high-intensity or low-intensity Ca2+ signals (see insets). From: Berridge: Nature, Volume 386(6627).April 24, 1997.759-760

Figure 2. Compartmentalization of Ca2+signals in neurons. a, External Ca2+ enters through receptor-operated channels (ROCs) or voltage-operated channels (VOCs), and the signal may be amplified by activating receptors for Ins(1,4,5)P3 on the spine apparatus. By integrating separate input signals, these receptors could act as coincidence detectors. b, In response to electrical signals (Delta V), the entry of Ca (2+) through VOCs stimulates neurotransmitter release. Such signals, arising through Ca2+entry, are usually very rapid in onset and decline. c, Localized Ca2+ release from the endoplasmic reticulum (ER) opens K (+) channels to modulate neuronal excitability. d, Proposed role of Ca2+ in memory consolidation. Entry of external Ca2+ can act locally by co-opting other signallingpathways, or it can act globally by flooding directly into the nucleus. Such a global signal might be amplified by release of Ca2+ from the internal stores. From: Berridge: Nature, Volume 395(6703).October 15, 1998.645-648

Possible Correlation of Calcium Overload with Cytotoxicity

Several pieces of evidence have shown that increase of intracellular calcium concentration ([Ca2+]i) is the primary cause of cytotoxic effects, including necrosis and apoptosis, of toxicants and stress induced responses

For exampleCardiotoxicity: oxidative stress, adriamycin, TCDDHepatotoxicity: acetaminophen, CCl4Immunotoxicity: glucocorticoids, tributyl tin, TCDDKidney toxicity: oxidative stress, heavy metalsNeurotoxicity: cyanide, lead, ischemia, excitatory amino acid

www.vetmed.ufl.edu/path/pbteach/ wlc/vem5161/cell/cell1.htm

Role of Calcium Overload in Cellular Injury

Relation of Ca2+ to early reversible injury- bleb and cytoskeletal alteration- cell volume changes- nuclear changes- codensation of mitochondria

Relation of Ca2+ to irreversible injury- swollen mitochondria- DNA strand break

Mechanisms of Ca2+ Induced Cell Injury

1. Alteration of cell signallingcalmodulin-dependent enzymes, protein kinase, phosphatase

2. Activation of degenerative enzymesprotease, phospholipase, endonuclease

3. Mitochondria damagecalcium overload, permeability transition, ATP depletion

4. Cytoskeletal alterationassociation of actin and actin binding protein

5. Gene expressionc-fos, cjun, c-myc

6. Othersphosphatidylserine exposure and macrophage recognitiontransglutaminase activation (apoptotic body formation)oxidative stress

Molecules

Regulate

Cellular Ca2+

Homeostasis

1. Cell Membrane Ca2+ Transporters

Calcium Channels (I)

Voltage Dependent Ca Channels (VOC)Receptor Operated Ca Channels (ROC)Ca Release Activated Ca Channel (CRAC)Second Messenger Operated Channels (SMOC)G Protein Operated Channels (GOC)

Calcium channel structure: Most voltage-gated calcium channels consist of several proteins non-covalently associated with each other. They are termed α1, α2-δ , β and γ subunits. α2 and γ carry complex carbohydrate moieties (glycoproteins). The channel is formed only by α1.

homepage.uibk.ac.at/.../c53701/ js/calcium_channels.html

a1-Subunit Structure: a1 subunit consists of about 2000 amino acid residues (molecular mass about 240,000 dalton) and carries all the structural features required for a calcium-selective, voltage-activated channel: the voltage sensors (positive charges in S4 helices), the selectivity filter (glutamate rgions in the S5-S6 linkers, illustrated in red), and the ion conducting pathway (comprised by the S5 + S5-S6-linker + S6 in the four repeats illustrated in blue). N, N-terminus, C, C-terminus of protein. The inset shows the possible arrangement of the transmembranne helices around a calcium-selective pore.

A molecular model of a synthetic calcium channel protein (shown as purple helices) complexed with the drug nifedipine. The drug is shown in the middle of the pore formed by the interior residues of the

protein helices. The orange spheres represent isoleucine residues that act as a

"gate" in the channel for the passage of calcium ions (yellow spheres). This model was generated by extensive computations which

involve Monte Carlo search and energy minization. It shows how the drug may close the gate and prevent the influx of calcium through the calcium channel. Such channel-

blocking drugs help reduce muscle contraction and are widely used in treating

cardiac disorders.Taken from Zhorov and Ananthanarayanan,

Biophys.J. 70: 22-37, 1996

Na/Ca Exchanger (NACA)

-Influx or efflux of Ca-Highly experessed in heart muscle, where it is major pathwayfor extrusion of Ca

-Involved in Ca reabsorption in kidney-6 isoforms expressed, for example, heart (NACA1), kidney(NACA2, 3), brain (NACA4-6)

-70kD (brain), 120kD (heart), 215kD (ROS) glycoprotein-Low calcium affinity (1-5µM), which could be increased bycalmodulin-dependent kinase but still in low affinity level

-Exchange 3Na with 1Ca-No specific inhibitor, may be inhibited by doxorubicin and amiloride etc.

2. Intracellular Ca2+ Release Channels

cADPR

(Calcium –inducedCalcium Release)

ExcitationContractionCoupling

www.john-libbey-eurotext.fr/.../ abc/58/2/147-56/images.htm

Purification of Ryanodine Receptor

RyanodineRyanodine Receptor (Receptor (RyRRyR))

• Classification of RyR GenesGene Locus Expressionryr1 19q13.1 Skeletal muscleryr2 1q42.1-q43 Ventrical muscleryr3 15q14-q15 Brain, non-muscle cells

• Modulators of RyR Channel ActivityAgents ryr1 ryr2 ryr3Ca2+ +/- +/- +/?Mg2+ - - ?ATP + + ?cADPR n.e. + +/?calmodulin - - ?ryanodine +/- +/- +/-caffeine + + -

Malignant Hyperthermia

Genetic analysis of MH started with studies of the animal model, the porcine stress syndrome. In the porcine model the gene locus was identified as a Halothane (Hal) sensitive locus. While the condition is recessively inherited in the pig, the human condition is transmitted as an autosomal dominant. The Hal locus mapped to porcine chromosome 6, and since the corresponding group of human genes is located on 19q this gave the first indication for a chromosomal location in man. Further support for this genomic site came from the simultaneous mapping of the ryanodine receptor gene (RYR1) to the same interval, and the finding of the causative mutation Arg 615Cys in the pig. MHS could be linked to 19q13.1 and RYR1 in a number of large families. At present about 18 mutations in RYR1 have been described to be associated with human MHS which altogether accounts for approximately 45 % of cases. However several MHS families could be formally excluded from linkage to RYR1. A combination of systemic linkage and testing of candidate genes has led to the identification of additional MH-loci. These are on Chromosomes 1, 3, 7, and perhaps 17.

• Increased metabolism• Increased oxygen consumption• Tachypnea• Tachycardia• Rigidity• Fever/profuse sweating.• Muscle damage.

Factors Couple to Phospholipase C Pathway

Via PLCβ – G protein linkedACh, Histamine, NA, 5-HT, ATP, PAF, TXA2, Glutamate, Angiotensin II, Vasopressin, Bradykinin, Substance P, Bombesin, Neuropeptide Y, Thrombin, Endothelin, TRH, GnRH, PTH, Glucagon, Cholecystokinin, ordorants, Light

Via PLCγ – tyrosine kinase linkedPDGF, EGF, antigen, FGF, ErbB2

Nature Reviews Neuroscience 2; 387-396 (2001)

www.biocarta.com/pathfiles/ pkcPathway.asp

Fig.1 Calcium waves and oscillations in rat chromaffin cells. Ca2+ values are coded according to the pseudocolor scale on the left, where the lowest values are coded blue. First line: in agonist-induced Ca2+ oscillations, Ca2+ increased initially in a defined subcellular region (pacemaker), and then spread to involve the whole cytosol. Second line: miniature Ca2+ spikes remained restricted to the pacemaker region. Fig. 2 Calcium oscillations in articularchondrocytes. Ca2+ values are coded according to the pseudocolor scale on the left, where the lowest values are coded blue. Ca2+ responses induced by extracellular ATP: after the first synchronuousCa2+ transient, chondrocytes undergo asynchronous Ca2+ oscillations.

www.univ.trieste.it/~nirdbbcm/ calcium.html

Calcium Channels (II)

Voltage Dependent Ca Channels (VOC)Receptor Operated Ca Channels (ROC)Ca Release Activated Ca Channel (CRAC)Second Messenger Operated Channels (SMOC)G Protein Operated Channels (GOC)

Other Calcium Channels and Capacitative Calcium Entry

TIPs Review, 15:71-83, 1994

Figure 2 | Architecture of TRP channels. Like the broader class of ion channel subunits, TRP channels comprise six membrane-spanning domains. S1–S6 are transmembrane domains. Lanthanum ions (La3+) and 2-aminodiethyldiphenyl borate (2-APB) often block these channels, although not specifically. Ankyrinrepeats are found in the amino termini of TRPC and TRPV channels.

Nature Reviews Neuroscience 2; 387-396 (2001)THE TRP ION CHANNEL FAMILY

Mammalian homologues of Drosophilatransient receptor potential (TRP) channel gene encode a family of at least 20 ion channel proteins. They are widely distributed in mammalian tissues, but their specific physiological functions are largely unknown. A common theme that links the TRP channels is their activation or modulation by phosphatidylinositol signal transduction pathway. Three subgroups comprise the TRP channel family; the best understood of these mediates responses to painful stimuli. Other proposed functions include repletion of intracellular calcium stores, receptor-mediated excitation and modulation of cell cycle.

3. Ca2+-ATPase (Calcium Pump)

atlas.biophys.uni-frankfurt.de/ barth/structure.html

Sarco/Endoplasmic Reticulum Calcium Pump(SERCAs; Ca2+-ATPase)-M.W. ~110 kD-transport Ca with high affinity and capacity-transport Ca at 2:1 ratio of ATP-regulated by phospholamban but not calmodulin-E~p formation during catalytic cycle-might be regulated by kinase phosphorylation-specific inhibitor: thapsigargin-non-specific inhibitors: vanadate, thio-reactiveagens, 4-aminpyridine

Plasma membrane Calcium Pump (Ca2+-ATPase)-protein isolated from erythrocyte by calmodulincolumn with ~138 kD M.W.-transport Ca at 1:1 ration of ATP in exchange of

H+-may be regulated by acid phospholipid, PUFA, proteolytic treatment, calmodulin

-can be regulated by cAMP-dependent phosphorylation

TOPICAL REVIEWCalcium pumps and keratinocytes: lessons from Darier’s diseaseand Hailey–Hailey disease

British Journal of Dermatology 2004; 150: 821–828.

Skin biopsy from Darier’s disease reveals a characteristicdyskeratosis with suprabasal acantholysis secondary to breakdown ofthe desmosome adhesion junctions. The epidermis shows hyperproliferativebudding beneath the dyskeratotic plug.

Desmosomes are junctional complexes that are formed among opposing plasma membranes. In some instances, however, the hemi-demosomes are formed between the epithelial cells and the basal lamina of the epithelium. These can be found between the interdigitating cellular bridges in squamous cell carcinomas (green arrowheads) as well as in adenocarcinomas. Each desmosome consists of an electron dense plate immediately underneath the plasma membrane (blue arrowheads) where tonofilaments converge (red arrowheads). The space between the plasma membranes, normally is 250 angstrom in width and is filled with electron dense material (browh arrowhead)

Low Power; Desmosome High Power; Desmosome

http://www.bioscience.org/atlases/tumpath/em/desmosom.htm

4. Mitochondria (Ca2+ reservoir)

www.sghms.ac.uk/depts/immunology/ ~dash/apoptosis/mito.html

Ca2+ Homeostasis Controlling Molecules

Target Molecules of Toxicants That Will Induce Intracellular Calcium Overload

1. Cell membranemembrane lytic compounds, peptide, ionophore, Na,K-ATPase inhibitors

2. [Ca2+] Regulating Proteinsheavy metals, SH-reactive compounds, specific inhibitors

3. Mitochondriaelectron transfer chain inhibitors, uncouplers, other mitochondria toxins

Biochemical Pharmacology 55:713-718 (1998)

Role of Nitric Oxide and Its Intracellular SignallingPathways in the Control of Ca2+ Homeostasis

Ca2+: A Signal for Life and Death

References:1. Calcium as a cellular regulator, E.Carafoli and C. Klee Eds,

Oxford University Press, New York, 1999.2. Calcium –medicated mechanisms in chemically induced cell

death, P. Nicotera, G. Bellomo and S. Orrenius, Annu. Rev. Pharmacol. Toxicol. 32:449-470, 1992.

3. Calcium-mediated cell injury and cell death, B.F. Trump and J.K. Berezesky, FASEB 9:219-228, 1995.

4. Molecular mechanisms of toxicant-induced immuno-suppresion: Role of second messangers, M.P. Holsapple, J.G. Karras, J.A. Burchiel, D.R. Davila, A.R. Schatz, N.E. Kaminski, Annu. Rev. Pharmacol. Toxicol. 36:131-159, 1996.

5. The role of calcium in regulation of apoptosis, D.J. McConkey and S. Orrenius, BBRC 239:357-366, 1997