Kinetic studies of Ca2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles

The time-course of Ca²⁺ release from sarcoplasmic reticulum isolated from muscles of normal pigs and those of pigs susceptible to malignant hyperthermia were investigated using stopped-flow spectrophotometry and arsenazo III as a Ca²⁺ indicator. Several methods were used to trigger Ca²⁺ release: (a) addition of halothane (e.g., 0.2 mM); (b) an increase of extravesicular Ca²⁺ concentration ([Ca₀²⁺]); (c) a combination of (a) and (b), and (d) replacement of ions (potassium gluconate with choline chloride) to produce membrane depolarization. The initial rates of Ca²⁺ release induced by either halothane or Ca²⁺ alone, or both, are at least 70% higher in malignant hyperthermic sarcoplasmic reticulum than in normal. The amount of Ca²⁺ released by halothane at low [Ca₀²⁺] in malignant hyperthermic sarcoplasmic reticulum is about twice as large as in normal sarcoplasmic reticulum. Membrane depolarization led to biphasic Ca²⁺ release in both malignant hyperthermic and normal sarcoplasmic reticulum, the rate constant of the rapid phase of Ca²⁺ release induced by membrane depolarization being significantly higher in malignant hyperthermic sarcoplasmic reticulum ($\text{k = 83}\text{s}{}^{\mathrm{?1}}$) than in normal ($\text{k = 37}\text{s}{}^{\mathrm{?1}}$). Thus, all types of Ca²⁺ release investigated (a, b, c and d) have higher rates in malignant hyperthermic sarcoplasmic reticulum than normal sarcoplasmic reticulum. These results suggest that the putative Ca²⁺ release channels located in the sarcoplasmic reticulum are altered in malignant hyperthermic sarcoplasmic reticulum.     

Mitochondria in cardiomyocyte Ca signaling

Ca²⁺ signaling is of vital importance to cardiac cell function and plays an important role in heart failure. It is based on sarcolemmal, sarcoplasmic reticulum and mitochondrial Ca²⁺ cycling. While the first two are well characterized, the latter remains unclear, controversial and technically challenging.In mammalian cardiac myocytes, Ca²⁺ influx through L-type calcium channels in the sarcolemmal membrane triggers Ca²⁺ release from the nearby junctional sarcoplasmic reticulum to produce Ca²⁺ sparks. When this triggering is synchronized by the cardiac action potential, a global [Ca²⁺]_i transient arises from coordinated Ca²⁺ release events. The ends of intermyofibrillar mitochondria are located within 20 nm of the junctional sarcoplasmic reticulum and thereby experience a high local [Ca²⁺] during the Ca²⁺ release process. Both local and global Ca²⁺ signals may thus influence calcium signaling in mitochondria and, reciprocally, mitochondria may contribute to the local control of calcium signaling. In addition to the intermyofibrillar mitochondria, morphologically distinct mitochondria are also located in the perinuclear and subsarcolemmal regions of the cardiomyocyte and thus experience a different local [Ca²⁺].Here we review the literature in regard to several issues of broad interest: (1) the ultrastructural basis for mitochondrion – sarcoplasmic reticulum cross-signaling; (2) mechanisms of sarcoplasmic reticulum signaling; (3) mitochondrial calcium signaling; and (4) the possible interplay of calcium signaling between the sarcoplasmic reticulum and adjacent mitochondria.Finally, this review discusses experimental findings and mathematical models of cardiac calcium signaling between the sarcoplasmic reticulum and mitochondria, identifies weaknesses in these models, and suggests strategies and approaches for future investigations.     

Effect of long chain unsaturated fatty acids on the calcium transport of sarcoplasmic reticulum

The effect of archidonic, oleic and linoleic acid on calcium uptake and release by sarcoplasmic reticulum isolated from longissimus dorsi muscle was investigated using a Ca²⁺ electrode. All three long chain fatty acids stimulated the release of Ca²⁺ from sacroplasmic reticulum when added after exogenous Ca²⁺ was accumulated by the vesicles, and also inhibited Ca²⁺ uptake when added before Ca²⁺. This inhibitory effect on the calcium transport by arachidonic, oleic and linoleic acid was prevented by bovine serum albumin through its ability to bind with the fatty acid. The order of effectiveness of the fatty acids in inhibiting calcium transport by isolated sarcoplasmic reticulum was $\text{arachidonic acid> oleic acid >}$ linoleic acid. Similar inhibition of calcium uptake and induction of calcium release by arachidonic acid was observed in muscle homogenate sarcoplasmic reticulum preparations. Both arachidonic and oleic acid stimulated the (Ca²⁺ + Mg²⁺)-ATPase activity of sarcoplasmic reticulum at low concentrations, but inhibited the (Ca²⁺ + Mg²⁺)-ATPase activity at high concentrations. The maximal (Ca²⁺ + Mg²⁺-ATPase activity observed with arachidonic acid was twice that obtained with oleic acid, but the concentration of arachidonic acid required was 3–4-times greater than that of oleic acid. The concentration of arachidonic acid required to give maximum stimulation of the (Ca²⁺ + Mg²⁺)-ATPase activity was 3.6-times greater than that needed for complete inhibition of calcium accumulation by the sacroplasmic reticulum. With oleic acid, however, the concentration required to give maximum stimulation of the (Ca²⁺ + Mg²⁺)-ATPase activity inhibited the sarcoplasmic reticulum Ca²⁺ accumulation by 72%. The present data support our hypothesis that, in porcine malignant hyperthermia, unsaturated fatty acids from mitochondrial membranes released by endogenous phospholipase A₂ would induce the sarcoplasmic reticulum to release calcium (Cheah K.S. and Cheah, A.M. (1981) Biochim. Biophys. Acta 634, 70–84).     

Effects of glucagon, insulin and cyclic-AMP on mitochondrial calcium uptake in the liver.

The effects of glucagon and insulin administration $\text{in vivo}$ on hepatic mitochondrial Ca²⁺ uptake were compared with the effects of these hormones when they were added directly to the perfused liver. Glucagon administration increased mitochondrial calcium uptake both $\text{in vivo}$ and in the perfused liver. In contrast, while injection of insulin into rats stimulated, addition of insulin to the perfusate, inhibited Ca²⁺ uptake. Cyclic AMP, when added to the perfusate, also increased the uptake of Ca²⁺ by mitochondria, subsequently isolated. The possible implications of the results are discussed.     

Differential regulation of intracellular calcium oscillations by mitochondria and gap junctions

Fluctuations of intracellular Ca²⁺ ([Ca²⁺]_i) regulate a variety of cellular functions. The classical Ca²⁺ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca²⁺]_i oscillations. Although mitochondria have recently been shown to absorb and release Ca²⁺ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca²⁺]_i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca²⁺]_i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca²⁺-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca²⁺]_i oscillations in NT2 cells. The [Ca²⁺]_i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol—but not Tg-induced [Ca²⁺]_i oscillations. Furthermore, carbachol-induced [Ca²⁺]_i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca²⁺]_i oscillations. These data suggest that the distinct patterns of [Ca²⁺]_i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.     

Mitochondrial Ca2+-Handling in Fast Skeletal Muscle Fibers from Wild Type and Calsequestrin-Null Mice

Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca²⁺-binding protein inside SR, calsequestrin. Mitochondrial free Ca²⁺-concentration ([Ca²⁺]_mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca²⁺ release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca²⁺]_mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca²⁺ concentration. The rise in [Ca²⁺]_mito during electrical stimulation occurred in 20−30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s⁻¹. Accordingly, frequency-dependent increase in [Ca²⁺]_mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca²⁺]_mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca²⁺]_mito transients were increased by inhibition of mitochondrial Na⁺/Ca²⁺ exchanger (mNCX). These results provide direct evidence for fast Ca²⁺ accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca²⁺-handling and a dependence of mitochondrial Ca²⁺-handling on intracellular (SR) and external Ca²⁺ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca²⁺-leakage.     

Mitochondrial calcium signalling and cell death: Approaches for assessing the role of mitochondrial Ca uptake in apoptosis

Local Ca²⁺ transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca²⁺ release to activate mitochondrial Ca²⁺ uptake and to evoke a matrix [Ca²⁺] ([Ca²⁺]_m) rise. [Ca²⁺]_m exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca²⁺ release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca²⁺ sensitivity of both the Ca²⁺ release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca²⁺ accumulation in various apoptotic paradigms, methods are available for buffering of [Ca²⁺], for dissipation of the driving force of the mitochondrial Ca²⁺ uptake and for inhibition of the mitochondrial Ca²⁺ transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca²⁺ handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca²⁺ uptake on cytosolic [Ca²⁺] and [Ca²⁺]_m in intact cultured cells.     

Involvement of mitochondria in intracellular calcium sequestration by rat gonadotropes

Intracellular Ca²⁺ ([Ca²⁺]_i) dynamics were studied in identified rat gonadotropes using the whole-cell patch-clamp technique in conjunction with Indo-1 photometry. The kinetics of depolarization-induced [Ca²⁺]_i transients vary with Ca²⁺ load. In addition to a rapid initial decay, large (> 500 nM) [Ca²⁺]_i transients have a slow plateau phase. Application of the mitochondrial inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly slows the decay of [Ca²⁺]_i transients, consistent with stopping uptake of Ca²⁺ by mitochondria. CCCP causes a small increase of [Ca²⁺]_i at rest. After a large Ca²⁺ entry the amount is much larger, consistent with release from a mitochondrial Ca²⁺ pool that fills during cytoplasmic Ca²⁺ loading. The rate of Ca²⁺ uptake by mitochondria is dependent upon [Ca ²⁺]_i. Consistent with previous studies, gonadotropin releasing hormone (GnRH) induces [Ca²⁺]_i oscillations. The mitochondrial inhibitors CCCP and cyanide (CN⁻) terminate these oscillations. The mitochondrial ATP-synthase inhibitor oligomycin reduces the frequency and increases the amplitude of the oscillations. In the presence of ruthenium red (a non-specific blocker of the mitochondrial Ca²⁺-uniporter) in the pipette, GnRH does not induce rhythmic [Ca²⁺]_i oscillations. We suggest that mitochondria play a significant role in the rapid clearance of cytosolic Ca²⁺ loads in gonadotropes and participate in GnRH-induced periodic [Ca²⁺]_i oscillations.     

Kinetic studies on sodium-dependent calcium uptake by myocardial cells and neuroblastoma cells in culture

Kinetic analyses were made on intracellular Na⁺-dependent Ca²⁺ uptake by myocardial cells and neuroblastoma cells (N-18 strain) in culture. Cells loaded with various concentrations of Na⁺ could be prepared by incubating them in Ca²⁺-free medium containing various concentrations of Na⁺. Cells pre-loaded with various concentrations of Na⁺ were incubated in medium containing Ca²⁺ and ⁴⁵Ca. The resulting ⁴⁵Ca uptake by the two types of cell depended greatly on the initial intracellular concentrations of Na⁺. Lineweaver-Burk plots of the initial rate of Ca²⁺ uptake against the external concentration of Ca²⁺ fitted well to straight lines obtained by linear regression (r > 0.95). This result shows that Ca²⁺ uptake by the two types of cell was achieved by a carrier-mediated transport system. This Na⁺-dependent Ca²⁺ uptake was accompanied by Na⁺ release and the ratio of Na⁺ release to Ca²⁺ uptake was close to 3 : 1. A comparison of the kinetic data between myocardial cells and N-18 cells suggested that N-18 cells possess a carrier showing the same properties as that of myocardial cells, i.e.: (1) a similar dependency on the intracellular concentration of Na⁺; (2) the coincidence of the apparent Michaelis constants for Ca²⁺ (0.1 mM); (3) the similarities of the K_i values for Co²⁺, Sr²⁺ and Mg²⁺ (Co²⁺ < Sr²⁺ < Mg²⁺) and (4) a similar dependency on pH. However, the maximal initial rate, V, of N-18 cells was about $\text{1}\text{100}$ that of myocardial cells. The rate of Na⁺-dependent Ca²⁺ uptake by non-excitable cells was much lower than that by myocardial cells.     

Heterogeneity of SH groups in sarcoplasmic reticulum.

The incorporation of [¹⁴C] N-ethylmaleimide reveals fast and slow-reacting sulfhydryl groups in sarcoplasmic reticulum. Two proteins react with the label: a fast-reacting glycoprotein recently isolated (Ikemoto, Cucchiaro and Garcia (1976) J. Cell Biol.$\text{70}$, 290a), and the Ca²⁺-ATPase. Labeling sarcoplasmic reticulum with a maleimide spin label gives a similar pattern. The spectra of maleimide-spin-labeled sarcoplasmic reticulum have both ‘strongly’ and ‘weakly’ immobilized components. Maleimide-spin-labeled purified Ca²⁺-ATPase, or sarcoplasmic reticulum labeled first with N-ethylmaleimide, and then with maleimide spin label, show spectra devoid of the ‘weakly’ immobilized component; the latter is enhanced in partially purified glycoprotein obtained from spin-labeled sarcoplasmic reticulum. This indicates that spectra from maleimide-spin-labeled sarcoplasmic reticulum do not reflect exclusively the state of the Ca²⁺-ATPase enzyme.     

Skeletal muscle mitochondrial phospholipase A2 and the interaction of mitochondria and sarcoplasmic reticulum in porcine malignant hyperthermia

Comparative studies were carried out on the Ca²⁺-transport systems of mitochondria and sarcoplasmic reticulum from longissimus dorsi muscle of genetically selected malignant hyperthermia-prone and normal pigs in order to identify the biochemical lesion responsible for the enhanced release of Ca²⁺ in the sarcoplasm occurring in porcine malignant hyperthermia. Mitochondria isolated from longissimus dorsi muscle of malignant hyperthermia-prone pigs contained a significantly (P < 0.001) higher amount of endogenous long-chain fatty acids. Similar amounts of endogenous mitochondrial phospholipase A₂ were observed in both types of pigs, but the total activity in malignant hyperthermia-prone pigs was at least twice that of normal. Spermine, a phospholipase A₂ inhibitor, lowered the activity in both types of mitochondria to a similar final level. Mitochondria of malignant hyperthermia-prone pigs showed a significantly (P < 0.001) higher oligomycin-insensitive (Ca²⁺ + Mg²⁺)-ATPase activity, but the Mg²⁺-ATPase and the (Ca²⁺ + Mg²⁺)-ATPase activities were similar in both types of pigs. Sarcoplasmic reticulum isolated from longissimus dorsi muscle of malignant hyperthermia-prone pigs showed a significantly higher (Ca²⁺ + Mg²⁺)-ATPase activity and a lower rate of Ca²⁺ uptake; the maximal amount and the rate of Ca²⁺ uptake by sarcoplasmic reticulum of malignant hyperthermia-prone pigs were half that of normal. Mitochondria from longissimus dorsi muscle of malignant hyperthermia-prone pigs inhibited the Ca²⁺-transport system of the sarcoplasmic reticulum of longissimus dorsi from both normal and malignant hyperthermia-prone pigs, but mitochondria from normal pigs had no influence on the sarcoplasmic reticulum from either type. Experimental evidence favours the concept that long-chain fatty acids released from skeletal muscle mitochondria by endogenous mitochondrial phospholipase A₂ are responsible for the enhanced release of Ca²⁺ from mitochondria (Cheah, K.S. and Cheah, A.M. (1981) Biochim. Biophys. Acta 634, 70–84), and also additional release of Ca²⁺ from sarcoplasmic reticulum into the sarcoplasm during porcine malignant hyperthermia syndrome.     

Evidence for an endogenous protein inhibitor of sarcoplasmic reticulum calcium pump in heart muscle

A cytosolic protein fraction, termed CPF-I, derived by (NH₄)₂ SO₄ fractionation of rabbit heart cytosol caused marked inhibition (up to 95%) of ATP-dependent Ca²⁺ uptake by cardiac sarcoplasmic reticulum. The inhibitory effect of CPF-I was concentration-dependent (50% inhibition with ～ 80–100 μg CPF-I) and heat labile. The inhibitor reduced the velocity of Ca²⁺ uptake without altering the apparent affinity of the transport system for Ca²⁺. Concomitant with the inhibition of Ca²⁺ uptake, Ca²⁺-sensitive ATP hydrolysis was also inhibited by CPF-I. The inhibitor did not cause release of Ca²⁺ from Ca²⁺-preloaded membrane vesicles. The inhibitor activity of CPF-I could be adsorbed to a DEAE cellulose column and could be eluted with a linear gradient of KCl. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum calcium pump in cardiac muscle and raises the intriguing possibility of its participation in the regulation of calcium pump $\text{in}\text{vivo}$.     

DDT inhibition of Ca-Mg ATPase from peripheral nerves and muscles of lobster, Homarus americanus

Sensitivity of CaMg ATPase from axonic plasma membrane (APM) and sarcoplasmic reticulum (SR) of lobster, $\text{Homarus}\text{,}\text{americanus}$, to DDT was studied. The CaMg ATPase found in SR with the high Ca²⁺ affinity is sensitive to DDT while the portion of ATPase related to the low Ca²⁺ affinity site is not inhibited by DDT. Also, DDT is more inhibitory against the CaMg ATPase prepared from APM than the one obtained from SR. The relationship between inhibition of the CaMg ATPase by DDT in the axonic nerve membrane and $\text{in}\text{,}\text{vivo}$ poisoning symptoms of the nervous system is discussed.     

Aggregate-prone desmin mutations impair mitochondrial calcium uptake in primary myotubes

Desmin, being a major intermediate filament of mature muscle cell, interacts with mitochondria within the cell and participates in mitochondria proper localization. The goal of the present study was to assess the effect of aggregate-prone and non-aggregate-prone desmin mutations on mitochondrial calcium uptake. Primary murine satellite cells were transduced with lentiviruses carrying desmin in wild type or mutant form, and were induced to differentiate into myotubes. Four mutations resulting in different degree of desmin aggregates formation were analyzed. Tail domain mutation Asp399Tyr has the mildest impact on desmin filament polymerization, rod domain mutation Ala357Pro causes formation of large aggregates composed of filamentous material, and Leu345Pro and Leu370Pro are considered to be the most severest in their impact on desmin polymerization and structure. For mitochondrial calcium measurement cells were loaded with rhod 2-AM. We found that aggregate-prone mutations significantly decreased [Ca²⁺]_mit, whereas non-aggregate-prone mutations did not decrease [Ca²⁺]_mit. Moreover aggregate-prone desmin mutations resulted in increased resting cytosolic [Ca²⁺]. However this increase was not accompanied by any alterations in sarcoplasmic reticulum calcium release. We suggest that the observed decline in [Ca²⁺]_mit was due to desmin aggregate accumulation resulting in the loss of desmin mitochondria interactions.     

Effects of ruthenium red on Ca2+ uptake and ATPase of sarcoplasmic reticulum of rabbit skeletal muscle

Ruthenium red, a powerful inhibitor of Ca²⁺ transport by mitochondria, does not inhibit the active Ca²⁺ uptake by sarcoplasmic reticulum isolated from rabbit skeletal muscle promoted by 5 mM ATP-Mg in the presence or absence of potassium oxalate. Although concentrations of ruthenium red up to 100 μM do not affect the active uptake of Ca²⁺, 25 μM of the inorganic dye inhibit the passive binding of Ca²⁺ by about 50%. This inhibitory effect is observed in sarcoplasmic reticulum even after its lipid fraction is extracted with acetone.Although active Ca²⁺ uptake by sarcoplasmic reticulum is not inhibited by ruthenium red, in the absence of oxalate it inhibits significantly the Ca²⁺-dependent ATPase activity but not the Mg²⁺-ATPase. However, if potassium oxalate is present, the Ca²⁺-stimulated ATPase is not sensitive to the dye. It is not clear how oxalate functions to protect the Ca²⁺-ATPase against the inhibitor effect of ruthenium red.The high sensitivity to ruthenium red of the Ca²⁺ transport mechanism in mitochondria as compared to the Ca²⁺ transport in sarcoplasmic reticulum may be useful in determining the extent to which each organelle functions in the cell to regulate intracellular free Ca²⁺.     

GT-repeat polymorphism in the heme oxygenase-1 gene promoter and the risk of carotid atherosclerosis related to arsenic exposure

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca²⁺ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP₃Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca²⁺ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP₃Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP₃Rs were located in the SR. [Ca²⁺]i, [Ca²⁺]_m and [Ca²⁺]_SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca²⁺]_SR, increased [Ca²⁺]_i and [Ca²⁺]_m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca²⁺ release from the SR into the mitochondria through IP₃Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.     

Calcium transport by pigeon erythrocyte membrane vesicles

Membrane vesicles from pigeon erythrocytes show a rapid, ATP-dependent accumulation of ⁴⁵Ca²⁺. Ca²⁺ accumulation ratios greater than or approximately equal to 10⁴ are readily attained. For ATP-dependent Ca²⁺ uptake, V is 1.5 mmol · 1^?1 · min^?1 at 27°C (approx. 0.9 nmol · mg^?1 protein · min^?1), [Ca²⁺]_{$\text{1}\text{2}$} is 0.18 μM, [ATP]_{$\text{1}\text{2}$} is 30–60 μM, the Ca²⁺ uptake rate depends on [Ca²⁺]² and the dependence of uptake rate on ATP concentration implies strong ATP-ATP cooperativity. The Arrhenius activation energy is 19.1 ± 1.4 kcal/mol and the pH optimum is approx. 6.9.     

The effect of calcium ion transport ATPase upon the passive calcium ion permeability of phospholipid vesicles

The uptake and release of Ca²⁺ by sarcoplasmic reticulum fragments and reconstituted ATPase vesicles was measured by a stopped-flow fluorescence method using chlortetracycline as Ca²⁺ indicator.Incorporation of the Ca²⁺ transport ATPase into phospholipid bilayers of widely different fatty acid composition increases their passive permeability to Ca²⁺ by several orders of magnitude. Therefore in addition to participating in active Ca²⁺ transport, the ($\text{Mg}{}^{\mathrm{2+}}\text{+ Ca}{}^{\mathrm{2+}}$)-activated ATPase also forms hydrophilic channels across the membrane. The relative insensitivity of the permeability effect of ATPase to changes in the fatty acid composition of the membrane is in accord with the suggestion that the Ca²⁺ channels arise by protein-protein interaction between four ATPase molecules. The reversible formation of these channels may have physiological significance in the rapid Ca²⁺ release from the sarcoplasmic reticulum during activation of muscle.     

Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes

Ca²⁺ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca²⁺-dependent enzymes. However, excessive mitochondrial Ca²⁺ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca²⁺ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca²⁺ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca²⁺ levels ([Ca²⁺]_c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca²⁺ content (Ca^2+_m) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca²⁺]_c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca²⁺ accumulation. Vasopressin caused a rapid (< 90 sec), but modest (< 2 fold) increase in Ca²⁺m that was not further increased during prolonged incubations, despite a sustained [Ca²⁺]_c elevation. By contrast, thapsigargin induced a net Ca²⁺ accumulation in mitochondria that continued for up to 30 min and reached Ca^2+_m levels 10–20 fold over basal. Accumulation of mitochondrial Ca²⁺ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca²⁺ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kineses and phosphatases. The results indicate that net mitochondrial Ca²⁺ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca²⁺ through artificial means, enabling mitochondria to function as a Ca²⁺ sink under these conditions. (Mol Cell Biochem 174: 173–179, 1997)     

Mitochondria Are Linked to Calcium Stores in Striated Muscle by Developmentally Regulated Tethering Structures

Bi-directional calcium (Ca²⁺) signaling between mitochondria and intracellular stores (endoplasmic/sarcoplasmic reticulum) underlies important cellular functions, including oxidative ATP production. In striated muscle, this coupling is achieved by mitochondria being located adjacent to Ca²⁺ stores (sarcoplasmic reticulum [SR]) and in proximity of release sites (Ca²⁺ release units [CRUs]). However, limited information is available with regard to the mechanisms of mitochondrial-SR coupling. Using electron microscopy and electron tomography, we identified small bridges, or tethers, that link the outer mitochondrial membrane to the intracellular Ca²⁺ stores of muscle. This association is sufficiently strong that treatment with hypotonic solution results in stretching of the SR membrane in correspondence of tethers. We also show that the association of mitochondria to the SR is 1) developmentally regulated, 2) involves a progressive shift from a longitudinal clustering at birth to a specific CRU-coupled transversal orientation in adult, and 3) results in a change in the mitochondrial polarization state, as shown by confocal imaging after JC1 staining. Our results suggest that tethers 1) establish and maintain SR–mitochondrial association during postnatal maturation and in adult muscle and 2) likely provide a structural framework for bi-directional signaling between the two organelles in striated muscle.