The effect of Ca2+ and acyl coenzyme A:lysophospholipid acyltransferase inhibitors on permeability properties of the liver mitochondrial inner membrane

We have reported previously that a number of metabolites and toxins which cause Ca2+ release from mitochondria do so by increasing the permeability of the inner membrane. The metabolic basis of this permeability change is proposed to be perturbation of a phospholipid deacylation-reacylation cycle which results in an accumulation of free fatty acids and lysophospholipids (see Broekemeier, K. M., Schmid, P. C., Schmid, H. H. O., and Pfeiffer, D. R. (1985) J. Biol. Chem. 260, 105-113 and references therein). This hypothesis predicts that inhibitors of acyl-CoA:lysophospholipid acyltransferase would be among those agents which increase membrane permeability and that their effects on permeability could occur in the absence of pyridine nucleotide oxidation or of an accumulation of glutathione disulfide. The hypolipidemic drugs WY-14643 and clofibric acid inhibit the mitochondrial acyl-CoA:lysophospholipid acyltransferase and have the predicted effects on mitochondrial permeability properties. The development of increased permeability due to WY-14643 and clofibric acid requires accumulated Ca2+ specifically, is sensitive to inhibitors of phospholipase A2, and results in a pattern of solute release and swelling which is typical of other Ca2+-releasing agents. Neither agent promotes pyridine nucleotide nor sulfhydryl glutathione oxidation in the absence of Ca2+. In addition, the swelling response to hypolipidemic drugs is not significantly inhibited by dithiothreitol. In the presence of Ca2+, both agents promote an accumulation of free fatty acids. The composition of these lipid degradation products suggests that mitochondria treated with hypolipidemic drugs retain an active lysophospholipase whereas this enzyme is inactivated by Ca2+-releasing agents which alter mitochondrial sulfhydryl groups.     

Continuous fluorometric assay of Ca2+ transport by liposomes with Quin 2 entrapped: effect of phospholipase A2 and unsaturated long-chain fatty acids

The amount of free calcium in the cytoplasm is important in stimulation coupled with a number of cellular functions. The putative ionophoretic action of membrane lipid metabolites on Ca2+ offers convenient explanation of the stimulation-coupled mobilization of cytoplasmic Ca2+. To analyze the ionophoretic action of the lipid metabolites, we devised a sensitive method to study Ca2+ transport that uses liposome-entrapped Quin 2. A calcium ionophore, A23187, increased the fluorescence intensity of the Ca2+-Quin 2 complex as a function of Ca2+ transport into liposomes. A similar Ca2+ flux into the liposomes was induced by phospholipase A2 (PLA2) and by various long-chain fatty acids in liposomes that consist of phospholipids containing unsaturated fatty acids. The potencies of the fatty acids for Ca2+ transport is inversely correlated with their melting points. The oxidized products of the unsaturated fatty acids increased the Ca2+ and nonspecific permeability of the biological membranes. These results suggest that stimulation-coupled PLA2 activation might mediates the mobilization of cytoplasmic Ca2+.     

Effect of lysophospholipids, arachidonic acid and other fatty acids on regulation of Ca2+ transport in permeabilized pancreatic islets.

The immediate reaction products of PLA2-mediated hydrolysis of phospholipids were tested for their ability to induce Ca2+ mobilization from internal stores in permeabilized ob/ob mouse pancreatic islets. Lysophospholipids and unsaturated fatty acids increased the free Ca2+ concentration in the incubation medium of permeabilized ob/ob mouse pancreatic islets. The potency of the lysophospholipids decreased in the following order: lysophosphatidylcholine = lysophosphatidylglycerol much greater than lysophosphatidylinositol greater than lysophosphatidylserine much greater than lysophosphatidylethanolamine. Arachidonic acid and palmitoleic acid had a potency comparable to lysophosphatidylinositol, while palmitic acid was ineffective. The Ca(2+)-mobilizing effect of inositol-1,4,5-trisphosphate (IP3) in permeabilized islet cells was additive to the lysophospholipid effect, indicating different sites of action. Both Ca(2+)-mobilizing effects were counteracted by the polyamine spermine, while the presence of Mg2+ shifted the Ca2+ concentrations to higher levels. Since not only an activation of a phospholipase C but also an activation of a phospholipase A2 with subsequent generation of lysophospholipids and free fatty acids is reported to occur in glucose-induced insulin secretion, the interaction of the phospholipase C reaction product IP3 with a lysophospholipid or an unsaturated fatty acid may affect the extent and duration of the rise in the free cytoplasmic Ca2+ concentration responsible for initiation of insulin secretion.     

Fluorimetric study of phospholipase A-induced efflux of Ca2+ from liposomes]

Artificial vesicles, liposomes, were prepared from the total fraction of phospholipids of rat liver mitochondria. Electron microscopy showed that the structure of liposomes depended on cation composition of the medium in which they were formed. Fluorescence of chlorotetracycline increased in the suspension of liposomes loaded with Ca+2 due to the formation of Ca+2-chlorotetracycline-phospholipid membrane complex. Incubation of liposome suspension with phospholipase A in the presence of EDTA resulted in a decrease of chlorotetracycline fluorescence indicating a break of the integrity of liposome membranes and Ca+2 efflux.     

The role of Ca2+ ions in a phage infection of Corynebacterium glutamicum

It was shown that neither uncouplers of oxidative phosphorylation, nor lack of Ca2+ ions affected the normal MC-2 phage absorption on Corynebacterium glutamicum cells, while the phage development was repressed under these conditions. Simultaneous measurement of Ca2+, K+ and H+ ion flows, as well as measurement of membrane potential showed that the addition of the phage into the experimental medium led to significant depolarization of the membrane from -160 mV to -100 mV due to the penetration of Ca2+ ions into the cells followed by K+ and H+ efflux. The (Ca2+) to (K+ + H+) ratio was shown to be 1 : 1. Phage DNA is supposed to be injected into the host cells as a positively charged (Ca2+-DNA) complex.     

Role of Ca2+ in phosphatidylinositol response and arachidonic acid release in formylated tripeptide- or Ca2+ ionophore A23187-stimulated guinea pig neutrophils

The role of Ca2+ in phospholipid metabolism and arachidonic acid release was studied in guinea pig neutrophils. The chemotactic peptide formylmethionyl-leucyl-phenyl-alanine (fMLP) activated [32P]Pi incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA) without any effects on the labeling of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). This activation was observed in Ca2+-free medium. Even in the neutrophils severely deprived of Ca2+ with EGTA and Ca2+ ionophore A23187, the stimulated labeling was not inhibited. When [3H]arachidonic acid-labeled neutrophils were stimulated by fMLP, a loss of [3H]arachidonic acid moiety in PI and the resultant increase in [3H]arachidonyl-diacylglycerol (DG), -PA, and free [3H]arachidonic acid was marked within 3 min. With further incubation, a loss of [3H]arachidonic acid in PC and PE became significant. These results suggest the activation of phospholipase C preceded the activation of phospholipase A2. In Ca2+-free medium, the decrease in [3H]arachidonyl-PI and the increase in [3H]arachidonyl-PA were only partially inhibited, although the release of [3H]arachidonic acid and a loss of [3H]arachidonyl-PC and -PE was completely blocked. These results show that PI-specific phospholipase C was not as sensitive to Ca2+ deprivation as arachidonic acid cleaving enzymes, phospholipase A2, and diacylglycerol lipase. Ca2+ ionophore A23187, which is known as an inducer of secretion, also stimulated [32P]Pi incorporation into PI and PA, although the incorporation into other phospholipids, such as PC and PE, was inhibited. This stimulated incorporation seemed to be caused by the activation of de novo synthesis of these lipids, because the incorporation of [3H]glycerol into PA and PI was also markedly stimulated by Ca2+ ionophore. But the chemotactic peptide did not increase the incorporation of [3H]glycerol into any glycerolipids including PI and PA. Thus, it is clear that fMLP mainly activates the pathway, PI leads to DG leads to PA, whereas Ca2+ ionophore activates the de novo synthesis of acidic phospholipids. When [3H]arachidonic acid-labeled neutrophils were treated with Ca2+ ionophore, the enhanced release of arachidonic acid and the accumulation of [3H]arachidonyl-DG, -PA with a concomitant decrease in [3H]arachidonyl-PC, -PE, and -PI were observed. Furthermore, the Ca2+ ionophore stimulated the formation of lysophospholipids, such as LPC, LPE, LPI, and LPA nonspecifically. These data suggest that Ca2+ ionophore releases arachidonic acid, unlike fMLP, directly from PC, PE, and PI, mainly by phospholipase A2. When neutrophils were stimulated by fMLP, the formation of LPC and LPE was observed by incubation for more than 3 min. Because a loss of arachidonic acid from PI occurred rapidly in response to fMLP, it seems likely the activation of PI-specific phospholipase C occurred first and was followed by the activation of phospholipase A2 when neutrophils are activated by fMLP...     

Lysophosphatidylcholine induces arachidonic acid release and calcium overload in cardiac myoblastic H9c2 cells.

Lysophosphatidylcholine (lyso-PC) and arachidonate are products of phosphatidylcholine hydrolysis by phospholipase A(2). In this study, the modulation of arachidonate release by exogenous lyso-PC in rat heart myoblastic H9c2 cells was examined. Incubation of H9c2 cells with lyso-PC resulted in an enhanced release of arachidonate in both a time- and dose-dependent fashion. Lyso-PC species containing palmitoyl (C(16:0)) or stearoyl (C(18:0)) groups evoked the highest amount of arachidonate release, while other lysophospholipid species were relatively ineffective. Cells treated with phospholipase A(2) inhibitors resulted in the attenuation of the enhanced arachidonate release in the presence of lyso-PC. Lyso-PC caused the translocation of phospholipase A(2) from the cytosol to the membrane fraction and induced an increase in Ca2+ flux from the medium into the cells. Nimodipine, a specific Ca(2+)-channel blocker, partially attenuated the lyso-PC-induced rise in intracellular Ca2+. Concurrent with Ca2+ influx, lyso-PC caused an enhancement of protein kinase C activity. The lyso-PC-induced arachidonate release was attenuated when cells were pre-incubated with specific protein kinase C and mitogen activated protein kinase kinase inhibitors. Taken together, these results strongly indicate that the lyso-PC-induced increases in levels of intracellular calcium and stimulation of protein kinase C lead to the activation of cytosolic phospholipase A(2) which results in the enhancement of arachidonate release in H9c2 cells.     

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.     

外源Ca2+对模拟微重力环境中鱼腥藻细胞质膜透性和光合特性的影响

以鱼腥藻为材料,研究了外源Ca^2 对模拟微重力环境中微藻细胞膜透性的影响。实验结果表明：提高培养基中的Ca^2 浓度可减轻由模拟微重力造成的膜透性增大,有助于稳定细胞膜结构和功能。同时,外源Ca^2 降低了藻细胞光系统Ⅱ（PSⅡ）的光化学效率（以荧光参数Fv/Fm 表示）下降的由度,表明外源Ca^2 对模拟微生重力环境下鱼腥藻细胞光合作用的损伤,有良好的防护效应。     

Role of Ca2+ and cyclic nucleotides in the control of prostaglandin E production in the rat anterior pituitary gland.

In an attempt to relate changes in the intracellular concentration of prostaglandin E to the secretion process, two agents known to increase cyclic nucleotide concentrations and hormone release were added to dispersed rat anterior pituitary cells. They caused increases in teh intracellular prostaglandin E concentrations. Increasing the K+ concentration in the medium (which stimulates hormone release) caused a rapid rise in prostaglandin E concentrations. The addition of the Ca2'onophore A23187 had a similar effect. The effects of changes in the K+ and Ca2+concentrations and the addition of EDTA were measured on the redistribution of radioactivity in pituitary glands prelabelled with [3H]arachidonic acid. Elevated K+ concentrations stimulated the transfer of label to prostaglandins and free arachidonic acid, suggesting an increased phospholipase A activity. On the other hand, the absence of extracellular CaCl2 and the addition of EDTA had the opposite effect, which could be cancelled by the addition of sufficient amounts. of CaCl2. It is concluded that the addition of agents that increase membrane permeability to bivalent cations probably results in an influx of Ca2+ and this appears to result in increased phospholipase A activity, which in turn leads to an increase in prostaglandin production.     

The role of glutathione in the retention of Ca2+ by liver mitochondria

Concentrations of rhein and nitrofurantoin in the micromolar range induce Ca2+ release and the development of increased inner membrane permeability in liver mitochondria. Both compounds inhibit the mitochondrial glutathione reductase causing a depletion of GSH and an accumulation of GSSG in energized mitochondria. Under these conditions, the compounds also alter the oxidation state of pyridine nucleotides, NADH becoming oxidized while NADPH remains reduced. Using rhein or nitrofurantoin, together with t-butyl-hydroperoxide and beta-hydroxybutyrate, it is possible to selectively alter the NAD/NADH, the NADP/NADPH, and the GSSG/GSH ratios and to determine the effect of these different states on the ability of Ca2+ to produce a permeable inner membrane. No correlation between pyridine nucleotide ratios and sensitivity to Ca2+ was observed. Mitochondria are stable to Ca2+ when the GSH content is high, but become permeable when Ca2+ is present and GSH is converted to GSSG. It is proposed that the GSSG/GSH ratio, by controlling the reduction state of critical sulfhydryl groups, regulates lysophospholipid acyltransferase activity and, therefore, the ability of mitochondria to remain impermeable upon activation of the intramitochondrial Ca2+ requiring phospholipase A2.     

Effect of the purified (Mg2+ + Ca2+)-activated ATPase of sarcoplasmic reticulum upon the passive Ca2+ permeability and ultrastructure of phospholipid vesicles.

The passive Ca2+ permeability of fragmented sarcoplasmic reticulum membranes is 10(4) to 10(61 times greater than that of liposomes prepared from natural or synthetic phospholipids. The contribution of membrane proteins to the Ca2+ permeability was studied by incorporating the purified [Ca2+ + Mg2+]-activated ATPase into bilayer membranes prepared from different phospholipids. The incorporation of the Ca2+ transport ATPase into the lipid phase increased its Ca2+ permeability to levels approaching that of sarcoplasmic reticulum membranes. The permeability change may arise from a reordering of the structure of the lipid phase in the environment of the protein or could represent a specific property of the protein itself. The calcium-binding protein of sarcoplasmic reticulum did not produce a similar effect. The increased rate of Ca2+ release from reconstituted ATPase vesicles is not a carrier-mediated process as indicated by the linear dependence of the Ca2+ efflux upon the gradient of Ca2+ concentration and by the absence of competition and countertransport between Ca2+ and other divalent metal ions. The increased Ca2+ permeability upon incorporation of the transport ATPase into the lipid phase is accompanied by similar increase in the permeability of the vesicles for sucrose, Na+, choline, and SO42- indicating that the transport ATPase does not act as a specific Ca2+ channel. Native sarcoplasmic reticulum membranes are asymmetric structures and the 75-A particles seen by freeze-etch electron microscopy are located primarily in the outer fracture face. In reconstituted ATPase vesicles the distribution of the particles between the two fracture faces is even, indicating that complete structural reconstitution was not achieved. The Ca2+ transport activity of reconstituted ATPase vesicles is also much less than that of fragmented sarcoplasmic reticulum. The density of the 40-A surface particles visible after negative staining of native or reconstituted vesicles is greater than that of the intramembranous particles and the relationship between these two structures remains to be established.     

Evidence for tight coupling of phospholipase activation and Ca2+ influx during acrosome reaction of golden hamster spermatozoa

1. Phospholipases have been proposed to play a key role in sperm acrosome reaction. To examine the activation mechanism of phospholipases and subsequently sperm fertilizing capacity. Ca2+ fluxes and phospholipid turnover (breakdown and synthesis) were investigated in golden hamster spermatozoa during acrosome reaction. 2. Upon exposure of the spermatozoa to 1.7 mM Ca2+, a net uptake by the cells occurred in two distinguishable phases. 3. Depletion of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at a time that an initial Ca2+ uptake was observed to reach almost steady-state, prevented the secondary Ca2+ uptake and acrosome reaction. 4. The time course of an initial Ca2+ uptake seemed to precede that of the acrosome reaction. 5. Incubation of the spermatozoa with Ca2+ in the presence of [3H]glycerol induced a rapid increase in labeling of phosphatidic acid, a key intermediate of phosphinositide turnover initiated by the action of phospholipase C, which appeared to parallel the time course of a first phase of Ca2+. 6. Phospholipase A2 activation, detected by lysophospholipid formation, slightly delayed the initial events of first Ca2+ uptake and phosphatidic acid production. 7. It is concluded that first Ca2+ entry into the cells, associated with phosphatidic acid production, activates a phospholipase A2, leading to the production of substances, like lysophospholipids and fatty acids, which may contribute to acrosome reaction.     

Stimulation of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by phospholipase D

Treatment of canine cardiac sarcolemmal vesicles with phospholipase D resulted in a large stimulation (up to 400%) of Na+-Ca2+ exchange activity. The phospholipase D treatment decreased the apparent Km (Ca2+) for the initial rate of Nai+-dependent Ca2+ uptake from 18.2 +/- 2.6 to 6.3 +/- 0.3 microM. The Vmax increased from 18.0 +/- 3.6 to 31.5 +/- 3.6 nmol of Ca2+/mg of protein/s. The effect was specific for Na+-Ca2+ exchange; other sarcolemmal transport enzymes ((Na+, K+)-ATPase; ATP-dependent Ca2+ transport) are inhibited by incubation with phospholipase D. Phospholipase D had little effect on the passive Ca2+ permeability of the sarcolemmal vesicles. After treatment with 0.4 unit/ml of phospholipase D (20 min, 37 degrees C), the sarcolemmal content of phosphatidic acid rose from 0.9 +/- 0.2 to 8.9 +/- 0.4%; simultaneously, Na+-Ca2+ exchange activity increased 327 +/- 87%. It is probable that the elevated phosphatidic acid level is responsible for the enhanced Na+-Ca2+ exchange activity. In a previous study (Philipson, K. D., Frank, J. S., and Nishimoto, A. Y. (1983) J. Biol. Chem. 258, 5905-5910), we hypothesized that negatively charged phospholipids were important in Na+-Ca2+ exchange, and the present results are consistent with this hypothesis. Stimulation of Na+-Ca2+ exchange by phosphatidic acid may be important in explaining the Ca2+ influx which accompanies the phosphatidylinositol turnover response which occurs in a wide variety of tissues.     

Evidence for proton countertransport by the sarcoplasmic reticulum Ca2(+)-ATPase during calcium transport in reconstituted proteoliposomes with low ionic permeability

To ascertain the coupling between Ca2+ and H+ fluxes during Ca2+ transport by the Ca2(+)-pumping ATPase of the sarcoplasmic reticulum, we used well characterized reconstituted proteoliposomes. The method for the functional reconstitution of the Ca2(+)-ATPase was an extension of our recently published procedure (Rigaud, J. L., Paternostre, M. T., and Bluzat, A. (1988) Biochemistry, 27, 2677-2688). The reconstituted vesicles which sustained high Ca2+ transport activities in the absence of Ca2+ precipitating anions exhibited low ionic passive permeability. Proton fluxes generated by external acid pulses have been monitored by using the fluorescence of the pH-sensitive probe pyranine trapped inside proteliposomes. When K+ was the only permeant ion, low proton-hydroxyl passive permeability was found (permeability coefficient congruent to 5 x 10(-5) cm s-1). In the presence of Cl-1 ions, a higher proton permeability was observed, presumably due to diffusion of HCl molecules. It was further demonstrated that systematic characterization of the passive permeability is essential for understanding and controlling the ATP-dependent Ca2+ accumulation in the reconstituted liposomes. The first line of evidence for Ca2(+)-H+ countertransport during operation of the Ca2(+)-ATPase came from Ca2+ uptake measurements. The ATP-dependent Ca2+ accumulation into proteoliposomes was shown to be critically dependent upon the ionic composition of the medium and the presence of ionophores. In K2SO4 medium a very low Ca2+ uptake was obtained which was only slightly affected by the presence of valinomycin. On the contrary, Ca2+ accumulation was increased 3-4-fold in the presence of the protonophore carbonyl-cyanide-p-trifluoromethoxy phenylhydrazone, indicating that a transmembrane pH gradient was built up during Ca2+ uptake that inhibited the transport activity of the pump. Accordingly, we found that Ca2+ loading capacity increased with internal buffer capacity. Finally in KCl medium, high Ca2+ accumulation was observed even in the absence of protonophore in agreement with a rapid dissipation of the pH gradient in the presence of chloride ions. Additional evidence that the Ca2+ pump of sarcoplasmic reticulum operated as a Ca2(+)-H+ countertransport was provided by measurements of ATP-dependent intraliposomal alkalinization using entrapped 8-hydroxyl-1,3,6-pyrene trisulfonate (pyranine) and accumulation of the weak acid acetate. In K2SO4 medium, transmembrane pH gradients of about 1 pH unit were generated with kinetics parallel to those of the Ca2+ uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chloride-dependent sarcoplasmic reticulum Ca2+ release correlates with increased Ca2+ activation of ryanodine receptors.

The mechanism by which chloride increases sarcoplasmic reticulum (SR) Ca2+ permeability was investigated. In the presence of 3 microM Ca2+, Ca2+ release from 45Ca(2+)-loaded SR vesicles prepared from procine skeletal muscle was increased approximately 4-fold when the media contained 150 mM chloride versus 150 mM propionate, whereas in the presence of 30 nM Ca2+, Ca2+ release was similar in the chloride- and the propionate-containing media. Ca(2+)-activated [3H]ryanodine binding to skeletal muscle SR was also increased (2- to 10-fold) in media in which propionate or other organic anions were replaced with chloride; however, chloride had little or no effect on cardiac muscle SR 45Ca2+ release or [3H]ryanodine binding. Ca(2+)-activated [3H]ryanodine binding was increased approximately 4.5-fold after reconstitution of skeletal muscle RYR protein into liposomes, and [3H]ryanodine binding to reconstituted RYR protein was similar in chloride- and propionate-containing media, suggesting that the sensitivity of the RYR protein to changes in the anionic composition of the media may be diminished upon reconstitution. Together, our results demonstrate a close correlation between chloride-dependent increases in SR Ca2+ permeability and increased Ca2+ activation of skeletal muscle RYR channels. We postulate that media containing supraphysiological concentrations of chloride or other inorganic anions may enhance skeletal muscle RYR activity by favoring a conformational state of the channel that exhibits increased activation by Ca2+ in comparison to the Ca2+ activation exhibited by this channel in native membranes in the presence of physiological chloride (< or = 10 mM). Transitions to this putative Ca(2+)-activatable state may thus provide a mechanism for controlling the activation of RYR channels in skeletal muscle.     

EGTA inhibits reverse uniport-dependent Ca2+ release from uncoupled mitochondria. Possible regulation of the Ca2+ uniporter by a Ca2+ binding site on the cytoplasmic side of the inner membrane

When rat liver mitochondria are allowed to accumulate Ca2+, treated with ruthenium red to inhibit reverse activity of the Ca2+ uniporter, and then treated with an uncoupler, they release Ca2+ and endogenous Mg2+ and undergo large amplitude swelling with ultrastructural expansion of the matrix space. These effects are not produced by Ca2+ plus uncoupler alone. Like other "Ca2+-releasing agents" (i.e. N-ethylmaleimide, t-butylhydroperoxide, oxalacetate, etc.), the development of nonspecific permeability produced by ruthenium red plus uncoupler requires accumulated Ca2+ specifically and is antagonized by inhibitors of phospholipase A2. The permeability responses are also antagonized by ionophore A23187, indicating that a rapid pathway for Ca2+ efflux from deenergized mitochondria is necessary to prevent the development of nonspecific permeability. EGTA can be substituted for ruthenium red to produce the nonspecific permeability change in Ca2+-loaded, uncoupler-treated mitochondria. The permeability responses to EGTA plus uncoupler again require accumulated Ca2+ specifically and are antagonized by inhibitors of phospholipase A2 and by ionophore A23187. The equivalent effects of ruthenium red and EGTA on uncoupled, Ca2+-containing mitochondria indicate that reducing the extramitochondrial Ca2+ concentration to the subnanomolar range produces inhibition of reverse uniport activity. It is proposed that inhibition reflect regulation of the uniporter by a Ca2+ binding site which is available from the cytoplasmic side of the inner membrane. EDTA cannot substitute for EGTA to induce nonspecific permeability in Ca2+-loaded, uncoupled mitochondria. Furthermore, EDTA inhibits the response to EGTA with an I50 value of approximately 10 microM. These data suggest that the uniporter regulatory site also binds Mg2+. The data suggest further that Mg2+ binding to the regulatory site is necessary to inhibit reverse uniport activity, even when the site is not occupied by Ca2+.     

The modulation of rat brain Na(+)-Ca2+ exchange by K+

The involvement of potassium ions in the Na(+)-Ca2+ exchange process was studied in rat brain synaptic plasma membrane (SPM) vesicles. Addition of equimolar [K+] to the intravesicular and the extravesicular medium led to a stimulation of the Na+ gradient-dependent Ca2+ influx; this stimulation was noticeable already at 0.5 mM and reached its maximum at 2 mM K+. The magnitude of the K+ stimulation was between 1.3-2.5-fold in different SPM preparations. K+ ions also stimulated the Na(+)-dependent Ca2+ efflux. K+ stimulation of Na(+)-Ca2+ exchange is of considerable specificity, since it is not mimicked by either Li+ or H+. The following lines of evidence suggest that K+ modulation of Na(+)-Ca2+ exchange involves the catalytic moiety of the transporter itself and not an unrelated K+ channel which modulates the membrane potential. 1) K+ stimulation of the transport process was conserved following reconstitution of the transporter into phospholipid-rich liposomes, an experimental condition which presumably separates the native membrane proteins among different vesicular structures. 2) K+ stimulation of Na+ gradient-dependent Ca2+ influx persists also when the build up of negative inside membrane potential is prevented by addition of carbonyl cyanide p-trifluoromethoxy phenylhydrazone which renders the membrane highly permeable to protons both in the native and the reconstituted preparation. 3) K+ stimulation of Na+ gradient-dependent Ca2+ influx is obtained also when tetraethylammonium chloride, 2,3-diaminopyridine and Cs+ are added to the Ca2+ uptake medium. Reconstituted SPM vesicles take up 86Rb+ in response to activation of Na+ gradient-dependent Ca2+ influx. The ratio of Ca2+ taken up by SPM vesicles in a Na+ gradient-dependent manner to the corresponding amounts of Rb+ taken up varies between 8-5 in different SPM preparations. If the stoichiometry of the process is 1 Rb+/1 Ca2+, then Rb+ cotransport is mediated by 10-20% of the transporters present in the preparation.     

Exogenous phospholipase enzymes mimic effects of phenylephrine on Ca2+ transport in hepatocytes

Phospholipase C from Clostridium perfringens induced the release of 45Ca2+ from isolated rat hepatocytes incubated at 0.1 mM extracellular Ca2+ with a time course similar to that for the action of phenylephrine. Under the conditions of these experiments, no significant damage to the plasma membrane was detected in the presence of phospholipase C. Little 45Ca2+ release was induced by bee venom phospholipase A2. At 1.3 mM extracellular Ca2+, both phospholipase enzymes stimulated the initial rate of 45Ca2+ exchange. Concentrations of phospholipase C comparable with those that stimulated 45Ca2+ release increased the rates of glucose release and O2 utilization by 70 and 20% respectively. An increase in the rate of O2 utilization but not glucose release was observed after the addition of phospholipase A2 to hepatocytes. The possible role for a cellular phospholipase C in the mechanism by which phenylephrine stimulates glycogenolysis in the liver cell is briefly discussed.     

Evidence that Na+/H+ exchange regulates receptor-mediated phospholipase A2 activation in human platelets

Data in the previous paper suggest that epinephrine can mobilize a small pool of arachidonic acid via an enzymatic pathway distinct from phospholipase C and that this pathway is blocked by perturbations that block Na+/H+ exchange. The present studies demonstrate that epinephrine and ADP stimulate a phosphatidylinositol-hydrolyzing phospholipase A2 activity in human platelets. This occurs even when measurable phospholipase C activation, platelet secretion, and secondary aggregation are blocked with the thromboxane A2 receptor antagonist SQ29548. Furthermore, perturbants of Na+/H+ exchange diminish lysophosphatidylinositol production in response to epinephrine, ADP, and thrombin, but not to the Ca2+ ionophore A23187. Artificial alkalinization of the platelet interior with methylamine reverses the effect of the Na+/H+ antiporter inhibitor, ethylisopropylamiloride, on thrombin-stimulated lysolipid production, suggesting that the alkalinization of the platelet interior which would occur secondary to activation of Na+/H+ exchange might play an important role in phospholipase A2 activation. In addition, treatment of platelets with methylamine increases the sensitivity of phospholipase A2 to activation by the Ca2+ ionophore A23187, suggesting that changes in pH and Ca2+ may regulate phospholipase A2 activity synergistically. Finally, epinephrine causes a prompt decrease in platelet-chlortetracyclin fluorescence even in the presence of cyclooxygenase inhibitors, suggesting that epinephrine is able to mobilize membrane-bound Ca2+ independent of phospholipase C activation. Taken together, the data suggest that epinephrine-provoked stimulation of phospholipase A2 activity may occur as a result of Ca2+ mobilization and a concomitant intraplatelet alkalinization resulting from accelerated Na+/H+ exchange.