Mitochondrial Ca influx and efflux rates in guinea pig cardiac mitochondria:Low and high affinity effects of cyclosporine A

Ca²⁺ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca²⁺ is controlled primarily by the mitochondrial Ca²⁺ uniporter and the mitochondrial Na⁺/Ca²⁺ exchanger, influencing NADH production through Ca²⁺-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca²⁺-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca²⁺ release. Here we selectively measure Ca²⁺ influx rate through the mitochondrial Ca²⁺ uniporter and Ca²⁺ efflux rates through Na⁺-dependent and Na⁺-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na⁺/Ca²⁺ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ_m loss, Ca²⁺ release, NADH oxidation, swelling) of high extramitochondrial Ca²⁺ additions, allowing mitochondria to tolerate total mitochondrial Ca²⁺ loads of > 400 nmol/mg protein. For Ca²⁺ pulses up to 15 μM, Na⁺-independent Ca²⁺ efflux through the permeability transition pore accounted for ~ 5% of the total Ca²⁺ efflux rate compared to that mediated by the mitochondrial Na⁺/Ca²⁺ exchanger (in 5 mM Na⁺). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ efflux at higher concentrations (IC₅₀ = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca²⁺ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca²⁺ load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Mitochondrial calcium transport: mechanisms and functions

Ca(2+)transport across the mitochondrial inner membrane is facilitated by transporters having four distinct sets of characteristics as well as through the Ca(2+)-induced mitochondrial permeability transition pore (PTP). There are two modes of inward transport, referred to as the Ca(2+)uniporter and the rapid mode or RaM. There are also two distinct mechanisms mediating outward transport, which are not associated with the PTP, referred to as the Na(+)-dependent and the Na(+)-independent Ca(2+)efflux mechanisms. Several important functions have been proposed for these mechanisms, including control of the metabolic rate for cellular energy (ATP) production, modulation of the amplitude and shape of cytosolic Ca(2+)transients, and induction of apoptosis through release of cytochrome c from the mitochondrial inter membrane space into the cytosolic space.The goals of this review are to survey the literature describing the characteristics of the mechanisms of mitochondrial Ca(2+)transport and their proposed physiological functions, emphasizing the more recent contributions, and to consider how the observed characteristics of the mitochondrial Ca(2+)transport mechanisms affect our understanding of their functions.     

The mitochondrial Na(+)/Ca(2+) exchanger

Powered by the steep mitochondrial membrane potential Ca(2+) permeates into the mitochondria via the Ca(2+) uniporter and is then extruded by a mitochondrial Na(+)/Ca(2+) exchanger. This mitochondrial Ca(2+) shuttling regulates the rate of ATP production and participates in cellular Ca(2+) signaling. Despite the fact that the exchanger was functionally identified 40 years ago its molecular identity remained a mystery. Early studies on isolated mitochondria and intact cells characterized the functional properties of a mitochondrial Na(+)/Ca(2+) exchanger, and showed that it possess unique functional fingerprints such as Li(+)/Ca(2+) exchange and that it is displaying selective sensitivity to inhibitors. Purification of mitochondria proteins combined with functional reconstitution led to the isolation of a polypeptide candidate of the exchanger but failed to molecularly identify it. A turning point in the search for the exchanger molecule came with the recent cloning of the last member of the Na(+)/Ca(2+) exchanger superfamily termed NCLX (Na(+)/Ca(2+)/Li(+) exchanger). NCLX is localized in the inner mitochondria membrane and its expression is linked to mitochondria Na(+)/Ca(2+) exchange matching the functional fingerprints of the putative mitochondrial Na(+)/Ca(2+) exchanger. Thus NCLX emerges as the long sought mitochondria Na(+)/Ca(2+) exchanger and provide a critical molecular handle to study mitochondrial Ca(2+) signaling and transport. Here we summarize some of the main topics related to the molecular properties of the Na(+)/Ca(2+) exchanger, beginning with the early days of its functional identification, its kinetic properties and regulation, and culminating in its molecular identification.     

Role of the choline exchanger in Na(+)-independent Mg(2+) efflux from rat erythrocytes

Two types of Na(+)-independent Mg(2+) efflux exist in erythrocytes: (1) Mg(2+) efflux in sucrose medium and (2) Mg(2+) efflux in high Cl(-) media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na(+)-independent Mg(2+) efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K(+),Cl(-)- and Na(+),K(+),Cl(-)-symport, Na(+)/H(+)-, Na(+)/Mg(2+)-, Na(+)/Ca(2+)- and K(+)(Na(+))/H(+) antiport, Ca(2+)-activated K(+) channel and Mg(2+) leak flux. We suggest that, in choline Cl medium, Na(+)-independent Mg(2+) efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg(2+) efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg(2+) to the same degree. The K(d) value for inhibition of [(14)C]choline efflux and for inhibition of Mg(2+) efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg(2+) efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg(2+) efflux was reduced to the same degree by these inhibitors as was the [(14)C]choline efflux.     

Ca(2+) efflux in mitochondria from the yeast Endomyces magnusii.

Calcium release pathways in Ca(2+)-preloaded mitochondria from the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca(2+) was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca(2+) release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca(2+) and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca(2+) efflux set in after uptake of approximately 150 nmol of Ca(2+)/mg of protein. The rate of this efflux was proportional to the Ca(2+) load and insensitive to aeration, protonophorous uncouplers, and Na(+) ions. Ca(2+) efflux was inhibited by La(3+), Mn(2+), Mg(2+), tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside and t-butyl hydroperoxide were without effect. Ca(2+) efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca(2+) efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na(+)-independent pathway, in many ways similar to the one in mammalian mitochondria.     

The permeability transition pore as a Ca(2+) release channel: new answers to an old question

Mitochondria possess a sophisticated array of Ca(2+) transport systems reflecting their key role in physiological Ca(2+) homeostasis. With the exception of most yeast strains, energized organelles are endowed with a very fast and efficient mechanism for Ca(2+) uptake, the ruthenium red (RR)-sensitive mitochondrial Ca(2+) uniporter (MCU); and one main mechanism for Ca(2+) release, the RR-insensitive 3Na(+)-Ca(2+) antiporter. An additional mechanism for Ca(2+) release is provided by a Na(+) and RR-insensitive release mechanism, the putative 3H(+)-Ca(2+) antiporter. A potential kinetic imbalance is present, however, because the V(max) of the MCU is of the order of 1400nmol Ca(2+)mg(-1) proteinmin(-1) while the combined V(max) of the efflux pathways is about 20nmol Ca(2+)mg(-1) proteinmin(-1). This arrangement exposes mitochondria to the hazards of Ca(2+) overload when the rate of Ca(2+) uptake exceeds that of the combined efflux pathways, e.g. for sharp increases of cytosolic [Ca(2+)]. In this short review we discuss the hypothesis that transient opening of the Ca(2+)-dependent permeability transition pore may provide mitocondria with a fast Ca(2+) release channel preventing Ca(2+) overload. We also address the relevance of a mitochondrial Ca(2+) release channel recently discovered in Drosophila melanogaster, which possesses intermediate features between the permeability transition pore of yeast and mammals.     

Intracellular sodium modulates mitochondrial calcium signaling in vascular endothelial cells

We have investigated the role of extramitochondrial Na(+) for the regulation of mitochondrial Ca(2+) concentration ([Ca(2+)](m)) in permeabilized single vascular endothelial cells. [Ca(2+)](m) was measured by loading the cells with the membrane-permeant Ca(2+) indicator fluo-3/AM and subsequent removal of cytoplasmic fluo-3 by surface membrane permeabilization with digitonin. An elevation of extramitochondrial Ca(2+) resulted in a dose-dependent increase in the rate of Ca(2+) accumulation into mitochondria (k(0.5) = 3 microm) via the mitochondrial Ca(2+) uniporter. In the presence of 10 mm extramitochondrial Na(+) ([Na(+)](em)), repetitive application of brief pulses of high Ca(2+) (2-10 microm) to simulate cytoplasmic [Ca(2+)] oscillations caused transient increases of [Ca(2+)](m) characterized by a fast rising phase that was followed by a slow decay. Removal of extramitochondrial Na(+) or inhibition of mitochondrial Na(+)/Ca(2+) exchange with clonazepam blocked mitochondrial Ca(2+) efflux and resulted in a net accumulation of Ca(2+) by the mitochondria. Half-maximal activation of mitochondrial Na(+)/Ca(2+) exchange occurred at [Na(+)](em) = 4.4 mm, which is well within the physiological range of cytoplasmic [Na(+)]. This study provides evidence that Ca(2+) efflux from the mitochondria in vascular endothelial cells occurs solely via Na(+)/Ca(2+) exchange and emphasizes the important role of intracellular Na(+) for mitochondrial Ca(2+) regulation.     

Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger

Although the Na(+)/H(+) exchanger (NHE) is considered to be involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the Na(+)/Ca(2+) exchanger, the exact mechanisms of its participation in Ca(2+) handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca(2+) movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N-isobutyl)amiloride (MIA). [Ca(2+)](i) in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca(2+)](i) and augmented the KCl-induced increase in [Ca(2+)](i) in a concentration-dependent manner. The MIA-induced increase in basal [Ca(2+)](i) was unaffected by extracellular Ca(2+), antagonists of the sarcolemmal (SL) L-type Ca(2+) channel, and inhibitors of the SL Na(+)/Ca(2+) exchanger, SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. However, the MIA-induced increase in basal [Ca(2+)](i) was attenuated by inhibitors of SL Na(+)-K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+) transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca(2+) concentration and attenuated by agents that inhibit SL L-type Ca(2+) channels, the SL Na(+)/Ca(2+) exchanger, SL Na(+)-K(+)-ATPase, and SR Ca(2+) release channels and the SR Ca(2+) pump. However, the effect of MIA on the KCl-induced increase in [Ca(2+)](i) remained unaffected by treatment with inhibitors of SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca(2+)](i), whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca(2+)](i) in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca(2+)](i) and that MIA-induced increases in basal [Ca(2+)](i), as well as augmentation of the KCl-induced increase in [Ca(2+)](i), in cardiomyocytes are regulated differentially.     

Molecular identity and functional properties of the mitochondrial na+/ca2+ exchanger

The mitochondrial membrane potential that powers the generation of ATP also facilitates mitochondrial Ca(2+) shuttling. This process is fundamental to a wide range of cellular activities, as it regulates ATP production, shapes cytosolic and endoplasmic recticulum Ca(2+) signaling, and determines cell fate. Mitochondrial Ca(2+) transport is mediated primarily by two major transporters: a Ca(2+) uniporter that mediates Ca(2+) uptake and a Na(+)/Ca(2+) exchanger that subsequently extrudes mitochondrial Ca(2+). In this minireview, we focus on the specific role of the mitochondrial Na(+)/Ca(2+) exchanger and describe its ion exchange mechanism, regulation by ions, and putative partner proteins. We discuss the recent molecular identification of the mitochondrial exchanger and how its activity is linked to physiological and pathophysiological processes.     

Discovery of a novel potent Na+/Ca2+ exchanger inhibitor: design, synthesis and structure-activity relationships of 3,4-dihydro-2(1H)-quinazolinone derivatives

Design, synthesis and structure-activity relationships for 3,4-dihydro-2(1H)-quinazolinone derivatives with the inhibitory activities of the Na(+)/Ca(2+) exchanger are discussed. These studies based on lead compound 1a lead to the discovery of a structurally novel and highly potent inhibitor against the Na(+)/Ca(2+) exchanger 4f (SM-15811), which directly inhibited the Na(+)-dependent Ca(2+) influx via the Na(+)/Ca(2+) exchanger in cardiomyocytes with a high potency.     

Ca(2+) sources for the exocytotic release of glutamate from astrocytes

Astrocytes can exocytotically release the gliotransmitter glutamate from vesicular compartments. Increased cytosolic Ca(2+) concentration is necessary and sufficient for this process. The predominant source of Ca(2+) for exocytosis in astrocytes resides within the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate and ryanodine receptors of the ER provide a conduit for the release of Ca(2+) to the cytosol. The ER store is (re)filled by the store-specific Ca(2+)-ATPase. Ultimately, the depleted ER is replenished by Ca(2+) which enters from the extracellular space to the cytosol via store-operated Ca(2+) entry; the TRPC1 protein has been implicated in this part of the astrocytic exocytotic process. Voltage-gated Ca(2+) channels and plasma membrane Na(+)/Ca(2+) exchangers are additional means for cytosolic Ca(2+) entry. Cytosolic Ca(2+) levels can be modulated by mitochondria, which can take up cytosolic Ca(2+) via the Ca(2+) uniporter and release Ca(2+) into cytosol via the mitochondrial Na(+)/Ca(2+) exchanger, as well as by the formation of the mitochondrial permeability transition pore. The interplay between various Ca(2+) sources generates cytosolic Ca(2+) dynamics that can drive Ca(2+)-dependent exocytotic release of glutamate from astrocytes. An understanding of this process in vivo will reveal some of the astrocytic functions in health and disease of the brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Calcium-dependent spontaneously reversible remodeling of brain mitochondria

An exposure of cultured hippocampal neurons expressing mitochondrially targeted enhanced yellow fluorescent protein to excitotoxic glutamate resulted in reversible mitochondrial remodeling that in many instances could be interpreted as swelling. Remodeling was not evident if glutamate receptors were blocked with MK801, if Ca(2+) was omitted or substituted for Sr(2+) in the bath solution, if neurons were treated with carbonylcyanide p-trifluoromethoxyphenylhydrazone to depolarize mitochondria, or if neurons were pretreated with cyclosporin A or N-methyl-4-isoleucine-cyclosporin (NIM811) to inhibit the mitochondrial permeability transition. In the experiments with isolated brain synaptic or nonsynaptic mitochondria, Ca(2+) triggered transient, spontaneously reversible cyclosporin A-sensitive swelling closely resembling remodeling of organelles in cultured neurons. The swelling was accompanied by the release of cytochrome c, Smac/DIABLO, Omi/HtrA2, and AIF but not endonuclease G. Depolarization with carbonylcyanide p-trifluoromethoxyphenylhydrazone or inhibition of the Ca(2+) uniporter with Ru360 prevented rapid onset of the swelling. Sr(2+) depolarized mitochondria but failed to induce swelling. Neither inhibitors of the large conductance Ca(2+)-activated K(+) channel (charybdotoxin, iberiotoxin, quinine, and Ba(2+)) nor inhibitors of the mitochondrial ATP-sensitive K(+) channel (5-hydroxydecanoate and glibenclamide) suppressed swelling. Quinine, dicyclohexylcarbodiimide, and Mg(2+), inhibitors of the mitochondrial K(+)/H(+) exchanger, as well as external alkalization inhibited a recovery phase of the reversible swelling. In contrast to brain mitochondria, liver and heart mitochondria challenged with Ca(2+) experienced sustained swelling without spontaneous recovery. The proposed model suggests an involvement of the Ca(2+)-dependent transient K(+) influx into the matrix causing mitochondrial swelling followed by activation of the K(+)/H(+) exchanger leading to spontaneous mitochondrial contraction both in situ and in vitro.     

Ca2+ dysregulation induces mitochondrial depolarization and apoptosis: role of Na+/Ca2+ exchanger and AKT

We previously reported that constitutively activated Galpha(q) (Q209L) expression in cardiomyocytes induces apoptosis through opening of the mitochondrial permeability transition pore. We assessed the hypothesis that disturbances in Ca(2+) handling linked Galpha(q) activity to apoptosis because resting Ca(2+) levels were significantly increased prior to development of apoptosis. Treating cells with EGTA lowered Ca(2+) and blocked both loss of mitochondrial membrane potential (an indicator of permeability transition pore opening) and apoptosis (assessed by DNA fragmentation). When cytosolic Ca(2+) and mitochondrial membrane potential were simultaneously measured by confocal microscopy, sarcoplasmic reticulum (SR)-driven slow Ca(2+) oscillations (time-to-peak approximately 4 s) were observed in Q209L-expressing cells. These oscillations were seen to transition into sustained increases in cytosolic Ca(2+), directly paralleled by loss of mitochondrial membrane potential. Ca(2+) transients generated by caffeine-induced release of SR Ca(2+) were greatly prolonged in Q209L-expressing cells, suggesting a decreased ability to extrude Ca(2+). Indeed, the Na(+)/Ca(2+) exchanger (NCX), which removes Ca(2+) from the cell, was markedly down-regulated at the mRNA and protein levels. Adenoviral NCX expression normalized cytosolic Ca(2+) levels and prevented DNA fragmentation in cells expressing Q209L. Interestingly, constitutively activated Akt, which rescues cells from Q209L-induced apoptosis, prevented the decrease in NCX expression, normalized cytosolic Ca(2+) levels and spontaneous Ca(2+) oscillations, shortened caffeine-induced Ca(2+) transients, and prevented loss of the mitochondrial membrane potential. Our findings demonstrate that NCX down-regulation and consequent increases in cytosolic and SR Ca(2+) can lead to Ca(2+) overloading-induced loss of mitochondrial membrane potential and suggest that recovery of Ca(2+) dysregulation is a target of Akt-mediated protection.     

alpha-Adrenoceptor stimulation-mediated negative inotropism and enhanced Na(+)/Ca(2+) exchange in mouse ventricle

Mechanisms underlying the negative inotropic response to alpha-adrenoceptor stimulation in adult mouse ventricular myocardium were studied. In isolated ventricular tissue, phenylephrine (PE), in the presence of propranolol, decreased contractile force by approximately 40% of basal value. The negative inotropic response was similarly observed under low extracellular Ca(2+) concentration ([Ca(2+)](o)) conditions but was significantly smaller under high-[Ca(2+)](o) conditions and was not observed under low-[Na(+)](o) conditions. The negative inotropic response was not affected by nicardipine, ryanodine, ouabain, or dimethylamiloride (DMA), inhibitors of L-type Ca(2+) channel, Ca(2+) release channel, Na(+)-K(+) pump, or Na(+)/H(+) exchanger, respectively. KB-R7943, an inhibitor of Na(+)/Ca(2+) exchanger, suppressed the negative inotropic response mediated by PE. PE reduced the magnitude of postrest contractions. PE caused a decrease in duration of the late plateau phase of action potential and a slight increase in resting membrane potential; time courses of these effects were similar to that of the negative inotropic effect. In whole cell voltage-clamped myocytes, PE increased the L-type Ca(2+) and Na(+)/Ca(2+) exchanger currents but had no effect on the inwardly rectifying K(+), transient outward K(+), or Na(+)-K(+)-pump currents. These results suggest that the sustained negative inotropic response to alpha-adrenoceptor stimulation of adult mouse ventricular myocardium is mediated by enhancement of Ca(2+) efflux through the Na(+)/Ca(2+) exchanger.     

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.     

An integrated model of cardiac mitochondrial energy metabolism and calcium dynamics

We present an integrated thermokinetic model describing control of cardiac mitochondrial bioenergetics. The model describes the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and mitochondrial Ca(2+) handling. The kinetic component of the model includes effectors of the TCA cycle enzymes regulating production of NADH and FADH(2), which in turn are used by the electron transport chain to establish a proton motive force (Delta mu(H)), driving the F(1)F(0)-ATPase. In addition, mitochondrial matrix Ca(2+), determined by Ca(2+) uniporter and Na(+)/Ca(2+) exchanger activities, regulates activity of the TCA cycle enzymes isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase. The model is described by twelve ordinary differential equations for the time rate of change of mitochondrial membrane potential (Delta Psi(m)), and matrix concentrations of Ca(2+), NADH, ADP, and TCA cycle intermediates. The model is used to predict the response of mitochondria to changes in substrate delivery, metabolic inhibition, the rate of adenine nucleotide exchange, and Ca(2+). The model is able to reproduce, qualitatively and semiquantitatively, experimental data concerning mitochondrial bioenergetics, Ca(2+) dynamics, and respiratory control. Significant increases in oxygen consumption (V(O(2))), proton efflux, NADH, and ATP synthesis, in response to an increase in cytoplasmic Ca(2+), are obtained when the Ca(2+)-sensitive dehydrogenases are the main rate-controlling steps of respiratory flux. These responses diminished when control is shifted downstream (e.g., the respiratory chain or adenine nucleotide translocator). The time-dependent behavior of the model, under conditions simulating an increase in workload, closely reproduces experimentally observed mitochondrial NADH dynamics in heart trabeculae subjected to changes in pacing frequency. The steady-state and time-dependent behavior of the model support the hypothesis that mitochondrial matrix Ca(2+) plays an important role in matching energy supply with demand in cardiac myocytes.     

Inhibition of sodium-calcium exchange by ceramide and sphingosine

Na(+)/Ca(2+) exchange activity in Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger was inhibited by the short chain ceramide analogs N-acetylsphingosine and N-hexanoylsphingosine (5-15 micrometer). The sphingolipids reduced exchange-mediated Ba(2+) influx by 50-70% and also inhibited the Ca(2+) efflux mode of exchange activity. The biologically inactive ceramide analog N-acetylsphinganine had only modest effects on exchange activity. Cells expressing the Delta(241-680) and Delta(680-685) deletion mutants of the Na(+)/Ca(2+) exchanger were not inhibited by ceramide; these mutants show defects in both Na(+)-dependent and Ca(2+)-dependent regulatory behavior. Another mutant, which was defective only in Na(+)-dependent regulation, was as sensitive to ceramide inhibition as the wild-type exchanger. Inhibition of exchange activity by ceramide was time-dependent and was accelerated by depletion of internal Ca(2+) stores. Sphingosine (2.5 micrometer) also inhibited the Ca(2+) influx and efflux modes of exchange activity in cells expressing the wild-type exchanger; sphingosine did not affect Ba(2+) influx in the Delta(241-680) mutant. The effects of the exogenous sphingolipids were reproduced by blocking cellular ceramide utilization pathways, suggesting that exchange activity is inhibited by increased levels of endogenous ceramide and/or sphingosine. We propose that sphingolipids impair Ca(2+)-dependent activation of the exchanger and that in cardiac myocytes, this process serves as a feedback mechanism that links exchange activity to the diastolic concentration of cytosolic Ca(2+).     

Thyroid hormones stimulate calcium transport systems in rat intestine

Thyroid hormone status influences calcium metabolism. To elucidate the mechanism of action of thyroid hormones on transcellular transport of calcium in rat intestine, Ca(2+) influx and efflux studies were carried out in brush border membrane vesicles (BBMV) and across the basolateral membrane (BLM) of enterocytes, respectively. Steady-state uptake of Ca(2+) into BBMV as well as Ca(2+) efflux from the BLM enterocytes was significantly increased in hyperthyroid (Hyper-T) rats and decreased in hypothyroid (Hypo-T) rats as compared to euthyroid (Eu-T) rats. Kinetic studies revealed that increase in steady state Ca(2+) uptake into BBMV from hyper-T rats was fraternized with decrease in Michaelis Menten Constant (K(m)), indicating a conformational change in Ca(2+) transporter. Further, this finding was supported by significant changes in transition temperature and membrane fluidity. Increased Ca(2+) efflux across enterocytes was attributed to sodium-dependent Ca(2+) exchange activity which was significantly higher in Hyper-T rats and lower in Hypo-T rats as compared to Eu-T rats. However, there was no change in Ca(2+)-ATPase activity of BLMs of all groups. Kinetic studies of Na(+)/Ca(2+) exchanger revealed that alteration in Na(+)-dependent Ca(2+) efflux was directly associated with maximal velocity (V(max)) of exchanger among all the groups. cAMP, a potent activator of Na(+)/Ca(2+) exchanger, was found to be significantly higher in intestinal mucosa of Hyper-T rats as compared to Eu-T rats. Therefore, the results of this study suggest that Ca(2+) influx across BBM is possibly modulated by thyroid hormones by mediating changes in membrane fluidity. Thyroid hormones activated the Na(+)/Ca(2+) exchange in enterocytes possibly via cAMP-mediated pathway.     

Feedback inhibition of sodium/calcium exchange by mitochondrial calcium accumulation

Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger were subjected to two periods of 5 and 3 min, respectively, during which the extracellular Na(+) concentration ([Na(+)](o)) was reduced to 20 mm; these intervals were separated by a 5-min recovery period at 140 mm Na(+)(o). The cytosolic Ca(2+) concentration ([Ca(2+)](i)) increased during both intervals due to Na(+)-dependent Ca(2+) influx by the exchanger. However, the peak rise in [Ca(2+)](i) during the second interval was only 26% of the first. The reduced rise in [Ca(2+)](i) was due to an inhibition of Na(+)/Ca(2+) exchange activity rather than increased Ca(2+) sequestration since the influx of Ba(2+), which is not sequestered by internal organelles, was also inhibited by a prior interval of Ca(2+) influx. Mitochondria accumulated Ca(2+) during the first interval of reduced [Na(+)](o), as determined by an increase in fluorescence of the Ca(2+)-indicating dye rhod-2, which preferentially labels mitochondria. Agents that blocked mitochondrial Ca(2+) accumulation (uncouplers, nocodazole) eliminated the observed inhibition of exchange activity during the second period of low [Na(+)](o). Conversely, diltiazem, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger, increased mitochondrial Ca(2+) accumulation and also increased the inhibition of exchange activity. We conclude that Na(+)/Ca(2+) exchange activity is regulated by a feedback inhibition process linked to mitochondrial Ca(2+) accumulation.