The effects of nitric oxide and peroxynitrite on the formation of prostaglandin and arachidonoyl-CoA formed from arachidonic acid in rabbit kidney medulla microsomes.
Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). To clarify factors deciding the metabolic fate of free AA into these two pathways, we investigated the effects of a nitric oxide (NO) donor 1-hydroxyl-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC7), and peroxynitrite (ONOO(-)) on the formation of PG and AA-CoA from high and low concentrations of AA (60 and 5 micro M) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 micro M [14C]-AA in 0.1M Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced GSH and hydroquinone) and cofactors of ACS (ATP, MgCl(2) and CoA). After incubation, PG (as total PGs) and AA-CoA were separated by selective extraction using petroleum ether and ethyl acetate. When 60 micro M AA was used as the substrate concentration, NOC7 stimulated the PG formation at 0.5 micro M, and inhibited it at 50 and 100 micro M, without affecting the AA-CoA formation. When 5 micro M AA was used as the substrate concentration, NOC7 showed no effect on the PG and AA-CoA formation up to 10 micro M or below, but enhanced the AA-CoA formation with a coincident decrease in the PG formation at 50 micro M or over. Experiments utilizing a NO antidote, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, revealed that the observed effects of NOC7 using 60 and 5 micro M AA are caused by NO. On the other hand, ONOO(-) stimulated the PG formation from 60 micro M AA, with no alteration in the AA-CoA formation at a concentration of 100 micro M, but when 5 micro M AA was used as the substrate concentration, it was without effect on the PG and AA-CoA formation. These findings indicate that actions of NO and ONOO(-) on the PG and AA-CoA formation by the kidney medulla microsomes may change depending on the substrate concentration. The effects of NO using 5 micro M AA were reversed by the addition of the superoxide generating system (xanthine-xanthine oxidase plus catalase), indicating that superoxide is a vital modulator of the action of NO. These results suggest that NO, but not ONOO(-), can be a regulator of the PG and AA-CoA formation at low substrate concentrations (close to the physiological concentration of AA), and that superoxide may play an important role in the action of NO.
Sakuma
Satoru
S
Department of Hygienic Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, 569-1094, Osaka, Japan. sakuma@gly.oups.ac.jp
Fujimoto
Yohko
Y
Katoh
Yuhsuke
Y
Fujita
Tadashi
T
D000214:Acyl Coenzyme A / Q000096:biosynthesis*; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D007679:Kidney Medulla / Q000648:ultrastructure; D008861:Microsomes / Q000187:drug effects* / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000378:metabolism / Q000494:pharmacology*; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D011453:Prostaglandins / Q000096:biosynthesis*; D011817:Rabbits; D014226:Triazenes / Q000494:pharmacology
C100973:(1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-3-aminopropyl)-3-methyl-1-triazene; D000214:Acyl Coenzyme A; D001095:Arachidonic Acids; D020030:Nitric Oxide Donors; D011453:Prostaglandins; D014226:Triazenes; D030421:Peroxynitrous Acid; D009569:Nitric Oxide