CLONING OF THE ALPHA-SUBUNIT OF G(S) PROTEIN FROM SPONTANEOUSLY HYPERTENSIVE RATS

Enhanced sodium reabsorption by the kidney has a significant role in t he development of genetic hypertension. In the spontaneously hypertens ive rat (SHR) model of genetic hypertension, the enhanced sodium reabs orption likely arises from abnormal hormonal regulation of tubular tra nsport. Since hormonal signaling pathways are coupled frequently via G TP binding proteins, one explanation for hormonal abnormalities in SHR would be a defect in a GTP binding protein or proteins. Recent work h as suggested that the regulation of Na+,K+-ATPase activity by cholera toxin-sensitive GTP binding proteins is abnormal in SHR. The purpose o f the present studies was to clone the alpha(s)-subunit, which is the subunit ADP ribosylated by cholera toxin, of G(s) protein to determine whether it is abnormal in SHR. Reverse transcription-polymerase chain reaction was able to detect mRNA for alpha(s) in both Wistar-Kyoto (W KY) rats and SHR. Northern analysis indicated that equivalent amounts of alpha(s) mRNA were present in WKY rats and SHR. S1 nuclease analysi s demonstrated that there was no difference in the amount of alpha sho rt and long forms between WKY rats and SHR. Subcloning and sequencing of polymerase chain reaction products from WKY rats and SHR indicated that the alpha(s) forms present in renal cortex were identical. ADP ri bosylation studies with cholera toxin demonstrated the presence of equ ivalent amounts of alpha(s) protein in WKY rats and SHR. Taken togethe r, these results suggest that the abnormal regulation of Na+,K+-ATPase activity by a cholera toxin-sensitive pathway in SHR does not arise f rom a defect in the alpha(s)-subunit.