Sensitivity of mitochondrial peptidyl-prolyl cis-trans isomerase, pyridine nucleotide hydrolysis and Ca2+ release to cyclosporine A and related compounds

Prooxidants activate a specific Ca2+ release pathway from mitochondria. Here we investigate the inhibitory potency of cyclosporine A and six related compounds with respect to peptidyl-prolyl cis-trans isomerase (PPIase), pyridine nucleotide hydrolysis and Ca2+ release. Whereas the absolute inhibitory potency of the compounds varies by about three orders of magnitude, a given compound is always most effective on PPIase, followed by pyridine nucleotide hydrolysis, and least effective in Ca2+ release inhibition. The data show that pyridine nucleotide hydrolysis is a prerequisite but not a consequence of Ca2+ release. They also strongly suggest that PPIase participates in the Ca2+ release mechanism from intact mitochondria by regulating the intramitochondrial NAD+ glycohydrolase, and thereby ascribe a physiological function to the protein. Furthermore, a complete lack of correlation between the inhibitory potencies described here and the reported immunosuppressive activities of the drugs is evident.     

Characterization of a high capacity calcium transport system in mitochondria of the yeast Endomyces magnusii

The Ca2+ transport system of Endomyces magnusii mitochondria has been shown previously to be activated by spermine. Here we report it to be regulated also by low, physiological ADP concentrations, by the intramitochondrial NADH/NAD+ ratio, and by Ca2+ ions. The combination of all these physiological modulators induced high initial rates of Ca2+ uptake and high Ca2+-buffering capacity of yeast mitochondria, enabling them to lower the medium [Ca2+] to approximately 0.2 microM. The mechanisms of stimulation by these agents are discussed.     

Mitochondrial malic enzymes. An association between NAD(P)+-dependent malic enzyme and cell renewal in Sprague-Dawley rat tissues

The activities of the mitochondrial NAD(P)+- and NADP+-dependent malic enzymes were measured in 11 tissues of the male Sprague-Dawley rat. The NAD(P)+-dependent malic enzyme was present in small intestinal mucosa, spleen, thymus, lung, and testis. Each of these tissues contain cells that are undergoing active rates of renewal. The NADP+-dependent malic enzyme was not confined to tissues undergoing cell renewal, and was present in mitochondria from brain, skeletal, and heart muscle, kidney, and lung and testis. Both enzymes were absent or at a low activity in normal and regenerating liver. The results support, and extent to nonneoplastic tissues, our proposal that cells which show active and sustained rates of renewal contain the NAD(P)+-dependent malic enzyme and have a unique intramitochondrial pathway for malate oxidation (Sauer, L.A., Dauchy, R.T., Nagel, W.O., and Morris, H.P. (1980) J. Biol. Chem. 255, 3844-3848).     

Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria

Expression of the human protooncogene bcl-2 protects neural cells from death induced by many forms of stress, including conditions that greatly elevate intracellular Ca2+. Considering that Bcl-2 is partially localized to mitochondrial membranes and that excessive mitochondrial Ca2+ uptake can impair electron transport and oxidative phosphorylation, the present study tested the hypothesis that mitochondria from Bcl-2-expressing cells have a higher capacity for energy-dependent Ca2+ uptake and a greater resistance to Ca(2+)-induced respiratory injury than mitochondria from cells that do not express this protein. The overexpression of bcl-2 enhanced the mitochondrial Ca2+ uptake capacity using either digitonin-permeabilized GT1-7 neural cells or isolated GT1-7 mitochondria by 1.7 and 3.9 fold, respectively, when glutamate and malate were used as respiratory substrates. This difference was less apparent when respiration was driven by the oxidation of succinate in the presence of the respiratory complex I inhibitor rotenone. Mitochondria from Bcl-2 expressors were also much more resistant to inhibition of NADH-dependent respiration caused by sequestration of large Ca2+ loads. The enhanced ability of mitochondria within Bcl-2-expressing cells to sequester large quantities of Ca2+ without undergoing profound respiratory impairment provides a plausible mechanism by which Bcl-2 inhibits certain forms of delayed cell death, including neuronal death associated with ischemia and excitotoxicity.     

Oxidative phosphorylation in intact hepatocytes: quantitative characterization of the mechanisms of change in efficiency and cellular consequences

Two mechanisms may affect the yield of the oxidative phosphorylation pathway in isolated mitochondria: (i) a decrease in the intrinsic coupling of the proton pumps (H+/2e- or H+/ATP), and (ii) an increase in the inner membrane conductance (proton or cation leak). Hence three kinds of modifications can occur and each of them have been characterized in isolated rat liver mitochondria (see preceding chapter by Rigoulet et al.). In intact isolated hepatocytes, these modifications are linked to specific patterns of bioenergetic parameters, i.e. respiratory flux, mitochondrial redox potential, DY, and phosphate potential. (1) The increase in H+/ATP stoichiometry of the mitochondrial ATP synthase, as induced by almitrine [20], leads to a decrease in mitochondrial and cytosolic ATP/ADP ratios without any change in the protonmotive force nor in the respiratory rate or redox potential. (2) In comparison to carbohydrate, octanoate metabolism by beta-oxidation increases the proportion of electrons supplied at the second coupling site of the respiratory chain. This mimics a redox slipping. Octanoate addition results in an increased respiratory rate and mitochondrial NADH/NAD ratio while protonmotive force and phosphate potential are almost unaffected. The respiratory rate increase is associated with a decrease in the overall apparent thermodynamic driving force (2deltaE'o - ndeltap) which confirms the 'redox-slipping-like' effect. (3) An increase in proton conductance as induced by the protonophoric uncoupler 2,4-dinitrophenol (DNP) leads to a decrease, as expected, in the mitochondrial NADH/NAD and ATP/ ADP ratios and in deltapsi while respiratory rate is increased. Thus, each kind of modification (proton leak, respiratory chain redox slipping or increase in H+/ATP stoichiometry of ATPase) is related to a specific set of bioenergetic parameters in intact cells. Moreover, these patterns are in good agreement with the data found in isolated mitochondria. From this work, we conclude that quantitative analysis of four bioenergetic parameters (respiration rate, mitochondrial NADH/ NAD ratio, protonmotive force and mitochondrial phosphate potential) gives adequate tools to investigate the mechanism by which some alterations may affect the yield of the oxidative phosphorylation pathway in intact cells.     

Neurotransmitter transporters as molecular targets for addictive drugs

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca2+ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.     

Cyclic ADP-ribose as an endogenous regulator of the non-skeletal type ryanodine receptor Ca2+ channel

The skeletal and cardiac isoforms of the ryanodine receptor Ca2+ channel (RyRC) constitute the Ca2+ release pathway in sarcoplasmic reticulum of skeletal and cardiac muscles, respectively. A direct mechanical and a Ca(2+)-triggered mechanism (Ca(2+)-induced Ca2+ release) have been respectively proposed to explain the in situ activation of Ca2+ release in skeletal and cardiac muscle. In non-muscle cells, however, where the RyRC also participates in Ca2+ signalling, the mechanism of RyRC activation is unknown. Cyclic adenosine 5'-diphosphoribose (cADPR), which is present in many mammalian tissues, has been reported to induce Ca2+ release from ryanodine-sensitive intracellular Ca2+ stores in sea urchin eggs. Here we provide evidence that cADPR directly activates the cardiac but not the skeletal isoform of the RyRC. This, together with results on sea urchin eggs, suggests that cADPR is an endogenous activator of the non-skeletal type of RyRC and may thus have a role similar to inositol 1,4,5-trisphosphate in Ca2+ signalling.     

Integrating cytosolic calcium signals into mitochondrial metabolic responses

Stimulation of hepatocytes with vasopressin evokes increases in cytosolic free Ca2+ ([Ca2+]c) that are relayed into the mitochondria, where the resulting mitochondrial Ca2+ ([Ca2+]m) increase regulates intramitochondrial Ca2+-sensitive targets. To understand how mitochondria integrate the [Ca2+]c signals into a final metabolic response, we stimulated hepatocytes with high vasopressin doses that generate a sustained increase in [Ca2+]c. This elicited a synchronous, single spike of [Ca2+]m and consequent NAD(P)H formation, which could be related to changes in the activity state of pyruvate dehydrogenase (PDH) measured in parallel. The vasopressin-induced [Ca2+]m spike evoked a transient increase in NAD(P)H that persisted longer than the [Ca2+]m increase. In contrast, PDH activity increased biphasically, with an initial rapid phase accompanying the rise in [Ca2+]m, followed by a sustained secondary activation phase associated with a decline in cellular ATP. The decline of NAD(P)H in the face of elevated PDH activity occurred as a result of respiratory chain activation, which was also manifest in a calcium-dependent increase in the membrane potential and pH gradient components of the proton motive force (PMF). This is the first direct demonstration that Ca2+-mobilizing hormones increase the PMF in intact cells. Thus, Ca2+ plays an important role in signal transduction from cytosol to mitochondria, with a single [Ca2+]m spike evoking a complex series of changes to activate mitochondrial oxidative metabolism.     

Dantrolene inhibition of sarcoplasmic reticulum Ca2+ release by direct and specific action at skeletal muscle ryanodine receptors

The skeletal muscle relaxant dantrolene inhibits the release of Ca2+ from the sarcoplasmic reticulum during excitation-contraction coupling and suppresses the uncontrolled Ca2+ release that underlies the skeletal muscle pharmacogenetic disorder malignant hyperthermia; however, the molecular mechanism by which dantrolene selectively affects skeletal muscle Ca2+ regulation remains to be defined. Here we provide evidence of a high-affinity, monophasic inhibition by dantrolene of ryanodine receptor Ca2+ channel function in isolated sarcoplasmic reticulum vesicles prepared from malignant hyperthermia-susceptible and normal pig skeletal muscle. In media simulating resting myoplasm, dantrolene increased the half-time for 45Ca2+ release from both malignant hyperthermia and normal vesicles approximately 3.5-fold and inhibited sarcoplasmic reticulum vesicle [3H]ryanodine binding (Ki approximately 150 nM for both malignant hyperthermia and normal). Inhibition of vesicle [3H]ryanodine binding by dantrolene was associated with a decrease in the extent of activation by both calmodulin and Ca2+. Dantrolene also inhibited [3H]ryanodine binding to purified skeletal muscle ryanodine receptor protein reconstituted into liposomes. In contrast, cardiac sarcoplasmic reticulum vesicle 45Ca2+ release and [3H]ryanodine binding were unaffected by dantrolene. Together, these results demonstrate selective effects of dantrolene on skeletal muscle ryanodine receptors that are consistent with the actions of dantrolene in vivo and suggest a mechanism of action in which dantrolene may act directly at the skeletal muscle ryanodine receptor complex to limit its activation by calmodulin and Ca2+. The potential implications of these results for understanding how dantrolene and malignant hyperthermia mutations may affect the voltage-dependent activation of Ca2+ release in intact skeletal muscle are discussed.     

Reversal of impaired oxidative phosphorylation and calcium overloading in the skeletal muscle mitochondria of CHF-146 dystrophic hamsters

Membrane-mediated excessive intracellular calcium accumulation (EICA) and diminished cellular energy production are the hallmarks of dystrophic pathobiology in Duchenne and Becker muscular dystrophies. We reported reversal of respiratory damage and Ca(2+)-overloading in the in vitro cardiac mitochondria from CHF-146 dystrophic hamsters (DH) with hereditary muscular dystrophy (Bhattacharya et al., 1993). Here we studied respiratory dysfunctions in the skeletal muscle mitochondria from young and old DH, and whether these abnormalities can be reversed by reducing [Ca2+] in the isolation medium, thereby lowering intramitochondrial Ca(2+)-overloading. Age- and sex-matched CHF-148 albino normal hamsters (NH) served as controls. As an index of EICA and cellular degeneration, Ca and Mg levels were assayed in the skeletal muscle and mitochondria. Mitochondria from young and old DH, isolated without EDTA (BE medium), revealed poor coupling of oxidative phosphorylation, diminished stimulated oxygen consumption rate, and lower respiratory control ratio and ADP/O ratios, compared to NH. Incorporation of 10 mM EDTA (Bo medium) in the isolation medium restored mitochondrial functions of the dystrophic organelles to a near-normal level, and reduced Ca(2+)-overloading. The mitochondrial Ca level in DH was significantly higher than in NH, irrespective of the medium. However, compared to Bo medium, the dystrophic organelles isolated in BE medium had lower Ca levels and markedly improved oxidative phosphorylation as seen in NH. Muscle Ca contents in the young and old DH were elevated relative to NH, showing a positive correlation with the increased mitochondrial Ca(2+)-sequestration. Dystrophic muscle also revealed Ca deposition with an abundance of Ca(2+)-positive and necrotic myofibers by light microscopy, and intramitochondrial Ca(2+)-overloading by electron microscopy, respectively. However, Mg levels in the muscle and mitochondria did not alter with age or dystrophy. These data parallel our observations in the heart, and suggest that functional impairments and Ca(2+)-overloading also occur in the skeletal muscle mitochondria of DH, and are indeed reversible if EICA is regulated by slow Ca(2+)-channel blocker therapy (Johnson and Bhattacharya, 1993).     

Glutathione is a cofactor for H2O2-mediated stimulation of Ca2+-induced Ca2+ release in cardiac myocytes

Reactive oxygen species are known to cause attenuation of cardiac muscle contraction. This attenuation is usually preceded by transient augmentation of twitch amplitude as well as cytosolic Ca2+. The present study examines the role of an endogenous antioxidant, glutathione in the mechanism of H2O2-mediated augmentation of Ca2+ release from the sarcoplasmic reticulum. Whole-cell patch-clamped single rat ventricular myocytes were dialyzed with the Cs+-rich internal solution containing 200 microM fura-2 and 2 mM glutathione (reduced form). After equilibration of the myocyte with intracellular dialyzing solution, Ca2+ current-induced Ca2+ release from the sarcoplasmic reticulum was monitored. Rapid perfusion with H2O2 (100 microM or 1 mM) for 20 s inhibited Ca2+ current, but enhanced the intracellular Ca2+ transients for 3-4 min. Thus, the efficacy of Ca2+-induced Ca2+ release mechanism was augmented in 71% of myocytes (n = 7). This enhancement ranged between 1.5- to threefold as the concentrations of H2O2 were raised from 100 microM to 1 mM. If glutathione were excluded from the patch pipette or replaced with glutathione disulfide, the enhancement of Ca2+-induced Ca2+ release was seen in only a minority (20%) of the myocytes. H2O2 exposure did not increase the basal intracellular Ca2+ levels, suggesting that the mechanism of H2O2 action was not mediated by inhibition of the sarcoplasmic reticulum Ca2+ uptake or activation of passive Ca2+ leak pathway. H2O2-mediated stimulation of Ca2+-induced Ca2+ release was also observed in myocytes dialyzed with dithiothreitol (0.5 mM). Therefore, reduced thiols support the action of H2O2 to enhance the efficacy of Ca2+-induced Ca2+ release, suggesting that redox reactions might regulate Ca2+ channel-gated Ca2+ release by the ryanodine receptor.     

Cyclo-oxygenase-2 in vascular smooth muscle

Prolonged heart ischaemia causes an inhibition of oxidative phosphorylation and an increase of Ca2+ in mitochondria. We investigated whether elevated Ca2+ induces changes in the oxidative phosphorylation system relevant to ischaemic damage, and whether Ca2+ and other inducers of mitochondrial permeability transition cause the release of cytochrome c from isolated heart mitochondria. We found that 5 microM free Ca2+ induced changes in oxidative phosphorylation system similar to ischaemic damage: increase in the proton leak and inhibition of the substrate oxidation system related to the release of cytochrome c from mitochondria. The phosphorylating system was not directly affected by high Ca2+ and ischaemia. The release of cytochrome c from mitochondria was caused by Ca2+ and 0.175-0.9 mM peroxynitrite but not by NO, and was prevented by cyclosporin A. Adenylate kinase and creatine kinase were also released after incubation of mitochondria with Ca2+, however, the activity of citrate synthase in the incubation medium with high and low Ca2+ did not change. The data suggest that release of cytochrome c and other proteins of intermembrane space may be due to the opening of the mitochondrial permeability transition pore, and may be partially responsible for inhibition of mitochondrial respiration induced by ischaemia, high calcium, and oxidants.     

Care tracker: a new approach to nursing care in ambulatory settings

It is becoming increasingly clear that mitochondrial Ca2+ uptake from and release into the cytosol has important consequences for neuronal and glial activity. Ca2+ regulates mitochondrial metabolism, and mitochondrial Ca2+ uptake and release modulate physiological and pathophysiological cytosolic responses. In glial cells, inositol 1,4,5-trisphosphate-dependent Ca2+ responses are faithfully translated into elevations in mitochondrial Ca2+ levels, which modifies cytosolic Ca2+ wave propagation and may activate mitochondrial enzymes. The location of mitochondria within neurones may partially determine their role in Ca2+ signalling. Neuronal death due to NMDA-evoked Ca2+ entry can be delayed by an inhibitor of the mitochondrial permeability transition pore, and mitochondrial dysfunction is being increasingly implicated in a number of neurodegenerative conditions. These findings are illustrative of an emerging realization by neuroscientists of the importance of mitochondrial Ca2+ regulation as a modulator of cellular energetics, endoplasmic reticulum Ca2+ release and neurotoxicity.     

Functional imaging of mitochondria in saponin-permeabilized mice muscle fibers

Confocal laser-scanning and digital fluorescence imaging microscopy were used to quantify the mitochondrial autofluorescence changes of NAD(P)H and flavoproteins in unfixed saponin-permeabilized myofibers from mice quadriceps muscle tissue. Addition of mitochondrial substrates, ADP, or cyanide led to redox state changes of the mitochondrial NAD system. These changes were detected by ratio imaging of the autofluorescence intensities of fluorescent flavoproteins and NAD(P)H, showing inverse fluorescence behavior. The flavoprotein signal was colocalized with the potentiometric mitochondria-specific dye dimethylaminostyryl pyridyl methyl iodide (DASPMI), or with MitoTrackerTM Green FM, a constitutive marker for mitochondria. Within individual myofibers we detected topological mitochondrial subsets with distinct flavoprotein autofluorescence levels, equally responding to induced rate changes of the oxidative phosphorylation. The flavoprotein autofluorescence levels of these subsets differed by a factor of four. This heterogeneity was substantiated by flow-cytometric analysis of flavoprotein and DASPMI fluorescence changes of individual mitochondria isolated from mice skeletal muscle. Our data provide direct evidence that mitochondria in single myofibers are distinct subsets at the level of an intrinsic fluorescent marker of the mitochondrial NAD-redox system. Under the present experimental conditions these subsets show similar functional responses.     

Membrane potential generation coupled to oxidation of external NADH in liver mitochondria

Oxidation of added NADH by rat liver mitochondria has been studied. It is found that exogenous NADH, when oxidized by rat liver mitochondria in sucrose hypotonic medium supplemented with Mg2+ and EGTA, generates a membrane potential (delta psi) even in the absence of added cytochrome c. ADP and phosphate decrease delta psi, the effect being reversed by oligomycin. Rotenone and myxothiazol do not inhibit delta psi generated by oxidation of exogenous NADH. Added cytochrome c increases the rate of the exogenous NADH oxidation and coupled delta psi formation. In sucrose isotonic medium, or in hypotonic medium without Mg2+, exogenous NADH fails to stimulate respiration and to form a membrane potential. In the presence of Mg2+, exogenous NADH appears to be effective in delta psi generation in isotonic sucrose medium if mitochondria were treated with digitonin. In isotonic KCl without Mg2+, oxidation of exogenous NADH is coupled to the delta psi formation and MgCl2 addition before mitochondria prevents this effect. In hypotonic (but not in isotonic) sucrose medium, Mg2+ makes a portion of the cytochrome c pool reducible by exogenous NADH or ascorbate. It is assumed that (i) hypotonic treatment or digitonin causes disruption of the outer mitochondrial membrane, and, as a consequence, desorption of the membrane-bound cytochrome c in a Mg2+-dependent fashion; (ii) incubation in isotonic KCI without Mg2+ results in swelling of mitochondrial matrix, disruption of the outer membrane and cytochrome c desorption whereas Mg2+ lowers the K+ permeability of the inner membrane and, hence, prevents swelling; (iii) desorbed cytochrome c is reduced by added NADH via NADH-cytochrome b5 reductase and cytochrome b5 or by ascorbate and is oxidized by cytochrome oxidase. The role of desorbed cytochrome c in oxidation of superoxide and cytoplasmic NADH as well as possible relations of these events to apoptosis are discussed.     

Results of surgical resection in patients over the age of 70 years with non small-cell lung cancer

Calcium release from the sarcoplasmic reticulum (SR) depending on depolarization of the transverse tubular membrane (TTM) caused by rapid ionic replacement was measured in skeletal muscle triadic vesicles using a stopped-flow apparatus and Fura-2, a membrane-impermeable Ca2+ indicator. Calcium release was triggered by an increase in the magnitude of depolarization. This Ca2+ release was inhibited by ruthenium red, digoxin and dantrolene, and enhanced by caffeine. Thus, Ca2+ release was found to occur through the SR Ca2+ release channel via TTM depolarization and to be able to cause skeletal muscle contraction. Calcium release curves could be divided into two phases. In contrast to other previous studies, in the fast phase the amount of released Ca2+ increased with an increase in the magnitude of depolarization but the Ca2+ release rate did not; on the other hand, in the slow phase the Ca2+ release rate increased but the amount of Ca2+ did not. Furthermore, the Ca2+ release rate was controlled by the luminal Ca2+ concentration of the SR only in the fast phase. These independent dual kinetics of Ca2+ release were explained by the calsequestrin regulation model.     

Use of topiramate, a new anti-epileptic as a mood stabilizer

2-Hydroxycarbazole was shown to induce Ca2+ release from skeletal muscle and cardiac muscle sarcoplasmic reticulum at concentrations between 100-500 microM. This release was blocked by both 1 mM tetracaine and 30 microM ruthenium red which inhibit the ryanodine receptor or by pre-treatment with 10 mM caffeine which depletes the ryanodine receptor-containing Ca2+ stores. This, in addition to the fact that 2-hydroxycarbazole has little effect on Ca2+ ATPase activity, indicates that it activates Ca2+ release through the ryanodine receptor. The apparent EC50 value for release from both skeletal muscle and cardiac muscle sarcoplasmic reticulum was approximately 200 microM and maximal release occurred at 400-500 microM, making it approximately 20 times more potent than caffeine. The dose-dependency in the extent of Ca2+ release induced by 2-hydroxycarbazole was also apparently highly cooperative for both preparations. That 2-hydroxycarbazole was able to mobilize Ca2+ from non-muscle cell microsomes and in intact TM4 cells (which contain ryanodine receptors), makes this compound a more potent and commercially available alternative to caffeine in studying the role of this intracellular Ca2+ channel in a variety of systems.     

Evidence for mitochondrial Ca(2+)-induced Ca2+ release in permeabilised endothelial cells

Generally most intracellular Ca2+ is stored in the endoplasmic reticulum (ER) and mitochondria. Recently a mitochondrial Ca(2+)-induced Ca2+ release (mCICR) mechanism, unconnected with ryanodine receptors (RyR's), has been shown in tumour cells. The existence of a mitochondrial Ca2+ release mechanism in BAE cells was investigated using saponin-permeabilised BAE cells. When buffered intracellular solution were 'stepped' from 10 nM to 10 microM free Ca2+, the mitochondrial inhibitors CN (2 mM), FCCP (1 microM), and RR (20 microM) significantly reduced total CICR by approximately 25%. The ER Ca(2+)-ATPase inhibitor thapsigargin (100 nM) had no effect. Furthermore, cyclosporin A (200 nM), an inhibitor of the mitochondrial permeability transition pore (PTP), abolished total CICR. Therefore, the novel ryanodine-caffeine insensitive CICR mechanism previously reported in BAE cells involves mitochondrial Ca2 release. It is proposed that in BAE cells, mCICR occurs via the mitochondrial PTP and may be physiologically important in endothelial cell Ca2+ signalling.     

Caffeine-induced release of intracellular Ca2+ from Chinese hamster ovary cells expressing skeletal muscle ryanodine receptor. Effects on full-length and carboxyl-terminal portion of Ca2+ release channels

The ryanodine receptor (RyR)/Ca2+ release channel is an essential component of excitation-contraction coupling in striated muscle cells. To study the function and regulation of the Ca2+ release channel, we tested the effect of caffeine on the full-length and carboxyl-terminal portion of skeletal muscle RyR expressed in a Chinese hamster ovary (CHO) cell line. Caffeine induced openings of the full length RyR channels in a concentration-dependent manner, but it had no effect on the carboxyl-terminal RyR channels. CHO cells expressing the carboxyl-terminal RyR proteins displayed spontaneous changes of intracellular [Ca2+]. Unlike the native RyR channels in muscle cells, which display localized Ca2+ release events (i.e., "Ca2+ sparks" in cardiac muscle and "local release events" in skeletal muscle), CHO cells expressing the full length RyR proteins did not exhibit detectable spontaneous or caffeine-induced local Ca2+ release events. Our data suggest that the binding site for caffeine is likely to reside within the amino-terminal portion of RyR, and the localized Ca2+ release events observed in muscle cells may involve gating of a group of Ca2+ release channels and/or interaction of RyR with muscle-specific proteins.     

Cyclosporin A-sensitive release of Ca2+ from mitochondria in intact thymocytes

Release of Ca2+ from mitochondria into cytosol in intact thymocytes was studied using the fluorescent dye Fluo-3. It was shown that the release of Ca2+ induced by the dithiol cross-linking agent phenylarsine oxide or by uncoupler was strongly inhibited by cyclosporin A, a specific inhibitor of the permeability transition pore (PTP) in mitochondria. Oxidative stress sensitized the pore so even partial uncoupling caused rapid cyclosporin A-sensitive release of Ca2+. The experiments on digitonin-permeabilized cells confirmed that uncoupling induced opening of the PTP, which forms the major pathway for rapid release of Ca2+ from thymocyte mitochondria.