No abstract

Dopamine inhibits Na؉ ,K ؉ -ATPase activity in renal tubule cells. This inhibition is associated with phosphorylation and internalization of the ␣ subunit, both events being protein kinase C-dependent. Studies of purified preparations, fusion proteins with site-directed mutagenesis, and heterologous expression systems have identified two major protein kinase C phosphorylation residues (Ser-11 and Ser-18) in the rat ␣ 1 subunit isoform. To identify the phosphorylation site(s) that mediates endocytosis of the subunit in response to dopamine, we have performed site-directed mutagenesis of these residues in the rat ␣ 1 subunit and expressed the mutated forms in a renal epithelial cell line. Dopamine inhibited Na ؉ ,K ؉ -ATPase activity and increased ␣ subunit phosphorylation and clathrin-dependent endocytosis into endosomes in cells expressing the wild type ␣ 1 subunit or the S11A ␣ 1 mutant, and both effects were blocked by protein kinase C inhibition. In contrast, dopamine did not elicit any of these effects in cells expressing the S18A ␣ 1 mutant. While Ser-18 phosphorylation is necessary for endocytosis, it does not affect per se the enzymatic activity: preventing endocytosis with wortmannin or LY294009 blocked the inhibitory effect of dopamine on Na ؉ ,K ؉ -ATPase activity, although it did not alter the increased ␣ subunit phosphorylation induced by this agonist.We conclude that dopamine-induced inhibition of Na ؉ ,K ؉ -ATPase activity in rat renal tubule cells requires endocytosis of the ␣ subunit into defined intracellular compartments and that phosphorylation of Ser-18 is essential for this process.

The (Na(+)+K+)ATPase, an integral membrane protein located in virtually all animal cells, couples the hydrolysis of ATP to the countertransport of Na+ and K+ ions across the plasma membrane. In neurons, a large portion of cellular energy is expended by this enzyme to maintain the ionic gradients that underlie resting and action potentials. Although neurotransmitter regulation of the enzyme in brain has been reported, such regulation has been characterized either as a nonspecific phenomenon or as an indirect effect of neurotransmitter-induced changes in ionic gradients. We report here that the neurotransmitter dopamine, through a synergistic effect on D1 and D2 receptors, inhibits the (Na(+)+K+)ATPase activity of isolated striatal neurons. Our data provide unequivocal evidence for regulation by a neurotransmitter of a neuronal ion pump. They also demonstrate that synergism between D1 and D2 receptors, which underlies many of the electrophysical and behavioural effects of dopamine in the mammalian brain, can occur on the same neuron. In addition, the results support the possibility that dopamine and other neurotransmitters can regulate neuronal excitability through the novel mechanism of pump inhibition.

Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic beta cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the beta cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics.