DYSFUNCTIONAL D1(A) RECEPTOR-G-PROTEIN COUPLING IN PROXIMAL TUBULES OF SPONTANEOUSLY HYPERTENSIVE RATS IS NOT DUE TO ABNORMAL G-PROTEINS

Background Dysfunctional dopamine neurotransmission and defective D1(A ) receptor-G protein coupling exist in renal proximal tubules (RPT) of the spontaneously hypertensive rat (SHR). Objective To determine whet her the G proteins in SHR are abnormal, preventing formation of agonis t high affinity sites in SHR, Methods We examined the expression level s of the alpha-subunits of G proteins, as well as D1(A) receptor recep tor coupling to exogenously added normal G proteins, in RPT of SHR and the normotensive Wister-Kyoto (WKY) rat, Results In the presence of 1 10 mmol/l NaCl, the D1(A) dopamine receptor-selective agonist SKF R-38 393 binds both to high- and to low-affinity sites on solubilized and r econstituted D1(A) receptors extracted from renal proximal tubules of normotensive Wistar-Kyoto (WKY) rats, In the spontaneously hypertensiv e rat (SHR), SKF R-38393 bound to a single site on the reconstituted r eceptor with affinity values corresponding to the low-affinity state o f the receptor, Western blot analyses indicated that the alpha-subunit of the guanine nucleotide binding protein (G-protein), G(s), was expr essed at similar levels, whereas G(o) alpha was not expressed in proxi mal tubule membranes from WKY rats and SHR. Pretreatment of proximal t ubule membranes with the alkylating agent N-ethylmaleimide in the pres ence of SKF R-38393 inactivated alpha-subunits of endogenous G-protein s, but not D1(A) receptors, resulting in loss of high-affinity binding sites in WKY rats, These N-ethylmaleimide-treated D1(A) receptors fro m WKY rats, when reconstituted with exogenous sources of G-proteins, w ere able to couple to these exogenous G-proteins, with complete restor ation of high-affinity sites, Moreover, the affinity values and the pr oportion of these hybrid sites were similar to those of untreated rece ptors, and these affinity sites were regulated by guanine nucleotide a nalogs, Reconstitution of D1(A) receptors from SHR with the same exoge nous G-proteins failed to similarly induce formation of the high-affin ity binding sites in the hybrid reconstituted systems, and SKF R-38393 continued to bind in a single low-affinity state of the receptor, Con clusion These results demonstrate that the absence of G-protein coupli ng in SHR is due to intrinsic defects within the receptor protein, rat her than to any abnormalities of the endogenous G-proteins themselves.