Ouabain-sensitive ATP Hydrolysis Research Articles

Altered corticosteroid control of the erythrocyte sodium-potassium pump in the spontaneously hypertensive rat.

Recent evidence suggests that corticosteroids may participate in the regulation of erythrocyte Na,K pump activity. To examine the possible role of mineral- and glucocorticoids in the physiological control of Na,K pump in vivo, 10-week-old Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were randomly assigned to four treatment groups (n = 10 for each): (a) sham operation, (b) bilateral adrenalectomy, (c) bilateral adrenalectomy followed by daily intraperitoneal (i.p.) injection of aldosterone, 10 micrograms/kg, (d) bilateral adrenalectomy followed by daily i.p. injections of dexamethasone 60 micrograms/kg. Fourteen days later all rats were sacrificed and the erythrocyte Na,K pump activity was assessed by two different assays: ouabain sensitive ATP hydrolysis in isolated membranes (ATPase) and 86Rb uptake by intact erythrocytes. SHR exhibited reduced Na,K pump activity as measured by ATPase (compared to WKY) and by 86Rb uptake (compared to WKY and SD rats). Adrenalectomy was associated with 22-44% reduction in ATPase in all three rat species (P less than 0.05-0.01). Adrenalectomized aldosterone or dexamethasone treated SHR, WKY and SD rats exhibited ATPase activity that was indistinguishable from the corresponding control groups. Similarly, 86Rb uptake was lower in adrenalectomized SD and WKY rats. This reduction could be at least partially prevented by daily treatment with either aldosterone or dexamethasone. In SHR adrenalectomy had no effect on 86Rb uptake whether accompanied by daily treatment with aldosterone or dexamethasone or not. These results suggest that the erythrocyte sodium potassium pump is corticosteroid dependent in normotensive rats. An abnormal response of the Na,K pump to corticosteroids is observed in SHR, with a dissociation between steroid stimulated enzymatic ATP hydrolysis and actual transmembrane pumping as measured by 86Rb uptake.

Activation by lithium ions of the inside sodium sites in (Na + + K +)-ATPase

1. 1. Purified pig kidney ATPase was incubated in 30–160 mM Tris-HCl with various monovalent cations. 130 mM LiCl stimulated a ouabain-sensitive ATP hydrolysis (about 5% of the maximal (Na + + K +) activity), whereas 160 mM Tris-HCl did not stimulate hydrolysis. Similar results were obtained with human red blood cell broken membranes. 2. 2. In the absence of Na + and with 130 mM LiCl, the ATPase activity as a function of KCl concentration showed an initial slight inhibition (50 μM KCl) followed by an activation (maximal at 0.2 mM KCl) and a further inhibition, which was total at 25 mM KCl. In the absence of LiCl, the rate of hydrolysis was not affected by any of the KCl concentrations investigated. 3. 3. The lithium-activation curve for ATPase activity in the absence of both Na + and K + had sigmoid characteristics. It also showed a marked dependence on the total LiCl + Tris-HCl concentration, being inhibited at high concentrations. This inhibition was more noticeable at low LiCl concentrations. 4. 4. In the absence of Na +, 130 mM Li + showed promoted phosphorylation of ATPase from 1 to 3 mM ATP in the presence of Mg 2+. In enzyme treated with N-ethylmaleimide, the levels of phosphorylation in Li +-containing solutions, amounted to 40% of those in Na +- and up to 7 times of those in K +-containing solutions. 5. 5. The total (Na + + K +)-ATPase activity was markedly inhibited at high buffer concentrations (Tris-HCl, Imidazole-HCl and tetramethylammonium-HEPES gave similar results) in cases when either the concentration of Na + or K + (or both) was below saturation. On the other hand, the maximal (Na + + K +)-ATPase activity was not affected (or very slightly) by the buffer concentration. 6. 6. Under standard conditions (Tris-HCl + NaCl = 160 mM) the Na +-activation curve of Na +-ATPase had a steep rise between 0 and 2.5 mM, a fall between 2.5 and 20 mM and a further increase between 20 and 130 mM. With 30 mM Tris-HCl, the curve rose more steeply, inhibition was noticeable at 2.5 mM Na + and was completed at 5 mM Na +. With Tris-HCl + NaCl = 280 mM, the amount of activation decreased and inhibition at intermediate Na + concentrations was not detected.

ATP hydrolysis associated with an uncoupled sodium flux through the sodium pump: evidence for allosteric effects of intracellular ATP and extracellular sodium

1. A method has been developed for regenerating [gamma(32)P]ATP of constant specific activity within resealed red cell ghosts, and for measuring its hydrolysis. The method may be used to follow the hydrolysis of ATP at concentrations down to 1 muM, and for periods long enough for the ATP at these very low concentrations to turn over several hundred times.2. Using this method we have been able to show that the ;uncoupled' efflux of Na caused by the Na pump when resealed red cell ghosts are incubated in (Na + K)-free media is associated with a hydrolysis of ATP. The stoicheiometry is roughly 2-3 Na ions expelled per molecule of ATP hydrolysed.3. Measurements of ATP hydrolysis and Na efflux as functions of intracellular ATP concentration have shown that uncoupled Na efflux, and its associated ATP hydrolysis, are saturated at intracellular ATP concentrations in the region of 1 muM.4. Measurement of ATP hydrolysis as a function of ATP concentration in resealed ghosts incubated in a K-containing medium gave a complicated activation curve suggesting the involvement of high-affinity (K(m)ca. 1 muM) and low-affinity (K(m)ca. 100 muM) sites.5. When resealed ghosts containing about 1 muM-ATP were incubated in a Na-free or in a high-Na medium, the addition of K to the medium reduced the rate of ouabain-sensitive ATP hydrolysis.6. Ouabain-sensitive ATP hydrolysis in resealed ghosts incubated in K-free choline media was inhibited by external Na at low concentrations (K(i) < 1 mM), but this inhibition was reversed as the external Na concentration was further increased.7. The results show that uncoupled Na efflux may be thought of as the transport mode associated with Na-ATPase activity, just as Na-K exchange is the transport mode associated with (Na + K)-ATPase activity. The significance of the differences between uncoupled Na efflux and Na-ATPase activity, on the one hand, and Na-K exchange and (Na + K)-ATPase activity, on the other, is discussed.

Increased ouabain-sensitive 86Rb+ uptake and sodium and potassium ion-activated adenosine triphosphatase activity in transformed cell lines.

During the log phase of growth both the active, ouabain-sensitive K+ uptake, measured as 86Rb+, and the sodium and potassium ion-activated ATPase ((Na+ + K+)-ATPase) activity of SV40-transformed 3T3 cells were 2.5-and 5,5-fold higher, respectively, than in untransformed 3T3 cells. A similar higher active K+ uptake was found for Rous sarcoma virus and SV40-transformed baby hamster kidney cells compared with untransformed BHK cells. The active K+ uptake in SV403T3 and normal 3T3 cells decreased when the growth rate of both cell types diminished. Reduction in ouabain-sensitive ATP hydrolysis only occurred later, however, when appreciable decreases in cell viability were seen. Arrhenius plots of the (Na+ + K+)-ATPase activity of SV403T3 cells indicated a discontinuity at 24 degrees, whereas no similar discontinuity was indicated for 3T3 cells. The consequences of elevated K+ transport and (Na+ + K+)-ATPase activity in transformed cells and the possibility that the increased activity might be related to differences inphospholipid fatty acyl chain fluidity are discussed.