Differential Regulation of Na-H Exchanger-3 by D₁ Receptors and G-αS in Normotensive and Spontaneously Hypertensive Rats

Abstract 1996 Poster Session II, Sunday, 5/2 (poster 182)

We have reported that an impaired ability of D₁ receptors to inhibit luminal Na-H exchanger (NHE) activity in renal proximal tubules and induce a natriuresis cosegregate with hypertension in the F2 generation of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rat (SHR) crossbreeds (J Clin Invest 97:2283, 1996). However, it is not clear if the defect is at the level of the receptor, G-proteins, or effector proteins. We, therefore, studied D₁ receptor, G-protein subunit, and NHE-3 expression in renal proximal tubules. In order to obviate any confounding contribution of second messengers, in some studies, we used renal brush border membrane vesicles (BBMV), a system devoid of cytoplasmic components and second messengers. D₁ receptor (SHR=16±6 U, n=3; WKY=13±3 U, n=3) and G-αS (SHR=16±4 U, n=5; WKY=15±2 U, n=5) expression were similar in both groups. Basal NHE activity was slightly greater in SHR (2.95 ±0.67 nmol 22Na/mg protein/min, n=12) than in WKY (2.08±0.33, n=11). NHE-3 protein expression in BBM was also slightly greater in SHR (57.3±3.2 density units[U], n=3) than in WKY (43.3±4 U, n=3). However, the non-hydrolyzable GTP, GTPγS, but not the inactive analogue, GDPβS, decreased NHE activity and increased NHE3 co-immunoprecipitated G-αS to a similar extent in WKY and SHR indicating normal function of G-proteins and NHE-3 in the SHR. The D₁ receptor function was impaired in SHR because fenoldopam increased NHE3 co-immunoprecipitated G-αS in WKY but not in SHR. Moreover, the inhibitory effects of D₁ agonists (SKF 81297, fenoldopam [5µM]) were less in SHR (- 25±3%, n=12) than in WKY (-59±5%, n=11). To determine further the interaction between G-αS, NHE-3, and the D₁ receptor, we studied immortalized renal proximal tubule cells (IRPTC) from WKY and SHR. D₁ receptor, G-αS, and NHE-3 expression were similar in SHR and WKY IRPTC. Neither fenoldopam (50 µM), time (5-120min), GTPγS nor GDPβS had any effect on NHE-3 expression in IRPTC of WKY and SHR. As in the BBM studies, GTPγS but not GDPβS, increased NHE-3 co-immunoprecipitable with G-αS to a similar extent in WKY (26±5 U, n=3) and SHR (25±4 U, n=3). However, as in BBM studies, fenoldopam (5 µM, n=5) increased NHE-3 co-immunoprecipitable with G-αS in WKY but not in SHR IRPTC. These studies suggest that the D₁ dopaminergic defect in renal proximal tubules in hypertension is proximal to G-protein subunits and effectors and presumably at the receptor level. Indeed, serine-phosphorylation of the D₁ receptor is increased in renal proximal tubules in the SHR and may be responsible for uncoupling the D₁ receptor from its G-protein/effector complex in hypertension.