Mechanism of sodium independent calcium efflux from rat liver mitochondria

On the basis of primarily two types of observations, it has been suggested that the Na+-independent Ca2+ efflux mechanism of rat liver mitochondria is a passive Ca2+-2H+ exchanger. First, when a pulse of acid is added to a suspension of mitochondria loaded with Ca2+, a pulse of intramitochondrial Ca2+ is often released, even in the presence of the inhibitor of mitochondrial Ca2+ influx, ruthenium red. Second, at a pH near 7, the stoichiometry of Ca2+ released to H+ taken up by Ca2+-loaded mitochondria, following treatment with ruthenium red, has been observed to be 1:2. This evidence for a Ca2+-2H+ exchanger is reexamined here by studying the release of Ca2+ upon acidification of the medium by addition of buffer, the dependence of liver mitochondrial Ca2+ efflux on external medium pH and intramitochondrial pH, and the Ca2+-Ca2+ exchange properties of the Ca2+ efflux mechanism. These studies show no pulse of mitochondrial Ca2+ efflux when pH is abruptly lowered by addition of buffer. The stoichiometry between Ca2+ and H+ fluxes is found to be highly pH dependent. The reported 1:2 stoichiometry between Ca2+ efflux and H+ influx is only observed at one pH. Furthermore, the rate of Ca2+ efflux from mitochondria is found to increase only very slightly at most as suspension pH is decreased. The rate of Ca2+ efflux is not found to increase with increasing intramitochondrial pH. Finally, no Ca2+-Ca2+ isotope exchange can be demonstrated over the Na+-independent efflux mechanism (i.e., in the presence of ruthenium red). It is concluded that these data do not support the hypothesis that the Na+-independent Ca2+ efflux mechanism is a passive Ca2+-2H+ exchanger.     

The effect of alkanols on Ca2+ transport in brain mitochondria.

Ethanol stimulates the Na(+)-dependent Ca2+ efflux in brain mitochondria and inhibits the Na(+)-independent Ca(2+)-efflux. Here, we studied the effects of n-alkanols on the various Ca2+ transport processes in brain mitochondria. Only short-chain alcohols (i.e. methanol, ethanol and propanol) stimulated Na+/Ca2+ exchange. The inhibition of H+/Ca2+ exchange was significant only with ethanol. Short-chain alcohols inhibit while long-chain alcohols activate the cyclosporin-sensitive Ca(2+)-efflux. These data suggest that the mechanism of the alkanols' effects on Na+/Ca2+ exchange, H+/Ca2+ exchange and the cyclosporin sensitive pore are entirely different. Alkanols have no effect on the electrogenic Ca2+ uniporter. Ethanol did not affect the apparent K0.5 for Na+ (7.5 mM) of the Na+/Ca2+ exchange. Similarly, the magnitude of the effect of ethanol did not depend on matrix Ca2+ concentration, suggesting that short-chain alkanols do not stimulate the rate of Na+/Ca2+ exchange by increasing the affinity of the carrier to Ca2+in or Na+out. High concentrations of K+, Mg2+ and Ca2+ enhanced the ethanol effect. It is possible that high surface potential attenuates the effect of ethanol. It is suggested that ethanol stimulation of Na+/Ca2+ exchange depends on the modulation of the surface dielectric constant.     

Alcohol stimulates Na+/Ca2+ exchange in brain mitochondria

Ethanol, at low concentrations, specifically stimulates the Na(+)-dependent Ca2(+)-efflux in brain mitochondria. In addition, at higher concentrations, ethanol inhibits the Na(+)-independent Ca2(+)-efflux. The electrogenic Ca(+)-uptake system is not affected by ethanol. The specific stimulation of Na+/Ca2+ exchange reaches a maximum of 60% stimulation, with half-maximal stimulation at 130 mM ethanol. The inhibition of the Na(+)-independent efflux is proportional to the ethanol concentration, becoming significant only above 200 mM, with 50% inhibition at 0.5 M. The inhibition of the Na(+)-independent efflux is, in large part, due to an inhibition of the activation of the Cyclosporin-sensitive pore. Long-term ethanol-feeding had no effect on the Ca2+ transport systems and their sensitivity to acute ethanol treatment. It is suggested that the stimulation of the Na(+)-dependent Ca2(+)-efflux, which is the dominant Ca2+ efflux pathway in brain mitochondria, contributes to the intoxicating effects of ethanol.     

Pathways for Ca2+ efflux in heart and liver mitochondria.

1. Two processes of Ruthenium Red-insensitive Ca2+ efflux exist in liver and in heart mitochondria: one Na+-independent, and another Na+-dependent. The processes attain maximal rates of 1.4 and 3.0 nmol of Ca2+.min-1.mg-1 for the Na+-dependent and 1.2 and 2.0 nmol of Ca2+.min-1.mg-1 for the Na+-independent, in liver and heart mitochondria, respectively. 2. The Na+-dependent pathway is inhibited, both in heart and in liver mitochondria, by the Ca2+ antagonist diltiazem with a Ki of 4 microM. The Na+-independent pathway is inhibited by diltiazem with a Ki of 250 microM in liver mitochondria, while it behaves as almost insensitive to diltiazem in heart mitochondria. 3. Stretching of the mitochondrial inner membrane in hypo-osmotic media results in activation of the Na+-independent pathway both in liver and in heart mitochondria. 4. Both in heart and liver mitochondria the Na+-independent pathway is insensitive to variations of medium pH around physiological values, while the Na+-dependent pathway is markedly stimulated parallel with acidification of the medium. The pH-activated, Na+-dependent pathway maintains the diltiazem sensitivity. 5. In heart mitochondria, the Na+-dependent pathway is non-competitively inhibited by Mg2+ with a Ki of 0.27 mM, while the Na+-independent pathway is less affected; similarly, in liver mitochondria Mg2+ inhibits the Na+-dependent pathway more than it does the Na+-independent pathway. In the presence of physiological concentrations of Na+, Ca2+ and Mg2+, the Na+-independent and the Na+-dependent pathways operate at rates, respectively, of 0.5 and 1.0 nmol of Ca2+.min-1.mg-1 in heart mitochondria and 0.9 and 0.2 nmol of Ca2+.min-1.mg-1 in liver mitochondria. It is concluded that both heart and liver mitochondria possess two independent pathways for Ca2+ efflux operating at comparable rates.     

Regulation of Ca2+ transport in brain mitochondria. I. The mechanism of spermine enhancement of Ca2+ uptake and retention

Spermine enhances electrogenic Ca2+ uptake and inhibits Na(+)-independent Ca2+ efflux in rat brain mitochondria. As a result, Ca2+ retention by brain mitochondria increases greatly and the external free Ca2+ level at steady-state can be lowered to physiologically relevant concentrations. The stimulation of Ca2+ uptake by spermine is more pronounced at low concentrations of Ca2+, effectively lowering the apparent Km for Ca2+ uptake from 3 microM to 1.5 microM. However, the apparent Vmax is also increased. At low Ca2+ concentrations, Ca2+ uptake is diffusion-limited. Spermine strongly inhibits Ca2+ binding to anionic phospholipids and it is suggested that this increases the rate of surface diffusion which reduces the apparent Km for uptake. The same effect could inhibit the Na(+)-independent efflux if the rate of efflux is limited by Ca2+ dissociation from the efflux carrier. In brain mitochondria (but not in liver) the spermine effect depends on the presence of ADP. In a medium that contains physiological concentrations of Pi, Mg+, K+, ADP and spermine, brain mitochondria sequester Ca2+ down to 0.1 microM and below, depending on the matrix Ca2+ load. Moreover, brain mitochondria under the same conditions buffer the external medium at 0.4 microM, a concentration at which the set point becomes independent of the matrix Ca2+ content. Thus, mitochondria appear to be capable of modulating calcium oscillations in brain cells.     

Osmoregulation of atrial myocytic ANP release: osmotransduction via cross-talk between L-type Ca2+ channel and SR Ca2+ release

Hyperosmolality has been known to increase ANP release. However, its physiological role in the regulation of atrial myocytic ANP release and the mechanism by which hyperosmolality increases ANP release are to be defined. The purpose of the present study was to define these questions. Experiments were performed in perfused beating rabbit atria. Hyperosmolality increased atrial ANP release, cAMP efflux, and atrial dynamics in a concentration-dependent manner. The osmolality threshold for the increase in ANP release was as low as 10 mosmol/kgH2O (approximately 3%) above the basal levels (1.55 +/- 1.71, 17.19 +/- 3.11, 23.15 +/- 5.49, 54.04 +/- 11.98, and 62.00 +/- 13.48% for 10, 20, 30, 60, and 100 mM mannitol, respectively; all P < 0.01). Blockade of sarcolemmal L-type Ca2+ channel activity, which increased ANP release, attenuated hyperosmolality-induced increases in ANP release (-13.58 +/- 4.68% vs. 62.00 +/- 13.48%, P < 0.001) and cAMP efflux but not atrial dynamics. Blockade of the Ca2+ release from the sarcoplasmic reticulum, which increased ANP release, attenuated hyperosmolality-induced increases in ANP release (13.44 +/- 7.47% vs. 62.00 +/- 13.48%, P < 0.01) and dynamics but not cAMP efflux. Blockades of Na+-K+-2Cl- cotransporter, Na+/H+ exchanger, and Na+/Ca2+ exchanger had no effect on hyperosmolality-induced increase in ANP release. The present study suggests that hyperosmolality regulates atrial myocytic ANP release and that the mechanism by which hyperosmolality activates ANP release is closely related to the cross-talk between the sarcolemmal L-type Ca2+ channel activity and sarcoplasmic reticulum Ca2+ release, possibly inactivation of the L-type Ca2+ channels.     

The Ba2+ sensitivity of the Na+-induced Ca2+ efflux in heart mitochondria: the site of inhibitory action

The Na+-induced Ca2+ release from rat heart mitochondria was measured in the presence of Ruthenium red. Ba2+ effectively inhibited the Na+-induced Ca2+ release. At 10 mM Na+ 50% inhibition was reached by 1.51 +/- 0.48 (S.D., n = 8) microM Ba2+ in the presence of 0.1 mg/ml albumin and by 0.87 +/- 0.25 (S.D., n = 3) microM Ba2+ without albumin. In order to inhibit, it was not required that Ba2+ ions enter the matrix. 140Ba2+ was not accumulated in the mitochondrial matrix space; further, in contrast to liver mitochondria, Ba2+ inhibition was immediate. The Na+-induced Ca2+ release was inhibited by Ba2+ non-competitively, with respect of the extramitochondrial Na+. The double inhibitor titration of the Na+-Ca2+ exchanger with Ba2+ in the presence and absence of extramitochondrial Ca2+ revealed that the exchanger possesses a common binding site for extramitochondrial Ca2+ and Ba2+, presumably the regulatory binding site of the Na+-Ca2+ exchanger, which was described by Hayat and Crompton (Biochem. J. 202 (1982) 509-518). All these observations indicate that Ba2+ acts at the cytoplasmic surface of the inner mitochondrial membrane. The inhibitory properties of Ba2+ on the Na+-dependent Ca2+ release in heart mitochondria are basically different from those found on Na+-independent Ca2+ release in liver mitochondria (Lukács, G.L. and Fonyó, A. (1985) Biochim. Biophys. Acta 809, 160-166).     

The isolation of lymphocyte mitochondria and their regulation of extramitochondrial free Ca2+ concentration.

1. A method for the isolation of functionally intact mitochondria from lymphocytes is described. It involves digitonin breakage of the plasma membrane, followed by differential centrifugation. The yield was 36 mg of mitochondrial protein/200 g of pig mesenteric lymph node (6 mg of mitochondrial protein/10(9) lymphocytes). The mitochondrial had a respiratory-control ratio of 2--3.5 with succinate as substrate. 2. Ca2+ transport by these mitochondria was investigated. They were able to regulate the extramitochondrial free [Ca2+] very precisely, by buffering any displacements from the steady-state. The exact extramitochondrial free [Ca2+] of this steady-state depended on the conditions of incubation. In a medium designed to resemble the cytoplasmic environment, with added Ca2+, lymphocyte mitochondria maintained a steady-state free [Ca2+] of 0.63 microM (pCa of 6.2). The rates of Ca2+ uptake and efflux under these conditions, with both lymphocyte and liver mitochondria, were very much lower than those in a less complex medium. 3. Lymphocyte mitochondria were shown to possess an Na+-independent Ruthenium Red-insensitive efflux pathway similar to that of liver mitochondria. Ruthenium Red totally inhibited the electrophoretic uniporter. Although Na+ had no effect on the steady-state maintained by lymphocyte mitochondria, they were shown to possess an Na+/H+ antiporter.     

The Ca 2+ pumps and the Na + / Ca 2+ exchangers

The Ca 2+ ATPases or Ca 2+ pumps transport Ca 2+ ions out of the cytosol, by using the energy stored in ATP. The Na + / Ca 2+ exchanger uses the chemical energy of the Na + gradient (the Na + concentration is much higher outside than inside the cell) to remove Ca 2+ from the cytosol. Ca 2+ pumps are found in the plasma membrane and in the endoplasmic reticulum of the cells. The pumps are probably present in the membrane of other organelles, but little experimental information is available on this matter. The Na + / Ca 2+ exchangers are located on the plasma membrane. A Na + / Ca 2+ exchanger was found in the mitochondria, but very little is known on its structure and sequence. These transporters control the Ca 2+ concentration in the cytosol and are vital to prevent Ca 2+ overload of the cells. Their activity is controlled by different mechanisms, that are still under investigation. A number of the possible isoforms for both types of proteins has been detected.© Kluwer Academic Publishers     

Effect of Quin-2 on 45Ca2+ uptake mediated by Na+i/Ca2+o exchange and 45Ca2+ efflux in rat brain synaptosomes: a requirement for [Ca2+]i

The Na+/Ca2+ exchanger of squid axons, barnacle muscle and sarcolemma requires micromolar intracellular calcium for activation in the Na+i/Ca2+o exchange mode ('reverse' Na+/Ca2+ exchange). The requirement for [Ca2+]i has been demonstrated with the use of intracellular calcium buffers, such as Quin-2, to inhibit Na+i/Ca2+o exchange. However, the inhibition of Na+i/Ca2+o exchange in mammalian nerve terminals loaded with Quin-2 has not been observed [7], suggesting a lower sensitivity to low [Ca2+]i for this system. In contrast, the results reported herein indicate that 45Ca2+ uptake in synaptosomes through Na+i/Ca2+o exchange is inhibited by Quin-2 much in the same way as it is in the squid, provided that synaptosomes are preincubated in low Ca2+ medium to avoid saturation of Quin-2. Under these conditions, 45Ca2+ efflux via Ca2+i/Ca2+o exchange is also inhibited. Our results indicate that the Na+i/Ca2+o and Ca2+i/Ca2+o modes of the Na+/Ca2+ exchanger from rat brain synaptosomes require intracellular calcium for activation. However, because no clear relationship between the observed [Ca2+]i values and the inhibition of Na+i/Ca2+o exchange has been found, it is suggested that localised submembrane calcium concentrations not detected by the [Ca2+]i probe might regulate the exchanger.     

Ca2+-release pathways from mitochondria of the yeast Endomyces magnusii

Ca2+-release pathways from Ca2+-preloaded mitochondria of the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca2+ was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Intensive aeration of the mitochondrial suspension rapidly inhibited the efflux of Ca2+ and induced its reuptake. The Ca2+ release was not affected by cyclosporin A, an inhibitor of the nonselective permeability transition of mammalian mitochondria. With acetate as the permeant anion, a spontaneous net Ca2+ efflux began after uptake of about 75% of the added cation. The rate of this efflux was insensitive to cyclosporin A, aeration, and Na+ and was proportional to the Ca2+ load. The Ca2+ release was inhibited by La3+, Mn2+, Mg2+, TPP+, and nigericin (in the presence of KCl) and activated by spermine and hypotonicity. We conclude that Ca2+ efflux from preloaded E. magnusii mitochondria is very similar to the Na+-independent specific pathway for Ca2+ release operative in mitochondria from nonexcitable mammalian tissues.     

Inhibitory analysis of the monovalent metals' cations interaction with the system of Na+-dependent Ca2+ efflux from the liver mitochondria

Kinetic analysis reveals the mainly competitive inhibition of Na+-dependent Ca2+ efflux from mitochondria by cations of monovalent metals. Potency of the inhibitory effect of metals' cations on Na+-dependent Ca2+ efflux from mitochondria matrix increases in such an order (I50, mM): Cs+ (137.11) < Rb+ (122.63) < Li+ (24.59) < Tl+ (0.541). The results of correlation analysis show that sodium ions translocation by mitochondrial exchanger and its inhibition by the cations of monovalent metals is determined by their affinity for the oxygen-containing ligands and are accompanied with the ions dehydration. Inhibition of the mitochondrial Na+/Ca2+ exchanger by monovalent metal cations is also accompanied with the inhibition of cooperative interactions of metal ions with the ionbinding centers during transport cycle, which can be one of the mechanisms of the inhibition of ions translocation by this ion-transporting system.     

Intracellular Ca2+ regulation in CD3 stimulated Jurkat T cells involves H+ fluxes.

Triggering the CD3/TCR complex of T lymphocytes induces a rapid rise in cytosolic free calcium followed by a slowly declining plateau. The level of this plateau depends on external pH, the more alkalinized media leading to higher values. Neither a pH-dependent binding of mAb, nor a perturbation of internal pH can account for this effect. In a sodium-free medium, or in the presence of dimethylamiloride Ca2+, elevation is accompanied by an acidification of the cells; both of them depend, to the same extent, on external calcium concentration. TPA inhibits CD3-, but not ionomycin-induced Ca2+ and H+ raises, indicating that it acts more probably on Ca2+ influx, rather than on its efflux. These results suggest that intracellular calcium could be regulated by a Ca2+/H+ ATPase which drives H+ in and Ca2+ out. In the presence of external Na+, H+ should return to the medium by the Na+/H+ exchanger.     

A mechanism of Ca2+ release from Ca2+ stores coupling to the Na+/Ca2+ exchanger in cultured smooth muscle cells.

We previously observed Ca2+ release from intracellular Ca2+ stores caused by reduction in extracellular Na+ concentration ([Na+]o). The purpose of this study was to determine whether lowering [Na+]o can elicit Ca2+ release from Ca2+ stores via the Na+/Ca2+ exchanger and to elucidate the mechanisms related to the Ca2+ release pathway in cultured longitudinal smooth muscle cells obtained from guinea pig ileum. Low [Na+]o-induced Ca2+ release was inhibited by antisense oligodeoxynucleotides for Na+/Ca2+ exchanger type 1 (anti-NCX). Application of anti-NCX to cells attenuated both the number of Ca2+ responding cells and the expression of the exchanger. Moreover, microinjection of heparin, a blocker of inositol 1,4,5-trisphosphate (IP3) receptors, into the cells inhibited low [Na+]o-induced Ca2+ release. These findings suggest that low [Na+]o-induced Ca2+ release occurs through an IP3-induced Ca2+ release mechanism due to changes in the Ca2+ flux regulated by the Na+/Ca2+ exchanger.     

Regulated release of Ca2+ from respiring mitochondria by Ca2+/2H+ antiport.

Simultaneous measurements of oxygen consumption and transmembrane transport of Ca2+, H+, and phosphate show that the efflux of Ca2+ from respiring tightly coupled rat liver mitochondria takes place by an electroneutral Ca2+/2H+ antiport process that is ruthenium red-insensitive and that is regulated by the oxidation-reduction state of the mitochondrial pyridine nucleotides. When mitochondrial pyridine nucleotides are kept in a reduced steady state, the efflux of Ca2+ is inhibited; when they are in an oxidized state, Ca2+ efflux is activated. These processes were demonstrated by allowing phosphate-depleted mitochondria respiring on succinate in the presence of rotenone to take up Ca2+ from the medium. Upon subsequent addition of ruthenium red to block Ca2+ transport via the electrophoretic influx pathway, and acetoacetate, to bring mitochondrial pyridine nucleotides into the oxidized state, Ca2+ efflux and H+ influx ensued. The observed H+ influx/Ca2+ efflux ratio was close to the value 2.0 predicted for the operation of an electrically neutral Ca2+/2H+ antiport process.     

Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A

Ca(2+) plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca(2+) is controlled primarily by the mitochondrial Ca(2+) uniporter and the mitochondrial Na(+)/Ca(2+) exchanger, influencing NADH production through Ca(2+)-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca(2+)-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca(2+) release. Here we selectively measure Ca(2+) influx rate through the mitochondrial Ca(2+) uniporter and Ca(2+) 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(+)/Ca(2+) exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ(m) loss, Ca(2+) release, NADH oxidation, swelling) of high extramitochondrial Ca(2+) additions, allowing mitochondria to tolerate total mitochondrial Ca(2+) loads of >400nmol/mg protein. For Ca(2+) pulses up to 15μM, Na(+)-independent Ca(2+) efflux through the permeability transition pore accounted for ~5% of the total Ca(2+) efflux rate compared to that mediated by the mitochondrial Na(+)/Ca(2+) exchanger (in 5mM Na(+)). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na(+)/Ca(2+) exchanger-mediated Ca(2+) efflux at higher concentrations (IC(50)=2μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~40% at 10μM cyclosporine A, while having no effect on the mitochondrial Ca(2+) uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca(2+) 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.     

Sustained Ca2+ transfer across mitochondria is Essential for mitochondrial Ca2+ buffering,sore-operated Ca2+ entry,and Ca2+ store refilling

Mitochondria have been found to sequester and release Ca2+ during cell stimulation with inositol 1,4,5-triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca2+ that sustain activity of capacitative Ca2+ entry (CCE). Procedures that prevent mitochondrial Ca2+ uptake inhibit local Ca2+ buffering and CCE, but it is not clear whether Ca2+ has to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca2+ efflux on the ability of mitochondria to buffer subplasmalemmal Ca2+, to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in endothelial cells. Upon the addition of histamine, the initial mitochondrial Ca2+ transient, monitored with ratio-metric-pericam-mitochondria, was largely independent of extracellular Ca2+. However, subsequent removal of extracellular Ca2+ produced a reversible decrease in [Ca2+]mito, indicating that Ca2+ was continuously taken up and released by mitochondria, although [Ca2+]mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Na+/Ca2+ exchanger with CGP 37157 increased [Ca2+]mito and abolished the ability of mitochondria to buffer subplasmalemmal Ca2+, resulting in an increased activity of BKCa channels and a decrease in CCE. Hence, CGP 37157 also reversibly inhibited ER refilling during cell stimulation. These effects of CGP 37157 were mimicked if mitochondrial Ca2+ uptake was prevented with oligomycin/antimycin A. Thus, during cell stimulation a continuous Ca2+ flux through mitochondria underlies the ability of mitochondria to generate subplasmalemmal microdomains of low Ca2+, to facilitate CCE, and to relay Ca2+ from the plasma membrane to the ER.     

Alterations in mitochondrial Ca2+ flux by the antibiotic X-537A (lasalocid-A).

A previous communication (Pereira da Silva, L., Bernardes, C.F. and Vercesi, A.E. (1984) Biochem. Biophys. Res. Commun. 124, 80-86) presented evidence that lasalocid-A, at concentrations far below those required to act as a Ca2+ ionophore, significantly inhibits Ca2+ efflux from liver mitochondria. In the present work we have studied the mechanism of this inhibition in liver and heart mitochondria. It was observed that lasalocid-A (25-250 nM), like nigericin, promotes the electroneutral exchange of K+ for H+ across the inner mitochondrial membrane and as a consequence can cause significant alterations in delta pH and delta psi. An indirect effect of these changes that might lead to inhibition of mitochondrial Ca2+ release was ruled out by experiments showing that the three observed patterns of lasalocid-A effect depend on the size of the mitochondrial Ca2+ load. At low Ca2+ loads (5-70 nmol Ca2+/mg protein), under experimental conditions in which Ca2+ release is supposed to be mediated by a Ca2+/2H+ antiporter, the kinetic data indicate that lasalocid-A inhibits the efflux of the cation by a competitive mechanism. The Ca2+/2Na+ antiporter, the dominant pathway for Ca2+ efflux from heart mitochondria, is not affected by lasalocid-A. At intermediate Ca2+ loads (70-110 nmol Ca2+/mg protein), lasalocid-A slightly stimulates Ca2+ release. This effect appears to be due to an increase in membrane permeability caused by the displacement of a pool of membrane bound Mg2+ possibly involved in the maintenance of membrane structure. Finally, at high Ca2+ loads (110-140 nmol Ca2+/mg protein) lasalocid-A enhances Ca2+ retention by liver mitochondria even in the presence of Ca2(+)-releasing agents such as phosphate and oxidants of the mitochondrial pyridine nucleotides. The maintenance of a high membrane potential under these conditions may indicate that lasalocid-A is a potent inhibitor of the Ca2(+)-induced membrane permeabilization. Nigericin, whose chemical structure resembles that of lasalocid-A, caused similar results.     

Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors

The plasma membrane Na+/Ca2+ exchanger (NCX) is almost certainly the major Ca2+ extrusion mechanism in cardiac myocytes. Binding of Na+ and Ca2+ ions to its large cytosolic loop regulates ion transport of the exchanger. We determined the solution structures of two Ca2+ binding domains (CBD1 and CBD2) that, together with an alpha-catenin-like domain (CLD), form the regulatory exchanger loop. CBD1 and CBD2 are very similar in the Ca2+ bound state and describe the Calx-beta motif. Strikingly, in the absence of Ca2+, the upper half of CBD1 unfolds while CBD2 maintains its structural integrity. Together with a 7-fold higher affinity for Ca2+, this suggests that CBD1 is the primary Ca2+ sensor. Specific point mutations in either domain largely allow the interchange of their functionality and uncover the mechanism underlying Ca2+ sensing in NCX.     

Characterisation of the serotonin efflux induced by cytosolic Ca2+ and Na+ concentration increase in human platelets.

BACKGROUND/AIM: The present study aimed at elucidating the mechanism(s) of serotonin (5-HT) efflux induced by thapsigargin from human platelets in the absence of extra-cellular Ca2+. METHODS: Efflux of pre-loaded radiolabeled serotonin was generally determined by filtration techniques. Cytosolic concentrations of Ca2+, Na+ and H+ were measured with appropriate fluorescent probes. RESULTS: 5-HT efflux from control or reserpine-treated platelets--where reserpine prevents 5-HT transport into the dense granules--was proportional to thapsigargin evoked cytosolic [Ca2+]c increase. Accordingly factors as prostacyclin, aspirin and calyculin which reduced [Ca2+]c-increase also inhibited the 5-HT efflux. Thapsigargin, which also caused a remarkable increase in cytosolic [Na+]c, promoted less 5-HT release, in parallel to lower [Na+]c and [Ca2+]c increase, when added to platelet suspensions containing low [Na+]. The Na+/H+ exchanger monensin increased the [Na+]c and induced 5-HT efflux without affecting the Ca2+ level. The 5-HT efflux induced by both [Ca2+] or [Na+]c increase did not depend on pH or membrane potential changes, whereas it decreased in the absence of extra-cellular K+, and increased in the absence of Cl- or Na+. CONCLUSION: Increases in [Ca2+]c and [Na+]c independently induce serotonin efflux through the outward directed plasma membrane serotonin transporter SERT. This event might be physiologically important at the level of capillaries or narrowed arteries where platelets are subjected to high shear stress which causes [Ca2+]c increase followed by 5-HT release which might exert vasodilatation.