Cloning of the alpha-subunit of GS protein from spontaneously hypertensive rats.
Enhanced sodium reabsorption by the kidney has a significant role in the development of genetic hypertension. In the spontaneously hypertensive rat (SHR) model of genetic hypertension, the enhanced sodium reabsorption likely arises from abnormal hormonal regulation of tubular transport. Since hormonal signaling pathways are coupled frequently via GTP binding proteins, one explanation for hormonal abnormalities in SHR would be a defect in a GTP binding protein or proteins. Recent work has suggested that the regulation of Na+,K(+)-ATPase activity by cholera toxin-sensitive GTP binding proteins is abnormal in SHR. The purpose of the present studies was to clone the alpha S-subunit, which is the subunit ADP ribosylated by cholera toxin, of GS 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 (WKY) rats and SHR. Northern analysis indicated that equivalent amounts of alpha S mRNA were present in WKY rats and SHR. S1 nuclease analysis demonstrated that there was no difference in the amount of alpha S short 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 ribosylation studies with cholera toxin demonstrated the presence of equivalent amounts of alpha S protein in WKY rats and SHR. Taken together, these results suggest that the abnormal regulation of Na+,K(+)-ATPase activity by a cholera toxin-sensitive pathway in SHR does not arise from a defect in the alpha S subunit.
Division of Nephrology, University of Texas Medical Branch, Galveston 77550.
R W
RW
Gurich
R E
RE
Beach
C R
CR
Caflisch
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002772:Cholera Toxin / Q000494:pharmacology; D003001:Cloning, Molecular; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D011918:Rats, Inbred SHR / Q000235:genetics*; D011921:Rats, Inbred WKY / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D017931:DNA Primers; D046911:Macromolecular Substances; D012333:RNA, Messenger; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D000254:Sodium-Potassium-Exchanging ATPase