The activity of the Na,K-ATPase can be sensitive to physiological changes in Cai. Intracellular proteins such as calnaktin, calmodulin, and protein kinase C could regulate the pump during transient changes in Cai. The mechanisms by which these proteins interact with the Na,K-ATPase, their distribution in different kinds of cells, and their role in regulating the Na,K-ATPase are not yet determined. Preliminary data indicate that an increase in Cai within the physiological range could be associated with either a stimulation or an inhibition of enzyme activity or a change in the affinity of the Na,K-ATPase for ouabain. The type of response probably depends on the kind of cell, its associated intracellular proteins, and its physiological state. Ca and intracellular proteins could play a key role in the regulation of the Na,K-ATPase by hormones.

We present evidence that Na-K-ATPase in the rat proximal tubule is directly activated by ANG II much faster than previously observed. Specifically, we show that a 2-min exposure to 0.1 and 1 nM ANG II slowed the rate of intracellular sodium accumulation in response to an increase in extracellular sodium added in the presence of gramicidin D. From these data, we show that ANG II directly stimulates Na-K-ATPase activity at rate-limiting concentrations of intracellular sodium. Under these same conditions, exposing proximal tubules to ANG II altered the amount of 32P incorporated into multiple phosphopeptides generated from a tryptic digest of the alpha-subunit of Na-K-ATPase. Na-K-ATPase was isolated from whole cell lysates by means of a ouabain-affinity column and then separated into its individual subunits by SDS-PAGE. Na-K-ATPase bound to the column in its E2 conformation and was eluted by altering its conformation to E1 using Na+ATP. Na-K-ATPase isolated from cells treated with ANG II eluted more easily from the ouabain-affinity column than Na-K-ATPase isolated from control cells, suggesting that ANG II decreased the affinity of Na-K-ATPase for ouabain. Thus ANG II rapidly stimulated the activity of Na-K-ATPase in 2 min or less by a mechanism that could involve changes in phosphorylation and conformation of Na-K-ATPase. We suggest that the physiological role for rapid direct activation of Na-K-ATPase is greater control of intracellular sodium during sodium reabsorption.

We earlier observed that treating rat proximal tubules with concentrations of angiotensin II (ANG II) that directly stimulate Na-K-ATPase activity changed how Na-K-ATPase subsequently eluted from an ouabain-affinity column. In this study we tested whether ANG II increases the rate of elution in response to ligands that trigger the decay of E(2)-P, which implies a change in functional properties of Na-K-ATPase, or by decreasing the amount subsequently eluted with SDS, which suggests a change in how Na-K-ATPase interacts with other proteins. We utilized a new digoxin-affinity column and novel lines of opossum kidney (OK) cells that coexpress the rat AT(1a) receptor and either the wild-type rat alpha(1)-isoform of Na-K-ATPase or a truncation mutant missing the first 32 amino acids of its NH(2) terminus. We characterized how rat kidney microsomes bind to and elute from the digoxin-affinity column and demonstrated that they are heterogeneous in the rate at which they release digoxin in response to ligands that trigger the decay of E(2)-P. Incubating OK cells with ANG II stimulated the ensuing elution of wild-type rat alpha(1)-subunit by increasing the kinetic response to ligands that cause a decay of E(2)-P without affecting the amount later eluted with SDS. In contrast, ANG II had no effect on the kinetic response of the truncation mutant but decreased the amount eluted with SDS. These data suggest that ANG II regulates both the kinetic properties of Na-K-ATPase and its interaction with other proteins by a mechanism(s) involving its NH(2) terminus.

This study is aimed at identifying the Na pump isoform composition of human erythroid precursor cells and mature human erythrocytes. We used purified and synchronously growing human erythroid progenitor cells cultured for 7-14 days. RNA was extracted from the progenitor cells on different days and analyzed by RT-PCR. The results showed that only the ␣1, ␣3, ␤2, and ␤3 subunit isoforms and the ␥ modulator were present. Northern analysis of the erythroid progenitor cells again showed that ␤2 but not ␤1 or ␣2 isoforms were present. The erythroid cells display a unique ␤ subunit expression profile (called ␤-profiling) in that they contain the message for the ␤2 isoform but not ␤1, whereas leukocytes and platelets are known to have the message for the ␤1 but not for the ␤2 isoform. This finding is taken to indicate that our preparations are essentially purely erythroid and free from white cell contamination. Western analysis of these cultured progenitor cells confirmed the presence of ␣1, ␣3, (no ␣2), ␤2, ␤3, and ␥ together now with clear evidence that ␤1 protein was also present at all stages. Western analysis of the Na pump from mature human erythrocyte ghosts, purified by ouabain column chromatography, has also shown that ␣1, ␣3, ␤1, ␤2, ␤3, and ␥ are present. Thus, the Na pump isoform composition of human erythroid precursor cells and mature erythrocytes contains the ␣1 and ␣3 isoforms of the ␣ subunit, the ␤1, ␤2, and ␤3 isoforms of the ␤ subunit, and the ␥ modulator.

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