The stoichiometry of the exchange catalysed by the mitochondrial calcium/sodium antiporter.

Rat heart mitochondria respiring on succinate in the presence of Ruthenium Red (to inhibit uptake on the Ca2+ uniporter) released Ca2+ on the calcium/sodium antiporter until a steady state was reached. Addition of the ionophore A23187 (which catalyses Ca2+/2H+ exchange) did not perturb this steady state. Thermodynamic analysis showed that if a Ca2+/nNa+ exchange with any value of n other than 2 was at equilibrium, addition of A23187 would cause an obvious change in extramitochondrial free [Ca2+]. Therefore the endogenous calcium/sodium antiporter of mitochondria catalyses electroneutral Ca2+/2Na+ exchange.     

Acetylcholine Release from Isolated Synaptic Vesicles Related to Ionic Permeability Changes: Continuous Detection with a Chemiluminescent Method

The effect of ionic permeability changes on acetylcholine (ACh) release from isolated cholinergic synaptic vesicles of Torpedo was studied using a chemiluminescent method for continuous ACh detection. Vesicles rendered freely permeable to potassium by valinomycin lost most of their ACh content in K+ media, if the accompanying anion was permeant; it thus appeared that ACh leakage occurred as the result of internal osmotic changes. Upon addition of ionophores that catalyse monovalent cation/H+ exchange (gramicidin D or a mixture of valinomycin plus protonophore FCCP), a rapid but transient ACh release was observed. Surprisingly, nigericin which also catalyses K+/H+ exchange, had no effect on ACh release. The divalent cation ionophore A23187 promoted ACh release only when calcium (and not magnesium) was introduced into the external medium in a millimolar concentration range. As the simultaneous addition of the protonophore FCCP and A23187 decreased this calcium-dependent ACh leakage, a releasing effect of A23187 through Ca2+/H+ exchange is suspected. The present results emphasise the role of internal protons for ACh retention inside synaptic vesicles.     

Uncoupler-stimulated release of Ca2+ from Ehrlich ascites tumor cell mitochondria

Ruthenium red-insensitive, uncoupler-stimulated release of Ca2+ from Ehrlich ascites tumor cell mitochondria is much slower than from rat liver mitochondria under comparable conditions. In the presence of Pi and at moderate or high Ca2+ loads, ruthenium red-insensitive Ca2+ efflux elicited with uncoupler is approximately 20 times more rapid for rat liver than Ehrlich cell mitochondria. This is attributed to resistance of tumor mitochondria to damage by Ca2+ due to a high level of endogenous Mg2+ that also attenuates Ca2+ efflux. Calcium release from rat liver and tumor mitochondria is inhibited by exogenous Mg2+. This applies to ruthenium red-insensitive spontaneous Ca2+ efflux associated with Ca2+ uptake and uncoupling, and (b) ruthenium red-insensitive Ca2+ release stimulated by uncoupling agent. The endogenous Mg2+ level of Ehrlich tumor mitochondria is approximately three times that of rat liver mitochondria. Endogenous Ca2+ is also much greater (six fold) in Ehrlich tumor mitochondria compared to rat liver. Despite the quantitative difference in endogenous Mg2+, the properties of internal Mg2+ are much the same for rat liver and Ehrlich cell mitochondria. Ehrlich ascites tumor mitochondria exhibit slow, metabolically dependent Mg2+ release and rapid limited release of Mg2+ during Ca2+ uptake. Both have been observed with rat liver and other types of mitochondria. The proportions of apparently "bound" and "free" Mg2+ (inferred from release by the ionophore, A23187) do not differ significantly between tumor and liver mitochondria. Thus, the endogenous Mg2+ of tumor mitochondria has no unusual features but is simply elevated substantially. Ruthenium red-insensitive Ca2+ efflux, when expressed as a function of the intramitochondrial Ca2+/Mg2+ ratio, is quite similar for tumor and rat liver. It is proposed, therefore, that endogenous Mg2+ is a major regulatory factor responsible for differences in the sensitivity to damage by Ca2+ and Ca2+ release by Ehrlich ascites tumor mitochondria compared to mitochondria from normal tissues.     

The use of phospholipid-impregnated millipore filters for recording nonelectrogenic cation flows in the presence of Men+/nH+ exchangers

It has been shown that with a cation (K+, Na+, Ca2+) concentration gradient on a Millipore filter impregnated with a decane solution of phospholipid, in the presence of a Men+/nH+ exchanger (nigericin, monensin, A23187), addition of a protonophore induces the formation of an electric potential positively charged on the side where the concentration of the cation is lower. The formation of the potential is induced by the hydrogen ion concentration gradient in the filter and in the unstirred layers as a result of the Men+/nH+ exchange. In such a system, with a pH gradient on the filter in the presence of monensin and valinomycin, a potential is generated with the plus on the side of the lower concentration of hydrogen. The effect is the result of the formation of a potassium ion concentration gradient in the unstirred layers in the course of the K+/H+ exchange. It is concluded that phospholipid-impregnated filters can be used for search and identification of electroneutral membrane ionophores of the Men+/nH+ exchanger type.     

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.     

Cation transport and specificity of ionomycin. Comparison with ionophore A23187 in rat liver mitochondria

Based on the effects of ionomycin upon mitochondrial respiration, ionomycin was shown to be an effective ionophore for Ca2+ in rat liver mitochondria. The ionomycin-induced efflux of Ca2+ across the inner membrane was more sensitive to loading the mitochondria with Ca2+ than was efflux catalyzed by A23187. At saturating concentrations of Ca2+, the turnover number for ionomycin was 3- to 5-fold greater than that of A23187. Ionomycin catalyzed the efflux of mitochondrial Mg2+ at rates comparable to those observed with A23187. Ionomycin also mediated an efflux of K+ provided that the mitochondria were depleted of their endogenous divalent metal ions. The apparent turnover numbers for K+ efflux suggest that ionomycin is more specific for divalent metal ions than A23187.     

Nutritional effects on mitochondrial bioenergetics. Alterations in calcium uptake by rat liver mitochondria

The uptake of Ca2+ by energized liver mitochondria was compared in normal fed as well as in protein-energy malnourished rats. In the presence of phosphate, mitochondria obtained from both groups were able to accumulate Ca2+ from the suspending medium and eject H+ during oxidation of common substrates which activate different segments of the respiratory chain. The rate of Ca2+ uptake was significantly lower in mitochondria from protein-energy malnourished rats. The rates of oxygen consumption and H+ ejection were decreased by 20-30% during oxidation of substrates at the three coupling sites. Similarly, mitochondria from protein-energy malnourished rats exhibit a 34% decrease in the maximal rate of Ca2+ uptake and a 25% lower capacity for Ca2+ load. The stoichiometric relationship of Ca2+/2e- remained unaffected. In steady state, with succinate as a substrate in the presence of rotenone and N-ethylmaleimide, mitochondria from normal fed and protein-energy malnourished rats showed a similar rate of Ca2+ uptake. Furthermore in both groups the stoichiometry of the H+/O ratio was close to 8.0 (H+/site ratio close to 4.0), and of Ca2+/site was close to 2.0. The diminished rate of Ca2+ uptake observed in mitochondria from protein-energy malnourished rats could be explained on the basis of a depressed rate of electron transport in the respiratory chain rather than by an effect at the level of the Ca2+ or H+ transport mechanism per se.     

The effect of endogenous phosphate on the H+/Mn2+ ratio and the state of Mn2+ in the mitochondrial matrix.

1. Kinetics and stoichiometry of H+ extrusion and reuptake and of Mn2+ uptake and release have been measured in respiring liver mitochondria in the absence of external added Pi. H+ and Mn2+ fluxes are parallel during aerobic cation uptake but not during uncoupler induced cation release. The H+/Mn2+ is 1.24. Addition of SH reagents, in concentrations inhibiting the Pi carrier, modifies the kinetics of H+ extrusion and of Mn2+ uptake and release. The slow phase of uncoupler induced Mn2+ release is diminished. The H+/Mn2+ is increased to 1.72. Addition of SH reagents, after the phase of aerobic uptake is completed, results in a significant reduction of the extent of uncoupler-induced Mn2+ release. The extent of reuptake of endogenous Pi during aerobic uptake of Mn2+ is about 8 nmol x mg protein-1. 2. Aerobic uptake of Mn2+ in the absence of external Pi results in an electron spin resonance spectrum which is the sum of two components. One, denoted as S, corresponds to Mn(H2O)2+(6). Another denoted as E, reflects spin exchange narrowing. In contrast to previous claims the following evidence suggests that the spin exchange component is due to Mn3(PO4)2 precipitate: (a) the dimension of the spin exchange spectrum is markedly reduced by abolition of Pi transport; (b) the spin exchange spectrum is released very slowly by addition of uncouplers under conditions where uncouplers cause a rapid deenergization of mitochondria, reuptake of H+ and release of cations; (c) the free matrix Mn2+ is released slowly after addition of uncoupler if there is a large spin exchange signal; howeover the free matrix Mn2+ is abolished rapidly by uncoupler when formation of the spin exchange signal is prevented by pretreatment with Ca2+; (d) the band width of the spin exchange fraction is independent of the Mn2+/protein ratio either under kinetic or steady state conditions; (e) the experimental spectrum recalls closely that obtained by computer simulation by assuming it as a combination of Mn(H2O)2+(6) and Mn3(PO4)2. 3. It is concluded that endogenous Pi affects the process of aerobic divalent cation uptake. A part of Mn2+ uptake in the absence of externally added anions, consists of a Mn3(PO4)2 precipitate. This accounts for a H+/Mn2+ ratio lower than 2.     

The H+/site ratio of mitochondrial electron transport.

The number of H+ ejected during passage of 2e- through each energy-conserving site of the mitochondrial respiratory chain (the H+/site ratio) was measured in three ways. In each case transmembrane movements of endogenous phosphate were minimized. (1) Measurement of the uptake of weak acids during loading of mitochondria with Ca2+ demonstrated that 2.0 weak acid anions were accumulated per Ca2+ ion. Since 1.7 to 2.0 Ca2+ ions were were taken up per site, these data correspond to an H+/site ratio of 3.5 to 4.0. (2) More direct measurement of H+ ejection using the oxygen pulse technique demonstrated that the H+/site ratio was 3.0. In these experiments phosphate movements were prevented by addition of N-ethylmaleimide to inhibit phosphate-hydroxide antiport, by washing the mitochondria to remove endogenous phosphate, or by working at 5 degrees C to reduce the rate of phosphate transport. When phosphate movements were allowed, H+/site ratios of 2.0 were observed. (3) Measurement of the initial steady rates of oxygen consumption and H+ ejection following addition of substrate to aerobic, substrate-limited mitochondria yielded H+/site ratios of 2.0, which were elevated to 4.0 when phosphate transport was prevented as described above. Previous determinations of the H+/site ratio were thus underestimates due to the unrecognized movements of endogenous phosphate; our results show that the H+/site ratio is at least 3.0 andmay be as high as 4.0.     

The influence of ATP-dependent K(+)-channel diazoxide opener on the opening of mitochondrial permeability transition pore in rat liver mitochondria

The influence of mitochondrial ATP-dependent K(+)-channel (K+(ATP)-channel) opener, diazoxide (DZ) on the mitochondrial permeability transition pore (MPTP) opening in rat liver mitochondria is studied. In the absence of DZ the MPTP opening leads to the increase in the rate of K(+)- and Ca(2+)-cycling supported by the simultaneous functioning of K(+)-channels and K+/H(+)-antiporter, and also Ca(2+)-uniporter together with MPTP as the cations influx and efflux pathways. Independent of MPTP opening, the activation of both constitutes of K(+)-cycle, K(+)-uptake as well as K+/H(+)-exchange, by DZ is observed. It is shown that the activation of transmembrane exchange of K+, combined with MPTP opening, results in partial inhibition of the latter. A simple methodical approach for the estimation of DZ influence on the open state of mitochondrial pore is proposed. It is shown that MPTP closure followed by Ca2+ reentry to the matrix is accompanied by the K+/H(+)-exchange inhibition which takes place in the same timeframes as the increase in matrix Ca2+ content. Relevant to physiological conditions, an important physiological function of MPTP is revealed, that is the maintenance of relatively low matrix level of Ca2+ accompanied by the acceleration of transmembrane ion exchange (K+ and Ca2+) which could strongly influence the energy state and energy-dependent processes in mitochondria.     

K+/H+ antiport in heart mitochondria

Heart mitochondria depleted of endogenous divalent cations by treatment with A23187 and EDTA swell in (a) K+ acetate or (b) K+ nitrate when an uncoupler is present. These mitochondria also exchange matrix 42K+ with external K+, Na+, or Li+ in a reaction that does not require respiration and is insensitive to uncouplers. Untreated control mitochondria do not swell in either medium nor do they show the passive cation exchange. Both the swelling and the exchange reactions are inhibited by Mg2+ and by quinine and other lipophilic amines. Swelling and exchange are both strongly activated at alkaline pH, and the exchange reaction is also increased markedly by hypotonic conditions. All of these properties correspond to those reported for a respiration-dependent extrusion of K+ from Mg2+-depleted mitochondria, a reaction attributed to a latent Mg2+- and H+-sensitive K+/H+ antiport. The swelling reactions are strongly inhibited by dicyclohexylcarbodiimide reacted under hypotonic conditions, but the exchange reaction is not sensitive to this reagent. Heart mitochondria depleted of Mg2+ show marked increases in their permeability to H+, to anions, and possibly to cations, and the permeability to each of these components is further increased at alkaline pH. This generalized increase in membrane permeability makes it likely that K+/H+ antiport is not the only pathway available for K+ movement in these mitochondria. It is concluded that the swelling, 42K+ exchange, and K+ extrusion data are all consistent with the presence of the putative K+/H+ antiport but that definitive evidence for the participation of such a component in these reactions is still lacking.     

The regulation of extramitochondrial steady state free Ca2+ concentration by rat insulinoma mitochondria

For the study of Ca2+ handling by mitochondria of an insulin secretory tissue, a method for the isolation of functionally intact insulinoma mitochondria is described. The mitochondria had a respiratory control ratio of 6.3 +/- 0.3 with succinate as a substrate. The regulation of extramitochondrial [Ca2+]o concentration by suspensions of insulinoma mitochondria was studied using Ca2+-selective minielectrodes. The mitochondria were found to maintain an ambient free Ca2+ concentration of about 0.3 and 0.9 microM in the absence or presence of Mg2+ (1 mM), respectively. The addition of Na+ resulted in a dose-dependent (half-maximal 4 mM Na+) increase in steady state [Ca2+]o. Na+ accelerated the ruthenium red-induced Ca2+ efflux, suggesting the existence of a Ca2+/2Na+ antiporter, as described in mitochondria of excitable tissues. Experiments were performed to study the effects of various agents on the steady state extramitochondrial free Ca2+. cAMP, 3-isobutyl-1-methylxanthine, and NADH were found to have no effect, whereas phosphoenolpyruvate induced a net Ca2+ efflux, the kinetic of which suggests deleterious effects on mitochondrial functions. A small decrease in pH (0.1 unit) of the incubation buffer resulted in an increase of the extramitochondrial Ca2+ steady state that was reversible upon restoration of the pH to its initial value. In conclusion, insulinoma mitochondria were able to maintain an extramitochondrial [Ca2+]o steady state in the submicromolar range that was markedly influenced by the ionic composition of the incubation medium. Thus, mitochondria may play a role in the regulation of cellular calcium homeostasis and insulin release.     

Induction of 2H+/Me2+ exchange in rat-liver mitochondria

The time dependency of CA2+ efflux from Ca2+-loaded rat liver mitochondria has been investigated. The rate of ruthenium-red-insensitive Ca2+ efflux is continuously increased during the retention as a result of induction of an electroneutral H+ Ca2+ exchange system. The activation of the Ca2+ efflux pathway takes place under the constant value of the membrane potential and is accompanied by oxidation of mitochondrial pyridine nucleotides. It has also been found that the ruthenium-red-insensitive H+/Sr2+ exchange occurs in mitochondria during Sr2+-induced oscillation of ion fluxes. The rate of H+/Sr2+ exchange is variable and depends on the stage of the oscillatory cycle.     

Participation of endogenous fatty acids in Ca2+ release activation from mitochondria

A correlation between the rate of H+/Ca2+ exchange and the content of free fatty acids in mitochondria has been found. Fatty acids were isolated from mitochondria with different activities of H+/Ca2+ exchange. It has been shown that these free fatty acids are able to induce Ca2+ release in exchange to protons after being added to freshly isolated mitochondria.     

Transport and control of Ca2+ by pigeon erythrocytes. I. Survey of some cell responses to a range of A23187 doses in the presence of Ca2+

Cells were subjected to a range of 45Ca2+ influx loads with A23187. We measured cell 45Ca2+ with time and A23187 dose, and the apparent 45Ca2+ influxes (identical to "J(in,app)") at Ca2+ steady state. We also measured endogeneous exchangeable and total cell Ca2+, which were 50 and 17-220 microM respectively. The effects of A23187 and Ca2+ on cell ATP, swelling, net Cl- permeability, and cell morphology were measured. These were modest and do not affect our conclusions. J(in,app) congruent to 3 X 10(-4) [A23187]2.9 X [Ca2+(o)]mumoles/l X min with 92-552 microM [Ca2+(o)] (identical to external Ca2+ concentration) and 0-7 microM A23187. J(in,app) was increased an order of magnitude by vanadate and is probably much less than the true influx. The least unlikely explanation found for the high [A23187] exponent, 2.9, was that most of the Ca2+ crossing the membrane is expelled by the pump before it can move deeper into the cell. Calcium pumping increased rapidly in response to increased influx, but the steady state cell 45Ca2+ was approximately proportional to J(in,app) rather than approximately constant between 10 and 120 mumoles/l X min with 184 microM [Ca2+(o)]. This is not the result expected from a simple feedback mechanism. At high A23187 doses the pump appears fully activated resulting in a linear relation between cell/medium 45Ca2+ and [A23187]-2. From the plot we calculated alpha identical to free/total exchangeable Ca2+ = 0.38 +/- 0.08 (n = 3) and a maximum pump rate, "Pmax" = 78 mumole/l X min. Pmax is underestimated insofar as J(in,app) is less than the true influx.     

Induction of acrosomal reaction and calcium uptake in ram spermatozoa by ionophores

Ram spermatozoa incubated in the presence of Ca2+ and the Ca2+-ionophore A23187 undergo a process which is known as the acrosome reaction. This reaction is characterized by fusion of the outer acrosomal membrane and the overlying plasma membrane to form mixed vesicles which can be seen in the electron microscope. As a result, the trypsin-like acrosin is released from the cells to the medium. The occurrence of the acrosome reaction was determined by following acrosin activity in the medium. After 2 h of incubation of the cells in the presence of ionophore and Ca2+, the released acrosin activity is related to the ionophores according to the sequence: A23187 greater than monensin greater than valinomycin greater than FCCP = without ionophore. The study of Ca2+ uptake by the cells revealed that Ca2+ enters the cell prior to the release of acrosin. Monensin can induce Ca2+ uptake and acrosin release only when Na+ is present in the incubation medium. There is no increase in Ca2+ uptake with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). We suggest that the Na+/H+ exchange induced by monensin causes an increase in intracellular Na which is the driving force for the Ca2+ entry via a Ca2+/Na+ antiporter. Since monensin can induce an increase in Ca2+ uptake only in the presence of Na+, FCCP enhances Ca2+ uptake in the presence of valinomycin, and A23187 is a Ca2+/2H+ exchanger, we suggest that alkalization of the intracellular space is involved in the acrosome reaction. Calcium uptake in the presence of monensin is not affected by the uncoupler FCCP, a result which indicates that Ca2+ is not accumulated in the mitochondria. Incubation of cells for 3 h in the absence of Ca2+ or ionophore caused a 3-fold increase in the rate of acrosin release when monensin and Ca2+ were added together. There was no change in this rate when A23187 was used. We suggest that during the preincubation time (known as capacitation) the permeability of the plasma membrane to Ca2+ is enhanced. This study shows that acrosin release and Ca2+ uptake can be used as a quantitative asay for the determination of the acrosome reaction.     

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. II. (Ca2+ + Mg2+)-ATPase-dependent Ca2+ transport activity

Purified plasma membrane vesicles from GH3 rat anterior pituitary cells exhibit a Mg2+-ATP-dependent Ca2+ transport activity. Concentrative uptake of Ca2+ is abolished by exclusion of either Mg2+ or ATP or by inclusion of the Ca2+ ionophore A23187. Furthermore, addition of A23187 to vesicles which have reached a steady state of ATP-supported Ca2+ accumulation rapidly and completely discharges accumulated cation. Ca2+ uptake is unaffected by treatment of vesicles with oligomycin, the uncoupler CCCP, or valinomycin and is greatly reduced in non-plasma membrane fractions. Likewise, Ca2+ accumulation is not stimulated by oxalate, consistent with the plasma membrane origin of this transport system. (Na+, K+)-ATPase participation in the Ca2+ transport process (i.e. via coupled Na+/Ca2+ exchange) was eliminated by omitting Na+ and including ouabain in the reaction medium. Ca2+ transport activity in GH3 vesicles has a similar pH dependence as that seen in a number of other plasma membrane systems and is inhibited by orthovanadate in the micromolar range. Inhibition is enhanced if the membranes are preincubated with vanadate for a short time. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ and ATP are 0.7 and 125 microM, respectively. The average Vmax is 3.6 nmol of Ca2+/min/mg of protein at 37 degrees C. Addition of exogenous calmodulin or calmodulin antagonists had no significant effect on these kinetic properties. GH3 plasma membranes also contain a Na+/Ca2+ exchange system. The apparent Km for Ca2+ is almost 10-fold higher in this system than that for ATP-driven Ca2+ uptake. When both processes are compared under similar conditions, the Vmax of the exchanger is approximately 2-3 times that of ATP-dependent Ca2+ accumulation. Similar results are obtained when purified plasma membranes from bovine anterior pituitary glands were investigated. It is suggested that both Na+/Ca2+ exchange and the (Ca2+ + Mg2+)-ATPase are important in controlling intracellular levels of Ca2+ in anterior pituitary cells.     

Stimulation of beta-adrenoceptors inhibits calcium-dependent potassium-channels in mouse macrophages

K+ efflux in mouse macrophages exhibited a rate constant (kK) of 0.67 +/- 0.04 (h)-1 (mean +/- SEM of 16 experiments). This was strongly stimulated by increasing concentrations of the Ca2+ ionophore A23187 up to a maximal value of 4.01 +/- 0.25 (h)-1 with an IC50 of 7.6 +/- 1.9 microM (mean +/- SEM of 6 experiments). Similar results were obtained with the Ca2+ ionophore ionomycin. Binding experiments with 3H-dihydroalprenolol revealed a high density of beta-adrenergic receptors (97.5 +/- 5.2 fmol/mg protein) with apparent dissociation constant of 2.03 +/- 0.06 nM. Isoproterenol at a concentration of 10(-6)-10(-5) M induced a two- to threefold stimulation of endogenous levels of cyclic AMP (cAMP). A23187-stimulated K+ efflux was partially inhibited by stimulation of adenylate cyclase with isoproterenol, forskolin or, PGE1; exogenous cAMP; and inhibition of phosphodiesterase with MIX (1-methyl-3-isobutylxanthine). Maximal inhibition of K+ efflux was obtained by simultaneous addition of isoproterenol and MIX. In dose-response curves, the isoproterenol-sensitive K+ efflux was half-maximally inhibited (IC50) with 2-5 X 10(-10) M of isoproterenol concentration. Propranolol was able to completely block the effect of isoproterenol, with an IC50 of about 1-2 X 10(-7) M. Isoproterenol and MIX were also able to partially inhibit ionomycin-stimulated K+ efflux. Isoproterenol and MIX did not inhibit A23187-stimulated K+ efflux in an incubation medium where NaCl was replaced by sucrose (or choline), suggesting the involvement of an Na+:Ca2+ exchange mechanism. Our results show that stimulation of beta-adrenoceptors in mouse macrophages counterbalances the opening of K+ channels induced by the calcium ionophore A23187. This likely reflects a decrease in cytosolic free calcium content via a cAMP-mediated stimulation of Na+:Ca2+ exchange.     

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.     

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.