Sakuma Satoru, Fujimoto Yohko, Katoh Yuhsuke, Fujita Tadashi
Department of Hygienic Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, 569-1094, Osaka, Japan.
Prostaglandins Leukot Essent Fatty Acids. 2003 May;68(5):343-9. doi: 10.1016/s0952-3278(03)00026-7.
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.
在生理条件下，少量游离花生四烯酸（AA）从膜磷脂中释放出来，环氧化酶（COX）和酰基辅酶A合成酶（ACS）竞争性地作用于这种脂肪酸，形成前列腺素（PGs）和花生四烯酰辅酶A（AA-CoA）。为了阐明决定游离AA代谢命运进入这两条途径的因素，我们研究了一氧化氮（NO）供体1-羟基-2-氧代-3-（N-甲基-3-氨丙基）-3-甲基-1-三氮烯（NOC7）和过氧亚硝酸根（ONOO⁻）对兔肾髓质微粒体中高浓度和低浓度AA（60和5 μM）形成PG和AA-CoA的影响。肾髓质微粒体在含有COX辅因子（还原型谷胱甘肽和对苯二酚）和ACS辅因子（ATP、MgCl₂和辅酶A）的0.1M Tris/HCl缓冲液（pH 8.0）中与60或5 μM [¹⁴C]-AA一起孵育。孵育后，通过用石油醚和乙酸乙酯进行选择性萃取来分离PG（作为总PGs）和AA-CoA。当使用60 μM AA作为底物浓度时，NOC7在0.5 μM时刺激PG形成，而在50和100 μM时抑制PG形成，且不影响AA-CoA形成。当使用5 μM AA作为底物浓度时，NOC7在10 μM及以下对PG和AA-CoA形成无影响，但在50 μM及以上时增强AA-CoA形成，同时PG形成减少。利用NO解毒剂羧基-2-苯基-4,4,5,5-四甲基咪唑啉-1-氧基3-氧化物进行的实验表明，使用60和5 μM AA时观察到的NOC7的作用是由NO引起的。另一方面，ONOO⁻刺激60 μM AA形成PG，在100 μM浓度下AA-CoA形成无变化，但当使用5 μM AA作为底物浓度时，它对PG和AA-CoA形成无影响。这些发现表明，NO和ONOO⁻对肾髓质微粒体中PG和AA-CoA形成的作用可能会根据底物浓度而改变。添加超氧化物生成系统（黄嘌呤-黄嘌呤氧化酶加过氧化氢酶）可逆转使用5 μM AA时NO的作用，表明超氧化物是NO作用的重要调节剂。这些结果表明，在低底物浓度（接近AA的生理浓度）下，NO而非ONOO⁻可作为PG和AA-CoA形成的调节剂，并且超氧化物可能在NO的作用中发挥重要作用。
