Dopamine inhibits Na',K+-ATPase activity in several renal tubule segments and thereby regulates urinary Na" excretion. We now show that a phosphopeptide of 31 amino acids, corresponding to residues 8-38 of the protein phosphatase inhibitor DARPP-32 (dopamine-and cAMPregulated phosphoprotein ofMr 32,000), mimics the inhibitory action of dopamine on Na',K+-ATPase activity in renal tubule cells from the ascending limb of the loop of Henle. The dephosphorylated form of the peptide Is ineffective. The results indicate that dopamine acts through a protein phosphorylation pathway to regulate the activity of an ion pump. In addition, the data suggest that inhibition of protein phosphatase 1 by phophorylated DARPl-32 is a component of the mechanism by which dopatine regulates urinary Na`excretion. Na+,K+-ATPase regulates a number of vital functions, including intracellular electrolyte homeostasis and pH, cell volume, membrane potential, cellular uptake of amino acids, and transcellular Na' transport (1). Na',K+-ATPase is particularly abundant in kidney and brain. Dopamine has been shown to inhibit the activity of this enzyme in neostriatal neurons (2) and in several segments of the rat renal nephron, including the proximal tubule (3, 4), the thick ascending limb (TAL) of the loop of Henle (5), and the cortical collecting tubule (6). The peripheral actions of dopamine are critical for the regulation of Na+ and extracellular volume homeostasis and for the regulation ofblood pressure (7). In fact, dopamine is the drug of choice in many clinical situations in which renal function is compromised. Therefore, an understanding of the cellular and molecular mechanisms by which dopamine regulates cellular Na' homeostasis is of interest both to cell biology and to clinical medicine.DARPP-32 (dopamine-and cAMP-regulated phosphoprotein of Mr 32,000) is localized to cells containing the D1 subclass of dopamine receptor, including medium-sized spiny neurons of the neostriatum (8-10) and renal tubule cells of the TAL (5). (In this report we have used the terminology D1 and D2 to classify dopamine receptors. In the kidney, the functional equivalents of the D1 and D2 dopamine receptors are often referred to as the DA1 and DA2 receptors, respectively.) Phosphorylation ofDARPP-32 on threonine-34 converts it into a potent inhibitor (K-10-9M) (11) of protein phosphatase 1, a major protein phosphatase in virtually all tissues (12). Although the biochemical mechanism of action of DARPP-32 has been established (11), its precise physiological role in signal transduction is not yet understood. Distinct neurotransmitter pathways regulate the phosphorylation (13) and dephosphorylation (14) of DARPP-32. However, there has been no direct evidence that DARPP-32 mediates any of the actions of dopamine or other neurotransmitters that regulate its state of phosphorylation. In this study, we present evidence that phosphorylated DARPP-32 mimics D1 dopamine receptor agonists in inhibiting Na',K+-ATPase activity in renal tubule segments from the medul...