Abstract
Inhibition of Na+,K+-ATPase activity by dopamine is an important mechanism by which renal tubules modulate urine sodium excretion during a high salt diet. However, the molecular mechanisms of this regulation are not clearly understood. Inhibition of Na+,K+-ATPase activity in response to dopamine is associated with endocytosis of its alpha- and beta-subunits, an effect that is protein kinase C-dependent. In this study we used isolated proximal tubule cells and a cell line derived from opossum kidney and demonstrate that dopamine-induced endocytosis of Na+,K+-ATPase and inhibition of its activity were accompanied by phosphorylation of the alpha-subunit. Inhibition of both the enzyme activity and its phosphorylation were blocked by the protein kinase C inhibitor bisindolylmaleimide. The early time dependence of these processes suggests a causal link between phosphorylation and inhibition of enzyme activity. However, after 10 min of dopamine incubation, the alpha-subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal from the plasma membrane. Dephosphorylation occurred in the late endosomal compartment. To further examine whether phosphorylation was a prerequisite for subunit endocytosis, we used the opossum kidney cell line transfected with the rodent alpha-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the alpha-subunit with a concomitant decrease in Na+,K+-ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent alpha-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser11 and Ser18). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the alpha-subunit is essential for Na+,K+-ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.
Highlights
Activators of protein kinase C (PKC), such as phorbol esters and diacylglycerol analogs, decrease Naϩ,Kϩ-ATPase activity in isolated rat renal PCT segments [7, 8] as well as the vectorial transport of sodium by isolated perfused PCTs [9]
Despite the information gained during the last few years on the regulation of Naϩ,Kϩ-ATPase activity, it is not known whether inhibition of enzyme activity in intact cells depends on the phosphorylation of the catalytic subunit, or whether such phosphorylation is necessary for subunit endocytosis in response to a physiologic agonist such as DA
In the present study, using intact renal PCT cells metabolically labeled with [32P]orthophosphate, we have examined whether dopamine phosphorylates the Naϩ,Kϩ-ATPase ␣-subunit and whether this effect is responsible for the decreased enzymatic activity and subunit endocytosis
Summary
Activators of PKC, such as phorbol esters and diacylglycerol analogs, decrease Naϩ,Kϩ-ATPase activity in isolated rat renal PCT segments [7, 8] as well as the vectorial transport of sodium by isolated perfused PCTs [9]. Inhibition of Na؉,K؉-ATPase activity in response to dopamine is associated with endocytosis of its ␣- and -subunits, an effect that is protein kinase C-dependent.
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