Different substrate utilization between prostaglandin endoperoxide H synthase-1 and -2 in NIH3T3 fibroblasts.
Recent studies suggested that prostaglandin endoperoxide H synthase- and prostaglandin endoperoxide H synthase-2 (PGHS-1 and PGHS-2) utilize different pools of arachidonic acid for synthesizing prostanoids. Using cultured murine NIH3T3 fibroblasts, we investigated the mechanism for the different utilization of arachidonic acid between PGHS-1 and -2. Histofluorescence staining for PGHS activity in intact cells demonstrated that quiescent 3T3 cells expressed only PGHS-1 activity and serum-activated 3T3 cells pretreated with aspirin expressed only PGHS-2 activity. Endogenous arachidonic acid released by calcium ionophore A23187 was not converted by PGHS-1 but exclusively converted by PGHS-2. In the cell free system, the kinetics of PGHS-1 were not so much different from those of PGHS-2. However, in intact cells, arachidonic acid at concentrations lower than 2.5 microM was converted by PGHS-2 alone but not by PGHS-1. Our findings indicated that this small amount of arachidonic acid as released by some stimuli is converted exclusively by PGHS-2. Furthermore, treating the PGHS-2-expressing cells with sodium selenite or ebselen, reducing agents of intracellular peroxides, only decreased PGHS-2 activity. We speculate that only PGHS-2 has been activated by intracellular peroxides and subsequently, it can convert the arachidonic acid released endogenously.
Shitashige
M
M
Department of Physiological Chemistry, Graduate School, Faculty of Dentistry, Tokyo Medical & Dental University, Japan.
Morita
I
I
Murota
S
S
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism; D001241:Aspirin / Q000494:pharmacology; D001393:Azoles / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002453:Cell Cycle; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D007150:Immunohistochemistry; D007476:Ionophores / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D054833:Isoindoles; D007700:Kinetics; D008054:Lipid Peroxides / Q000032:analysis; D008565:Membrane Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016566:Organoselenium Compounds / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D018038:Sodium Selenite / Q000494:pharmacology; D013379:Substrate Specificity
D000975:Antioxidants; D001393:Azoles; D007476:Ionophores; D007527:Isoenzymes; D054833:Isoindoles; D008054:Lipid Peroxides; D008565:Membrane Proteins; D016566:Organoselenium Compounds; D016718:Arachidonic Acid; D000001:Calcimycin; C042986:ebselen; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; C496512:Ptgs1 protein, mouse; D018038:Sodium Selenite; D001241:Aspirin