Cytochrome P450-derived arachidonic acid metabolism in the rat kidney: Characterization of selective inhibitors

Abstract

We characterized the inhibitory activity of several acetylenic and olefinic compounds on cytochrome P450 (CYP)-derived arachidonic acid ω- hydroxylation and epoxidation using rat renal cortical microsomes and recombinant CYP proteins. Among the acetylenic compounds, 6-(2- propargyloxyphenyl-)hexanoic acid (PPOH) and N-methylsulfonyl-6-(2- propargyloxyphenyl)hexanamide were found to be potent and selective inhibitors of microsomal epoxidation with IC50 values of 9 and 13 μM, respectively. On the other hand, 17-octadecynoic acid inhibited both ω- hydroxylation and epoxidation of arachidonic acid with IC50 values of 7 and 5 μM, respectively. The olefinic compounds N-methylsulfonyl-12,12- dibromododec-11-enamide (DDMS) and 12,12-dibromododec-11-enoic acid (DBDD) exhibited a high degree of selectivity inhibiting microsomal ω-hydroxylation with an IC50 value of 2 μM, whereas the IC50 values for epoxidation were 60 and 51 μM for DDMS and DBDD, respectively. Studies using recombinant rat CYP4A isoforms showed that PPOH caused a concentration-dependent inhibition of ω-hydroxylation and 11,12-epoxidation by CYP4A3 or CYP4A2 but had no effect on CYP4A1-catalyzed ω-hydroxylase activity. On the other hand, DDMS inhibited both CYP4A1- and CYP4A3- or CYP4A2-catalyzed arachidonic acid oxidations. Inhibition of microsomal activity by PPOH, but not DDMS, was time- and NADPH-dependent, a result characteristic of a mechanism-based irreversible inhibitor. These studies provide information useful for evaluating the role of the CYP-derived arachidonic acid metabolites in the regulation of renal function and blood pressure.