Simultaneous measurement of prostaglandin and arachidonoyl CoA formed from arachidonic acid in rabbit kidney medulla microsomes: the roles of Zn2+ and Cu2+ as modulators of formation of the two products.
Under physiological conditions, small amounts of free arachidonic acid (AA) is released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) act competitively on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). To date, there is no information about the factors deciding the metabolic fate of free AA into these two pathways. In this study, we tried to establish a method for the simultaneous measurement of PG and AA-CoA synthesis from exogenous AA in microsomes from rabbit kidney medulla. The kidney medulla microsomes were incubated with [14C]-AA in 0.1 M-Tris/HCI buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl2 and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. When 60 microM AA was used as the substrate, indomethacin (an inhibitor of COX) and triacsin C (an inhibitor of ACS) reduced only PG and AA-CoA formation, respectively. On the other hand, when 5 microM AA was used as the substrate, indomethacin and triacsin C came to increase significantly the AA-CoA and PG formation, respectively. Thus, the experiments utilizing indomethacin and triacsin C revealed that the incubation using 60 microM AA can simultaneously detect the changes in the activities of COX and ACS caused by drugs, while the incubation using 5 microM AA can detect the changes in the product formation elicited by the resulting shunt of AA. Further, using these incubation conditions, the effects of Zn2+ and Cu2+ on the PG and AA-CoA formation were examined. Zn2+ inhibited the AA-CoA synthesis from 60 microM AA without affecting the PG synthesis. In contrast, when 5 microM AA was used as the substrate, a significant increase in the PG formation was observed in the presence of this ion, indicating that drug actions on the PG formation from AA by the kidney medulla microsomes may change depending on the substrate concentration. On the other hand, Cu2+ increased PG synthesis and inhibited AA-CoA synthesis from both 60 and 5 microM AA. These results suggest that the simultaneous measurements of PG and AA-CoA formation by the kidney medulla microsomes under high (60 microM) and low (5 microM) substrate concentrations can investigate the direct and indirect actions of drugs on the COX and ACS activities, and are useful for clarifying the haemostatic control of the metabolic fate of AA into the two enzymatic pathways. Furthermore, this study showed that Zn2+ and Cu2+ can modulate PG and AA-CoA formation by affecting COX activity, ACS activity, and/or the AA flow into the two enzymatic pathways.
Sakuma
S
S
Department of Hygienic Chemistry, Osaka University of Pharmaceutical Sciences, Takatsuki, Japan. sakuma@oysun01.oups.ac.jp
Fujimoto
Y
Y
Kitao
A
A
Sakamoto
H
H
Nishida
H
H
Fujita
T
T
D000214:Acyl Coenzyme A / Q000096:biosynthesis*; D000818:Animals; D000975:Antioxidants; D016718:Arachidonic Acid / Q000494:pharmacology; D003066:Coenzyme A Ligases / Q000378:metabolism*; D003300:Copper / Q000494:pharmacology; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000493:pharmacokinetics; D006873:Hydroquinones / Q000493:pharmacokinetics; D007213:Indomethacin / Q000494:pharmacology; D007679:Kidney Medulla / Q000648:ultrastructure; D008297:Male; D008861:Microsomes / Q000737:chemistry; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011453:Prostaglandins / Q000096:biosynthesis*; D011817:Rabbits; D014226:Triazenes / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D000214:Acyl Coenzyme A; D000975:Antioxidants; D016861:Cyclooxygenase Inhibitors; D004791:Enzyme Inhibitors; D006873:Hydroquinones; D011453:Prostaglandins; D014226:Triazenes; D016718:Arachidonic Acid; C034613:triacsin C; D003300:Copper; D011451:Prostaglandin-Endoperoxide Synthases; D003066:Coenzyme A Ligases; D005978:Glutathione; D015032:Zinc; C031927:hydroquinone; D007213:Indomethacin