Inhibition of NADPH-cytochrome P450 reductase and glyceryl trinitrate biotransformation by diphenyleneiodonium sulfate.
We reported previously that the flavoprotein inhibitor diphenyleneiodonium sulfate (DPI) irreversibly inhibited the metabolic activation of glyceryl trinitrate (GTN) in isolated aorta, possibly through inhibition of vascular NADPH-cytochrome P450 reductase (CPR). We report that the content of CPR represents 0.03 to 0.1% of aortic microsomal protein and that DPI caused a concentration- and time-dependent inhibition of purified cDNA-expressed rat liver CPR and of aortic and hepatic microsomal NADPH-cytochrome c reductase activity. Purified CPR incubated with NADPH and GTN under anaerobic, but not aerobic conditions formed the GTN metabolites glyceryl-1,3-dinitrate (1,3-GDN) and glyceryl-1,2-dinitrate (1,2-GDN). GTN biotransformation by purified CPR and by aortic and hepatic microsomes was inhibited > 90% after treatment with DPI and NADPH. DPI treatment also inhibited the production of activators of guanylyl cyclase formed by hepatic microsomes. We also tested the effect of DPI on the hemodynamic-pharmacokinetic properties of GTN in conscious rats. Pretreatment with DPI (2 mg/kg) significantly inhibited the blood pressure lowering effect of GTN and inhibited the initial appearance of 1,2-GDN (1-5 min) and the clearance of 1,3-GDN. These data suggest that the rapid initial formation of 1,2-GDN is related to mechanism-based GTN biotransformation and to enzyme systems sensitive to DPI inhibition. We conclude that vascular CPR is a site of action for the inhibition by DPI of the metabolic activation of GTN, and that vascular CPR is a novel site of GTN biotransformation that should be considered when investigating the mechanism of GTN action in vascular tissue.
Department of Pharmacology and Toxicology, Queen's University, Kingston, Ontario, Canada.
J J
JJ
McGuire
D J
DJ
Anderson
B J
BJ
McDonald
R
R
Narayanasami
B M
BM
Bennett
D000818:Animals; D001011:Aorta / Q000201:enzymology; D001711:Biotransformation / Q000187:drug effects; D006439:Hemodynamics / Q000187:drug effects; D007275:Injections, Intravenous; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D005996:Nitroglycerin / Q000493:pharmacokinetics*; D009861:Onium Compounds / Q000008:administration & dosage / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D009861:Onium Compounds; C007517:diphenyleneiodonium; D019001:Nitric Oxide Synthase; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D005996:Nitroglycerin