Relations among transepithelial sodium transport, potassium exchange, and cell volume in rabbit ileum

The relation between active transepithelial Na transport across rabbit ileum and 42K exchange from the serosal solution across the basolateral membranes has been explored. Although 42K influx across the basolateral membranes is inhibited by ouabain and by complete depletion of cell Na, it is not affected when transepithelial Na transport is abolished (i.e. in the presence of an Na-free mucosal solution) or stimulated (i.e. when glucose or alanine is added to the mucosal solution). We are unable to detect any relation between the ouabain-sensitive Na-K exchange mechanism responsible for the maintenance of intracellular Na and K concentrations and active transcellular Na transport. In addition, the maintenance of cell volume (water content) does not appear to be dependent upon transepithelial Na transport or the ouabain- sensitive Na-K exchange pump. Although the results of these studies cannot be considered conclusive, they raise serious questions regarding the role of the Na-K exchange pump, located at the basolateral membranes, in active transepithelial Na transport and the maintenance of cell volume.     

Phorbol esters augment polyamine transport by influencing Na(+)-K+ pump in murine leukemia cells.

In this report, we elucidate the role of Na(+)-K+ pump in the regulation of polyamine spermidine (Spd) transport in murine leukemia (L 1210) cells in culture. Ouabain, known to bind extracellularly to the alpha-subunit of the Na(+)-K+ pump, inhibits the pump activity. The L 1210 cells were found to possess ouabain binding sites at 7.5 fmol/10(6) cells. Ouabain significantly inhibited the Spd uptake in a dose-dependent manner. The maximum inhibition of Spd uptake by ouabain was observed beyond 200 microM. Spd transport was inversely correlated with the [3H]ouabain binding to L 1210 cells: an increase in the saturation of ouabain binding to L 1210 cells resulted in a decrease of the Spd uptake process. Treatment of L 1210 cells with protein kinase C activator phorbol esters increased the Spd transport and, also, ouabain-sensitive 86Rb+ uptake, a measure of the activity of the Na(+)-K+ pump. H-7, a protein kinase C inhibitor, significantly inhibited the ouabain-sensitive 86Rb+ uptake by L 1210 cells. Phorbol esters stimulated the level, but not the rate, of 22Na+ influx. Addition of H-7 to L 1210 cells inhibited the 22Na+ influx process. A concomitant phorbol ester-induced increase in 22Na+ influx, [14C]Spd uptake, together with the functioning of Na(+)-K+ pump, indicates the role of the "Na+ cycle" in the regulation of the polyamine transport process.     

Reversibility and partial reactions of the Na(+)-K+ pump of rat erythrocytes

An assay was developed to characterize the kinetic parameters of the Na(+)-K+ pump of rat erythrocytes under conditions as physiological as possible. Changes in the red cell Na+ and Rb+ content were determined in Na+ media (containing 2.5 mM inorganic phosphate (PO4) as a function of cell Na+ (2-8 mmol/l) and extracellular Rb+ (0.2-5 mM). Evaluation of the data revealed that under these conditions the Na(+)-K+ pump mediates, in addition to forward running 3 Nai+: 2 Rbo+ exchange, 1 Ki+:Rbo+ exchange and pump reversal (3 Nao+:2 Ki+ exchange). The two latter modes of Na(+)-K+ pump operation are accelerated by PO4 and lowering of cell Na+. At physiological cation and PO4 concentrations, 1Ki+:Rbo+ exchange contributes by 30-60% to total ouabain-sensitive Rb+ uptake. Thereby, the stoichiometry of ouabain-sensitive Na+ net-extrusion to Rb+ uptake is reduced to values between 1.0 and 0.5. Only at cell Na+ contents above 20 mmol/l the Na+:Rb+ stoichiometry approaches the value of 3:2 = 1.5. At certain constellations of Nai+ and Rbo+ the Na(+)-K+ pump cannot perform any net-transport of Na+ and K+ (Rb+). These equilibrium points are not far from those expected from thermodynamic considerations. The results demonstrate that in normal rat erythrocytes the reversible reaction cycle of the Na(+)-K+ pump runs in several modes of operation. The "abnormal" modes complicate the interpretation of unidirectional fluxes mediated by the Na(+)-K+ pump.     

The influence of virus transformation and cell population density on some membrane properties of mouse fibroblasts in culture

The influence of cell population density and simian virus 40 transformation on the activity of the Na-K pump was studied in mouse fibroblasts cultured in medium supplemented with fetal bovine serum. The activity of the Na-K pump was determined from K+ influx, ethacrynate-sensitive K+ influx, (Na+ + K+)-ATPase assay, and the determinations of intracellular potassium and sodium ion concentrations in these cells. The activity of the Na-K pump was found to decrease in density-inhibited cultures of normal fibroblasts (designated as 3T3 cells), while in the virus-transformed cells (SV3T3) the activity remained fairly constant at all cell population densities.     

Cyclic AMP stimulation of Na-K pump activity in quiescent swiss 3T3 cells

Recently, we have found that an increase in the intracellular level of cAMP acts as a mitogenic signal for Swiss 3T3 cells (Rozengurt et al., Proc. Natl. Acad, Sci. USA, 78:4392, 1981). The results presented in this paper demonstrate that addition of cAMP-elevating agents to confluent and quiescent cultures of Swiss 3T# causes a marked increase in the rate of 86Rb+ uptake but has no effect on the rate of cation efflux. The stimulation of ion uptake is mediated by the Na-K pump as shown by the ouabain sensitivity of the 86Rb+ fluxes. The increase in Na-K pump activity occurs whether cAMP is generated endogenously by stimulation of adenylate cyclase activity by cholera toxin, adenosine agonists, or PGE1 or added exogenously as 8BrcAMP. The stimulatory effect of these compounds on 86Rb+ uptake is potentiated by inhibitors of cyclic nucleotide phosphodiesterase activity. Cholera toxin stimulates the Na-K pump in a dose-dependent manner; half-maximal effect is achieved at 0.7 ng/ml. The stimulation of ouabain-sensitive 86Rb+ uptake by cAMP-elevating agents reaches a maximum after 2-3 h of incubation. This contrasts with the rapid (within minutes) stimulation of the Na-K pump caused by serum and other mitogenic agents. Further, cAMP-elevating agents fail to increase Na+ influx into 3T3 cells whereas serum causes a marked increase in Na+ influx, under identical experimental conditions. These findings suggest that the stimulation of Na-K pump activity caused by increased cAMP levels contrasts mechanistically with the rapid control of pump activity by serum which is primarily mediated by increased Na+ entry into the cells.     

Phospholemman overexpression inhibits Na+-K+-ATPase in adult rat cardiac myocytes: relevance to decreased Na+ pump activity in postinfarction myocytes.

Messenger RNA levels of phospholemman (PLM), a member of the FXYD family of small single-span membrane proteins with putative ion-transport regulatory properties, were increased in postmyocardial infarction (MI) rat myocytes. We tested the hypothesis that the previously observed reduction in Na+-K+-ATPase activity in MI rat myocytes was due to PLM overexpression. In rat hearts harvested 3 and 7 days post-MI, PLM protein expression was increased by two- and fourfold, respectively. To simulate increased PLM expression post-MI, PLM was overexpressed in normal adult rat myocytes by adenovirus-mediated gene transfer. PLM overexpression did not affect the relative level of phosphorylation on serine68 of PLM. Na+-K+-ATPase activity was measured as ouabain-sensitive Na+-K+ pump current (Ip). Compared with control myocytes overexpressing green fluorescent protein alone, Ip measured in myocytes overexpressing PLM was significantly (P < 0.0001) lower at similar membrane voltages, pipette Na+ ([Na+]pip) and extracellular K+ ([K+]o) concentrations. From -70 to +60 mV, neither [Na+]pip nor [K+]o required to attain half-maximal Ip was significantly different between control and PLM myocytes. This phenotype of decreased V(max) without appreciable changes in K(m) for Na+ and K+ in PLM-overexpressed myocytes was similar to that observed in MI rat myocytes. Inhibition of Ip by PLM overexpression was not due to decreased Na+-K+-ATPase expression because there were no changes in either protein or messenger RNA levels of either alpha1- or alpha2-isoforms of Na+-K+-ATPase. In native rat cardiac myocytes, PLM coimmunoprecipitated with alpha-subunits of Na+-K+-ATPase. Inhibition of Na+-K+-ATPase by PLM overexpression, in addition to previously reported decrease in Na+-K+-ATPase expression, may explain altered V(max) but not K(m) of Na+-K+-ATPase in postinfarction rat myocytes.     

Urea Inhibits the Na-K Pump in Human Erythrocytes

Recent studies have established that urea alters the activity of several volume-sensitive cation transport pathways. However, it has remained unclear whether urea has any effect on transport pathways that are not volume-sensitive. We examined the effect of urea on Na-K pump in the human erythrocytes. In cells from nine subjects, 500 mm urea inhibited 52 ± 10% of the pump activity measured as the ouabain-sensitive (OS) K influx. Urea inhibited the OS K influx reversibly, in a concentration-dependent manner. [³H] oubain binding, a measure of the number of Na-K pump sites remained unchanged with urea. Urea decreased the V _max for ouabain-sensitive K influx, but did not alter the apparent K _m for external K. Furthermore, urea did not alter the apparent K _m for intracellular Na. The ion turnover per pump site was decreased in the presence of urea. Thus, physiologically relevant urea concentration inhibit the Na-K pump in human erythrocyte. The inhibition of the Na-K pump by urea suggests that the effects of urea may not be limited to volume-sensitive transporters, but may be more widespread. Received: 2 March 1998/Revised: 15 June 1998     

The effect of hyperthermia on the sodium-potassium pump in Chinese hamster ovary cells

The effect of hyperthermia on the Na+-K+ pump was determined by measuring influx and efflux of 86Rb+ in Chinese hamster ovary cells from 31 to 50 degrees C. The maximum initial rate of ouabain-sensitive influx increased with temperature between 31 and 45 degrees C although Km increased significantly above 37 degrees C, implying a diminished affinity of the transport protein for its substrate. The changes in the kinetics of influx at temperatures up to 45 degrees C were rapidly reversible on return to 37 degrees C. Above 45 degrees C an irreversible decrease in 86Rb+ uptake was observed. Efflux of 86Rb+ increased from 31 to 40 degrees C but above 43 degrees C showed a small but significant decrease. The study of 86Rb+ influx after varying times of exposure to elevated temperatures showed that the Na+-K+ pump remains functional in cells which are reproductively dead. We have shown that although the kinetics of K+ transport are sensitive to temperature changes in the range used in clinical hyperthermia, the inactivation of the Na+-K+ pump is not a primary event in cell killing.     

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.     

Ouabain-Insensitive Sodium Movements in the Human Red Blood Cell

Red blood cells exposed to ouabain are capable of net Na outflux against an electrochemical gradient; the net outflux is inhibited by the diuretic, furosemide. In ouabain-treated cells, both the unidirectional Na outflux and the unidirectional Na influx are inhibited by furosemide. Furosemide also inhibits the ouabain-sensitive Na-Na exchange accomplished by the Na-K pump in K-free solutions. From the interaction of extracellular K, furosemide, and ouabain with the transport system, it seems possible that the ouabain-insensitive Na outflux is accomplished by the same mechanism that is responsible for the ouabain-sensitive Na-K exchange. The ouabain-insensitive Na outflux is increased by extracellular Na, and the influx increases as the intracellular Na increases. In fresh cells, high extracellular K concentrations decrease the ouabain-insensitive Na outflux and increase the ouabain-insensitive Na influx. When the rate constant for sodium outflux and the rate constant for sodium influx in ouabain-treated cells are plotted against the extracellular K concentration, the curves obtained are mirror images of each other. In starved cells, extracellular K increases the ouabain-insensitive Na outflux as does extracellular Na, and it has little effect on the Na influx.     

Exogeneous diacylglycerols downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes.

In this study, cell permeable diacylglycerols, sn-1,2-dioctanoglycerol (DiC8), and sn-1-oleoyl-2-acetylglycerol (OAG) were found to downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes. Both DiC8 and OAG decreased the binding of [3H]ouabain to intact oocytes while phorbol esters did not appreciably influence the same. These diacylglycerols inhibited the amiloride-sensitive 22Na+ influx and ouabain-sensitive 86Rb+ uptake in the oocytes. Furthermore, DiC8 prevented the 22Na+ efflux from the oocytes preloaded with 22Na+. Addition of H-7 to DiC8- and OAG-treated oocytes stimulated the pump activity curtailed by the two latters. The impairment of Na(+)-K+ pump activity by diacylglycerols suggests that protein kinase C activators may stimulate endocytosis of membrane-coupled Na(+)-K+ ATPase.     

Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+ -K+ -ATPase

Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca(2+)](i) homeostasis primarily by its direct inhibitory effects on Na(+)/Ca(2+) exchange.     

Insulin and glucagon stimulation of (Na+-K+)-ATPase transport activity in isolated rat hepatocytes

The effects of insulin and glucagon on the (Na+-K+)-ATPase transport activity in freshly isolated rat hepatocytes were investigated by measuring the ouabain-sensitive, active uptake of 86Rb+. The active uptake of 86Rb+ was increased by 18% (p less than 0.05) in the presence of 100 nM insulin, and by 28% (p less than 0.005) in the presence of nM glucagon. These effects were detected as early as 2 min after hepatocyte exposure to either hormone. Half-maximal stimulation was observed with about 0.5 nm insulin and 0.3 nM glucagon. The stimulation of 86Rb+ uptake by insulin occurred in direct proportion to the steady state occupancy of a high affinity receptor by the hormone (the predominant insulin-binding species in hepatocytes at 37 degrees C. For glucagon, half-maximal response was obtained with about 5% of the total receptors occupied by the hormone. Amiloride (a specific inhibitor of Na+ influx) abolished the insulin stimulation of 86Rb+ uptake while inhibiting that of glucagon only partially. Accordingly, insulin was found to rapidly enhance the initial rate of 22Na+ uptake, whereas glucagon had no detectable effect on 22Na+ influx. These results indicate that monovalent cation transport is influenced by insulin and glucagon in isolated rat hepatocytes. In contrast to glucagon, which appears to enhance 86Rb+ influx through the (Na+-K+)-ATPase without affecting Na+ influx, insulin stimulates Na+ entry which in turn may increase the pump activity by increasing the availability of Na+ ions to internal Na+ transport sites of the (Na+-K+)-ATPase.     

Estradiol-induced expression of N(+)-K(+)-ATPase catalytic isoforms in rat arteries: gender differences in activity mediated by nitric oxide donors

We tested the hypothesis that previously demonstrated gender differences in ACh-induced vascular relaxation could involve diverse Na(+)-K(+)-ATPase functions. We determined Na(+)-K(+)-ATPase by measuring arterial ouabain-sensitive 86Rb uptake in response to ACh. We found a significant increase of Na+ pump activity only in aortic rings from female rats (control 206 +/- 11 vs. 367 +/- 29 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.01). Ovariectomy eliminated sex differences in Na(+)-K(+)-ATPase function, and chronic in vivo hormone replacement with 17beta-estradiol restored the ACh effect on Na(+)-K(+)-ATPase. Because ACh acts by enhancing production of NO, we examined whether the NO donor sodium nitroprusside (SNP) mimics the action of ACh on Na(+)-K(+)-ATPase activity. SNP increased ouabain-sensitive 86Rb uptake in denuded female arteries (control 123 +/- 7 vs. 197 +/- 12 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.05). Methylene blue (an inhibitor of guanylate cyclase) and KT-5823 (a cGMP-dependent kinase inhibitor) blocked the stimulatory action of SNP. Exposure of female thoracic aorta to the Na+/K+ pump inhibitor ouabain significantly decreased SNP-induced and ACh-mediated relaxation of aortic rings. At the molecular level, Western blot analysis of arterial tissue revealed significant gender differences in the relative abundance of catalytic isoforms of Na(+)-K(+)-ATPase. Female-derived aortas exhibited a greater proportion of alpha2-isoform (44%) compared with male-derived aortas. Furthermore, estradiol upregulated the expression of alpha2 mRNA in male arterial explants. Our results demonstrate that enhancement of ACh-induced relaxation observed in female rats may be in part explained by 1) NO-dependent increased Na(+)-K(+)-ATPase activity in female vascular tissue and 2) greater abundance of Na(+)-K(+)-ATPase alpha2-isoform in females.     

Influence of chronic alterations of salt intake and aging on the kinetic of red cell Na+ and K+ transport in Sprague-Dawley rats

The kinetics of Na+ and K+ (Rb)+ transport mediated by the Na(+)-K+ pump and Na(+)-K+ cotransport system (assessed as a function of Rb+o and Na+i) as well as the magnitude of cation leaks were determined in red cells of young male rats subjected to chronic salt deprivation or salt loading (0.1% and 8% NaCl diet). These salt intake alterations induced moderate kinetic changes of the Na(+)-K+ pump which did not result in significant changes of ouabain-sensitive (OS) Rb+ uptake or Na+ net extrusion at in vivo Na+i and K+o concentrations because a decreased affinity for Na+i in salt-loaded animals was compensated by an increased maximal transport rate. High furosemide-sensitive (FS) Rb+ uptake in red cells of salt-deprived rats was caused by an increase of both the maximal transport rate and the affinity for Rb+o. Cation leaks were also higher in salt-deprived than in salt-loaded rats. In three age groups of rats fed a 1% NaCl diet FS Rb+ uptake (but not FS Na+ net uptake) rose with age due to an increasing maximal transport rate whereas the affinity of the cotransport system for Rbo+ did not change. The age-dependent changes in the kinetics of the Na(+)-K+ pump resulted in a slight decrease of OS Rb+ uptake with age that was not paralleled by corresponding Na+ net extrusion. No major age-related changes of cation leaks were found. Thus some intrinsic properties of red cell transport systems can be altered by salt intake and aging.     

Properties of Na-K pump in primary cultures of kidney cells.

Activities related to Na-K transport were measured in cell cultures of ground squirrel kidney cortex in order to compare these cells with those of intact kidney and of continuous cell lines. A microsomal preparation containing plasma membrane Na,K-ATPase from fresh kidney showed twice the activity of a similar preparation from 72-hour cultured cells. Na,K-ATPase of homogenates of 72-hour cells showed one-third to one-fourth the specific activity of that from 6-hour cultured cells. The associated K-dependent phosphatase activity also declined as a function of time in culture. The ouabain-sensitive influx of K into 6-hour cultured cells was twice as great as the K influx into 72-hour cells. The number of sites binding 3H-ouabain in intact cultured cells declined 81% on a cell protein basis between 6 and 72 hours in culture. This decline in ouabain binding sites was relatively greater than that of K influx, so that the K turnover number increased over this same time period. The decline in ouabain-sensitive K influx during culture was complementary to an increase in furosemide-sensitive K influx. Measurements of unidirectional and net K fluxes showed that there were three components of K influx into 3-day cultured cells: ouabain-sensitive Na:K exchange, furosemide-sensitive K:K exchange, and K diffusion. In the 6-hour cultures, however, there was no furosemide-sensitive K:K exchange. Thus, after three days in culture ground squirrel kidney cells lose a feature characteristic of the original parent cells (high Na,K-ATPase activity), and gain a feature common to many undifferentiated cultured cells (furosemide-sensitive K:K exchange).     

Modulation of functional and optimal (Na+-K+)ATPase activity during the cell cycle of neuroblastoma cells

Functional and optimal activities of the (Na+-K+)ATPase, as determined by ouabain-sensitive K+ influx in intact cells and ATP hydrolysis in cell homogenates respectively, have been measured during the cell cycle of neuroblastoma (clone Neuro-2A) cells. The cells were synchronized by selective detachment of mitotic cells. The ouabain-sensitive K+ influx decreased more than fourfold from 1.62 +/- 0.11 nmoles/min/10(6) cells to 0.36 +/- 0.25 nmoles/min/10(6) cells on passing from mitosis to early G1 phase. On entry into S phase a transient sixfold increase to 2.07 +/- 0.30 nmoles/min/10(6) cells was observed, followed by a rapid decline, after which the active K+ influx rose again steadily from 1.03 +/- 0.25 nmoles/min/10(6) cells in early S phase to 2.10 +/- 0.92 nmoles/min/10(6) cells just prior to the next mitosis. The ouabain-insensitive component rose linearly through the cycle in the same manner as the protein content/cell. Combining total K+ influx values with efflux data obtained previously showed that net loss of K+ occurred with transition from mitosis to G1 phase while net accumulation occurred with entry into S. Throughout mid-S phase net K+ flux was virtually zero, but a large net influx occurred again just before the next mitosis. The (Na+-K+)ATPase activity measured in cell homogenates decreased rapidly from mitosis to G1 phase and increased steadily throughout S phase, but the transient activation on entry into S phase was not observed. Complete inhibition of the (Na+-K+)ATPase mediated K+ influx by ouabain (5 mM) prevents the cells from entering S phase, while partial inhibition by lower concentrations of ouabain (0.2 and 0.5 mM; km = 0.17 mM) causes partial blockage in G1 and, to a lesser extent, a reduced rate of progression through the rest of the cell cycle. We conclude that the transient increase in (Na+-K+)ATPase mediated K+ influx at the G1/S transition is a prerequisite for entry into S phase, while maintenance of adequate levels of K+ influx is necessary for normal rate of progression through the rest of the cell cycle.     

Inhibition of sodium for sodium exchange by phlorizin in frog sartorius muscle

The effects of phlorizin (2 X 10(-3) mol X l-1) on the Na transport of frog (Rana esculenta) sartorius muscle were investigated in glucose-free medium. Phlorizin decreased the rate coefficient of 24Na efflux by about 40%. The degree of inhibition was comparable to that caused by ouabain (10(-4) mol X l-1). Phlorizin could evoke a further reduction in the 24Na efflux also in the presence of ouabain. The intracellular Na content of the phlorizin-treated muscles remained unchanged, in contrast to a 60% increase induced by ouabain. 42K uptake was not affected by phlorizin. Data indicate that the ouabain-sensitive Na-K pump was not involved in the action of phlorizin. At the same time, phlorizin failed to alter the residual 24Na efflux measured in Li-Ringer solution containing ouabain. When Na: Na exchange was restored by replacing Na into the washout solution in the presence of ouabain, the increase of 24Na efflux was significantly diminished by phlorizin. Phlorizin reduced the 24Na uptake into a compartment with a half time of 6 min by about 40% without affecting the intracellular compartment. The results suggest that phlorizin inhibits the ouabain-insensitive Na: Na exchange in a superficial Na compartment.     

Red cell sodium in DOCA-salt hypertension: a Brattleboro study.

The alteration of red cell Na+ content (Na+i), its causes and the possible relationship to the development of DOCA-salt hypertension were studied in Brattleboro rats. A pronounced hypertension developed in heterozygous (non-DI) animals that synthesize vasopressin (VP) although no substantial Na+i elevation was observed in their erythrocytes. In contrast, Na+i rose progressively in red cells of homozygous VP-deficient (DI) rats in which only marginal increase of systolic blood pressure was found after six weeks of DOCA-salt regimen. DOCA-salt treatment of non-DI rats did not cause major alterations in ouabain-resistant (OR) net Na+ uptake or ouabain-sensitive (OS) net Na+ extrusion but moderately increased furosemide-sensitive (FS) Rb+ uptake. The same treatment of DI rats doubled Na+i by an increased OR net Na+ uptake (due to a major elevation in both Na(+)-K+ cotransport and Na+ leak). Consequently, OS net Na+ extrusion was augmented in red cells of these animals. This was accompanied by an about threefold elevated FS Rb+ uptake. It can be concluded that a) the alterations of OR and/or OS Na+ or K+ transport observed in erythrocytes of Brattleboro DI rats are not essential for the development of severe DOCA-salt hypertension, b) red cell ion transport abnormalities revealed in DOCA-salt treated DI rats might be rather ascribed to cell potassium depletion, and c) increased inward Na(+)-K+ cotransport and Na+ leak causes red cell Na+i elevation that stimulates Na(+)-K+ pump activity.     

Seasonal changes in cation transport in red blood cells of grey squirrel (Sciurus carolinensis) in relation to thermogenesis and cellular adaptation to cold.

1. Unidirectional influx of 42K was measured in red cells of grey squirrels at seasonal intervals over two years. 2. Na/K pump-related (i.e. ouabain-sensitive) K influx at 37 degrees C was maximal in cells collected in January and was more than three times greater than cells collected in summer. Na/K pump activity, maximized by loading the cells with Na, exhibited a similar difference. 3. At 5 degrees C in fresh cells, ouabain-sensitive K influx, expressed as per cent of that at 37 degrees C, was highest in March. In Na-loaded cells it was lowest in summer. 4. Passive "leak" K influx (i.e., the residual influx remaining in presence of ouabain and bumetanide) was highest in October, and declined progressively to the summer months, when it was only 27% of that in October. 5. Cotransport (i.e., bumetanide-sensitive K influx) exhibited the same seasonal pattern as Na/K pump activity in fresh cells. 6. Net gain of Na in cells stored at 5 degrees C for three days in March was less than half of that in January or summer. 7. High transport activity in January may correlate with a requirement for increased non-shivering thermogenesis. However, red cells of grey squirrels exhibit maximum resistance to low temperature in March and at this time resemble the red cells of hibernating mammals.