Restoration of prostacyclin synthase in vascular smooth muscle cells after aspirin treatment: regulation by epidermal growth factor.
Prostacyclin is a potent vasodilator and inhibitor of platelet aggregation and plays an important role in maintenance of vascular homeostasis. Aspirin irreversibly inactivates prostacyclin synthetase by acetylating the enzyme. Recovery of the enzyme following inactivation by aspirin was studied in rat aorta smooth muscle cells in tissue culture. Confluent cultures superfused with [14C]arachidonic acid, synthesized prostacyclin (PGI2) together with prostaglandins E2, D2, and F2 alpha. Brief treatment with physiological levels of aspirin (0.2 mM) completely inactivated prostacyclin synthesis. Following aspirin removal and addition of fresh growth medium, PGI2 synthesis recovered rapidly with a T 1/2 of only 30-40 min, compared to a doubling time of 24-30 hr for the cells. Recovery of PGE2, PGD2, and PGF2a synthesis paralleled that of PGI2, confirming that cyclooxygenase rather than endoperoxide-prostacyclin isomerase was the labile component. Recovery of PGE2 synthesis after aspirin was blocked by cycloheximide but not by actinomycin D. Recovery of aspirin-inactivated cells required a non-dialyzable component present in serum. All samples tested, including fetal bovine, new-born calf, human, and guinea pig, showed the activity. Fresh serum also induced a cycloheximide-sensitive 2- to 3-fold increase in cyclooxygenase levels in resting confluent cells within 1 to 2 hr. Serum factor was also required to restore PG synthesis after aspirin-inactivation in other cells, including 3T3 mouse fibroblasts, SV40-3T3 and K-Balb 3T3 transformed mouse fibroblasts, NRK rat kidney cells, and REF-9 rat embryonic fibroblasts. The activity was thermolabile, and was completely removed from the medium by growing cells.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D001241:Aspirin / Q000494:pharmacology*; D001769:Blood; D003513:Cycloheximide / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D011464:Epoprostenol / Q000096:biosynthesis* / Q000378:metabolism; D019746:Intramolecular Oxidoreductases; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D001095:Arachidonic Acids; D016718:Arachidonic Acid; D004815:Epidermal Growth Factor; D003577:Cytochrome P-450 Enzyme System; D003513:Cycloheximide; D011464:Epoprostenol; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase; D001241:Aspirin