290 Biochimica el Biol~'sica Acta. 1136 (1992)290-296 © 1992 F_lsevict Science Pulflishets B.V.All fights reserved 0167-4889/92/$05.00 B3AMCR "3-238 Guanine nucleotide regulatory protein levels and function in spontaneously hypertensive rat vascular smith-muscle cells Catherine J. Clark a, Graeme Milligan b, Alastair R. McLellan a and John M.C Connell ~ a MRC Blood PressureUnit, Western Infnnm3; Gkttgow (UK) and s M~r Ftzarm~cologyGroup, Departmentof Biochemistr); Unirodly of Glazgo~; Glasgow(UK) (Received 12March1992) Key wards: Adenylyt cyclase: G protein; Spontaneously hypertensive rat;(Ralvascular smooth-muscle cell) We compared G-protein levelsand function in membranes from vascular srnooth-muscle cells (VSMC) derived from mesenteric arteries from SHR, WKY and Wistar rats. Basal adenylyl cyclaseactivily was significantly reduced in SHR membranescompared with W'tstar, but was similar to WKY. Isopmtereno| stinmlation(20-4 M) was signil-u:antb" lower in SHR membranescompared to WKY, but was similarto that in Wislar, whichwas also significantly lowerthan WKY. Forskolin(10-4 M} and NaF (10 -2 M), resulted in a higher stimulatoryresponse in SHR membranes. Biphasic effects of GTP on isoproterenol-stimulated membranes demonstrated unaltered G, function in SHR membranes. No significant differenceswere seen in the levels of G~a (44- and 42-kDa forms), G~2a and ~e 13-subunitin immunobIotling studies of the membranes.;unounts of Goa/GHa and Gi3a were also unchanged, in conclusion,there are differences in adenylyl cyclaseresponses in SHR VSMC membraneswhich are not a consequenceof altered levels of G-proteins, but may reflect genetic difference~ rather than effects of hypertension. Introduction Guanine nucleotide regulatory proteins (G proteins) are ke~v components in cellular signalling processes [1]; they transduce cellular signals between occupied re- ceptors and second messenger generating systems, such as adenylyl cyclase [2-7] and ph~p~olipase C (PLC) [8]. Generation of ojclic AMP (cAMP) by adenylyl cyclase is under dual control from stimulatory (G~) and inh~itory (G~) G proteins, while generation of diacyl- glycerol (DG) and inositol 1,4,5-trisphosphate (IP 3) by PLC in response to receptor activation is mediated by a G protein(s) genetically termed Gp. Abnormalities cf adenyly! cyclase and PLC activa- Correspondence to: IM.C ConnelL MRC Blood Pressure Unit, WesternInfirmar/~ Glasgow, GI 1 6NT,UK. Abbreviations: Ab, antibod5 ACA, adenylyl cyclase activity;,DG. diacytglycerot: G protein, guanine nucleotide regulatoryero~ein: HRP, horseradish pero.~dase: IgG. immunoglobulin G; IP~,inosdol 1,4~fi-tfisphosphatc; PLC, pho~hoiip~,se C; RMM, rat and mouse meintenance diet: SDS-PAGE.sodiumdodecylsulphaYe poiyacrsl- a.~ide gel clectrophoresis: S~E.,slandarderror of 1he mean;S'.-IR, .~pontaneously hypetlensive rat; ~'7"uS, Tris.buffered saline; VSMC, '~,ascular snmoth-rnusc|c cells;WKY, Wislar-Kyoto. tion have been reported in vascular (including vascular smooth-muscle of resistance vessels) [9-18] and non- vascular tissues [19-21] from the spontaneottsly hyper- tensive rat (SHR). in particular, reduced arterial relax- ation in response to/3-adrenoceptor stimulation udng isolated femoral artery material from the SHR has been observed [22], which parallels reduced adenylyl cyclase activity (ACA) response to this and other st~- ulatory ligands. The mechanism underlying this re- duced responsiveness has not been examined in detail. Similar changes in ACA have been noted in human non-insulin-dependent diabetes meilitus [23], and in animal models of diabetes which are insulin-resistant [24]. in these studies~ major changes in actMty and expression of both stimulatory and inlu'bitory G pro- teins have been reported which contribute to the ob- served changes in ACA. Human essential hyper- tension, and its animal model, the SHR, are both insulin-resistant states with hyperinsulinaemia, in this way, they resemble human diabetes and animal models of the ~ndition, and we wished to examine in this study whether abnormal ACA responses could be ob- served in cultured vascular tissue from the SHR and to test the hypothesis that changes in ACA might be a consequenc,~ . of altered activity or expression of G s or G i spe.cies.