Characterization of the adrenal 11 beta-hydroxylase in inbred salt-sensitive and resistant rats

Rats have been bred for susceptibility and resistance to the hypertensive effect of dietary salt (S/JR & R/JR). S/JR have an abnormal adrenal steroid 11 beta,18-hydroxylase activity resulting in increased production of 18-OH-DOC. S/JR also produce increased quantities of 19-nor-DOC, which may be related, since the 11 beta,18-hydroxylase also catalyzes the 19-hydroxylation of DOC, a pivotal step in 19-nor-DOC biosynthesis. The purpose of the present studies was to further characterize the mutant S/JR adrenal steroid 11 beta,18-hydroxylase. Preliminary studies are also presented on assessing the renal 19-desmolase, the last step in 19-nor-DOC biosynthesis. Adrenal glands were harvested from R/JR and S/JR and prepared for incubation studies, protein immunoblotting, and RNA analysis. Kidneys from Sprague-Dawley rats were also used for isolated renal perfusion studies. Both S/JR and R/JR strains had a single immunostaining band for 11 beta,18-hydroxylase at 51,000 molecular weight which were equal in intensity. Both strains had a single RNA transcript at 4.3 kilobases which hybridized with equal intensity to the bovine cDNA (pB11-9). The Km for 11 beta- and 18-hydroxylation was identical within strains but was different between strains. The Km for 19-hydroxylation was different between S/JR and R/JR, and was much greater than 11 beta- and 18-hydroxylation in both strains. This suggests that the catalytic site for 19-hydroxylation is different than that for 11 beta- and 18-hydroxylation and that the S/JR enzyme binds the substrate with higher affinity than the R/JR enzyme. In the isolated perfusion studies the rat kidneys converted 80% of 19-oxo-DOC to either 19-oic-DOC or 19-nor-DOC. These data demonstrate that the difference in S/JR enzyme activity is probably due to a point mutation in the enzyme or to a change in a regulatory protein.