Reversed Na+/Ca2+ exchange contributes to Ca2+ influx and respiratory burst in microglia

Phagocytosis and the ensuing NADPH-mediated respiratory burst are important aspects of microglial activation that require calcium ion (Ca(2+)) influx. However, the specific Ca(2+) entry pathway(s) that regulates this mechanism remains unclear, with the best candidates being surface membrane Ca(2+)-permeable ion channels or Na(+)/Ca(2+) exchangers. In order to address this issue, we used quantitative real-time RT-PCR to assess mRNA expression of the Na(+)/Ca(2+) exchangers, Slc8a1-3/NCX1-3, before and after phagocytosis by rat microglia. All three Na(+)/Ca(2+) exchangers were expressed, with mRNA levels of NCX1 > NCX3 > NCX2, and were unaltered during the one hour phagocytosis period. We then carried out a biophysical characterization of Na(+)/Ca(2+) exchanger activity in these cells. To investigate conditions under which Na(+)/Ca(2+) exchange was functional, we used a combination of perforated patch-clamp analysis, fluorescence imaging of a Ca(2+) indicator (Fura-2) and a Na(+) indicator (SBFI), and manipulations of membrane potential and intracellular and extracellular ions. Then, we used a pharmacological toolbox to compare the contribution of Na(+)/Ca(2+) exchange with candidate Ca(2+)-permeable channels, to the NADPH-mediated respiratory burst that was triggered by phagocytosis. We find that inhibiting the reversed mode of the Na(+)/Ca(2+) exchanger with KB-R7943, dose dependently reduced the phagocytosis-stimulated respiratory burst; whereas, blockers of store-operated Ca(2+) channels or L-type voltage-gated Ca(2+) channels had no effect. These results provide evidence that Na(+)/Ca(2+) exchangers are potential therapeutic targets for reducing the bystander damage that often results from microglia activation in the damaged CNS.     

Na+/Ca2+ exchange in isolated smooth muscle cells demonstrated by the fluorescent calcium indicator fura-2

Fura-2, a novel fluorescent indicator of cytoplasmic calcium concentrations ([Ca2+i]), was 'loaded' into smooth muscle cells isolated from guinea pig taenia coli. Resting cells maintained a stable [Ca2+i] of 107 +/- 26 nM (n = 13), which could be perturbed with ionomycin. [Ca2+i] was elevated by stimulation of the cells with carbachol or 50 mM KCl. Reduction of the plasmalemmal Na+ concentration gradient by inhibition of the Na+/K+-ATPase with ouabain markedly elevated [Ca2+i]; this elevation was dependent on extracellular Ca2+. [Ca2+i] was also increased by replacement of the extracellular Na+ with an organic cation.     

Role of Na+/Ca2+ exchange in regulating cytosolic Ca2+ in cultured human pulmonary artery smooth muscle cells

A rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMC) is an important stimulus for cell contraction, migration, and proliferation. Depletion of intracellular Ca²⁺ stores opens store-operated Ca²⁺ channels (SOC) and causes Ca²⁺ entry. Transient receptor potential (TRP) cation channels that are permeable to Na⁺ and Ca²⁺ are believed to form functional SOC. Because sarcolemmal Na⁺/Ca²⁺ exchanger has also been implicated in regulating [Ca²⁺]_cyt, this study was designed to test the hypothesis that the Na⁺/Ca²⁺ exchanger (NCX) in cultured human PASMC is functionally involved in regulating [Ca²⁺]_cyt by contributing to store depletion-mediated Ca²⁺ entry. RT-PCR and Western blot analyses revealed mRNA and protein expression for NCX1 and NCKX3 in cultured human PASMC. Removal of extracellular Na⁺, which switches the Na⁺/Ca²⁺ exchanger from the forward (Ca²⁺ exit) to reverse (Ca²⁺ entry) mode, significantly increased [Ca²⁺]_cyt, whereas inhibition of the Na⁺/Ca²⁺ exchanger with KB-R7943 (10 µM) markedly attenuated the increase in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. Store depletion also induced a rise in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. Removal of extracellular Na⁺ or inhibition of the Na⁺/Ca²⁺ exchanger with KB-R7943 attenuated the store depletion-mediated Ca²⁺ entry. Furthermore, treatment of human PASMC with KB-R7943 also inhibited cell proliferation in the presence of serum and growth factors. These results suggest that NCX is functionally expressed in cultured human PASMC, that Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange contributes to store depletion-mediated increase in [Ca²⁺]_cyt, and that blockade of the Na⁺/Ca²⁺ exchanger in its reverse mode may serve as a potential therapeutic approach for treatment of pulmonary hypertension. sodium-calcium exchange; calcium homeostasis; vascular smooth muscle     

Importance of Ca2+ influx by Na+/Ca2+ exchange under normal and sodium-loaded conditions in mammalian ventricles

Na⁺/Ca²⁺ exchange (NCX) is a major Ca²⁺ extrusion system in cardiac myocytes, but can also mediate Ca²⁺ influx and trigger sarcoplasmic reticulum Ca²⁺ release. Under conditions such as digitalis toxicity or ischemia/reperfusion, increased [Na⁺]_i may lead to a rise in [Ca²⁺]_i through NCX, causing Ca²⁺ overload and triggered arrhythmias. Here we used an agent which selectively blocks Ca²⁺ influx by NCX, KB-R7943 (KBR), and assessed twitch contractions and Ca²⁺ transients in rat and guinea pig ventricular myocytes loaded with indo-1. KBR (5 M) did not alter control steady-state twitch contractions or Ca²⁺ transients at 0.5 Hz in rat, but significantly decreased them in guinea pig myocytes. When cells were Na⁺-loaded by perfusion of strophanthidin (50 M), the addition of KBR reduced diastolic [Ca²⁺]_i and abolished spontaneous Ca²⁺ oscillations. In guinea pig papillary muscles exposed to substrate-free hypoxic medium for 60 min, KBR (10 M applied 10 min before and during reoxygenation) reduced both the incidence and duration of reoxygenation-induced arrhythmias. KBR also enhanced the recovery of developed tension after reoxygenation. It is concluded that (1) the importance of Ca²⁺ influx via NCX for normal excitation-contraction coupling is species-dependent, and (2) Ca²⁺ influx via NCX may be critical in causing myocardial Ca²⁺ overload and triggered activities induced by cardiac glycoside or reoxygenation.     

Cyclic nucleotide regulation of store-operated Ca2+ influx in airway smooth muscle

Sarcoplasmic reticulum (SR) Ca2+ release and plasma membrane Ca2+ influx are key to intracellular Ca2+ ([Ca2+]i) regulation in airway smooth muscle (ASM). SR Ca2+ depletion triggers influx via store-operated Ca2+ channels (SOCC) for SR replenishment. Several clinically relevant bronchodilators mediate their effect via cyclic nucleotides (cAMP, cGMP). We examined the effect of cyclic nucleotides on SOCC-mediated Ca2+ influx in enzymatically dissociated porcine ASM cells. SR Ca2+ was depleted by 1 microM cyclopiazonic acid in 0 extracellular Ca2+ ([Ca2+]o), nifedipine, and KCl (preventing Ca2+ influx through L-type and SOCC channels). SOCC was then activated by reintroduction of [Ca2+]o and characterized by several techniques. We examined cAMP effects on SOCC by activating SOCC in the presence of 1 microM isoproterenol or 100 microM dibutryl cAMP (cell-permeant cAMP analog), whereas we examined cGMP effects using 1 microM (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO nitric oxide donor) or 100 microM 8-bromoguanosine 3',5'-cyclic monophosphate (cell-permeant cGMP analog). The role of protein kinases A and G was examined by preexposure to 100 nM KT-5720 and 500 nM KT-5823, respectively. SOCC-mediated Ca2+ influx was dependent on the extent of SR Ca2+ depletion, sensitive to Ni2+ and La3+, but not inhibitors of voltage-gated influx channels. cAMP as well as cGMP potently inhibited Ca2+ influx, predominantly via their respective protein kinases. Additionally, cAMP cross-activation of protein kinase G contributed to SOCC inhibition. These data demonstrate that a Ni2+/La3+-sensitive Ca2+ influx in ASM triggered by SR Ca2+ depletion is inhibited by cAMP and cGMP via a protein kinase mechanism. Such inhibition may play a role in the bronchodilatory response of ASM to clinically relevant drugs (e.g., beta-agonists vs. nitric oxide).     

Reverse Na+/Ca2+-exchange mediated Ca2+-entry and noradrenaline release in Na+-loaded peripheral sympathetic nerves

[³H]noradrenaline ([³H]NA) released from sympathetic nerves in the isolated main pulmonary artery of the rabbit was measured in response to field stimulation (2 Hz, 1 ms, 60 V for 3 min) in the presence of uptake blockers (cocaine, 3 × 10⁻⁵ M and corticosterone, 5 × 10⁻⁵ M). The [³H]NA-release was fully blocked by the combined application of the selective and irreversible ‘N-type’ voltage-sensitive Ca²⁺-channel (VSCC)-blocker ω-conotoxin (ω-CgTx) GVIA (10⁻⁸ M) and the ‘non-selective’ VSCC-blocker aminoglycoside antibiotic neomycin (3 × 10⁻³ M). Na⁺-loading (Na⁺-pump inhibition by K⁺-free perfusion) was required to elicit further NA-release after blockade of VSCCs (ω-CgTx GVIA + neomycin). In K⁺-free solution, in the absence of functioning VSCCs (ω-CgTx GVIA + neomycin), the fast Na⁺-channel activator veratridine (10⁻⁵ M) further potentiated the nerve-evoked release of [³H]NA. This NA-release was significantly inhibited by KB-R7943, and fully blocked by $\text{C}{\text{a}}_{\text{o}}^{2+}-\text{removal}$. However, Li⁺-substitution was surprisingly ineffective. The non-selective K⁺-channel blocker 4-aminopyridine (4-AP, 10⁻⁴ M) also further potentiated the nerve-evoked release of NA in K⁺-free solution. This potentiated release was concentration-dependently inhibited by KB-R7943, significantly inhibited by Li⁺-substitution and abolished by $\text{C}{\text{a}}_{\text{o}}^{2+}-\text{removal}$.It is concluded that in Na⁺-loaded sympathetic nerves, in which the VSCCs are blocked, the reverse Na⁺/Ca²⁺-exchange-mediated Ca²⁺-entry is responsible for transmitter release on nerve-stimulation. Theoretically we suppose that the fast Na⁺-channel and the exchanger proteins are close to the vesicle docking sites.     

Salt-sensitive hypertension is triggered by Ca2+ entry via Na+/Ca2+ exchanger type-1 in vascular smooth muscle

Excessive salt intake is a major risk factor for hypertension. Here we identify the role of Na(+)/Ca(2+) exchanger type 1 (NCX1) in salt-sensitive hypertension using SEA0400, a specific inhibitor of Ca(2+) entry through NCX1, and genetically engineered mice. SEA0400 lowers arterial blood pressure in salt-dependent hypertensive rat models, but not in other types of hypertensive rats or in normotensive rats. Infusion of SEA0400 into the femoral artery in salt-dependent hypertensive rats increases arterial blood flow, indicating peripheral vasodilation. SEA0400 reverses ouabain-induced cytosolic Ca(2+) elevation and vasoconstriction in arteries. Furthermore, heterozygous NCX1-deficient mice have low salt sensitivity, whereas transgenic mice that specifically express NCX1.3 in smooth muscle are hypersensitive to salt. SEA0400 lowers the blood pressure in salt-dependent hypertensive mice expressing NCX1.3, but not in SEA0400-insensitive NCX1.3 mutants. These findings indicate that salt-sensitive hypertension is triggered by Ca(2+) entry through NCX1 in arterial smooth muscle and suggest that NCX1 inhibitors might be useful therapeutically.     

ATP stimulation of Na+/Ca2+ exchange in cardiac sarcolemmal vesicles

In cardiacsarcolemmal vesicles, MgATP stimulatesNa⁺/Ca²⁺exchange with the following characteristics:1) increases 10-fold the apparentaffinity for cytosolic Ca²⁺;2) a Michaelis constant for ATP of~500 µM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory;4) not observed in the presence of20 µM eosin alone but reinstated when vanadate is added;5) mimicked by adenosine5'-O-(3-thiotriphosphate), without the need for vanadate, but not by ,-methyleneadenosine 5'-triphosphate; and 6) notaffected by unspecific protein alkaline phosphatase but abolished by aphosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLCeffect is counteracted by phosphatidylinositol. In addition, in theabsence of ATP,L--phosphatidylinositol4,5-bisphosphate (PIP₂) was ableto stimulate the exchanger activity in vesicles pretreated with PI-PLC.This MgATP stimulation is not related to phosphorylation of thecarrier, whereas phosphorylation appeared in the phosphoinositides,mainly PIP₂, thatcoimmunoprecipitate with the exchanger. Vesicles incubated with MgATPand no Ca²⁺ show a markedsynthesis ofL--phosphatidylinositol4-monophosphate (PIP) with little production ofPIP₂; in the presence of 1 µM Ca²⁺, the net synthesis of PIP issmaller, whereas that of PIP₂increases ninefold. These results indicate thatPIP₂ is involved in the MgATPstimulation of the cardiacNa⁺/Ca²⁺exchanger through a fast phosphorylation chain: aCa²⁺-independent PIP formationfollowed by a Ca²⁺-dependentsynthesis of PIP₂.

     

Activation of Ca2+-activated Cl- channels by store-operated Ca2+ entry in arterial smooth muscle cells does not require reverse-mode Na+/Ca2+ exchange

The main purpose of this study was to characterize the stimulation of Ca(2+)-activated Cl(-) (Cl(Ca)) by store-operated Ca(2+) entry (SOCE) channels in rabbit pulmonary arterial smooth muscle cells (PASMCs) and determine if this process requires reverse-mode Na(+)/Ca(2+) exchange (NCX). In whole-cell voltage clamped PASMCs incubated with 1 μmol/L nifedipine (Nif) to inhibit Ca(2+) channels, 30 μmol/L cyclopiazonic acid (CPA), a SERCA pump inhibitor, activated a nonselective cation conductance permeable to Na(+) (I(SOC)) during an initial 1-3 s step, ranging from-120 to +60 mV, and Ca(2+)-activated Cl(-) current (I(Cl(Ca))) during a second step to +90 mV that increased with the level of the preceding hyperpolarizing step. Niflumic acid (100 μmol/L), a Cl(Ca) channel blocker, abolished I(Cl(Ca)) but had no effect on I(SOC), whereas the I(SOC) blocker SKF-96365 (50 μmol/L) suppressed both currents. Dual patch clamp and Fluo-4 fluorescence measurements revealed the appearance of CPA-induced Ca(2+) transients of increasing magnitude with increasing hyperpolarizing steps, which correlated with I(Cl(Ca)) amplitude. The absence of Ca(2+) transients at positive potentials following a hyperpolarizing step combined with the observation that SOCE-stimulated I(Cl(Ca)) was unaffected by the NCX blocker KB-R7943 (1 μmol/L) suggest that the SOCE/Cl(Ca) interaction does not require reverse-mode NCX in our conditions.     

Cyclic GMP stimulates Na+/Ca2+ exchange in vascular smooth muscle cells in primary culture.

We examined the effect of cGMP on Na+/Ca2+ exchange in rat aortic smooth muscle cells (VSMCs) in primary culture. The intracellular Ca2+ concentration [( Ca2+]i) was raised by adding ionomycin to VSMCs incubated at high extracellular pH (pH0) (pH0 = 8.8) and high extracellular Mg2+ (Mg2+0) (Mg2+0 = 20 mM), conditions that inhibit activity of the sarcolemmal Ca2+ pump. 45Ca2+ efflux observed under these conditions was mostly extracellular Na+ (Na+0)-dependent and thus presumably catalyzed by the Na+/Ca2+ exchanger. Brief treatment of VSMCs with 8-bromo-cGMP or atrial natriuretic peptide increased this Na+0-dependent 45Ca2+ efflux by about 50%. The 8-bromo-cGMP treatment did not significantly influence total cell Na+, membrane potential, and cell pH. Conversely, when VSMCs were loaded with Na+ and then exposed to a Na+0-free medium, the rate of 45Ca2+ uptake into VSMCs increased as cell Na+ increased. Prior treatment of VSMCs with 8-bromo-cGMP accelerated 45Ca2+ uptake by up to 60% without influencing Na+ loading itself. Treatment of VSMCs with 25 microM 2,5-di-(tert-butyl)-1,4-benzohydroquinone, an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, induced a transient elevation of [Ca2+]i. 8-Bromo-cGMP stimulated the rate of recovery phase of this Ca2+ transient measured in the high pHo/high Mg2+o medium. All these results indicate that cGMP stimulates Na+/Ca2+ exchange in VSMCs.     

Mitogen-induced activation of Na+/H+ exchange in vascular smooth muscle cells involves janus kinase 2 and Ca2+/calmodulin

The sodium/proton exchanger type 1 (NHE-1) plays an important role in the proliferation of vascular smooth muscle cells (VSMC). We have examined the regulation of NHE-1 by two potent mitogens, serotonin (5-HT, 5-hydroxytryptamine) and angiotensin II (Ang II), in cultured VSMC derived from rat aorta. 5-HT and Ang II rapidly activated NHE-1 via their G protein-coupled receptors (5-HT(2A) and AT(1)) as assessed by proton microphysiometry of quiescent cells and by measurements of intracellular pH on a FLIPR (fluorometric imaging plate reader). Activation of NHE-1 was blocked by inhibitors of phospholipase C, CaM, and Jak2 but not by pertussis toxin or inhibitors of protein kinase C. Immunoprecipitation/immunoblot studies showed that 5-HT and Ang II induce phosphorylation of Jak2 and induce the formation of signal transduction complexes that included Jak2, CaM, and NHE-1. The cell-permeable Ca(2+) chelator BAPTA-AM blocked activation of Jak2, complex formation between Jak2 and CaM, and tyrosine phosphorylation of CaM, demonstrating that elevated intracellular Ca(2+) is essential for those events. Thus, mitogen-induced activation of NHE-1 in VSMC is dependent upon elevated intracellular Ca(2+) and is mediated by the Jak2-dependent tyrosine phosphorylation of CaM and subsequent increased binding of CaM to NHE-1, similar to the pathway previously described for the bradykinin B(2) receptor in inner medullary collecting duct cells of the kidney [Mukhin, Y. V., et al. (2001) J. Biol. Chem. 276, 17339-17346]. We propose that this pathway represents a fundamental mechanism for the rapid regulation of NHE-1 by G(q/11) protein-coupled receptors in multiple cell types.     

Role of Na+/H+ exchange in thrombin- and arachidonic acid-induced Ca2+ influx in platelets

Platelet activation is accompanied by an increase of cytosolic free Ca2+ concentration, [Ca2+]i, (due to both extracellular Ca2+ influx and Ca2+ movements from the dense tubular system) and an Na+ influx associated with H+ extrusion. The latter event is attributable to the activation of Na+/H+ exchange, which requires Na+ in the extracellular medium and is inhibited by amiloride and its analogs. The present study was carried out to determine whether a link exists between Ca2+ transients (measured by the quin2 method and the 45CaCl2 technique) and Na+/H+ exchange activation (studied with the pH-sensitive intracellular probe, 6-carboxyfluorescein) during platelet stimulation. Washed human platelets, stimulated with thrombin and arachidonic acid, showed: (1) a large and rapid [Ca2+]i rise, mostly due to a Ca2+ influx through the plasma membrane; (2) a marked intracellular alkalinization. Both phenomena were markedly inhibited in the absence of extracellular Na+ or in the presence of an amiloride analog (EIPA). Monensin, a cation exchanger which elicits Na+ influx and alkalinization, and NH4Cl, which induces alkalinization only, were able to evoke an increase in [Ca2+]i, mostly as an influx from the extracellular medium. Our results suggest that Ca2+ influx induced by thrombin and arachidonic acid in human platelets is strictly dependent on Na+/H+-exchange activation.     

Ca2+ influx stimulated by vasopressin is mediated by phosphoinositide hydrolysis in rat smooth muscle cells

The mechanism of Ca2+ influx stimulated by arginine vasopressin (AVP) was studied in cultured rat smooth muscle cells. AVP stimulated 45Ca2+ influx even in the presence of nifedipine, a Ca2+ antagonist that inhibits voltage-dependent Ca2+ channel. NaF, a GTP-binding protein activator, mimicked the AVP-stimulated 45Ca2+ influx. The 45Ca2+ influx stimulated by a combination of AVP and NaF was not additive. The affinity of AVP receptor was decreased by guanosine 5'-O-(3-thiotriphosphate). Pertussis toxin failed to affect the AVP-stimulated 45Ca2+ influx. AVP did not stimulate cAMP production, but increased inositol trisphosphate generation. Both AVP-stimulated 45Ca2+ influx and inositol trisphosphate generation were inhibited by neomycin, a phospholipase C inhibitor, in a dose-dependent manner, and the patterns of both inhibitions were similar. These results suggest that, in rat smooth muscle cells, AVP-stimulated Ca2+ influx is mediated exclusively through phosphoinositide hydrolysis.     

'Slow' K+-stimulated Ca2+ influx is mediated by Na+-Ca2+ exchange: a pharmacological study

K+-stimulated 45Ca2+ influx was measured in rat brain presynaptic nerve terminals that were predepolarized in a K+-rich solution for 15 s prior to addition of 45Ca2+. This 'slow' Ca2+ influx was compared to influx stimulated by Na+ removal, presumably mediated by Na+-Ca2+ exchange. The K+-stimulated Ca2+ influx in predepolarized synaptosomes, and the Na+-removal-dependent Ca2+ influx were both saturating functions of the external Ca2+ concentration; and both were half-saturated at 0.3 mM Ca2+. Both were reduced about 50% by 20 microM Hg2+, 20 microM Cu2+ or 0.45 mM Mn2+. Neither the K+-stimulated nor the Na+-removal-dependent Ca2+ influx was inhibited by 1 microM Cd2+, La3+ or Pb2+, treatments that almost completely inhibited K+-stimulated Ca2+ influx in synaptosomes that were not predepolarized. The relative permeabilities of K+-stimulated Ca2+, Sr2+ or Ba2+ influx in predepolarized synaptosomes (10:3:1) and the corresponding selectivity ratio for Na+-removal-dependent divalent cation uptake (10:2:1) were similar. These results strongly suggest that the K+-stimulated 'slow' Ca2+ influx in predepolarized synaptosomes and the Na+-removal-dependent Ca2+ influx are mediated by a common mechanism, the Na+-Ca2+ exchanger.     

Endothelin stimulates Na+/H+ exchange in vascular smooth muscle cells

The effect of endothelin (ET) on the intracellular pH (pHi) of vascular smooth muscle cells (VSMC), was investigated using a fluorescent pH indicator 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF). ET at concentrations of over 10(-9) M caused dose-dependent transient acidification followed by Na(+)-dependent and amiloride-sensitive alkalization of the cells due to stimulation of Na+/H+ exchange. The alkalization induced by ET was Ca2(+)-dependent and was inhibited by a calcium channel blocker, nicardipine. Pretreatment with H-7, an inhibitor of protein kinase C, also inhibited the ET-induced cell alkalization. These results indicate that ET stimulates Na+/H+ exchange, resulting in alkalization of VSMC and that this ET-induced cell-alkalization is probably linked to Ca2+ influx and activation of protein kinase C.     

Na+/Ca2+ exchange in coated microvesicles.

Coated microvesicles isolated from bovine neurohypophyses could be loaded with Ca2+ in two different ways, either by incubation in the presence of ATP or by imposition of an outwardly directed Na+ gradient. Na+, but not K+, was able to release Ca2+ accumulated by the coated microvesicles. These results suggest the existence of an ATP-dependent Ca2+-transport system as well as of a Na+/Ca2+ carrier in the membrane of coated microvesicles similar to that present in the membranes of secretory vesicles from the neurohypophysis. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ of the ATP-dependent uptake was 0.8 microM. The average Vmax. was 2 nmol of Ca2+/5 min per mg of protein. The total capacity of microvesicles for Ca2+ uptake was 3.7 nmol/mg of protein. Both nifedipine (10 microM) and NH4Cl (50 mM) inhibited Ca2+ uptake. The ATPase activity in purified coated-microvesicles fractions from brain and neurohypophysis was characterized. Micromolar concentrations of Ca2+ in the presence of millimolar concentrations of Mg2+ did not change enzyme activity. Ionophores increasing the proton permeability across membranes activated the ATPase activity in preparations of coated microvesicles from brain as well as from the neurohypophysis. Thus the enzyme exhibits properties of a proton-transporting ATPase. This enzyme seems to be linked to the ion accumulation by coated microvesicles, although the precise coupling of the proton transport to Ca2+ and Na+ fluxes remains to be determined.