Intracellular sodium modulates the state of protein kinase C phosphorylation of rat proximal tubule Na+,K+-ATPase.

The natriuretic hormone dopamine and the antinatriuretic hormone noradrenaline, acting on alpha-adrenergic receptors, have been shown to bidirectionally modulate the activity of renal tubular Na+,K+-adenosine triphosphate (ATPase). Here we have examined whether intracellular sodium concentration influences the effects of these bidirectional forces on the state of phosphorylation of Na+,K+-ATPase. Proximal tubules dissected from rat kidney were incubated with dopamine or the alpha-adrenergic agonist, oxymetazoline, and transiently permeabilized in a medium where sodium concentration ranged between 5 and 70 mM. The variations of sodium concentration in the medium had a proportional effect on intracellular sodium. Dopamine and protein kinase C (PKC) phosphorylate the catalytic subunit of rat Na+,K+-ATPase on the Ser23 residue. The level of PKC induced Na+,K+-ATPase phosphorylation was determined using an antibody that only recognizes Na+,K+-ATPase, which is not phosphorylated on its PKC site. Under basal conditions Na+,K+-ATPase was predominantly in its phosphorylated state. When intracellular sodium was increased, Na+,K+-ATPase was predominantly in its dephosphorylated state. Phosphorylation of Na+,K+-ATPase by dopamine was most pronounced when intracellular sodium was high, and dephosphorylation by oxymetazoline was most pronounced when intracellular sodium was low. The oxymetazoline effect was mimicked by the calcium ionophore A23187. An inhibitor of the calcium-dependent protein phosphatase, calcineurin, increased the state of Na+,K+-ATPase phosphorylation. The results imply that phosphorylation of renal Na+,K+-ATPase activity is modulated by the level of intracellular sodium and that this effect involves PKC and calcium signalling pathways. The findings may have implication for the regulation of salt excretion and sodium homeostasis.