Coupling of Li+ distribution to the plasma membrane potential of rat cortical synaptosomes

The effect of the plasma membrane potential delta psi p on the transport rate and steady state distribution of Li+ was assessed in rat cortical synaptosomes. Up to 15 mM Li+ failed to saturate Li+ influx into polarized synaptosomes in a Na+-based medium with 3 mM external K+. Veratridine increased and tetrodotoxin, ouabain, or high external K+ decreased the rate of Li+ influx. At steady state, Li+ was concentrated about 3-fold in resting synaptosomes at 0.3 to 1 mM Li+ externally. Subsequent depolarization of the plasma membrane by veratridine or high external K+ induced an immediate release of Li+. When graded depolarizations were imposed onto the plasma membrane by varying concentrations of ouabain, veratridine, or external K+, steady state distribution of Li+ was linearly related with K+ distribution or electrochemical activity coefficients. It was concluded that uptake rate and steady state distribution of Li+ depend significantly on delta psi p. However, Li+ gradients were lower than predicted from delta psi p, suggesting that (secondary) active transport systems counteracted passive equilibration by uphill extrusion of Li+. The electrochemical potential difference delta mu Li+ maintained at a delta psi p of -72 mV was calculated to 4.2 kJ/mol of Li+. At physiological external K+, Li+ was not actively transported by the sodium pump. The ouabain sensitivity resulted from the coupling of Li+ uptake to the pump-dependent K+ diffusion potential. In low K+ and K+-free media, however, active transport of Li+ by the sodium pump contributed to total uptake. In the absence of K+, Li+ substituted for K+ in generating a delta psi p of -64 mV maximally, as calculated from TPMP+ distribution at 40 mM external Li+. Since Li+ gradients were far too low to account for a diffusion potential, it was assumed that Li+ gave rise to an electrogenic pump potential.     

(Ca2+ + Mg2+)-ATPase activity associated with the maintenance of a Ca2+ gradient by sarcoplasmic reticulum at submicromolar external [Ca2+]. The effect of hypothyroidism

The formation and maintenance of Ca2+-filling levels by sarcoplasmic reticulum vesicles from euthyroid (control) and hypothyroid skeletal muscle were investigated using the Ca2+-indicator quin-2, at [Ca2+] in the medium [( Cao2+]) of 0.05-0.3 microM. Rapid ATP-dependent Ca2+ uptake resulted in a steady-state Ca2+-filling level, Cai2+, within one minute. This Ca2+ gradient was maintained for at least three minutes, during which less than 20% of the ATP was consumed. Cai2+ was maximal (120 nmol/mg) for [Cao2+] greater than 0.3 microM and decreased to 40 nmol/mg at [Cao2+] of 0.05 microM. Preparations from both experimental groups showed qualitatively and quantitatively the same relationship between Cai2+ and [Cao2+] at steady state, despite a significantly lower Ca2+-pump content of hypothyroid sarcoplasmic reticulum, which resulted in a 25% lower maximal (Ca2+ + Mg2+)-ATPase activity. Maintenance of the steady state, at all levels of Cai2+, was associated with net ATP consumption by the Ca2+ pump and cycling of Ca2+, which processes were 30% slower in the hypothyroid group as compared to the control group. Determination of the passive efflux of Ca2+, as well as the fraction of leaky or unsealed sarcoplasmic reticulum fragments, excluded either of these possibilities as an explanation for the relatively high (Ca2+ + Mg2+)-ATPase rates at steady state. On the basis of these and previously reported results, it is concluded that the maintenance of a Ca2+ gradient by sarcoplasmic reticulum under physiological conditions with respect to external [Ca2+] and the concentrations of ATP, ADP and Pi, is associated with the cycling of Ca2+ coupled to net ATP hydrolysis. Using the obtained data it is calculated that the sarcoplasmic reticulum may account for 20% of the resting metabolic rate in skeletal muscle. Consequently, together with the previously reported lower sarcoplasmic reticulum content of skeletal muscle in hypothyroidism, we calculate that about one third of the decrease in basal metabolic rate in this thyroid state can be related to the alterations of the sarcoplasmic reticulum.     

Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca2+ concentration

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca²⁺-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-β-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-β-cyclodextrin complexes restored the Ca²⁺-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca²⁺, annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca²⁺ concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca²⁺ concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.     

Effect of maitotoxin on cytosolic Ca2+ levels and membrane potential in purified rat brain synaptosomes

In this study, the effects of the marine toxin maitotoxin on cytosolic Ca2+ levels and membrane potential in rat brain synaptosomes were evaluated. Maitotoxin (10 ng/ml) caused a remarkable increase of intrasynaptosomal Ca2+ levels monitored by the fluorescent probe fura-2. This increase was prevented by the removal of external Ca2+ ions. Tetrodotoxin, as well as the removal of extracellular Na+ ions, failed to affect maitotoxin-induced increase of intrasynaptosomal Ca2+ levels. Also the complete removal of all monovalent and divalent cations, except Ca2+ ions, from the incubation medium (0.32 M sucrose substitution), was unable to prevent the effect of maitotoxin on intrasynaptosomal Ca2+ levels. Maitotoxin (0.3-10 ng/ml), produced a dose-dependent depolarization of synaptosomal membranes, which required the presence of extracellular Ca2+ ions. The substitution of extracellular Na+ with choline or the removal of all cations from the incubation medium and their replacement with an isotonic concentration of sucrose (0.32 M), did not prevent the depolarizing effect exerted by maitotoxin. Also under these two ionic conditions, the effect of maitotoxin on membrane potential was critically dependent on the presence of 1 mM extracellular Ca2+. The depolarizing effect exerted by maitotoxin on synaptosomal membrane potential was also observed when extracellular Ca2+ ions were substituted with an equimolar concentration of Ba2+ or Sr2+ ions. In summary, these results appear to suggest that, in presence of 1 mM extracellular Ca2+ ions, maitotoxin depolarizes synaptosomal plasmamembrane by promoting the influx of extracellular Ca2+ ions. This enhanced influx of Ca2+ causes an increase of intrasynaptosomal Ca2+ levels.     

Ca2+ transport by the synaptosomal plasma membrane Ca2+-ATPase and the effect of thioridazine

Thioridazine inhibits the activity of the synaptic plasma membrane Ca(2+)-ATPase from pig brain and slightly decreases the rate of Ca(2+) accumulation by synaptic plasma membrane vesicles in the absence of phosphate. However, in the presence of phosphate, thioridazine increases the rate of Ca(2+) accumulation into synaptic plasma membrane vesicles. Phosphate anions diffuse through the membrane and form calcium phosphate crystals, reducing the free Ca(2+) concentration inside the vesicles and the rate of Ca(2+) leak. The higher levels of Ca(2+) accumulation obtained in the presence of thioridazine could be explained by a reduction of the rate of slippage on the plasma membrane ATPase.     

The interaction of ethanol with reconstituted synaptosomal plasma membrane Ca2+ -ATPase

The primary effect of ethanol is on the central nervous system. However, the molecular mechanisms responsible for the physiological symptoms of ethanol intoxication are still unknown. Low concentrations of ethanol were observed to stimulate the activity of the calcium pump from reconstituted synaptosomal plasma membrane Ca2+ -ATPase (PMCA), and ethanol inhibited Ca2+ -ATPase activity at concentrations above 5%. The greatest stimulating effect was obtained with 5% (v/v) ethanol and was lipid-dependent, being 74% when the protein had been reconstituted in phosphatidylcholine (PC) and less when the reconstituted protein had previously been activated by calmodulin or after removal of a 9-kDa autoinhibitory site by controlled trypsinization. Stimulation of the pump by ethanol was lower for the native or trypsin-digested protein in the presence of phosphatidylserine than in PC. These results suggest a direct ethanol-protein interaction, because the activating effect depended on the state of Ca2+ -ATPase (native or truncated, or in presence of calmodulin). The activating mechanism of ethanol may involve opening an autoinhibitory domain located close to the calmodulin binding domain.     

Simultaneous measurement of Ca2+ transients and of membrane depolarizations in synaptosomes.

Calcium and membrane potential sensitive dyes have been widely used to study the biochemical effects of the intracellular calcium concentration and of the membrane potential on diverse biochemical processes. However, due to the discontinuous measurement techniques applied, it was until now impossible to get an insight into the sequence and dynamics of the induced biological reactions. In order to study the relationship between the intracellular calcium concentration and the membrane potential, an apparatus was developed capable of measuring both biological processes simultaneously. Potassium chloride induced changes of the synaptosomal membrane potential and of the intracellular calcium concentration are presented.     

The role of ceramide in the modulation of intracellular Ca2+ levels by interleukin 1 beta in rat cortical synaptosomes

We recently demonstrated that the pro-inflammatory cytokine, interleukin 1beta (IL-1beta) elevates intracellular free Ca2+ levels ([Ca2+]i) in rat cortical synaptosomes in a manner involving activation of the IL-1 receptor and stimulation of p42 mitogen-activated protein (MAP) kinase. We now report that the effects of IL-1beta on [Ca2+]i are mimicked by the sphingolipid metabolite ceramide. In cortical synaptosomes ceramide elevates [Ca2+]i in a p42 MAP kinase-dependent manner, and we conclude that the effects of IL-1beta on Ca2+ homeostasis involve ceramide as an upstream component of the p42 MAP kinase pathway.     

Effects of rapid buffers on Ca2+ diffusion and Ca2+ oscillations.

Based on realistic mechanisms of Ca2+ buffering that include both stationary and mobile buffers, we derive and investigate models of Ca2+ diffusion in the presence of rapid buffers. We obtain a single transport equation for Ca2+ that contains the effects caused by both stationary and mobile buffers. For stationary buffers alone, we obtain an expression for the effective diffusion constant of Ca2+ that depends on local Ca2+ concentrations. Mobile buffers, such as fura-2, BAPTA, or small endogenous proteins, give rise to a transport equation that is no longer strictly diffusive. Calculations are presented to show that these effects can modify greatly the manner and rate at which Ca2+ diffuses in cells, and we compare these results with recent measurements by Allbritton et al. (1992). As a prelude to work on Ca2+ waves, we use a simplified version of our model of the activation and inhibition of the IP3 receptor Ca2+ channel in the ER membrane to illustrate the way in which Ca2+ buffering can affect both the amplitude and existence of Ca2+ oscillations.     

Proton permeability of the plasma membrane of rat cortical synaptosomes

1. Transmembrane pH gradients (acidic inside) and electrical gradients (negative inside) were estimated in cortical synaptosomes from the distribution of the weak base methylamine and the lipophilic cation tetraphenylphosphonium, respectively. 2. Acidic interior pH gradients were produced by outwardly directed K+ gradients in Na+-free media. External K+ accelerated the dissipation of preformed H+ gradients. The appearance of H+ in the medium was directly demonstrated by pH-stat titration of a weakly buffered medium. Amiloride failed to inhibit K+-induced H+ release. 3. Elevating K+ in the absence of Na+ did not affect the endogenous contents of noradrenaline, dopamine, and serotonin, as determined by high-performance liquid chromatography with electrochemical detection. 4. H+ diffusion potentials were generated when outwardly directed H+ gradients were imposed onto the plasma membrane indicating an electrogenic H+ efflux which is not coupled to other ions. 5. At low K+ in the Na+-free sucrose medium, the plasma membrane potential Em (derived from distribution of tetraphenylphosphorium cation) did not approach a value for EK, the K+ equilibrium potential (calculated from K+ gradients). The deviation of Em from EK could be quantitatively described by a modified constant-field equation, taking a relative H+/K+ permeability coefficient of 12,400 into consideration. 6. It is concluded that synaptosomes have a H+ conductance pathway in their plasma membrane in addition to the Na+/H+ antiporter. H+ influx is driven by and leads to a reduction of Em. K+/H+ exchange resulted from the electrical coupling of K+ and H+ fluxes via parallel K+ and H+ channels. Since the Na+/H+ antiporter counteracts passive equilibration of H+ under physiological conditions, a continuous cycling of H+ across the plasma membrane will take place. A possible physiological role of the H+ leak in pHi regulation is discussed.     

Regulation of Ca2+ efflux in rat liver mitochondria. Role of membrane potential

The paper analyzes the relationship between membrane potential (delta psi), steady state pCao (-log [Ca2+] in the outer aqueous phase) and rate of ruthenium-red-induced Ca2+ efflux in liver mitochondria. Energized liver mitochondria maintain a pCao of about 6.0 in the presence of 1.5 mM Mg2+ and 0.5 mM Pi. A slight depression of delta psi results in net Ca2+ uptake leading to an increased steady state pCao. On the other hand, a more marked depression of delta psi results in net Ca2+ efflux, leading to a decreased steady-state pCao. These results reflect a biphasic relationship between delta psi and pCao, in that pCao increases with the increase of delta psi up to a value of about 130 mV, whereas a further increase of delta psi above 130 mV results in a decrease of pCao. The phenomenon of Ca2+ uptake following a depression of delta psi is independent of the tool used to affect delta psi whether by inward K+ current via valinomycin, or by inward H+ current through protonophores or through F1-ATP synthase, or by restriction of e- flow. The pathway for Ca2+ efflux is considerably activated by stretching of the inner membrane in hypotonic media. This activation is accompanied by a decreased pCao at steady state and by an increased rate of ruthenium-red-induced Ca2+ efflux. By restricting the rate of e- flow in hypotonically treated mitochondria, a marked dependence of the rate of ruthenium-red-induced Ca2+ efflux on the value of delta psi is observed, in that the rate of Ca2+ efflux increases with the value of delta psi. The pCao is linearly related to the rate of Ca2+ efflux. Activation of oxidative phosphorylation via addition of hexokinase + glucose to ATP-supplemented mitochondria, is followed by a phase of Ca2+ uptake, which is reversed by atractyloside. These findings support the view that Ca2+ efflux in steady state mitochondria occurs through an independent, delta psi-controlled pathway and that changes of delta psi during oxidative phosphorylation can effectively modulate mitochondrial Ca2+ distribution by inhibiting or activating the delta psi-controlled Ca2+ efflux pathway.     

Calcium transport in brain synaptosomes during depolarization. The role of potential-dependent channels and Na+/Ca2+ metabolism

The contribution of Ca2+ channels and Na+/Ca2+ exchange to Ca2+ uptake in rat brain synaptosomes upon long- (t greater than or equal to 30 s) and short-term (t less than 30 s) depolarization by high K+ was studied by measuring the 45Ca content and free Ca2+ concentration (from Quin-2 fluorescence). At 37 degrees C, the system responsible for the K+-stimulated uptake of 45Ca (t greater than or equal to 30 s) and the Na+/Ca+ exchanger are characterized by a similar concentration dependence of external Ca2+ (Ca0(2+] and K0+ as well as by an equal sensitivity to verapamil (Ki = approximately 20-40 microM) and La2+ (Ki = approximately 50 microM). These data and the results from predepolarization suggest that the 45Ca entry into synaptosomes at t greater than or equal to 30 s is due to the activation of Na+/Ca+ exchange caused by its electrogenic component, while the insignificant contribution of Ca2+ channels can be accounted for by their inactivation. At low temperatures (2-4 degrees C) which decelerate the inactivation, the initial phase of 45Ca uptake is fully provided for by Ca2+ channels, showing a lower (as compared to the exchanger) affinity for Ca0(2+) (K0.5 greater than 1 mM)m a greater sensitivity to La3+ (Ki = approximately 0.2-0.3 microM) and verapamil (Ki = approximately 2-3 microM); these channels are fully inactivated by predepolarization with K0+, ouabain and batrachotoxin. The Ca2+ channels can be related to T-type channels, since they are not blocked by nicardipine and niphedipine.     

Short-time effects of neuroactive steroids on rat cortical Ca2+-ATPase activity

Recent experimental evidence indicates that some steroid hormones, apart from their well-documented genomic actions, could produce non-genomic rapid effects, and are potent modulators of the plasma membrane proteins, including voltage- and ligand-operated ion channels or G protein-coupled receptors. Neuroactive steroids, 17beta-estradiol, testosterone, pregnenolone sulfate and dehydroepiandrosterone sulfate, after a short-time incubation directly modulated the activity of plasma membrane Ca2+-ATPase purified from synaptosomal membranes of rat cortex. The sulfate derivatives of dehydroepiandrosterone and pregnenolone applied at concentrations of 10-11-10-6 M, showed an inverted U-shape potency in the regulation of Ca2+-ATPase activity. At physiologically relevant concentrations (10-8-10-9 M) a maximal enhancement of the basal activity reached 200%. Testosterone (10-11-10-6 M) and 17beta-estradiol (10-12-10-9 M) caused a dose-dependent increase in the hydrolytic ability of Ca2+-ATPase, and the activity with the highest concentration of steroids reached 470% and 200%, respectively. All examined steroids decreased the stimulatory effect of a naturally existing activator of the calcium pump, calmodulin. The present study strongly suggests that the plasma membrane calcium pump could be one of the possible membrane targets for a non-genomic neuroactive steroid action.     

Effect of external K+, Ca2+, and Ba2+ on membrane potential and ionic conductance in rat astrocytes

Summary 1. The purpose of this study was (a) to identify if astrocytes show a similar non-Nernstian depolarization in low K⁺ or low Ca²⁺ solutions as previously found in human glial and glioma cells, and (b) to analyze the influence of the K⁺ conductance on the membrane potential of astrocytes.2. The membrane potential (E_m) and the ionic conductance were studied with whole-cell patch-clamp technique in neonatal rat astrocytes (5–9 days in culture) and in human glioma cells (U-251MG).3. In 3.0 mM K⁺, E_m was –75 ± 1.0 mV (mean ± SEM,n=39) in rat astrocytes and –79 ± 0.7 mV (n=5) in U-251MG cells. In both cell types E_m changed linearly to the logarithm of [K⁺]₀ between 3.0 and 160 mM K⁺. K⁺ free medium caused astrocytes to hyperpolarize to –93 ± 2.7 mV (n=21) and U-251MG cells to depolarize to –27 ± 2.1 mV (n=3).4. The I-E curve did not show inward rectification in astrocytes at this developmental stage. The slope conductance (g) exhibited only a small decrease (–19%) in K⁺ free solution and no significant change in 160 mM K⁺.5. Ba²⁺ (1.0 mM) depolarized astrocytes to –45 ± 2.9 mV (n=11), decreasing the slope conductance (g) by 42.4 ± 8.3% (n=11). Ca²⁺ free solution depolarized astrocytes to –53 ± 3.4 mV (n=12) and resulted in a positive shift of the I-E curve, increasing g by 15.3 ± 8.2% (n=8).6. Calculations indicated that a block of K⁺ channels explains the depolarizing effect of Ba²⁺. The effects of K⁺ free or Ca²⁺ free solutions on E_m can be explained by a transformation of K⁺ channels to non-specific leakage channels. That astrocytes show a different reaction to low K⁺ than glioma cells can be related to the lack of inwardly rectifying K⁺ channels in astrocytes at this developmental stage.     

Effects of plasma membrane oxidoreductases on Ca2+ mobilization and protein phosphorylation in rat brain synaptosomes

We have investigated the possible role of plasma membrane oxidoreductases in the Ca²⁺ export mechanisms in rat brain synaptic membranes. Ca²⁺ efflux in nerve terminals is controlled both by a high-affinity/low capacity Mg-dependent ATP-stimulated Ca²⁺ pump and by a low affinity/high capacity ATP-independent Na⁺-Ca²⁺ exchanger. Both Ca²⁺ efflux mechanisms were strongly inhibited by pyridine nucleotides, in the order NADP>NAD>NADPH>NADH with IC₅₀ values of ca. 10 mM for NADP and ca. 3 mM for the other agents in the case of the ATP-driven Ca²⁺ pump and with IC₅₀ values between 8 and 10 mM for the Na⁺-Ca²⁺ exchanger. Oxidizing agents such as DCIP and ferricyanide inhibited the ATP-driven Ca²⁺ efflux mechanism but not the Na⁺-Ca²⁺ exchanger. In addition, full activation of plasma membrane oxidoreductases requires both an acceptor and an electron donor; therefore the combined effects of both substrates added together were also studied. When plasma membrane oxidoreductases of the synaptic plasma membrane were activated in the presence of both NADH (or NADPH) and DCIP or ferricyanide, the inhibition of the ATP-driven Ca²⁺ pump was optimal; by contrast, the pyridine nucleotide-mediated inhibition of the Na⁺-Ca²⁺ exchanger was partially released when both substrates of the plasma membrane oxidoreductases were present together. Furthermore, the activation of plasma membrane oxidoreductases also strongly inhibited intracellular protein phosphorylation in intact synaptosomes, mediated by eithercAMP-dependent protein kinase, Ca²⁺ calmodulin-dependent protein kinases, or protein kinase C.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - SDS sodium dodecyl sulfate - EGTA ethylenglycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid - DCIP dichlorophenol-indophenol     

Bile acids mobilise internal Ca2+ independently of external Ca2+ in rat hepatocytes

In the present study, we investigated the possible role of external Ca2+ in the rise of the cytosolic Ca+ concentration induced by the monohydroxy bile acid taurolithocholate in isolated rat liver cells. The results showed that: (a) the bile acid promotes the same dose-dependent increase in the cytosolic Ca+ concentration (half-maximal effect at 23 microM) in hepatocytes incubated in the presence of 1.2 mM Ca2+ or 6 microM Ca2+; (b) taurolithocholate is able to activate the Ca2(+)-dependent glycogen phosphorylase a by 6.3-fold and 6.0-fold in high and low Ca2+ media, respectively; (c) [14C]taurolithocholate influx is not affected by external Ca2+, and 45Ca2+ influx is not altered by taurolithocholate. These results establish that the effects of taurolithocholate on cell Ca2+ do not require extracellular Ca2+ and are consistent with the view that monohydroxy bile acids primarily release Ca2+ from the endoplasmic reticulum in the liver.     

Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium.

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.     

Mitochondrial membrane potential modulates regulation of mitochondrial Ca2+ in rat ventricular myocytes

Although recent studies focused on the contribution of mitochondrial Ca2+ to the mechanisms of ischemia-reperfusion injury, the regulation of mitochondrial Ca2+ under pathophysiological conditions remains largely unclear. By using saponin-permeabilized rat myocytes, we measured mitochondrial membrane potential (DeltaPsi(m)) and mitochondrial Ca2+ concentration ([Ca2+](m)) at the physiological range of cytosolic Ca2+ concentration ([Ca2+](c); 300 nM) and investigated the regulation of [Ca2+](m) during both normal and dissipated DeltaPsi(m). When DeltaPsi(m) was partially depolarized by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, 0.01-0.1 microM), there were dose-dependent decreases in [Ca2+](m). When complete DeltaPsi(m) dissipation was achieved by FCCP (0.3-1 microM), [Ca2+](m) remained at one-half of the control level despite no Ca2+ influx via the Ca2+ uniporter. The DeltaPsi(m) dissipation by FCCP accelerated calcein leakage from mitochondria in a cyclosporin A (CsA)-sensitive manner, which indicates that DeltaPsi(m) dissipation opened the mitochondrial permeability transition pore (mPTP). After FCCP addition, inhibition of the mPTP by CsA caused further [Ca2+](m) reduction; however, inhibition of mitochondrial Na+/Ca2+ exchange (mitoNCX) by a Na+-free solution abolished this [Ca2+](m) reduction. Cytosolic Na(+) concentrations that yielded one-half maximal activity levels for mitoNCX were 3.6 mM at normal DeltaPsi(m) and 7.6 mM at DeltaPsi(m) dissipation. We conclude that 1) the mitochondrial Ca2+ uniporter accumulates Ca2+ in a manner that is dependent on DeltaPsi(m) at the physiological range of [Ca2+](c); 2) DeltaPsi(m) dissipation opens the mPTP and results in Ca2+ influx to mitochondria; and 3) although mitoNCX activity is impaired, mitoNCX extrudes Ca2+ from the matrix even after DeltaPsi(m) dissipation.     

The membrane potential modulates the ATP-dependent Ca2+ pump of cardiac sarcolemma

The effect of membrane potential on the activity of the ATP-dependent Ca2+ pump of isolated canine ventricular sarcolemmal vesicles was investigated. The membrane potential was controlled by the intravesicular and extravesicular concentration of K+, and the initial rates of Ca2+ uptake both in the presence and the absence of valinomycin were determined. The rate of Ca2+ uptake was stimulated by a inside-negative potential induced in the presence of valinomycin. The valinomycin-dependent stimulation was enhanced by the addition of K+ channel blocker, tetraethylammonium ion or Ba2+. The electrogenicity of cardiac sarcolemmal ATP-dependent Ca2+ pump is suggested from the increase of Ca2+ uptake by negative potential induced by valinomycin.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.