Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase.

The first step towards ATP synthesis by the Ca2-ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35 degrees C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10-fold, whereas it had no effect on the apparent affinity for Mg2+. In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations. Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+. The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.     

Ruthenium red inhibits mitochondrial Na+ and K+ uniports induced by magnesium removal.

Removal of bound magnesium from the outer surface of the inner mitochondrial membrane opens up a Na+ and Li+ selective electrophoretic uniport pathway whereas simultaneous depletion of intramitochondrial magnesium induces an electrogenic K+ flux as well. In order to clarify the nature of these cation movements we tested the effect of ruthenium red, a potent and specific inhibitor of the mitochondrial Ca2+ uniporter on different Na+ and K+ uniport-associated phenomena. Ruthenium red efficiently inhibited mitochondrial swelling and depolarization induced by either EDTA in a NaCl-based medium (Na+ uniport) or by EDTA plus A23187 in a KCl-based medium (K+ uniport). For both cation uniports half-maximal inhibition was attained at a ruthenium red concentration as low as 40 nM. Complete inhibition was found above 200 nM. Neither the Na+/H+ nor the K+/H+ exchange was affected by ruthenium red. In light of these observations the possibility is raised that the electrogenic Na+ and K+ fluxes provoked by magnesium reduction or depletion may be mediated through the Ca2+ uniporter. It is suggested that intactness of the mitochondrial magnesium pools is necessary for maintaining the Ca2+ selectivity of the Ca2+ uniporter, and alterations of the membrane-associated magnesium content would make this transport route available also for monovalent cations.     

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 respiration of brain mitochondria and its regulation by monovalent cation transport.

The Na+ and K+ permeability properties of rat brain mitochondria were determined to explain the influences of these cations upon respiration. A new procedure for isolating exceptionally intact mitochondria with minimal contamination by synaptosomes was developed for this purpose. Respiration was uncoupled by Na+ and less so by K+. Uncoupling was maximal in the presence of EDTA plus Pi and was decreased by Mg2+. Maximal uncoupler-stimulated respiration rates were inhibited by Na+ but largely unaffected by K+. The inhibition by Na+ was relatively insensitive to Mg2+. Membrane Na+ and K+ conductances as well as neutral exchanges (Na+/H+ and K+/H+ antiport activities) were determined by swelling measurements and correlated with metabolic effects of the cations. Cation conductance, i.e. electrophoretic Na+ or K+ permeation, was increased by EDTA (Na+ greater than K+) and decreased by Mg2+. Magnesium preferentially suppressed Na+ conductance so as to reverse the cation selectivity (K+ greater than Na+). Neutral cation/H+ exchange rates (Na+ greater than K+) were not influenced by chelator or Mg2+. The extent of cation-dependent uncoupling of respiration correlated best with the inner membrane conductance of the ion according to an empirical relationship derived with the model K+ conductor valinomycin. The metabolic influences of Na+ and K+ can be explained in terms of coupled flow of these ions with protons and their effect upon the H+ electrochemical gradient although alternative possibilities are discussed. These in vitro studies are compared to previous observations in situ to assess their physiological significance.     

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.     

Fast efflux of Ca2+ mediated by the sarcoplasmic reticulum Ca2(+)-ATPase.

The Ca2(+)-ATPase found in the light fraction of sarcoplasmic reticulum vesicles can be phosphorylated by Pi, forming an acylphosphate residue at the catalytic site of the enzyme. This reaction was inhibited by the phenothiazines trifluoperazine, chlorpromazine, imipramine, and fluphenazine and by the beta-adrenergic blocking agents propranolol and alprenolol. The inhibition was reversed by raising either the Pi or the Mg2+ concentration in the medium and was not affected by the presence of K+. Phosphorylation of the Ca2(+)-ATPase by Pi was also inhibited by ruthenium red and spermidine. These compounds compete with Mg2+, but, unlike the phenothiazines, they did not compete with Pi at the catalytic site, and the inhibition was abolished when K+ was included in the assay medium. The efflux of Ca2+ from loaded vesicles was greatly increased by the phenothiazines and by propranolol and alprenolol. In the presence of 200 microM trifluoperazine, the rate of Ca2+ efflux was higher than 3 mumol of Ca2+/mg of protein/10 s. The activation of efflux by these drugs was antagonized by Pi, Mg2+, K+, Ca2+, ADP, dimethyl sulfoxide, ruthenium red, and spermidine. The increase of Ca2+ efflux caused by trifluoperazine was not correlated with binding of the drug to the membrane lipids. It is concluded that the Ca2+ pump can be uncoupled by different drugs, thereby greatly increasing the efflux of Ca2+ through the ATPase. Displacement of these drugs by the natural ligands of the ATPase blocks the efflux through the uncoupled pathway and limits it to a much smaller rate. Thus, the Ca2(+)-ATPase can operate either as a pump (coupled) or as a Ca2+ channel (uncoupled).     

Effect of Organic and Inorganic Calcium Channel Blockers on γ-Amino-n-Butyiic Acid Release Induced by Monensin and Veratrine in the Absence of External Calcium

The effects of two organic Ca2+ antagonists (verapamil and nitrendipine) and of two inorganic Ca2+ channel blockers (Co2+ and ruthenium red) on the Na+-dependent release of gamma-amino-n-butyric acid (GABA) triggered by veratrine and monensin in the absence of external Ca2+ were studied in mouse brain synaptosomes. Ca2+-independent release of GABA stimulated by the Na+ channel activator veratrine was inhibited with micromolar concentrations of verapamil and nitrendipine. In contrast, GABA release induced by the Na+ ionophore monensin was insensitive to the organic Ca2+ antagonists. Verapamil also failed to modify A23187-stimulated release of GABA in the presence of Ca2+ but inhibited high K+-induced release of the transmitter. Co2+ partially diminished veratrine-induced release but did not change monensin-induced release. Releasing responses to monensin and veratrine were insensitive to ruthenium red, which inhibited the Ca2+-dependent component of GABA release evoked by high K+ depolarization. These data demonstrate that the mechanism of inducing GABA release is different for veratrine and monensin, as evidenced by their differing sensitivities to inhibition by Ca2+ channel antagonists and organic Ca2+ blockers. It is concluded that voltage-sensitive Ca2+ channels of the presynaptic membrane are not involved in the inhibitory action of Ca2+ antagonists on the Na+-dependent, Ca2+-independent mechanism of GABA release.     

Photosensory transduction in the flagellated alga, Euglena gracilis I. Action of divalent cations, Ca2+ antagonists and Ca2+ ionophore on motility and photobehavior.

1. The flagellated alga, Euglena gracilis, swims forward essentially in a straight path under constant light intensity. Strong motility of the cells can be supported by Mg2+ alone but optimum motility is found in the presence of Mg2+, Ca2+ and K+. 2. Ca2+, Co2+, Mn2+ and Ba2+ induce a concentration-dependent increase in the rate at which the cells change the direction of their swimming path (a klinokinesis). Ni2+ immobilizes the flagellum. 3. On perception of a reduction ('step-down stimulus') in blue light intensity in their environment, Euglena rotate in place (tumble) for a finite period (the step-down photophobic response). 4. The duration of the tumbling is enhanced in the presence of divalent cations following the series Ca2+ greater than Ba2+ greater than Mn2+ greater than Co2+ greater than Mg2+ = Ni2+ = 0. 5. Neither the tumbling response in the presence of low concentrations of Ca2+ or the Ca2+-stimulated response is altered by verapamil (a Ca2+ conductance antagonist). The Ca2+ conductance/active transport antagonist, ruthenium red, is also inactive. 6. The Ca2+ ionophore, A23187, has little effect on flagellar activity in the absence of extracellular Ca2+. However, in the presence of A23187, Ca2+ induces a specific light-independent, concentration-dependent discontinuous tumbling response of the cells. 7. The data support a role for Ca2+ and Mg2+ in control of flagellar activity. However, blue light-induced tumbling behavior would not appear to be the direct result of a light-mediated alteration in the Ca2+ conductance of the flagellar membrane to affect flagellar reorientation. The results are discussed in connection with previous theories on control of flagella activity in green alga.     

Order of release of ADP and Pi from phosphoenzyme with bound ADP of Ca2+-dependent ATPase from sarcoplasmic reticulum and of Na+, K+-dependent ATPase studied by ADP-inhibition patterns

The inhibition of Ca2+-dependent ATPase from SR [EC 3.6.1.3] by ADP was of mixed type under both low Ca2+ and high Mg2+ concentration and high Ca2+ and low Mg2+ concentrations. On the other hand, the inhibition of Na+, K+-dependent ATPase [EC 3.6.1.3] by ADP was of competitive type in the presence of low and high K+ concentrations. These results suggest that ADP is released before Pi from the phosphoenzyme with bound ADP (EPADP) in the case of Ca2+-ATPase, but that Pi is released before ADP in the case of Na+, K+-ATPase.     

Calcium transport mechanisms in dog red blood cells studied from measurements of initial flux rates

Ca2+ efflux from dog red blood cells loaded with Ca2+ using the A23187 ionophore could be separated into two main components: (1) Mg- and ATP-dependent (active transport) and (2) dependent on external Na (K1/2 around 15 mM); at 80 microM internal free Ca the relative magnitudes of these fluxes were 70% and 30% respectively. The Na-dependent Ca2+ efflux had the following additional properties: (i) it was partially inhibited by ATP depletion or preincubation with vanadate, but it was not affected by Mg2+ depletion; (ii) it failed to be stimulated by external monovalent cations other than Na: (iii) it was stimulated by reduction in the internal Na+ concentration. Both active and Na-dependent Ca2+ efflux remained unchanged in hypotonic solutions or in solutions with alkaline pH (8.5). In cells containing ATP and Mg2+, external Ca2+ inhibited Ca2+ efflux (K1/2 around 1 mM); on the other hand, in Mg-free dog red cells external Ca2+ stimulated Ca2+ efflux (K1/2 about 30 microM). In Mg-depleted red cells incubated in the absence of external Na2+, Ca2+ influx as a function of external Ca2+ followed a monotonically saturable function (K1/2 around 20 microM): addition of Na resulted in (i) inhibition of Ca2+ influx and (ii) a sigmoid relationship between flux and external Ca2+. Intracellular Ca2+ stimulated the external Na-dependent Ca2+ efflux along a sigmoid curve (K1/2 around 30 microM); on the other hand the Ca pump had a biphasic response to internal Ca2+: stimulation at low internal Ca2+ (K1/2 between 1 and 10 microM), followed by a decline at internal Ca2+ concentrations higher than 50 microM.     

Ruthenium red-sensitive and -insensitive release of Ca2+ from uncoupled heart mitochondria

The uncoupler-induced release of accumulated Ca2+ from heart mitochondria can be separated into two components, one sensitive and one insensitive to ruthenium red. In mitochondria maintaining reduced NAD(P)H pools and adequate levels of endogenous adenine nucleotides, the release of Ca2+ following addition of an uncoupler is virtually all inhibited by ruthenium red and can be presumed to occur via reversal of the Ca2+ uniporter. When ruthenium red is added to block efflux via this pathway, high rates of Ca2+ efflux can still be induced by an uncoupler, provided either NADH is oxidized or mitochondrial adenine nucleotide pools are depleted by prior treatment. This ruthenium red-insensitive Ca2+-efflux pathway is dependent on the level of Ca2+ accumulated and is accompanied by swelling of the mitochondria and loss of endogenous Mg2+. Loss of Ca2+ by this relatively nonspecific pathway is strongly inhibited by Sr2+ and by nupercaine, as well as by oligomycin and exogenous adenine nucleotides. The loss of Ca2+ from uncoupled heart mitochondria occurs via a combination of these two mechanisms except under conditions chosen specifically to limit efflux to one or the other pathway.     

Selective inhibition by ionophore A23187 of the enzyme isomerization in the catalytic cycle of Na+, K+ -ATPase

The effect of an ionophore A23187 on the purified Na+,K+-ATPase from the outer medulla of pig kidney was investigated. When the enzyme was pretreated with A23187 in the presence of Na+ and K+, the ATPase activity was inhibited almost completely. When the pretreatment was performed in the presence of Na+ and absence of K+, formation of the phosphoenzyme (EP) from ATP was only slightly retarded. The steady state level of EP thus formed was not altered, but EP decomposition was strongly inhibited. Under these conditions, the accumulated EP was sensitive to ADP and insensitive to K+. On the other hand, when the pretreatment was performed in the absence of Na+ and presence of K+, EP formation following simultaneous addition of Na+ and ATP was extremely slow, but the steady state level of EP was not substantially altered. When the pretreatment was performed in the absence of Na+ and presence of K+, EP formation from Pi was unaffected, and the EP formed was in rapid equilibrium with Pi of the medium. These results demonstrate that A23187 selectively inhibits isomerization of the enzyme between the high Na+ and low K+ affinity form and the low Na+ and high K+ affinity form in the catalytic cycle, whether or not the enzyme is phosphorylated. This inhibition is quite similar to the A23187-induced inhibition of the enzyme isomerization in the catalytic cycle of the Ca2+ -ATPase from sarcoplasmic reticulum (Hara, H., and Kanazawa, T. (1986)J. Biol. Chem.261, 16584-16590). These findings suggest that some common mechanism, which is involved in the enzyme isomerization, between these two transport ATPases is strongly disturbed by A23187.     

Inhibition of red cell Ca2+-ATPase by vanadate

1. The Mg2+- plus Ca2+-dependent ATPase (Ca2+-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate. 2. Several natural activators of Ca2+-ATPase (Mg2+, K+, Na+ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate. 3. Among the ligands tests, K+, in combination with Mg2+, had the most pronounced effect on inhibition by vanadate. 4. Under conditions optimal for inhibition of Ca2+-ATPase, the K 1/2 for vanadate was 1.5 microM and inhibition was nearly complete at saturating vanadate concentrations. 5. There are similarities between the kinetics of inhibition of red cell Ca2+-ATPase and (Na+ + K+)-ATPase prepared from a variety of sources; however, (Na+ + K+)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.     

(Na+ + K+)-dependent adenosine triphosphatase. Regulation of inorganic phosphate, magnesium ion, and calcium ion interactions with the enzyme by ouabain

1. (Na+ + K+)-dependent adenosine triphosphatase was phosphorylated on the alpha-subunit by Pi in the presence of Mg2+. Phosphorylation was stimulated by ouabain. The interactions of Pi, Mg2+, and ouabain with the enzyme could be explained by a random terreactant scheme in which the binding of each ligand to the enzyme increased the affinities for the other two. Dissociation constants of all steps of this scheme were estimated. 2. In the presence of Pi and ouabain and without added Mg2+, the phosphoenzyme was formed. Because this could be prevented by ethylenediaminetetraacetic acid, but not ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, phosphoenzyme formation under these conditions was probably dependent on traces of endogenous Mg2+. The ability of this Mg2+ to support phosphorylation could be explained by the large increase in the enzyme's affinity for Mg2+ by ouabain. 3. In the absence of ouabain, Ca2+ did not support phosphorylation and inhibited Mg2+-dependent phosphorylation. At lower concentrations, Ca2+ was competitive with Mg2+. With increasing Ca2+ concentration, negative cooperativity was observed, suggesting the existence of multiple divalent cation sites with equivalent affinities for Mg2+, but varying affinities for Ca2+. 4. In the presence of ouabain, the maximum inhibition of Mg2+-dependent phosphorylation by Ca2+ was 50%. With saturating Pi, Mg2+, and ouabain, the number of sites binding ouabain was equal to the number of sites phosphorylated. Although Ca2+ halved phosphorylation and reduced the affinity for ouabain about 100-fold, it did not affect the number of ouabain sites. 5. We suggest that the enzyme is an alpha-oligomer and that the half-of-the-sites reactivity for phosphorylation in the presence of Pi, Mg2+, ouabain, and optimal Ca2+ is caused by (a) ouabain-induced increase in the affinities of both protomers for Mg2+ and (b) the inability of Ca2+ to replace Mg2+ on one of the protomers.     

Interactions of physiological ligands with the Ca pump and Na/Ca exchange in squid axons

We have studied the interaction of physiological ligands other than Nai and Cai with the Ca pump and Na/Ca exchange in internally dialyzed squid axons. The results show the following. (a) Internal Mg2+ is an inhibitor of the Nao-dependent Ca efflux. At physiological Mg2+i (4 mM), the inhibition amounts to approximately 50%. The inhibition is partial and noncompetitive with Cai, and is not affected by Nai or ATP. The ATP-dependent uncoupled efflux is unaffected by Mgi up to 20 mM. Both components of the Ca efflux require Mg2+i for their activation by ATP. (b) At constant membrane potential, Ki is an important cofactor for the uncoupled Ca efflux. (c) Orthophosphate (Pi) activates the Nao-dependent Ca efflux without affecting the uncoupled component. Activation by Pi occurs only in the presence of Mg-ATP or hydrolyzable ATP analogues. Pi under physiological conditions has no effect on the uncoupled component; nevertheless, at alkaline pH, it inhibits the Ca pump, probably by product inhibition. (d) ADP is a potent inhibitor of the uncoupled Ca efflux. The Nao-dependent component is inhibited by ADP only at much higher ADP concentrations. These results indicate that (a) depending on the concentration of Ca2+i, Na+i Mg2+i, and Pi, the Na/Ca carrier can operate under a low- or high-rate regime; (b) the interactions of Mg2+i, Pi, Na+i, and ATP with the carrier are not interdependent; (c) the effect of Pi on the carrier-mediated Ca efflux resembles the stimulation of the Nao-dependent Ca efflux by internal vanadate; (d) the ligand effects on the uncoupled Ca efflux are of the type seen in the Ca pump in red cells and the sarcoplasmic reticulum.     

Effect of hemolysate on calcium inhibition of the (Na+ + K+)-ATPase of human red blood cells

The sensitivity of the (Na+ + K+)-ATPase in human red cell membranes to inhibition by Ca2+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca2+ causing 50% inhibition of the (Na+ + K+)-ATPase is shifted from greater than 50 microM free Ca2+ in the absence of hemolysate to less than 10 microM free Ca2+ when hemolysate diluted 1:60 compared to in vivo concentrations is added to the assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the (Na+ + K+)-ATPase to Ca2+ in a dose-dependent fashion, causing over 80% inhibition of the (Na+ + K+)-ATPase at 10 microM free Ca2+ at the highest concentration of the extract tested. The active factor in this membrane extract is Ca2+-dependent, because it had no effect on the (Na+ + K+)-ATPase in the absence of Ca2+. Trypsin digestion prior to the assay destroyed the ability of this protein extract to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+.     

Calcium ionophoretic activity of chemically synthesized oligomeric derivatives of prostaglandin B1

Chemically synthesized dimers, trimers and tetramers of 15-dehydroprostaglandin B1 and 16,16'-dimethyl-15-dehydroprostaglandin B1 facilitate the release of Ca2+ from isolated rat liver mitochondria. The parent monomeric prostaglandins had no significant activity. The rate of release was stimulated by exogenous K+ or Na+, suggesting an antiport exchange of monovalent cations for intra-mitochondrial Ca2+. The activity depended upon the presence of ruthenium red, which prevented recycling of Ca2+; comparison of the activity with A23187 and carbonyl cyanide p-trifluoromethoxyphenylhydrazone indicated that the prostaglandin B1 oligomers were functioning as ionophores and the release of Ca2+ was not caused by an uncoupling of oxidative phosphorylation. The oligomers caused a major decrease in the membrane potential but only when the mitochondria were preloaded with exogenous Ca2+, and even then, the Ca2+ efflux was completed before the membrane potential decreased to less than 90 mV. The oligomeric molecules were able to form supramolecular aggregates in the presence of Ca2+ as detected by light scattering. They extracted Ca2+ into an organic phase, and translocated Ca2+ from one aqueous domain to another across an organic barrier; K+ and Na+ modulated these processes. The prostaglandin B1 derivatives also translocated Rb+ from one aqueous phase to another across an organic barrier when Ca2+ was translocated.     

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.     

Ouabain-insensitive Na+ stimulation of a microsomal Mg2+ -ATPase in gills of sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested.     

Uptake, retention, and efflux of Ca2+ by mitochondrial preparations from skeletal muscle

Functionally intact mitochondria, substantially free of contamination, were isolated from rabbit gastrocnemius muscle after protease digestion and their Ca2+-handling properties examined. When judged by their capacity to retain large Ca2+ loads and the magnitude of basal and Na+-stimulated Ca2+ effluxes, the most suitable isolation method was digestion of finely minced muscle in buffered isoosmotic KCl with low levels (0.4 mg/g) of trypsin or the bacterial protease nagarse, followed by differential centrifugation. Polytron disruption of skeletal muscle in both sucrose- and KCl-based media released mitochondria deficient in cytochrome c. Use of the divalent ion chelator EDTA rather than EGTA in the isolation medium sharply reduced Ca2+-dependent respiratory control and tolerance of the mitochondria to Ca2+ loads, probably by removing Mg2+ essential to membrane integrity. ADP-dependent respiratory control was not altered in mitochondria prepared in an EDTA-containing isolation medium. Purification of mitochondria on a Percoll density gradient did not improve their Ca2+-handling ability despite removal of minor contaminants. Mitochondria prepared by the protease method could accumulate micromole loads of Ca2+/mg while maintaining a low basal Ca2+ efflux. Addition of BSA to the assay medium slightly improved Ca2+ retention but was not essential either during isolation or assay. Ca2+-dependent state 3 respiration was maximal at pH 6.5-7.0 while respiratory control and Ca2+/O were optimal at pH 7.0-7.5. Neither Pi nor oxaloacetate induced Ca2+ release from loaded mitochondria when monitored for 30 min after ruthenium red addition. Na+-stimulated Ca2+ efflux had sigmoidal kinetics with a Hill coefficient of 3. Since skeletal muscle mitochondria can be isolated and assayed in simple media, functional deficiencies of mitochondria from diseased muscle are unlikely to be masked.