Cytochrome P-450-dependent HETEs: profile of biological activity and stimulation by vasoactive peptides

M. A. Carroll, M. Balazy, P. Margiotta, D. D. Huang, J. R. Falck and J. C. McGiff Department of Pharmacology, New York Medical College, Valhalla 10595, USA. The cytochrome P-450 pathway is capable of metabolizing arachidonic acid to omega- and subterminal hydroxylase metabolites, 16-, 17-, 18-, 19-, and 20-hydroxyeicosatetraenoic acids (P-450 HETEs). We have quantitated, by gas chromatography-mass spectrometry (GC/MS), endogenous HETEs exiting the rabbit isolated perfused kidney elicited by hormonal stimulation. Kidneys were perfused with Krebs-Henseleit solution containing indomethacin (2.8 microM) to prevent further metabolism of HETEs by cyclooxygenase. Phenylephrine (2-3 microM) was added to the perfusate to raise perfusion pressure to approximately 80 mmHg. Angiotensin II (ANG II), arginine vasopressin (AVP), and bradykinin (BK) were injected into the renal artery and perfusates collected throughout the vasoactive response. After addition of an internal standard, deuterated 19-HETE, perfusates were extracted and purified and P-450 HETEs were derivatized for GC/MS analysis. Under basal conditions, 16-, 18-, 19-, and 20-HETEs were released (range: 50-270 pg/ml), 19-HETE being the highest and fivefold greater than 16-HETE, the lowest. Injection of 50 ng ANG II increased by two- to sixfold P-450 HETE release associated with an increase of 40 +/- 11 mmHg in perfusion pressure. An equipressor dose of AVP (50 ng) did not release P-450 HETEs nor did a 5-micrograms dose of the vasodilator peptide BK, which decreased perfusion pressure by 22 +/- 6 mmHg. Authentic 19- and 20-HETE isomers resulted in dose-dependent dilation, as did 18(R)- and 16(R)-HETEs, whereas their enantiomers and 17-HETE isomers were without effect on perfusion pressure. The vasodilator effects of 18(R)- and 16(R)-HETEs, like 20- and 19-HETEs, were inhibited by indomethacin. Furthermore, P-450 HETEs exhibited both regio- and stereoselective inhibition of proximal tubule adenosine triphosphatase (ATPase) activity. The (S) enantiomers of 16- and 17-HETE potently inhibited activity, whereas their (R) isomers and other P-450 HETEs had negligible effects on ATPase activity. The quantity of HETEs released from the kidney, either under basal conditions or when stimulated by ANG II, and their biological profile suggest that subterminal HETEs may participate in renal mechanisms affecting vasomotion and tubular transport.