1. ¹Georgetown University Medical Center, Washington, Distr. Columbia, USA
2. ²Case Western Reserve University School of Medicine, Cleveland, Ohio, USA
3. ³University of Virginia Health Sciences Center, Charlottesville, Virginia, USA

Correspondence: P Yu, Division of Pediatric Nephrology, Department of Pediatrics, Georgetown University Medical Center, 3800 Reservoir Road, NW, Washington, DC 20007-2197, USA. E-mail: yup@georgetown.edu

Received 19 September 2005; Revised 14 April 2006; Accepted 9 May 2006; Published online 19 July 2006.

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 D₁R 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 (21.01.5 density units, DU) than in normotensive rats (7.42.9 DU). The increased basal serine phosphorylation of D₁Rs 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 (174%) and alter D₁R cell surface expression in intact cells from SHRs. Alkaline phosphatase treatment of RPTC membranes decreased D₁R phosphorylation and enhanced fenoldopam stimulation of adenylyl cyclase activity (266%) 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.