Pathway for uncoupler-induced calcium efflux in rat liver mitochondria: inhibition by ruthenium red

The rate of uncoupler-induced Ca2+ efflux from rat liver mitochondria is increased by acetate and decreased by phosphate. This effect depends on a shift of the apparent Km, which is increased by phosphate and decreased by acetate, while the Vmax is not modified. The modification of the apparent Km by permeant anions presumably reflects changes in the concentration of matrix free Ca2+. A major part of uncoupler-induced Ca2+ efflux is sensitive to Ruthenium Red, the specific inhibitor of the Ca2+ uniporter , but an apparent insensitivity is observed when the H+ permeability is rate limiting in the process of Ca2+ efflux. The titer of uncoupler required for maximal stimulation of Ca2+ efflux increases with the Ca2+ load and may be 1-2 orders of magnitude higher than that required for maximal stimulation of respiration. On the other hand, when the uncoupler concentration is raised above 10(-6) M, the process of Ca2+ efflux becomes again Ruthenium Red insensitive. The Ruthenium Red inhibition of uncoupler-induced Ca2+ efflux is time dependent, in that the degree of inhibition exerted by low amounts of Ruthenium Red increases with the incubation time. Since the inhibition of the rate of Ca2+ influx undergoes a parallel relief, it is possible that Ruthenium Red moves from the cytosolic to the matrix side of the inner membrane. It is concluded that, in native mitochondria, uncoupler-induced Ca2+ efflux occurs via reversal of the uniport Ca2+ carrier, and not through activation of an independent pathway.     

Properties of a new calcium ion antagonist on cellular uptake and mitochondrial efflux of calcium ions.

Compound YS 035 [NN-bis-(3,4-dimethoxyphenethyl)-N-methylamine] is a new synthetic compound capable of inhibiting Ca2+ uptake by different cells. The inhibition of Ca2+ uptake by muscle cells isolated from chicken embryo is dose-dependent in the compound YS 035 concentration range 10-30 microM. The new compound also inhibits Ca2+ entry into rat brain synaptosomes and less effectively into baby-hamster kidney cells. Compound YS 035 partially inhibits the slow Ca2+ release induced by Ruthenium Red and the rapid Na+-dependent efflux from heart mitochondria. The inhibition of the Na+/Ca2+ exchange appears to be of a non-competitive type with an apparent Ki of 28 microM. The new Ca2+ antagonist totally inhibits the Ca2+ efflux from liver mitochondria induced by Ruthenium Red, but it does not affect the release induced by uncoupler, respiratory inhibitor or chelator, nor the mitochondrial ATP synthesis and membrane potential. The properties shown by the new compound indicate it to be a Ca2+ antagonist and a useful tool for studies on the mitochondrial Ca2+ transport.     

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.     

Parallel efflux of Ca2+ and Pi in energized rat liver mitochondria.

Addition of Ruthenium Red to energized rat liver mitochondria that have previously accumulated Ca2+ and phosphate from the external medium induces a parallel efflux of both these ions. Mersalyl or dithioerythritol, which decrease Ruthenium Red-insensitive Ca2+ efflux, also decrease phosphate efflux to the same extent. Conversely diazenedicarboxylic acid bis(NN-dimethylamide) (DDBA), which increases the Ruthenium Red-induced Ca2+ efflux concurrently increases phosphate release. Dithioerythritol and DDBA, reducing and oxidizing agents of thiol groups respectively, modify Ca2+ and Pi efflux without penetrating the mitochondrial inner membrane. Under all the adopted conditions the membrane potential is preserved. The release of resting respiration and the parallel efflux of Mg2+ and adenine nucleotides, events closely correlated to Ca2+ cycling, are equally prevented either by mersalyl, which inhibits phosphate transport, or dithioerythritol; DDBA has the opposite effect. These findings and the observation that suggest that Ca2+ and phosphate transport in energized liver mitochondria are closely related and dependent on the redox state of membrane-bound thiol groups.     

Modulation of Ca2+ efflux from heart mitochondria.

The efflux of Ca2+ from rat heart mitochondria has been examined by using Ruthenium Red to inhibit active uptake after predetermined loadings with Ca2+. The efflux is proportional to the internal Ca2+ load; it is increased by Na+ applied when the mitochondria are respiring and this effect is inhibited by oligomycin. The efflux of Ca2+ is diminished by ATP and by ADP, with the latter the more effective. Both active uptake and efflux of Ca2+ are slowed by bongkrekic acid; this action has a time lag. The lower efflux found with the nucleotides and with bongkrekic acid seems to correspond to the more condensed state seen in the electron microscope when these agents are applied [Stoner & Sirak (1973) J. Cell Biol. 56, 51-64, 65-73]. The results are discussed in relation to the less-permeable state being contingent upon nucleotide binding to the membrane.     

Oxaloacetate- and acetoacetate-induced calcium efflux from mitochondria occurs by reversal of the uptake pathway.

1. Addition of oxaloacetate or acetoacetate to isolated rat liver mitochondria results in an efflux of Ca2+. Concomitant with this efflux is an immediate oxidation of endogenous nicotinamide nucleotides, a fall in the mitochondrial membrane potential and an increase in the rate of respiration. The primary effect in this sequence may be either (a) physiologically important stimulation of a Ca2+-efflux carrier, followed by Ca2+ re-uptake, a fall in membrane potential and increased respiration, or (b) physiologically unimportant damage to mitochondrial integrity, followed by a fall in membrane potential, increased respiration and Ca2+ efflux. 2. Ruthenium Red and EGTA will restore the increased respiratory rate to one approximating to the control rate of respiration. However, addition of lanthanide, at a concentration which inhibits the uptake but not the normal efflux of Ca2+, inhibits the rate of Ca2+ efflux induced by oxaloacetate or acetoacetate. Therefore the observed efflux is occurring by a reversal of the uptake pathway (uniporter) and thus follows the fall in membrane potential. 3. From these results we conclude that the decrease in membrane potential and increase in the rate of respiration seen during oxaloacetate- or acetoacetate-induced Ca2+ efflux cannot be accounted for by rapid Ca2+ cycling, but are due to damage to mitochondrial integrity.     

The inhibition of mitochondrial calcium transport by lanthanides and Ruthenium Red

An EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-quench technique was developed for measuring initial rates of (45)Ca(2+) transport by rat liver mitochondria. This method was used in conjunction with studies of Ca(2+)-stimulated respiration to examine the mechanisms of inhibition of Ca(2+) transport by the lanthanides and Ruthenium Red. Ruthenium Red inhibits Ca(2+) transport non-competitively with K(i) 3x10(-8)m; there are 0.08nmol of carrier-specific binding sites/mg of protein. The inhibition by La(3+) is competitive (K(i)=2x10(-8)m); the concentration of lanthanide-sensitive sites is less than 0.001nmol/mg of protein. A further difference between their modes of action is that lanthanide inhibition diminishes with time whereas that by Ruthenium Red does not. Binding studies showed that both classes of inhibitor bind to a relatively large number of external sites (probably identical with the ;low-affinity' Ca(2+)-binding sites). La(3+) competes with Ruthenium Red for most of these sites, but a small fraction of the bound Ruthenium Red (less than 2nmol/mg of protein) is not displaced by La(3+). The results are discussed briefly in relation to possible models for a Ca(2+) carrier.     

Stimulation of mitochondrial calcium ion efflux by thiol-specific reagents and by thyroxine. The relationship to adenosine diphosphate retention and to mitochondrial permeability.

Respiring rat heart mitochondria were loaded with Ca2+ and then treated with Ruthenium Red. The factors affecting the subsequent Ca2+-efflux were studied. Addition of rotenone or antimycin led to a decline of efflux except at pH values above 7.2, provided the load was less than about 80 nmol per mg of protein. Oligomycin reversed the effect of the respiratory inhibitors. Independently of respiration, efflux was stimulated by the uncoupler trifluoromethyltetrachlorbenzimadazole, by mersalyl and by thyroid hormones. The stimulated efflux could be diminished by ADP, with Mg2+ as cofactor if efflux was rapid. With respiration in progress, efflux could be stimulated by N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoate). The effects of mersalyl and of thyroid hormones could be diminished with dithiothreitol. In the absence of stimulating agents, the Ca2+ efflux was proportional to the load up to some critical amount, this critical amount was decreased by the agents. Thyroxine and mersalyl caused not only loss of Ca2+, but also simultaneous, but not necessarily proportional, loss of internal adenine nucleotides. Both efflux rates were kept at a low value by bongkrekic acid added before the stimulating agent. It is concluded that Ca2+ efflux is a measure of a permeability controlled by the binding of ADP (an Mg2+) to the inner membrane, and that this in turn depends on the maintenance of certain thiol gropus in a reduced form by a reaction that uses NADH and ATP and the energy-linked transhydrogenase.     

Effect of Ca2+, peroxides, SH reagents, phosphate and aging on the permeability of mitochondrial membranes

The mechanism by which a number of agents such as hydroperoxides, inorganic phosphate, azodicarboxylic acid bis(dimethylamide) (diamide), 2-methyl-1,4-naphthoquinone (menadione) and aging, induce Ca2+ release from rat liver mitochondria has been analyzed by following Ca2+ fluxes in parallel with K+ fluxes, matrix swelling and triphenylmethylphosphonium fluxes (as an index of transmembrane potential). Addition of hydroperoxides causes a cycle of Ca2+ efflux and reuptake and an almost parallel cycle of delta psi depression. The hydroperoxide-induced delta psi depression is biphasic. The first phase is rapid and insensitive to ATP and is presumably due to activation of the transhydrogenase reaction during the metabolization of the hydroperoxides. The second phase is slow and markedly inhibited by ATP and presumably linked to the activation of a Ca2+-dependent reaction. The slow phase of delta psi depression is paralleled by matrix K+ release and mitochondrial swelling. Nupercaine and ATP reduce or abolish also K+ release and swelling. Inorganic phosphate, diamide, menadione or aging also cause a process of Ca2+ efflux which is paralleled by a slow delta psi depression, K+ release and swelling. All these processes are reduced or abolished by Nupercaine and ATP. The slow delta psi depression following addition of hydroperoxide and diamide is largely reversible at low Ca2+ concentration but tends to become irreversible at high Ca2+ concentration. The delta psi depression increases with the increase of hydroperoxide, diamide and menadione concentration, but is irreversible only in the latter case. Addition of ruthenium red before the hydroperoxides reduces the extent of the slow but not of the rapid phase of delta psi depression. Addition of ruthenium red after the hydroperoxides results in a slow increase of delta psi. Such an effect differs from the rapid increase of delta psi due to ruthenium-red-induced inhibition of Ca2+ cycling in A23187-supplemented mitochondria. Metabolization of hydroperoxides and diamide is accompanied by a cycle of reversible pyridine nucleotide oxidation. Above certain hydroperoxide and diamide concentrations the pyridine nucleotide oxidation becomes irreversible. Addition of menadione results always in an irreversible nucleotide oxidation. The kinetic correlation between Ca2+ efflux and delta psi decline suggests that hydroperoxides, diamide, menadione, inorganic phosphate and aging cause, in the presence of Ca2+, an increase of the permeability for protons of the inner mitochondrial membrane. This is followed by Ca2+ efflux through a pathway which is not the H+/Ca2+ exchange.     

Characterization of Ca2+ transport in rat renal brush-border membranes and its modulation by phosphatidic acid.

The Ca2+ transport process by isolated renal brush-border membranes was characterized and the influence of the acidic phospholipid phosphatidic acid (PtdA) on this transport process was assessed. Ca2+ uptake by brush-border membranes exhibited saturation kinetics. It was inhibitable by a variety of multivalent cations, as well as by Ca2+-entry inhibitors, including verapamil, Ruthenium Red and gentamicin. It was selective for Ca2+ compared with Mg2+. This process was also electrophoretic since generation of K+ and anion-diffusion potentials, negative inside the vesicle, increased Ca2+ uptake. Elevations in PtdA content of brush-border membranes by either exogenous addition or endogenous generation of PtdA by incubating brush-border membranes with MgATP2- elevated the rate of Ca2+ uptake. This ATP effect could not be attributed to (Ca2+ + Mg2+)-dependent ATPase or contaminating membrane fragments. PtdA also increased the magnitude and rate of Ca2+ efflux from brush-border membranes preloaded with Ca2+. These modulations in uptake and efflux were not observed with phosphatidylcholine or phosphatidylinositol. In summary, these results are consistent with the presence of an electrophoretic uniport system for Ca2+ in renal brush-border membranes, and demonstrate that PtdA uniquely among phospholipids tested appears to facilitate transmembrane flux of Ca2+ across this membrane preparation.     

In dialyzed squid axons Ca2+i activates Ca2+o-Na+i and Na+o-Na+i exchanges in the absence of Ca chelating agents

We used internally dialyzed squid axons to explore whether the reported activatory effect of Ca2+i on the partial reactions of the Na+-Ca2+ exchange (essential activator) is secondary to the presence of Ca2+ chelating agents in the internal medium. The effect of Ca2+i pulses on both the reverse (Ca2+o-dependent Na+ efflux) and Na+-Na+ exchange (Na+o-dependent Na+ efflux) modes of the Na+-Ca2+ exchange was studied in axons dialyzed without EGTA. For these experiments a substantial inhibition of the Ca2+ buffer capacity of the axoplasm was achieved by the use of Ruthenium red (10-20 microM), cyanide (1 mM) and vanadate (1 mM) in the dialysis solution. Our results indicate that the Ca2+i requirement of the reverse and Na+-Na+ exchange can not be explained by a direct inhibition of the Na+-Ca2+ exchanger by EGTA. In fact, both modes of operation of the exchanger can be activated by internal Ca2+ ions in the complete absence of Ca2+ chelating agents thus indicating that the 'catalytic' effect of Ca2+i on the Na+-Ca2+ exchanger is a real phenomenon.     

Ruthenium red-insensitive Ca2+ uptake and release by mitochondria

Calcium uptake by rat liver mitochondria driven by an artificial pH gradient is ruthenium red insensitive, electrically neutral, and inhibited by the local anesthetic, nupercaine. This pH-driven Ca2+ transport is also inhibited by NH3, Pi, and acetate. Direct measurements of Pi indicate it is not translocated with Ca2+ during pH-driven Ca2+ uptake. Calcium is therefore not transported by a Ca2+-Pi symport mechanism. Ruthenium red-insensitive Ca2+ efflux is similar in its inhibition by nupercaine and its kinetics, and is also electroneutral. This suggests that the Ca2+ uptake described here occurs via reversal of the principal pathway of mitochondrial Ca2+ release. From the available data, pH-driven Ca2+ uptake (and presumably Ca2+ efflux) is hypothesized to occur by Ca2+ symport with unidentified anions. Protons may move counter to Ca2+ or reversibly dissociate from cotransported anions, which therefore couples Ca2+ transport to the pH gradient.     

Bax affects intracellular Ca2+ stores and induces Ca2+ wave propagation

In the present study, we evaluated proapoptotic protein Bax on mitochondria and Ca2+ homeostasis in primary cultured astrocytes. We found that recombinant Bax (rBax, 10 and 100 ng/ml) induces a loss in mitochondrial membrane potential (Delta Psi m). This effect might be related to the inhibition of respiratory rates and a partial release of cytochrome c, which may change mitochondrial morphology. The loss of Delta Psi m and a selective permeabilization of mitochondrial membranes contribute to the release of Ca2+ from the mitochondria. This was inhibited by cyclosporin A (5 microM) and Ruthenium Red (1 microg/ml), indicating the involvement of mitochondrial Ca2+ transport mechanisms. Bax-induced mitochondrial Ca2+ release evokes Ca2+ waves and wave propagation between cells. Our results show that Bax induces mitochondrial alteration that affects Ca2+ homeostasis and signaling. These changes show that Ca2+ signals might be correlated with the proapoptotic activities of Bax.     

Stimulation of calcium-ion efflux from liver mitochondria by sodium ions and its response to ADP and energy state.

The Ruthenium Red-insensitive efflux of Ca2+ from previously loaded rat liver mitochondria was studied as a function of the added Na+ concentration and ADP present. Stimulation of Ca2+ efflux is sigmoidally dependent on the Na+ concentration; maximal stimulation of efflux was observed with 12--15 mM-NaCl. Na+-stimulated Ca2+ efflux from liver mitochondria is about one-tenth that from cardiac mitochondria. No synergistic effect of K+ on the Na+-stimulated efflux was found. The alkali-metal cations other than Na+ did not stimulate efflux and did not prevent stimulation by Na+. In the absence of Na+, Ca2+ efflux was diminished by added ADP, but the Na+-stimulated efflux was made correspondingly greater as ADP concentration was increased to 16 microM. The Na+-stimulated Ca2+ efflux was inhibited by 70% by oligomycin and was not observed in the presence of antimycin. It is suggested that failure to observe Na+-stimulation of Ca2+ efflux from liver mitochondria by some investigators is attributable to a high basal efflux existing before addition of the Na+ salt.     

The entrapment of the Ca2+ indicator arsenazo III in the matrix space of rat liver mitochondria by permeabilization and resealing. Na+-dependent and -independent effluxes of Ca2+ in arsenazo III-loaded mitochondria.

The permeabilization-resealing technique [Al-Nasser & Crompton, Biochem. J. (1986) 239, 19-29] has been applied to the entrapment of arsenazo III in the matrix compartment of rat liver mitochondria. The addition of 10 mM-arsenazo III to mitochondria permeabilized with Ca2+ partially restores the inner-membrane potential (delta psi) and leads to the recovery of 3.9 nmol of arsenazo III/mg of protein in the matrix when the mitochondria are washed three times. The recovery of entrapped arsenazo III is increased 2-fold by 4 mM-Mg2+, which also promotes repolarization. ATP with or without Mg2+ decreased arsenazo III recovery. Under all conditions, less arsenazo III than [14C]sucrose is entrapped, in particular in the presence of ATP. The amount of arsenazo III entrapped is proportional to the concentration of arsenazo III used as resealant, and is equally distributed between heavy and light mitochondria. Arsenazo III-loaded permeabilized and resealed (PR) mitochondria develop delta psi values of 141 +/- 3 mV. PR mitochondria retain arsenazo III and [14C]sucrose for more than 2 h at 0 degrees C. At 25 degrees C, and in the presence of Ruthenium Red, PR mitochondria lose arsenazo III and [14C]sucrose at equal rates, but Ca2+ efflux is more rapid; this indicates that Ca2+ is released by an Na+-independent carrier in addition to permeabilization. The Na+/Ca2+ carrier of PR mitochondria is partially (60%) inhibited by extramitochondrial free Ca2+ stabilized with Ca2+ buffers; maximal inhibition is attained with 2 microM free Ca2+. A similar inhibition occurs in normal mitochondria with 3.5 nmol of matrix Ca2+/mg of protein, but the inhibition is decreased by increased matrix Ca2+. The data suggest the presence of Ca2+ regulatory sites on the Na+/Ca2+ carrier that change the affinity for matrix free Ca2+.     

The effects of manganese and changes in internal calcium on Na-Ca exchange fluxes in the intact squid giant axon.

The effects of manganese chloride were studied on Na-Ca exchange fluxes from intact squid axons. Ca uptakes and Cao-dependent sodium efflux were inhibited half-maximally by 3-7 mM MnCl2. Mn inhibition appears less during Nao-Cai exchange (half-maximal inhibition; 30 mM) than that during Cao-Nai exchange, even when both fluxes were activated with 100 mM Na. The effects of changes in [Ca2+i], effected by Ca-EGTA injection or inhibition of mitochondrial Ca uptake by ruthenium red, were examined on the reverse (Cao-Nai) exchange mode. Ca-EGTA mixtures, designed to raise [Ca2+i] above 2 microM, inhibited Cao-Nai exchange fluxes. Ruthenium red inhibited mitochondrial Ca buffering to effect increases in Cai in the absence of Ca chelators; it activated Nao-Cai exchange fluxes but had little effect on Cao-Nai exchange despite similar reported Km for Cai. The results reflect the difficulty in demonstrating the stimulatory effect of [Ca2+i] on Cao-Nai exchange fluxes in intact axons.     

Direct evidence for a role of intramitochondrial Ca2+ in the regulation of oxidative phosphorylation in the stimulated rat heart. Studies using 31P n.m.r. and ruthenium red.

1. The concentrations of free ATP, phosphocreatine (PCr), Pi, H+ and ADP (calculated) were monitored in perfused rat hearts by 31P n.m.r. before and during positive inotropic stimulation. Data were accumulated in 20 s blocks. 2. Administration of 0.1 microM-(-)-isoprenaline resulted in no significant changes in ATP, transient decreases in PCr, and transient increases in ADP and Pi. However, the concentrations of all of these metabolites returned to pre-stimulated values within 1 min, whereas cardiac work and O2 uptake remained elevated. 3. In contrast, in hearts perfused continuously with Ruthenium Red (2.5 micrograms/ml), a potent inhibitor of mitochondrial Ca2+ uptake, administration of isoprenaline caused significant decreases in ATP, and also much larger and more prolonged changes in the concentrations of ADP, PCr and Pi. In this instance values did not fully return to pre-stimulated concentrations. Administration of Ruthenium Red alone to unstimulated hearts had minor effects. 4. It is proposed that, in the absence of Ruthenium Red, the transmission of changes in cytoplasmic Ca2+ across the mitochondrial inner membrane is able to maintain the phosphorylation potential of the heart during positive inotropic stimulation, through activation of the Ca2+-sensitive intramitochondrial dehydrogenases (pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases) leading to enhanced NADH production. 5. This mechanism is unavailable in the presence of Ruthenium Red, and oxidative phosphorylation must be stimulated primarily by a fall in phosphorylation potential, in accordance with the classical concept of respiratory control. However, the full oxidative response of the heart to stimulation may not be achievable under such circumstances.     

Identification of the Ca2+-release activity and ryanodine receptor in sarcoplasmic-reticulum membranes during cardiac myogenesis.

Ca2+-induced Ca2+ release and pH-induced Ca2+ release activities were identified in sarcoplasmic-reticulum (SR) vesicles isolated from adult- and fetal-sheep hearts. Ca2+-induced Ca2+ release and pH-induced Ca2+ release appear to proceed via the same channels, since both phenomena are similarly inhibited by Ruthenium Red. Ca2+ release from fetal SR vesicles is inhibited by higher concentrations of Ruthenium Red than is that from adult membranes. Both fetal and adult SR vesicles bind ryanodine. Fetal SR shows higher ryanodine-binding capacity than adult SR vesicles. Scatchard analysis of ryanodine binding revealed only one high-affinity binding site (Kd 6.7 nM) in fetal SR vesicles compared with two distinct binding sites (Kd 6.6 and 81.5 nM) in the adult SR vesicles. SR vesicles isolated from fetal and adult hearts were separated on discontinuous sucrose gradients into light (free) and heavy (junctional) SR vesicles. Heavy SR vesicles isolated from adult hearts exhibited most of the Ca2+ release activities. In contrast, Ca2+-induced Ca2+ release, pH-induced Ca2+ release and ryanodine receptors were detected in both light and heavy fetal SR. These results suggest that fetal SR may not be morphologically and functionally as well differentiated as that of adult cardiac muscle and that it may contain a greater number of Ca2+-release channels than that present in adult SR membranes.     

Ca2+ transport by intact synaptosomes: the voltage-dependent Ca2+ channel and a re-evaluation of the role of sodium/calcium exchange

The verapamil-sensitive Ca2+ channel in the synaptosomal plasma membrane is investigated. Verapamil is without effect on Ca2+ uptake or steady-state content in synaptosomes with a polarized plasma membrane, but completely inhibits the additional Ca2+ uptake following plasma-membrane depolarization by high [K+], by veratridine plus ouabain or by high concentrations of the permeant cation tetraphenylphosphonium. Verapamil-insensitive Ca2+ influx and steady-state content are identical in polarized and depolarized synaptosomes, even though the Na+ electrochemical potential is greatly decreased in the latter, indicating that Na+/Ca2+ exchange is not a significant mechanism for Ca2+ efflux under these conditions. A transient Na+-dependent Ca2+ efflux can only be observed on addition of Na+ to Na+-depleted depolarized synaptosomes. While 0.2 mM verapamil decreases the ate of 86Rb+ efflux and 22Na+ entry during depolarization induced by veratridine plus ouabain, the final steady-state Na+ accumulation is not inhibited. Ca2+ efflux from synaptosomes following mitochondrial depolarization does not occur by a verapamil-sensitive pathway.     

Abnormal membrane properties of the sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia: modes of action of halothane, caffeine, dantrolene, and two other drugs

The role of sarcoplasmic reticulum (SR) in malignant hyperthermia (MH) was studied using the heavy microsomal fraction prepared from semitendinosus muscles of both normal and genetically MH-susceptible pigs. In the presence of ATP, SR was loaded with 70 nmol Ca2+/mg SR protein. Under these conditions, MH-SR demonstrated Ca2+-induced Ca2+ release (Ca-ICaR) and halothane-induced Ca2+ release (halothane-ICaR; halothane concentrations as low as 10 microM). Normal SR did not demonstrate these release phenomena. Dantrolene inhibited the halothane-ICaR, but did not inhibit the Ca-ICaR. Ruthenium red and tetracaine inhibited both types of Ca2+ release. From the measurement of passive Ca2+ efflux, it was shown that dantrolene did not affect the Ca2+ permeability of the SR itself, but suppressed only the halothane-induced increment of the permeability. The membrane order parameter of the SR, as measured by the spin-probe EPR technique, indicated that halothane disordered the lipid bilayer of MH-SR to a greater extent than it did of normal SR. This halothane disordering effect on MH-SR was antagonized by dantrolene. Ruthenium red and tetracaine did not antagonize the halothane disordering effect. These results raise the possibility that halothane could disturb the structure of the lipoprotein complex in MH-SR in such a way that it could open the Ca2+-release channels. The Ca2+ thus released further opens the channel through the Ca-ICaR mechanism in a positive feedback fashion, thus triggering the MH syndrome. The efficacy of dantrolene in ameliorating the MH syndrome might be related to the inhibition of this halothane effect.