Title

D1 dopamine receptor hyperphosphorylation in renal proximal tubules in hypertension

Document Type

Journal Article

Publication Date

9-1-2006

Journal

Kidney International

Volume

70

Issue

6

DOI

10.1038/sj.ki.5001708

Keywords

D receptor (D R) 1 1; Protein phosphatase activity; Serine phosphorylation

Abstract

A defect in the coupling of the D1 receptor (D1R) to its G protein/effector complex in renal proximal tubules plays a role in the pathogenesis of spontaneous hypertension. As there is no mutation of the D 1R gene in the spontaneously hypertensive rat (SHR), we tested the hypothesis that the coupling defect is associated with constitutive desensitization/phosphorylation of the D1R. The following experiments were performed: (1) Cell culture and membrane preparations from rat kidneys and immortalized rat renal proximal tubule cells (RPTCs); (2) immunoprecipitation and immunoblotting; (3) cyclic adenosine 3′,5′ monophosphate and adenylyl cyclase assays; (4) immunofluorescence and confocal microscopy; (5) biotinylation of cell surface proteins; and (6) in vitro enzyme dephosphorylation. Basal serine-phosphorylated D1Rs in renal proximal tubules, brush border membranes, and membranes from immortalized RPTCs were greater in SHRs (21.0 ± 1.5 density units, DU) than in normotensive rats (7.4 ± 2.9 DU). The increased basal serine phosphorylation of D 1Rs in SHRs was accompanied by decreased expression of D1R at the cell surface, and decreased ability of a D1-like receptor agonist (fenoldopam) to stimulate cyclic adenosine 3′,5′ monophosphate (cAMP) production. Increasing protein phosphatase 2A activity with protamine enhanced the ability of fenoldopam to stimulate cAMP accumulation (17 ± 4%) and alter D1R cell surface expression in intact cells from SHRs. Alkaline phosphatase treatment of RPTC membranes decreased D 1R phosphorylation and enhanced fenoldopam stimulation of adenylyl cyclase activity (26 ± 6%) in SHRs. Uncoupling of the D1R from its G protein/effector complex in renal proximal tubules in SHRs is caused, in part, by increased D1R serine phosphorylation. © 2006 International Society of Nephrology.

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