Sodium influx rate and ouabain-sensitive rubidium uptake in isolated guinea pig atria.

1. Ouabain-sensitive 86Rb+ uptake by tissue preparations has been used as an estimate of Na+ pump activity. This uptake, however, may be a measure of the Na+ influx rate, rather than capacity of the Na+ pump, since intracellular Na+ concentration is a determinant of the active Na+/Rb+ exchange reaction under certain conditions. This aspect was examined by studying the effect of altered Na+ influx rate on ouabain-sensitive 86Rb+ uptake in atrial preparations of guinea pig hearts. 2. Electrical stimulation markedly enhanced ouabain-sensitive 86Rb+ uptake without affecting nonspecific, ouabain-insensitive uptake. Paired-pulse stimulation studies indicate that the stimulation-induced enhancement of 86Rb+ uptake is due to membrane depolarizations, and hence related to the rate of Na+ influx. 3. Alterations in the extracellular Ca2+ concentration failed to affect the 86Rb+ uptake indicating that the force of contraction does not influence 86Rb+ uptake. 4. Reduced Na+ influx by low extracellular Na+ concentration decreased 86Rb+ uptake, and an increased Na+ influx by a Na+-specific ionophore, monensin, enhanced 86Rb+ uptake in quiescent atria. 5. Grayanotoxins, agents that increase transmembrane Na+ influx, and high concentrations of monensin appear to have inhibitory effects on ouabain-sensitive 86Rb+ uptake in electrically stimulated and in quiescent atria. 6. Electrical stimulation or monensin enhanced ouabain binding to (Na+ + K+)-ATPase and also increased the potency of ouabain to inhibit 86Rb+ uptake indicating that the intracellular Na+ available to the Na+ pump is increased under these conditions. 7. The ouabain-sensitive 86Rb+ uptake in electrically stimulated atria was less sensitive to alterations in the extracellular Na+ concentration, temperature and monensin than that in quiescent atria. 8. These results indicate that the rate of Na+ influx is the primary determinant of ouabain-sensitive 86Rb+ uptake in isolated atria. Electrical stimulation most effectively increases the Na+ available to the Na+ pump system. The ouabain-sensitive 86Rb+ uptake by atrial preparations under electrical stimulation at a relatively high frequency seems to represent the maximal capacity of the Na+ pump in this tissue.     

Na entry and Na-K pump activity in murine,hamster, and human cells - effect of monensin,serum, platelet extract,and viral transformation

The relationship between Na entry and the activity of the Na-K pump has been investigated in a variety of cell types by testing the effect of the Naionophore monensin, mitogenic stimulation with serum and oncogenic transformation by SV₄₀ and polyoma virus. We found that addition of monensin increases intracellular Na in quiescent cultures of murine, hamster, and human cells. In each case, the rise in intracellular Na by monensin is associated with an increase in the activity of the Na-K pump, which was measured as ouabain-inhibitable ⁸⁶Rb uptake. The addition of serum to quiescent cultures stimulates ⁸⁶Rb uptake in all cell types studied. Serum alone causes an increase in intracellular potassium with no consistent change in intracellular Na. In the presence of the Na-K pump inhibitor ouabain, serum causes a marked increase in intracellular Na, with little change in intracellular K. This pattern is interpreted as indicating that the primary effect of serum is to increase Na entry into the cells. A low concentration of monensin (0.2 μg/ml) mimics the effect of serum on ion fluxes and content, which supports the conclusion that serum and monensin stimulate ⁸⁶Rb uptake in the same manner, namely by increasing Na entry into the cells. In addition, a partially purified platelet extract stimulates Na entry and ⁸⁶Rb uptake in quiescent 3T3 cells. Finally 3T3 cells transformed by SV₄₀ or polyoma virus exhibit a higher rate of Na entry and of Na-K pump activity than their untransformed 3T3 counterparts. All these results indicate that the rate of Na entry plays an important role in the regulation of the activity of the Na-K pump and that an increase in Na and K movements is a rapid response elicited by serum in a variety of cell types.     

Control by calcium of protein discharge and membrane permeability to potassium in the rat lacrimal gland

Discharge of protein from slices of rat exorbital lacrimal gland was stimulated by 10^?5 M carbachol. This response was blocked by 10^?4 M atropine or by the omission of extracellular calcium. In the latter case, secretion could be restored by the reintroduction of calcium to the medium. Carbachol (10^?5 M) also stimulated the release of ⁸⁶Rb (a marker for potassium) from the slices. This effect was completely blocked by 10^?4 M atropine. The initial transient release of ⁸⁶Rb was only partially inhibited by Ca removal, but the later sustained phase of release was completely blocked. As with protein secretion, this effect of Ca removal could be reversed by re-introduction of Ca to the medium. It is concluded that activation of cholinergic receptors in the lacrimal gland stimulates protein discharge and increases potassium permeability by mechanisms utilizing extracellular calcium ions.     

Analysis of angiotensin-stimulated sodium transport in cultured smooth muscle cells from rat aorta

Angiotensin peptides (AI, AII, AIII) increased the rate of Na+ accumulation by smooth muscle cells (SMC) cultured from rat aorta. The stimulatory effect of AII on Na+ uptake was observed when Na+ exodus via the Na+/K+ pump was blocked either by ouabain or by the removal of extracellular K+. AII was at least ten times more potent than AIII and about 100 times more potent than AI in stimulating Na+ uptake. Saralasin had little effect on Na+ uptake by itself but almost completely blocked the increase caused by AII. The stimulation of net Na+ entry by AI, but not AII, was prevented by protease inhibitors. The stimulation of Na+ uptake was almost completely blocked by amiloride. Tetrodotoxin, which prevented veratridine from increasing Na+ uptake, had no effect on the response to AII. Angiotensin increased the rate of ouabain-sensitive 86Rb+ uptake (Na+/K+ pump activity) but had no effect on ouabain-sensitive ATPase activity in frozen-thawed SMC or in microsomal membranes isolated from cultured SMC. The stimulation of ouabain-sensitive 86Rb+ uptake by AII was blocked by saralasin. Omitting Na+ from the external medium prevented AII from increasing 86Rb+ uptake. AII had no effect on cell volume or cyclic AMP levels in the cultured SMC. These results suggest that angiotensin peptides activate an amiloride-sensitive Na+ transporter which supplies the Na+/K+ pump with more Na+, its rate-limiting substrate.     

Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR- 106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3- h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Agonist-specific regulation of [Na+]i in pancreatic acinar cells

In a companion paper (Zhao, H., and S. Muallem. 1995), we describe the relationship between the major Na+,K+, and Cl- transporters in resting pancreatic acinar cells. The present study evaluated the role of the different transporters in regulating [Na+]i and electrolyte secretion during agonist stimulation. Cell stimulation increased [Na+]i and 86Rb influx in an agonist-specific manner. Ca(2+)-mobilizing agonists, such as carbachol and cholecystokinin, activated Na+ influx by a tetraethylammonium-sensitive channel and the Na+/H+ exchanger to rapidly increase [Na+]i from approximately 11.7 mM to between 34 and 39 mM. As a consequence, the NaK2Cl cotransporter was largely inhibited and the activity of the Na+ pump increased to mediate most of the 86Rb(K+) uptake into the cells. Secretin, which increases cAMP, activated the NaK2Cl cotransporter and the Na+/H+ exchanger to slowly increase [Na+]i from approximately 11.7 mM to an average of 24.6 mM. Accordingly, secretin increased total 86Rb uptake more than the Ca(2+)- mobilizing agonists and the apparent coupling between the NaK2Cl cotransport and the Na+ pump. All the effects of secretin could be attributed to an increase in cAMP, since forskolin affected [Na+]i and 86Rb fluxes similar to secretin. The signaling pathways mediating the effects of the Ca(2+)-mobilizing agonists were less clear. Although an increase in [Ca2+]i was required, it was not sufficient to account for the effect of the agonists. Activation of protein kinase C stimulated the NaK2Cl cotransporter to increase [Na+]i and 86Rb fluxes without preventing the inhibition of the cotransporter by Ca(2+)-mobilizing agonists. The effects of the agonists were not mediated by changes in cell volume, since cell swelling and shrinkage did not reproduce the effect of the agonists on [Na+]i and 86Rb fluxes. The overall findings of the relationships between the various Na+,K+, and Cl- transporters in resting and stimulated pancreatic acinar cells are discussed in terms of possible models of fluid and electrolyte secretion by these cells.     

Effect of extracellular sodium removal upon 86Rb outflow from pancreatic islet cells

The present study was undertaken to characterize the effect of extracellular Na+ removal on 86Rb outflow from perifused rat pancreatic islets. Complete Na+ omission inhibited 86Rb outflow whether the islets were perifused in the presence or in the absence of extracellular Ca2+. Ouabain (1 mM) did not reduce the inhibitory effect of Na+ deprivation, whilst diphenylhydantoin (72.9 microM) mimicked the Na+-removal-induced fall in 86Rb outflow. Glucose (16.7 mM) lost its capacity to inhibit 86Rb outflow when the perifusate was deprived of extracellular Na+. These results indicate that Na+ omission reproduces the inhibitory effect of glucose on 86Rb outflow. The reduction in 86Rb outflow recorded after Na+ deprivation could be mediated by an intracellular acidification and/or a decrease in the intracellular Na+ activity. It is tempting to speculate that the capacity of glucose to reduce the B-cell Na+ content may participate in the process by which the sugar decreases K+ permeability.     

Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. II. Changes in Na+ and Ca2+ fluxes, Na+/K+ pump activity, and intracellular pH

The amphibian tetradecapeptide, bombesin, and structurally related peptides caused a marked increase in ouabain-sensitive 86Rb+ uptake (a measure of Na+/K+ pump activity) in quiescent Swiss 3T3 cells. This effect occurred within seconds after the addition of the peptide and appeared to be mediated by an increase in Na+ entry into the cells. The effect of bombesin on Na+ entry and Na+/K+ pump activity was concentration dependent with half-maximal stimulation occurring at 0.3-0.4 nM. The structurally related peptides litorin, gastrin-releasing peptide, and neuromedin B also stimulated ouabain-sensitive 86Rb+ uptake; the relative potencies of these peptides in stimulating the Na+/K+ pump were comparable to their potencies in increasing DNA synthesis (Zachary, I., and E. Rozengurt, 1985, Proc. Natl. Acad. Sci. USA., 82:7616-7620). Bombesin increased Na+ influx, at least in part, through an Na+/H+ antiport. The peptide augmented intracellular pH and this effect was abolished in the absence of extracellular Na+. In addition to monovalent ion transport, bombesin and the structurally related peptides rapidly increased the efflux of 45Ca2+ from quiescent Swiss 3T3 cells. This Ca2+ came from an intracellular pool and the efflux was associated with a 50% decrease in total intracellular Ca2+. The peptides also caused a rapid increase in cytosolic free calcium concentration. Prolonged pretreatment of Swiss 3T3 cells with phorbol dibutyrate, which causes a loss of protein kinase C activity (Rodriguez-Pena, A., and E. Rozengurt, 1984, Biochem. Biophys. Res. Commun., 120:1053-1059), greatly decreased the stimulation of 86Rb+ uptake and Na+ entry by bombesin implicating this phosphotransferase system in the mediation of part of these responses to bombesin. Since some activation of monovalent ion transport by bombesin was seen in phorbol dibutyrate-pretreated cells, it is likely that the peptide also stimulates monovalent ion transport by a second mechanism.     

Insulin action on cardiac glucose transport. Studies on the role of the Na+/K+ pump

Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na+/K+ pump. 86Rb+-uptake by cardiac cells was found to be linear up to 2 min with a steady-state reached by 40-60 min, and was used to monitor the activity of the Na+/K+ pump. Ouabain (10(-3) mol/l) inhibited the steady-state uptake of 86Rb+ by more than 90%. Both, the ouabain-sensitive and ouabain-insensitive 86Rb+-uptake by cardiac cells were found to be unaffected by insulin treatment under conditions where a significant stimulation of 3-O-methylglucose transport occurred. 86Rb+-uptake was markedly reduced by the presence of calcium and/or magnesium, but remained unresponsive towards insulin treatment. Inhibition of the Na+/K+ pump activity by ouabain and a concomitant shift in the intracellular Na+ :K+ ratio did not affect basal or insulin stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na+/K+ pump and the glucose transport system.     

Effects of Ca2+ on the sodium pump observed in cardiac myocytes isolated from guinea pigs

It is presently unknown whether Ca2+ plays a role in the physiological control of Na+/K+-ATPase or sodium pump activity. Because the enzyme is exposed to markedly different intra- and extracellular Ca2+ concentrations, tissue homogenates or purified enzyme preparations may not provide pertinent information regarding this question. Therefore, the effects of Ca2+ on the sodium pump were examined with studies of [3H]ouabain binding and 86Rb+ uptake using viable myocytes isolated from guinea-pig heart and apparently maintaining ion gradients. In the presence of K+, a reduction of the extracellular Ca2+ increased specific [3H]ouabain binding observed at apparent binding equilibria: a half-maximal stimulation was observed when extracellular Ca2+ was lowered to about 50 microM. The change in [3H]ouabain binding was caused by a change in the number of binding sites accessible by ouabain instead of a change in their affinity for the glycoside. Ouabain-sensitive 86Rb+ uptake was increased by a reduction of extracellular Ca2+ concentration. Benzocaine in concentrations reported to reduce the rate of Na+ influx failed to influence the inhibitory effect of Ca2+ on glycoside binding. When [3H]ouabain binding was at equilibrium, the addition of Ca2+ decreased and that of EGTA increased the glycoside binding. Mn2+, which does not penetrate the cell membrane, had effects similar to Ca2+. In the absence of K+, cells lose their tolerance to Ca2+. Reducing Ca2+ concentration prevented the loss of rod-shaped cells but failed to affect specific [3H]ouabain binding observed in the absence of K+. These results indicate that a large change in extracellular Ca2+ directly affects the sodium pump in cardiac myocytes isolated from guinea pigs.     

Formyl peptide stimulation of superoxide anion release from lung macrophages: sodium and potassium involvement

We examined the role of the monovalent cations Na+ and K+ in the events encompassing the release of O-2 by alveolar macrophages after stimulation with formyl methionyl phenylalanine (FMP). This was accomplished by determining the effect of changing the extracellular [Na+] and/or [K+] on FMP-stimulated O-2 production; and measuring 22Na+, 42K+ and 86Rb+ influx and efflux and intracellular [K+] for control and FMP-stimulated alveolar macrophages. Stimulated O-2 production was relatively insensitive to changes in extracellular K+ or Na+ concentrations until the [Na+] was decreased below 35 mM. At 4 mM [Na+], the rate of O-2 production remained at 75% of the maximal rate observed at physiological concentrations of [Na+]. Both influx and efflux of 22Na+ were stimulated above control rates by FMP. The increased rates of fluxes lasted for a few minutes suggesting a transient increase in membrane permeability to Na+. Ouabain partially inhibited 22Na+ efflux but had no effect on O-2 release. The influx of 86Rb+ and 42K+ was not altered by the addition of FMP but was virtually abolished in the presence of 10 microM ouabain or 1 mM quinine. In the presence of extracellular calcium, FMP-stimulated a prolonged (greater than 20 minutes) increase in 86Rb+ or 42K+ efflux which was inhibitable by 1 mM quinine. In the absence of extracellular calcium, FMP stimulation of K+ efflux was greatly diminished and was not affected by quinine, although quinine still inhibited O-2 production under these conditions. It was also observed that there was a loss of intracellular K+ when cells were stimulated by FMP in the presence of Ca+2, but not in the absence of Ca+2. Taken together, these results suggest a minimal direct role, if any, for K+ in the events that lead to FMP-stimulated O-2 release by alveolar macrophages.     

Stimulation of 45Ca efflux from rat lacrimal gland slices by carbachol and epinephrine

The effects of carbachol (10^?5M) and epinephrine (10^?5M) on efflux of ⁴⁵Ca from rat exorbital lacrimal gland slices were examined. Both carbachol and epinephrine stimulated a transient release of ⁴⁵Ca from the tissue. The quantity of Ca released was estimated to be of the order of 0.5 μmol/g. Release of ⁴⁵Ca by one agonist prevented subsequent release of ⁴⁵Ca by a different agonist. These data support the hypothesis put forth previously that in the lacrimal gland muscarinic or α-adrenergic receptor activation causes a transient increase in membrane permeability to K by triggering the release of a sizable intracellular pool of Ca common to both receptors.     

Effects of Prolonged Depolarization on the Nicotinic Acetylcholine Receptors of PC12 Cells

To determine whether prolonged depolarization and/or changes in intracellular Ca2+ concentrations stimulate adaptive responses of neuronal nicotinic acetylcholine receptors, PC12 pheochromocytoma cells were grown in medium containing various concentrations of K+. Nicotinic receptor function was determined as carbachol-stimulated uptake of 86Rb+. Cells were exposed to 50 mM K+ for up to 4 days and then allowed to repolarize for 60 min. Under these conditions, no changes in basal or carbachol-stimulated uptake of 86Rb+ were observed. Furthermore, neither the time course of carbachol-stimulated uptake or the carbachol concentration dependence of 86Rb+ uptake was altered. Finally, concurrent depolarization did not affect the functional down-regulation produced by chronic exposure of the cells to carbachol. Thus, neuronal nicotinic acetylcholine receptors on PC12 cells do not appear to be regulated by depolarization or prolonged elevation of the intracellular Ca2+ level.     

Cyclosporin A inhibits long-term activation of Na+,K+ pump in phytohemagglutinin-stimulated human lymphocytes

The effect of the immunosuppressive drug cyclosporin A (CsA) on the K (Rb) influx, intracellular K and Na contents, and on the major parameters of lymphocyte activation have been investigated in human peripheral blood lymphocytes activated by phytohemagglutinin (PHA). CsA suppressed protein, RNA, DNA syntheses and cell proliferation by 49.8 +/- 4.3, 67.6 +/- 10.1, 60.4 +/- 5.3 and 60.0 +/- 5.1%, respectively (n = 10) within 48 h. It also inhibited the late long-term Na+,K+ pump activation, as determined from the ouabain-sensitive Rb uptake, and prevented the increase in the intracellular K content at the late stages of G0/G1/S progression. Cyclosporin A did not affect the early transient pump activation, the dynamics, of ouabain-resistant influxes and the intracellular Na content in PHA-activated lymphocytes. When added 1 h after PHA, CsA neither affected the activation of the pump-mediated Rb influxes nor the increase in the intracellular K content. It is concluded that in activated human lymphocytes, the long-term activation of Na+,K+ pump associated with the mitogen-induced blast transformation, as well as the late increase in K content depend on the T-cell growth factor interleukin-2.     

Effects of monensin on metabolism of isolated rat islets of Langerhans.

Monensin, a univalent ionophore, is a carboxylic acid produced by Streptomyces cinnamonensis. It will complex various alkali-metal ions, but most readily binds Na+. Because of interest in the possible role of Na+ in the regulation of insulin secretion, we examined its effects on several aspects of the metabolism of isolated rat islets of Langerhans. The ionophore inhibited glucose-stimulated insulin release in a concentration-dependent manner, completely inhibiting secretion evoked by 20 mM-glucose at concentrations as low as 0.1 microM in static incubations. In perifusion experiments, both phases of insulin release were equally affected. Monensin (0.1 microM) had no significant effect on glucose oxidation as measured by the generation of 14CO2 from [14C]glucose. Monensin increased the rate of 22Na+ efflux from preloaded islets and net 22Na+ uptake over 30 min, in the absence of changes in islet volume or extracellular space. The ionophore increased the Rb+/K+ permeability of islet cells, as shown by its inhibition of 86Rb+ retention and stimulation of 86Rb+ efflux. At 0.1 microM, monensin abolished glucose-stimulated 45Ca2+ uptake by islets during 5 min incubations, and stimulated 45Ca2+ efflux from preloaded islets perifused with Ca2+-free medium, even in the complete absence of extracellular Na+. Studies of the uptake of 14C-labelled 5,5-dimethyloxazolidine-2,4-dione showed that 0.1 microM-monensin increased net intracellular pH from 7.05 to 7.13. 7 Monensin has widespread, complex, effects on the secretory responses and ion handling by the B cells, which are difficult to interpret in terms solely of actions as a Na+ ionophore.     

Volume regulation by human lymphocytes. Role of calcium

Human peripheral blood lymphocytes regulate their volumes in hypotonic solutions. In hypotonic media in which Na+ is the predominant cation, an initial swelling phase is followed by a regulatory volume decrease (RVD) associated with a net loss of cellular K+. In media in which K+ is the predominant cation, the rapid initial swelling is followed by a slower second swelling phase. 86Rb+ fluxes increased during RVD and returned to normal when the original volume was approximately regained. Effects similar to those induced by hypotonic stress could also be produced by raising the intracellular Ca++ level. In isotonic, Ca++- containing media cells were found to shrink upon addition of the Ca++ ionophore A23187 in K+-free media, but to swell in K+-rich media. Exposure to Ca++ plus A23187 also increased 86Rb+ fluxes. Quinine (75 microM), an inhibitor of the Ca++-activated K+ pathway in other systems blocked RVD, the associated K+ loss, and the increase in 86Rb+ efflux. Quinine also inhibited the volume changes and the increased 86Rb fluxes induced by Ca++ plus ionophore. The calmodulin inhibitors trifluoperazine, pimozide and chlorpromazine blocked RVD as well as Ca++ plus A23187-induced volume changes. Trifluoperazine also prevented the increase in 86Rb+ fluxes and K+ loss induced by hypotonicity. Chlorpromazine sulfoxide, a relatively ineffective calmodulin antagonist, was considerably less potent as an inhibitor of RVD than chlorpromazine. It is suggested than an elevation in cytoplasmic [Ca++], triggered by cell swelling, increases the plasma membrane permeability to K+, the ensuing increased efflux of K+, associated anions, and osmotically obliged water, leading to cell shrinking (RVD).     

Potassium/proton exchange in brush-border membrane of rat ileum

Summary These experiments were designed to determine whether proton-driven⁸⁶Rb uptake was present in apical membrane vesicles prepared from rat ileum. The uptake of⁸⁶Rb was approximately 300 to 350% greater in the presence of a 100-fold H⁺ gradient than in its absence and was greater at 1, 2 and 5 minutes (overshoot) than that at 90 minutes. Proton-driven⁸⁶Rb uptake was decreased by 20% in TMA-nitrate compared to that in TMA-gluconate. 0.3mm amiloride did not significantly inhibit proton-driven⁸⁶Rb uptake; in contrast, proton-driven²²Na uptake was significantly inhibited by 0.3mm amiloride by 34%. Similarly, 25mm KCl inhibited proton-driven⁸⁶Rb uptake more than that of²²Na, while the inhibition of proton-driven²²Na uptake by 25mm NaCl was greater than that of⁸⁶Rb. In additional studies intravesicular acidification measured by acridine orange fluorescence was demonstrated in the presence of an out-wardly directed K gradient. These studies demonstrate that a proton gradient stimulates⁸⁶Rb uptake and a K gradient induces intravesicular acidification; and that these fluxes are mediated by a K/H exchange distinct from Na/H exchange which is also present in this membrane. We conclude that a specific exchange process for K/H is located in ileal apical membrane vesicles.     

Sodium-pump activity and its inhibition by extracellular calcium in cardiac myocytes of guinea pigs

Myocardial sodium-pump activity was examined from ouabain-sensitive 86Rb+ uptake using myocytes isolated from guinea-pig heart. Either sodium loading or the sodium ionophore, monensin, increased 86Rb+ uptake by over 400%, indicating that the amount of Na+ available to the pump is the primary determinant of its activity, and that the sodium pump has a substantial reserve capacity in quiescent myocytes. Moreover, the degree of the above stimulation is markedly higher than corresponding values reported with multicellular preparations, suggesting that diffusion barriers make it impossible to observe the capacity of the sodium pump in the latter preparations. Removal of extracellular Ca2+ increased ouabain-sensitive 86Rb+ uptake, probably by enhancing turnover of the sodium pump rather than increasing availability of Na+ to the pump.     

Vacuolar ATPase driven potassium transport in highly metastatic breast cancer cells

Breast cancer is the second leading cause of death in women and thus has received a great deal of attention by researchers. Recent studies suggested decreased occurrence of cancer in patients treated with cardiac glycosides (CGs) for heart conditions. Because CGs induce their cellular effects via the Na⁺, K⁺ ATPase (Na–K), we treated four breast cancer cell lines (MCF-7, T47D, MDA-MB453, and MDA-MB231) and a non-cancerous breast ductal epithelial cell line (MCF-10A) with ouabain, a well-characterized CG, and measured cell proliferation by measuring bromodeoxyuridine incorporation. Ouabain (1 μM) decreased cell proliferation in all cell lines studied except MDA-MB453 cells. Western blot of Na–K α and β subunits showed α1, α3, and β1 expression in all cell lines except MDA-MB453 cells where Na–K protein and mRNA were absent. Potassium uptake, measured as rubidium (⁸⁶Rb) flux, and intracellular potassium were both significantly higher in MDA-MB453 cells compared to MCF-10A cells. RT-qPCR suggested a 7 fold increase in voltage-gated potassium channel (KCNQ2) expression in MDA-MB453 cells compared to MCF-10A cells. Inhibition of KCNQ2 prevented cell growth and ⁸⁶Rb uptake in MDA-MB453 cells but not in MCF-10A cells. All cancer cells had significantly higher vacuolar H-ATPase (V-ATPase) activity than MCF-10A cells. Inhibition of V-ATPase decreased ⁸⁶Rb uptake and intracellular potassium in MDA-MB453 cells but not in MCF-10A cells. The findings point to the absence of Na–K, high hERG and KCNQ2 expression, elevated V-ATPase activity and sensitivity to V-ATPase inhibitors in MDA-MB453. We conclude that cancer cells exhibit fundamentally different metabolic pathways for maintenance of intracellular ion homeostasis.     

Vasopressin rapidly stimulates NA entry and NA-K pump activity in quiescent cultures of mouse 3T3 cells

Addition of (Arg) vasopressin to quiescent cultures of Swiss 3T3 cells rapidly stimulates an ouabain-sensitive ⁸⁶Rb uptake. In contrast the hormone has no significant effect on the rate of efflux of this cation from preloaded cells. The stimulation of ⁸⁶Rb uptake is cycloheximide-insensitive, occurs within minutes of hormone addition and results from an increase in the Vmax of the uptake system. Vasopressin stimulates ion uptake in a concentration-dependent fashion (1-100 ng/ml); oxytocin also stimulated the Na-K pump but at significantly higher concentrations. The stimulation of the Na-K pump by vasopressin is apparently mediated by an increase in Na entry into the cells, since the hormone (1) strikingly shifts the concentration dependence on Na⁺ of the Na-K pump, (2) increases ²²Na uptake, and (3) increases intracellular Na contents when the efflux of this ion is blocked by ouabain. Since vasopressin is a potent mitogen for Swiss 3T3 cells, the results provide further evidence in support of a possible role of monovalent ion fluxes in signalling the initiation of growth stimulation.