Radioactive 86rubidium influx into red blood cells in essential hypertension in relation to the plasma renin activity

Changes in several mechanisms of sodium transport across the cell membranes are described in essential hypertension. We studied ouabain-sensitive and insensitive 86Rb+ influx into the red blood cells (RBC) of 16 healthy controls and 51 patients with essential hypertension (EH) divided according to their plasma renin activity (PRA) in 3 groups: 11 patients with high PRA (HREH), 18 patients with normal PRA (NREH) and 22 patients with low PRA (LREH). In addition to studying 86RB+ uptake by patients RBC, we tested also the effect of the patients' sera on 86Rb+ influx into the RBC of healthy subjects. Red blood cells of patients with HREH and NREH had lower ouabain-sensitive 86Rb+ influx in comparison with controls. No significant differences were found between these hypertensive groups. In contrast 86Rb+ uptake by the RBC of LREH patients was always higher than in controls or HREH and NREH. It was chiefly the ouabain-sensitive component that was raised, but some increase in ouabain-insensitive 86Rb+ influx also could be seen. The serum of patients with HREH and NREH, when incubated with RBC of healthy controls, lowered their ouabain-sensitive 86Rb+ influx. The decrease was more pronounced in NREH than in HREH group. Plasma from LREH patients increased both ouabain-sensitive and ouabain-insensitive 86Rb+ influx into the control RBC. These findings indicate that there may be differences in the sodium/potassium transport mechanisms across the cell membrane in various kinds of EH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of Ion Metabolism in Reaggregated Brain Cell Cultures

Mouse brain cell reaggregates have been used to study changes in sodium- and potassium-dependent ouabain-sensitive adenosine phosphohydrolase (Na+, K+-ATPase) activity and in 86Rb+ uptake and exit during development. Na+, K+-ATPase activity in these cultures has two ouabain-inhibitable components, both of which increased severalfold between day 3 and day 17 in culture. This increase, however, was less than that in developing brain. Little change in either total or extracellular water or in the equilibrium levels of Na+ and K+ occurred during development. The uptake of 86Rb+ measured a 10-min incubation showed only a modest increase during culture, whereas the exit of 86Rb+ from reaggregates preloaded with the tracer increased approximately fourfold. The exit consisted of both K+-independent and K+-stimulated components and the K+-stimulated fraction contributed most of the developmental change. When uptake rates were corrected for the contribution of the developmental changes in exit, these rates were found to increase as well. The 86Rb+ uptake correlated closely with the activity of the Na+,K+-ATPase during development. The pattern of developmental changes in enzyme activity and 86Rb+ uptake and exit suggest that, while little change in the steady-state levels of the ions occurred, the rates of ion movement increase markedly.     

Density-related changes of potassium (86Rb) uptake by amphibian endothelial cells

Potassium influx has been investigated in XTH-2 cells, a line derived from tadpole heart endothelia. In this line, the density at which the cultures become confluent is clearly separated from the density at which growth arrest takes place. Density-related changes in K+ influx were monitored by determining the uptake of 86Rb into well adhering cells kept in culture medium. The main observations were 1) 86Rb uptake is highest in single cells, and on confluency it reaches a low level, which is kept constant at higher cell density regardless of whether the cultures are stationary or still in logarithmic growth phase; 2) the relative amount of 86Rb taken up via the Na+ -K+ -2Cl- cotransport pathway and via the Na+/K+ pump changes from low cell density to confluent cultures; 86Rb uptake of single cells is nearly insensitive to ouabain, a maximum of ouabain sensitivity is reached around confluency, whereas piretanide-sensitive 86Rb uptake is highest in single cells and seems to reach a minimum at the onset of confluency; 3) the variations in Na+/K+ pumping rate reflect neither differences in the amount of enzyme present nor changes in enzyme repartition between apical and basolateral plasma membranes; they seem to result from either "masking" or "unmasking" of the enzyme; 4) no alterations in K+ uptake occur that would be characteristic of the "stationary growth phase." The only changes that seem to be related to arrest of proliferation are concerned with the Na+/K+-ATPase, which achieves an extraordinary susceptibility to stimulation by monensin and exhibits an increase in PNPPase activity.     

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.     

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)     

Effects of 20-HETE on Na+ transport and Na+ -K+ -ATPase activity in the thick ascending loop of Henle

Previous studies have indicated that 20-hydroxyeicosatetraenoic acid (20-HETE) inhibits Na+ transport in the medullary thick ascending loop of Henle (mTALH), but the mechanisms involved remain uncertain. The present study compared the effects of 20-HETE with those of ouabain and furosemide on intracellular Na+ concentration ([Na+]i), Na+ -K+ -ATPase activity, and 86Rb+ uptake, an index of Na+ transport, in mTALH isolated from rats. Ouabain (2 mM) increased, whereas furosemide (100 microM) decreased, [Na+]i in the mTALH of rats. Ouabain and furosemide inhibited 86Rb+ uptake by 91 and 30%, respectively. 20-HETE (1 microM) had a similar effect as ouabain and increased [Na+]i from 19 +/- 1 to 30 +/- 1 mM. 20-HETE reduced Na+ -K+ -ATPase activity by 30% and 86Rb+ uptake by 37%, but it had no effect on 86Rb+ uptake or [Na+]i in the mTALH of rats pretreated with ouabain. 20-HETE inhibited 86Rb+ uptake by 12% and increased [Na+]i by 19 mM in mTALH pretreated with furosemide. These findings indicate that 20-HETE secondarily inhibits Na+ transport in the mTALH of the rat, at least, in part by inhibiting the Na+ -K+ -ATPase activity and raising [Na+]i.     

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.     

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.     

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.     

Membrane associated events during proliferative inhibition of granulocyte precursors

A new way of assessing the significance of intracellular signals that may regulate cellular proliferation, would be to analyze possible 'second messengers' when proliferation is slowed down, rather than stimulated. Therefore, we examined proliferating mononuclear blood cells from leukaemic patients which had been exposed to an inhibitory ox leucocyte extract. The extract decreased 3H-thymidine incorporation in leukaemic cells in short-term cultures. The inhibition was not cell-line specific, but was nevertheless non-toxic and not due to endotoxin. The K+ flux into the leukaemic cells was assessed with 86Rb+, a K+ analogue. An inverse relationship was found between 86Rb+ uptake and 3H-thymidine incorporation. The increased 86Rb+ influx was probably due to leakage or exchange mechanisms other than the Na+/K+ membrane pump, as suggested by ouabain inhibition experiments. However, the long lag time (greater than 45 min) between addition of inhibitor and a marked increase in 86Rb+ uptake does not support a role for the K+ flux as an early mediator of the inhibitory signal.     

Furosemide and Ca2+ affect 86Rb+ efflux from pancreatic beta-cells by different mechanisms

The interaction between furosemide, calcium and D-glucose on the 86Rb+ efflux from beta-cell-rich mouse pancreatic islets was investigated in a perifusion system with high temporal resolution. Raising the glucose concentration from 4 to 20 mM induced an initial decrease in 86Rb+ efflux, which was followed by a steep increase and then a secondary decrease. Removal of extracellular calcium increased the 86Rb+ efflux at 4 mM D-glucose but reduced it at 20 mM. The initial biphasic changes in 86Rb+ efflux induced by 20 mM D-glucose were inhibited by calcium deficiency. Furosemide (100 microM) reduced the 86Rb+ efflux rate both at 4 and 20 mM D-glucose and the magnitudes appeared to be similar at either glucose concentration. Furosemide (100 microM) reduced the glucose-induced (10 mM) 45Ca+ uptake but did not affect the basal (3 mM D-glucose) 45Ca+ uptake. However, the ability of furosemide (100 microM) to reduce the 86Rb+ efflux at a high glucose concentration (20 mM) was independent of extracellular calcium. The inhibitory effects of furosemide and calcium deficiency on the 86Rb+ efflux rate appeared to be additive. It is concluded that the effect of furosemide on 86Rb+ efflux is not secondary to reduced calcium uptake and that the effects of furosemide and calcium deficiency are mediated by different mechanisms. The effect of furosemide is compatible with inhibition of loop diuretic-sensitive co-transport of Na+, K+ and Cl- and the effect of calcium deficiency with reduced activity of calcium-regulated potassium channels.     

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.     

Induction of 86Rb fluxes by Ca2+ and volume changes in thymocytes and their isolated membranes

Cell swelling and elevated intracellular Ca2+ increase K+ permeability in lymphocytes. Experiments were performed to test whether these effects can also be elicited in isolated plasma membrane vesicles. Rabbit thymocytes, used as a source of membrane vesicles, were found to regain their volume after swelling in hypotonic, low-K+ media. This regulatory volume decrease (RVD) was inhibited by quinine and trifluoperazine, but not affected by ouabain. Both efflux and uptake of K+ (86Rb) were stimulated by hypotonicity. Addition of A23187 plus Ca2+ also increased 86Rb fluxes. Ca2+- and volume-induced 86Rb fluxes were also studied in isolated membranes. A plasma membrane-rich vesicle fraction, enriched over 11-fold in 5'-nucleotidase, was isolated from thymocytes. The vesicles were about 35% inside-out and trapped 86Rb in an osmotically active compartment of approximately 1.3 microliter/mg protein. Equilibrium exchange fluxes of 86Rb in the vesicles were unaffected by Ca2+ with or without A23187. Calmodulin had no effect on 86Rb permeability but stimulated ATP-dependent Ca2+ accumulation. Hypotonic swelling increased both uptake and efflux of 86Rb from vesicles. However, this increase was not blocked by either quinine or trifluoperazine, was not specific for K+ (86Rb), and is probably unrelated to RVD. It is concluded that components essential for the volume- and Ca2+-induced changes in K+ permeability are lost or inactivated during membrane isolation. An intact cytoarchitecture may be required for RVD.     

In squid nerve fibers monovalent activating cations are not cotransported during Na+/Ca2+ exchange

Squid axons display a high activity of Na+/Ca2+ exchange which is largely increased by the presence of external K+, Li+, Rb+ and NH+4. In this work we have investigated whether this effect is associated with the cotransport of the monovalent cation along with Ca2+ ions. 86Rb+ influx and efflux have been measured in dialyzed squid axons during the activation (presence of Ca2+i) of Ca2+o/Na+i and Ca2+i/Ca2+o exchanges, while 86Rb+ uptake was determined in squid optic nerve membrane vesicles under equilibrium Ca2+/Ca2+ exchange conditions. Our results show that although K+o significantly increases Na+i-dependent Ca2+ influx (reverse Na+/Ca2+ exchange) and Rb+i stimulates Ca2+o-dependent Ca2+ efflux (Ca2+/Ca2+ exchange), no sizable transport of rubidium ions is coupled to calcium movement through the exchanger. Moreover, in the isolated membrane preparation no 86Rb+ uptake was associated with Ca2+/Ca2+ exchange. We conclude that in squid axons although monovalent cations activate the Na+/Ca2+ exchange they are not cotransported.     

Potassium transport in nonpigmented epithelial cells of ocular ciliary body: inhibition of a Na+, K+, Cl- cotransporter by protein kinase C.

The mechanisms by which 86Rb+ (used as a tracer for K+) enters human nonpigmented ciliary epithelial cells were investigated. Ouabain-inhibitable bumetanide-insensitive 86Rb+ transport accounted for approximately 70-80% of total, whereas bumetanide-inhibitable ouabain-insensitive uptake accounted for 15-25% of total. K+ channel blockers such as BaCl2 reduced uptake by approximately 5%. Bumetanide inhibited 86Rb+ uptake with an IC50 of 0.5 microM, while furosemide inhibited with an IC50 of about 20 microM. Bumetanide-inhibitable 86Rb+ uptake was reduced in Na(+)-free or Cl(-)-free media, suggesting that Na+ and Cl- were required for optimal uptake via this mechanism. These characteristics are consistent with a Na+, K+, Cl- cotransporter in NPE cells. Treatment of NPE cells for 15 min with phorbol 12-myristate, 13-acetate (PMA), an activator of protein kinase C, caused a 50-70% decrease in 86Rb+ uptake via the Na+, K+, Cl- cotransporter. Other 86Rb+ uptake mechanisms were not affected. 86Rb+ uptake via the Na+, K+, Cl- cotransporter could be inhibited by other phorbol esters and by dioctanoylglycerol, an analog of diacylglycerol, but not by 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C. Staurosporine, a protein kinase C inhibitor, blocked phorbol ester inhibition of 86Rb+ uptake. These data suggest that a Na+, K+, Cl- cotransporter in NPE cells is inhibited by activation of protein kinase C.     

Localization of the phlorizin site on Na, K-ATPase in red cell membranes.

Phlorizin at 2 X 10(-4) M inhibited Na+ and Rb+-activated ATPase activities in human red cell membranes by 43%. It inhibited the 86Rb uptake activity of erythrocytes by only 15%. 86Rb uptake into resealed ghosts was inhibited strongly when phlorizin and ATP were preloaded in the ghosts before resealing. Na,K-ATPase activity in the resealed ghosts was also inhibited in the presence of phlorizin inside but not outside the ghosts. These findings suggested that the phlorizin site is located inside the cell.     

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.     

Sodium and rubidium uptake in cells transformed by Rous sarcoma virus

Rates of uptake and intracellular concentrations of monovalent cations were measured in virus-transformed and nontransformed chick embryo (CE) cells. Uptake of 22Na+ into cells transformed by the BH strain of Rous sarcoma virus (RSV-BH) (CE-BH) was about double the rate of uptake into CE cells, or cells transformed by the Schmidt-Ruppin strain (RSV-SR): CE-SR. Likewise, the rate of efflux of 22Na+ was greater in CE-BH cells than in CE or CE-SR cells. The greater permeability of CE-BH cells to Na+ was apparent in higher intracellular Na+ concentrations. Experiments with cells exhibiting temperature-dependent transformation showed that new RNA and protein synthesis was a requirement for the acquisition of increased Na+ permeability, suggesting that the change is an indirect effect of the virus-coded transformation-inducing protein. Rates of 86Rb+ uptake, used as a measure of K+ influx, were indistinguishable in CE, CE-BH, and CE-SR cells. Also, equilibrium intracellular levels of 86Rb+ were similar in transformed and nontransformed cells, as were observed concentrations of K+. Also, no differences in ATPase activity, as indicated by ouabain binding or temperature sensitivity, were observed. We conclude that monovalent cations play no direct role in RSV-induced transformation, although the higher levels of Na+ in CE-BH cells may be responsible for other distinguishing biochemical features of these cells.     

Insulin stimulation of (Na+,K+)-adenosine triphosphatase-dependent 86Rb+ uptake in rat adipocytes

Insulin stimulated the uptake of 86Rb+ (a K+ analog) in rat adipocytes and increased the steady state concentration of intracellular potassium. Half-maximal stimulation occurred at an insulin concentration of 200 pM. Both basal- and insulin-stimulated 86Rb+ transport rates depended on the concentration of external K+, external Na+, and were 90% inhibited by 10(-3) M ouabain and 10(-3) M KCN, indicating that the hormone was activating the (Na+,K+)-ATPase. Insulin had no effect on the entry of 22Na+ or exit of 86Rb+. Kinetic analysis demonstrated that insulin acted by increasing the maximum velocity, Vmax, of 86Rb+ entry. Inhibition of the rate of Rb+ uptake by ouabain was best described by a biphasic inhibition curve. Scatchard analysis of ouabain binding to intact cells indicated binding sites with multiple affinities. Only the rubidium transport sites which exhibited a high affinity for ouabain were stimulated by insulin. Stimulation required insulin binding to an intact cell surface receptor, as it was reversible by trypsinization. We conclude that the uptake of 86Rb+ by the (Na+,K+)-ATPase is an insulin-sensitive membrane transport process in the fat cell.     

CSF-1 stimulates Na+K+-ATPase mediated 86Rb+ uptake in mouse bone marrow-derived macrophages

86Rb+ was used as an isotopic tracer for the measurement of K+-uptake into quiescent murine bone marrow-derived macrophages. 86Rb+ uptake was inhibited by ouabain indicating a Na+K+-ATPase is being measured. In support of this finding, increased sensitivity to ouabain inhibition was seen when the K+ content of the medium was reduced. A purified colony stimulating factor (CSF-1) was shown to stimulate the ouabain-sensitive 86Rb+ uptake in a dose-dependent manner. Such colony stimulating factor stimulation of 86Rb+ (K+) influx was rapid, with a maximal effect seen 10 minutes after growth factor addition followed by a gradual decrease. Thus increased Na+K+-ATPase activity was an early response of macrophages to the colony stimulating factor.