H+ extrusion by an apical vacuolar-type H+-ATPase in rat renal proximal tubules

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The C-terminal 165 amino acids of the plasma membrane Ca(2+)-ATPase confer Ca2+/calmodulin sensitivity on the Na+,K(+)-ATPase alpha-subunit.

The C-terminal 165 amino acids of the rat brain plasma membrane (PM) Ca(2+)-ATPase II containing the calmodulin binding auto-inhibitory domain was connected to the C-terminus of the ouabain sensitive chicken Na+,K(+)-ATPase alpha 1 subunit. Expression of this chimeric molecule in ouabain resistant mouse L cells was assured by the high-affinity binding of [3H]ouabain. In the presence of Ca2+/calmodulin, this chimeric molecule exhibited ouabain inhibitable Na+,K(+)-ATPase activity; the putative chimeric ATPase activity was absent in the absence of Ca2+/calmodulin and activated by Ca2+/calmodulin in a dose-dependent manner. Furthermore, this chimeric molecule could bind monoclonal IgG 5 specific to the chicken Na+,K(+)-ATPase alpha 1 subunit only in the presence of Ca2+/calmodulin, suggesting that the epitope for IgG 5 in this chimera is masked in the absence of Ca2+/calmodulin and uncovered in their presence. These results propose a direct interaction between the calmodulin binding auto-inhibitory domain of the PM Ca(2+)-ATPase and the specific regions of the Na+,K(+)-ATPase alpha 1 subunit that are structurally homologous to the PM Ca(2+)-ATPase. A comparison of the deduced amino acid sequences revealed several possible regions within the Na+,K(+)-ATPase that might interact with the auto-inhibitory domain of the PM Ca(2+)-ATPase.     

The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.     

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.     

Adenosine triphosphatases during maturation of cultured human skeletal muscle cells and in adult human muscle.

Na+/K(+)-ATPase, Mg(2+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase are examined in cultured human skeletal muscle cells of different maturation grade and in human skeletal muscle. Na+/K(+)-ATPase is investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase). SR Ca(2+)-ATPase is examined by ELISA, Ca(2+)-dependent phosphorylation and its activities on ATP and 3-O-methylfluorescein phosphate. Na+/K(+)-ATPase and SR Ca(2+)-ATPase are localized by immunocytochemistry. The activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase show a good correlation with the other assayed parameters of these ion pumps. All ATPase parameters investigated increase with the maturation grade of the cultured muscle cells. The number of ouabain-binding sites and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-MFPase are significantly higher in cultured muscle cells than in muscle. The Mg(2+)-ATPase activity, the content of SR Ca(2+)-ATPase and the activities of SR Ca(2+)-ATPase and Ca(2+)-dependent 3-O-MFPase remain significantly lower in cultured cells than in muscle. The ouabain-binding constant and the molecular activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase are equal in muscle and cultured cells. During ageing of human muscle the activity as well as the concentration of SR Ca(2+)-ATPase decrease. Thus the changes of the activities of the ATPases are caused by variations of the number of their molecules. Na+/K(+)-ATPase is localized in the periphery of fast- and slow-twitch muscle fibers and at the sarcomeric I-band. SR Ca(2+)-ATPase is predominantly confined to the I-band, whereas fast-twitch fibers are much more immunoreactive than slow-twitch fibers. The presence of cross-striation for Na+/K(+)-ATPase and SR Ca(2+)-ATPase in highly matured cultured muscle cells indicate the development and subcellular organization of a transverse tubular system and SR, respectively, which resembles the in vivo situation.     

Evidence against parallel operation of sodium/calcium antiport and ATP-driven calcium transport in plasma membrane vesicles from kidney tubule cells

The present study aimed to clarify the existence of a Na+/Ca2+ antiport device in kidney tubular epithelial cells discussed in the literature to represent the predominant mechanistic device for Ca2+ reabsorption in the kidney. Inside-out oriented plasma membrane vesicles from tubular epithelial cells of guinea-pig kidney showed an ATP-driven Ca2+ transport machinery similar to that known to reside in the plasma membrane of numerous cell types. It was not affected by digitalis compounds which otherwise are well-documented inhibitors of Ca2+ reabsorption. The vesicle preparation contained high, digitalis-sensitive (Na+ + K+)-ATPase activities indicating its origin from the basolateral portion of plasma membrane. The operation of a Na+/Ca2+ antiport device was excluded by the findings that steep Ca2+ gradients formed by ATP-dependent Ca2+ accumulation in the vesicles were not discharged by extravesicular Na+, and did not drive 45Ca2+ uptake into the vesicles via a Ca2+-45Ca2+ exchange. The ATP-dependent Ca2+ uptake into the vesicles became increasingly depressed with time by extravesicular Na+. This was not due to an impairment of the Ca2+ pump itself, but caused by Na+/Ca2+ competition for binding sites on the intravesicular membrane surface shown to be important for high Ca2+ accumulation in the vesicles. Earlier observations on Na+-induced release of Ca2+ from vesicles pre-equilibrated with Ca2+, seemingly favoring the existence of a Na+/Ca2+ antiporter in the basolateral plasma membrane, were likewise explained by the occurrence of Na+/Ca2+ competition for binding sites. The weight of our findings disfavors the transcellular pathway of Ca2+ reabsorption through tubule epithelium essentially depending on the operation of a Na+/Ca2+ antiport device.     

Ion metabolism in malaria-infected erythrocytes

Malaria parasites of the genus Plasmodium spend much of their asexual life cycle inside the erythrocytes of their vertebrate hosts. Parasites presumably have to exploit metabolic and transport mechanisms to adapt themselves to the host erythrocyte's physicochemical environment. This review surveys the metabolism and transport of Ca2+, alkali cations, and H+ in malaria-infected erythrocytes. The Ca2+ content of Plasmodium-infected erythrocytes increases as the parasite matures. An increase in the influx of extracellular Ca2+ into infected erythrocytes is evident at later stages of parasite development. In infected erythrocytes, Ca2+ is almost exclusively localized in the parasite compartment and changes but little in the cytosol of the host cell. The importance of Ca2+ in supporting the growth of intraerythrocytic parasites and the invasion of erythrocytes by the merozoite has been assessed by depletion of extracellular Ca2+ with chelators, or by disturbance of the metabolism and transport of Ca2+ with a variety of Ca2+ modulators. Membranes of malaria-infected erythrocytes change their permeability to alkali cations. Hence, levels of K+ decrease and levels of Na+ increase in the cytosol of infected erythrocytes. Intraerythrocytic parasites maintain a high K+, low Na+ state, suggesting a mechanism for transporting K+ inward and Na+ outward against concentration gradients of the alkali cations across the parasite plasma membrane and/or the parasitophorous vacuole membrane (PVM). Concomitantly, P. falciparum can grow in Na(+)-enriched human erythrocytes. Experimental evidence suggests that Plasmodium possesses in its plasma membrane a proton pump which is very sensitive to orthovanadate, carbonylcyanide m-chlorophenylhydrazone, a protonophore, and dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, but is only slightly sensitive to inhibitors of bacterial and mitochondrial respiration, such as antimycin A, CN-, or N3-, and ouabain, a Na+, K(+)-ATPase inhibitor. By operating this proton pump, parasites extrude H+ and thus generate an electrochemical gradient of protons (an internal negative membrane potential and a concentration gradient of protons) across the parasite plasma membrane. The electrochemical gradient apparently drives inward movement of Ca2+ and, possibly, glucose from the cytosol of infected erythrocytes. Little is known about the transport properties of the PVM. Recent sequence studies suggest that Plasmodium contains a cation-transporting ATPase which exhibits a high homology to the Ca2(+)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of the Na+/H+ exchange system in cardiac cells in relation to the control of the internal Na+ concentration. A molecular basis for the antagonistic effect of ouabain and amiloride on the heart

Cardiac cells in culture (from rat and chick heart) have a membrane Na+/H+ exchange system that is inhibited by amiloride (K0.5 = 5 microM) and by its more potent N-5-disubstituted derivatives dimethylamiloride (K0.5 = 300 nM) and ethylisopropylamiloride (K0.5 = 30 nM). The properties of the cardiac Na+/H+ exchange system are similar to those found for the Na+/H+ exchanger in other cellular types. The Na+/H+ exchange system is a major pathway for Na+ uptake by cardiac cells. Ouabain which inhibits the (Na+,K+)-ATPase, a major pathway for Na+ efflux, is known to provoke Na+ accumulation and to stimulate 45Ca2+ entry via the Na+/Ca2+ exchange mechanism, thereby producing an inotropic effect. N-5-Disubstituted amiloride derivatives, by blocking Na+ entry into cardiac cells, antagonize both ouabain-induced intracellular Na+ accumulation and the ouabain-induced acceleration of 45Ca2+ uptake.     

Regulation of Na+-K+-ATPase: effect of Mg and Ca ions

The participation of Mg2+ and Ca2+ in complicated mechanisms of Na+, K(+)-ATPase regulation is discussed in the survey. The regulatory actions of Mg2+ on Na+, K(+)-ATPase such as its participation in phosphorylation and dephosphorylation of the enzyme, ADP/ATP-exchange inhibition, cardiac glycosides and vanadate binding with the enzyme, conformational changes induction during ATPase cycle are reviewed in detail. Some current views of mechanisms of above mentioned Mg2+ regulatory effects are discussed. The experimental evidence of Ca2+ immediate influence on the functional activity of Na+, K(+)-ATPase (catalytic, transport and glycoside-binding) are given. It's noted that these effects are based on the conformational changes in the enzyme and also on the phase transition in membrane induced by Ca2+. Unimmediate action of Ca2+ on Na+, K(+)-ATPase is also discussed, especially due to its effect on other membrane systems functionally linked with Na(+)-pump (for instance, due to Na+/Ca(+)-exchanger activation). It's concluded that Mg2+ and Ca2+ as "universal regulators" of the cell effectively influence the functional activity and conformational states of Na+, K(+)-ATPase.     

The effect of alkanols on Ca2+ transport in brain mitochondria.

Ethanol stimulates the Na(+)-dependent Ca2+ efflux in brain mitochondria and inhibits the Na(+)-independent Ca(2+)-efflux. Here, we studied the effects of n-alkanols on the various Ca2+ transport processes in brain mitochondria. Only short-chain alcohols (i.e. methanol, ethanol and propanol) stimulated Na+/Ca2+ exchange. The inhibition of H+/Ca2+ exchange was significant only with ethanol. Short-chain alcohols inhibit while long-chain alcohols activate the cyclosporin-sensitive Ca(2+)-efflux. These data suggest that the mechanism of the alkanols' effects on Na+/Ca2+ exchange, H+/Ca2+ exchange and the cyclosporin sensitive pore are entirely different. Alkanols have no effect on the electrogenic Ca2+ uniporter. Ethanol did not affect the apparent K0.5 for Na+ (7.5 mM) of the Na+/Ca2+ exchange. Similarly, the magnitude of the effect of ethanol did not depend on matrix Ca2+ concentration, suggesting that short-chain alkanols do not stimulate the rate of Na+/Ca2+ exchange by increasing the affinity of the carrier to Ca2+in or Na+out. High concentrations of K+, Mg2+ and Ca2+ enhanced the ethanol effect. It is possible that high surface potential attenuates the effect of ethanol. It is suggested that ethanol stimulation of Na+/Ca2+ exchange depends on the modulation of the surface dielectric constant.     

A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.     

Relationship of hepatic cholate transport to regulation of intracellular pH and potassium.

Modulation of hepatic cholate transport by transmembrane pH-gradients and during interferences with the homeostatic regulation of intracellular pH and K+ was studied in the isolated perfused rat liver. Within the concentration range studied uptake into the liver was saturable and appeared to be associated with release of OH- and uptake of K+. Perfusate acidification ineffectually stimulated uptake. Application of NH4Cl caused intracellular alkalinization, release of K+ and stimulation of cholate uptake, withdrawal of NH4Cl resulted in intracellular acidification, regain of K+ and inhibition of cholate uptake. Inhibition of Na+/H(+)-exchange with amiloride reduced basal release of acid equivalents into the perfusate, initiated K(+)-release, and inhibited both, control cholate uptake and its recovery following intracellular acidification. K(+)-free perfusion caused K(+)-release and inhibited cholate uptake. K(+)-readmission resulted in brisk K(+)-uptake and recovery of cholate transport. Both effects were inhibited by amiloride. Interference with cholate transport through modulation of pH homeostasis by diisothiocyanostilbenedisulfonate (DIDS) could not be demonstrated because DIDS affected bile acid transport directly. Biliary bile acid secretion was stimulated by intracellular alkalinization and by activation of K(+)-transport. Uncoupling of the mutual interference between pH-dependent cholate uptake and K(+)-transport by amiloride indicates tertiary active transport of cholate. In this, Na+/K(+)-ATPase provides the transmembrane Na(+)-gradient to sustain Na+/H(+)-exchange which maintains the transmembrane pH-gradient and thus supports cholate uptake. Effects of canalicular bile acid secretion are consistent with a saturable, electrogenic transport.     

Insulin stimulates the translocation of Na+/K(+)-dependent ATPase molecules from intracellular stores to the plasma membrane in frog skeletal muscle.

The mechanism of the stimulation of Na+/K+ transport by insulin in frog skeletal muscle was studied. The ouabain-binding capacity in detergent-treated plasma membranes of insulin-exposed muscles was increased 1.9-fold compared with that of controls. Na+/K(+)-ATPase activity was found in an intracellular 'light fraction' (fraction II) prepared by using anion-exchange chromatography. Marker enzyme activities for plasma and Golgi membranes were not detected in this fraction. The specific activity of Na+/K(+)-ATPase in fraction II from insulin-exposed muscles was 58% of that in an identical fraction from control muscles. No significant difference in the protein yield of the plasma membrane preparation was observed between these two groups. In parallel with the decrease in the Na+/K(+)-ATPase activity in fraction II from insulin-exposed muscles, the ouabain-binding capacity in this fraction was also decreased. The addition of saponin to fraction II increased both Na+/K(+)-ATPase activity and ouabain binding, indicating that some of the Na+/K(+)-ATPase is located in sealed vesicles. These findings support the view that insulin stimulates the translocation of Na+/K(+)-ATPase molecules from fraction II to the plasma membrane.     

Evidence from 23Na NMR studies for the existence of sodium-channels in the brush border membrane of the renal proximal tubule

A fast Na+-exchange is shown to be present in isolated renal brush border membranes. The lower limit of the rate constant for this process, calculated from the 23Na-NMR spectrum is 580 sec-1. The actual exchange rate may be higher. A fast 7Li exchange is also shown to be present in the isolated membrane vesicles. The characteristic overshoot of the Na+ dependent D-glucose cotransport and Na+/H+ antiport can be demonstrated. The fact that neither treatment with papain, nor lowering of the temperature to 5 degrees C affected the 23Na-NMR spectra obtained in the renal brush border membrane vesicles is consistent with the possibility that the fast Na+-exchange occurs through a channel mechanism.     

The Na+-K+-ATPase as self-adhesion molecule and hormone receptor

Thanks to the homeostasis of the internal milieu, metazoan cells can enormously simplify their housekeeping efforts and engage instead in differentiation and multiple forms of organization (tissues, organs, systems) that enable them to produce an astonishing diversity of mammals. The stability of the internal milieu despite drastic variations of the external environment (air, fresh or seawater, gastrointestinal fluids, glomerular filtrate, bile) is due to transporting epithelia that can adjust their specific permeability to H(2)O, H(+), Na(+), K(+), Ca(2+), and Cl(-) over several orders of magnitude and exchange substances with the outer milieu with exquisite precision. This exchange is due to the polarized expression of membrane proteins, among them Na(+)-K(+)-ATPase, an oligomeric enzyme that uses chemical energy from ATP molecules to translocate ions across the plasma membrane of epithelial cells. Na(+)-K(+)-ATPase presents two types of asymmetries: the arrangement of its subunits, and its expression in one pole of the epithelial cell ("polarity"). In most epithelia, polarity consists of the expression of Na(+)-K(+)-ATPase towards the intercellular space and arises in part from the interaction of the extracellular segment of the β-subunit with another β-subunit present in a Na(+)-K(+)-ATPase molecule expressed by a neighboring cell. In addition to enabling the Na(+)-K(+)-ATPase to transport ions and water vectorially, this position exposes its receptors to ouabain and analogous cardiotonic steroids, which are present in the internal milieu because these were secreted by endocrine cells.     

Na(+), Cl(-), Ca(2+) and Zn(2+) transport by fish gills: retrospective review and prospective synthesis

The secondary active Cl(-) secretion in seawater (SW) teleost fish gills and elasmobranch rectal gland involves basolateral Na(+),K(+)-ATPase and NKCC, apical membrane CFTR anion channels, and a paracellular Na(+)-selective conductance. In freshwater (FW) teleost gill, the mechanism of NaCl uptake is more controversial and involves apical V-type H(+)-ATPase linked to an apical Na(+) channel, apical Cl(-)-HCO-3 exchange and basolateral Na(+),K(+)-ATPase. Ca(2+) uptake (in FW and SW) is via Ca(2+) channels in the apical membrane and Ca(2+)-ATPase in the basolateral membrane. Mainly this transport occurs in mitochondria rich (MR) chloride cells, but there is a role for the pavement cells also. Future research will likely expand in two major directions, molded by methodology: first in physiological genomics of all the transporters, including their expression, trafficking, operation, and regulation at the molecular level, and second in biotelemetry to examine multivariable components in behavioral physiological ecology, thus widening the integration of physiology from the molecular to the environmental levels while deepening understanding at all levels.     

Characterization of calcium transport by basal plasma membranes from human placental syncytiotrophoblast.

We have studied the mechanisms involved in calcium (Ca2+) transport through the basal plasma membranes (BPM) of the syncytiotrophoblast cells from full-term human placenta. These purified membranes were enriched 25-fold in Na+/K(+)-adenosine triphosphate (ATPase), 37-fold in [3H] dihydroalprenolol binding sites, and fivefold in alkaline phosphatase activity compared with the placenta homogenates. In the absence of ATP and Mg2+, a basal Ca2+ uptake was observed, which followed Michaelis-Menten kinetics, with a Km Ca2+ of 0.18 +/- 0.05 microM and Vmax of 0.93 +/- 0.11 nmol/mg/min. The addition of Mg2+ to the incubation medium significantly decreased this uptake in a concentration-dependent manner, with a maximal inhibition at 3 mM Mg2+ and above. The Lineweaver-Burk plots of Ca2+ uptake in the absence and in the presence of 1 mM Mg2+ suggest a noncompetitive type of inhibition. Preloading the BPM vesicles with 5 mM Mg2+ had no significant effect on Ca2+ uptake, eliminating the hypothesis of a Ca2+/Mg2+ exchange mechanism. This ATP-independent Ca2+ uptake was not sensitive to 10(-6) M nitrendipine nor to 10(-4) M verapamil. An ATP-dependent Ca2+ transport was also detected in these BPM, whose Km Ca2+ was 0.09 +/- 0.02 microM and Vmax 3.4 +/- 0.2 nmoles/mg/3 min. This Ca2+ transport requires Mg2+, the optimal concentration of Mg2+ being approximately 1 mM. Preincubation of the membrane with 10(-6) M calmodulin strongly enhanced the initial ATP-dependent Ca2+ uptake. Finally, no Na+/Ca2+ exchange process could be demonstrated.     

Activation of sodium-proton exchange is not a prerequisite for Ca2+ mobilization and aggregation in human platelets

Recently it has been suggested [(1987) Nature 325, 456-458; (1987) FEBS Lett. 212, 123-126] that the activation of Na+/H+ exchange is a prerequisite for platelet aggregation and the development of the Ca2+ signal. As direct evidence for the role of the Na+/H+-exchange pathway the inhibition of the Ca2+ signal by EIPA, a specific inhibitor of Na+/H+ exchange, was offered. Here we demonstrate that low concentrations of EIPA (below 1 microM) completely block Na+/H+ exchange while EIPA inhibits aggregation or Ca2+ mobilization only in concentrations 100-times greater than 1 microM. Moreover, another amiloride analogue, CBDMB, developed to act predominantly on Na+/Ca2+ exchange, does not affect Na+/H+ exchange in platelets but blocks aggregation and Ca2+ mobilization. We conclude that while Na+/H+ exchange has a fundamental role in platelet functions it is not prerequisite for the development of Ca2+ signal and aggregation.     

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.