Selective inhibition of arachidonic acid epoxidation in vivo.

Cytochrome P450 (CYP)-derived arachidonic acid metabolites, including epoxyeicosatrienoic acids (EETS) and 20-HETE, have been implicated in the regulation of renal function and vascular tone. Studying the function of specific CYP arachidonate metabolites has been hampered due to lack of selective inhibitors and difficulty in their solubilization. We have identified MS-PPOH as a potent and selective inhibitor of CYP-catalyzed arachidonate epoxidation in vitro. We used 2-hydroxypropyl-beta-cyclodextrin as a vehicle in order to administer MS-PPOH in vivo. One hour after administration, MS-PPOH (5 mg, IV bolus) significantly inhibited arachidonic acid epoxidation in rat renal cortical microsomes (vehicle-282 +/- 12 pmol/mg/min, MS-PPOH-206 +/- 10 pmol/mg/min, p < 0.05) but had no effect on 20-HETE formation (vehicle-383 32 pmol/mg/min, MS-PPOH-367 +/- 9 pmol/mg/min). The inhibitory effect lasts at least for 6 hours. There was no inhibition of 20-HETE synthesis at any time point. We also examined the effect of MS-PPOH on renal excretiry function. Three hours after MS-PPOH administration to anesthetized rats, urine flow rate became significantly higher (vehicle-275 +/- 16 microl/hour, MS-PPOH-406 +/- 44 microl/hour, p < 0.05). Sodium excretion rate was also significantly higher (vehicle-28.7 +/- 4 micromol/hour, MS-PPOH-63.3 +/- 10 micromol/hour, p < 0.05) but potassium excretion rate was not affected (vehicle-65.5 +/- 5 micromol/hour, MS-PPOH-79.2 +/- 2 micromol/hour). These results suggest that MS-PPOH may be useful as a selective inhibitor of CYP-catalyzed arachidonic acid epoxidation in vivo, and implicate EETs and anti-diuretic and anti-natriuretic in the regulation of renal function.

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

We characterized the inhibitory activity of several acetylenic and olefinic compounds on cytochrome P450 (CYP)-derived arachidonic acid omega-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 microM, respectively. On the other hand, 17-octadecynoic acid inhibited both omega-hydroxylation and epoxidation of arachidonic acid with IC50 values of 7 and 5 microM, 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 omega-hydroxylation with an IC50 value of 2 microM, whereas the IC50 values for epoxidation were 60 and 51 microM for DDMS and DBDD, respectively. Studies using recombinant rat CYP4A isoforms showed that PPOH caused a concentration-dependent inhibition of omega-hydroxylation and 11, 12-epoxidation by CYP4A3 or CYP4A2 but had no effect on CYP4A1-catalyzed omega-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.

Determinants of renal vasoconstriction after systemic inhibition of nitric oxide synthesis in rats.

The effects of NG-nitro-L-arginine (L-NNA, 10 mg/kg i.v.) on renal hemodynamics were examined in control rats, rats in which renal perfusion pressure was prevented from rising after L-NNA by constricting the abdominal aorta, and rats in which tubuloglomerular feedback was inhibited by furosemide pretreatment, ureteral ligation, or both interventions combined. In control rats, L-NNA increased (P < 0.05) renal vascular resistance (274 +/- 27%) along with systemic arterial (54 +/- 4%) and renal perfusion (54 +/- 5%) pressures and decreased (P < 0.05) renal blood flow (57 +/- 4%). In rats in which renal perfusion pressure was prevented from increasing along with systemic arterial pressure (54 +/- 4%), the L-NNA-induced elevation of renal vascular resistance (173 +/- 27%) was less intense (P < 0.05). In another study, where renal perfusion pressure was fixed at pre-L-NNA levels, L-NNA-induced increases in renal vascular resistance (130 +/- 20%) were attenuated (P < 0.05) further with furosemide pretreatment (52 +/- 12%), with ureteral ligation (75 +/- 10%), and with furosemide pretreatment and ureteral ligation combined (32 +/- 8%). These data suggest that vasoconstrictor mechanisms linked to tubuloglomerular feedback and perfusion pressure elevation contribute to renal vasoconstriction after systemic inhibition of nitric oxide synthesis with L-NNA.