A NOVEL INHIBITORY EFFECT ON PROSTACYCLIN SYNTHESIS OF COUPLING-FACTOR-6 EXTRACTED FROM THE HEART OF SPONTANEOUSLY HYPERTENSIVE RATS

The possible presence of an unknown prostacyclin synthesis inhibitory substance has been reported in some strains of rats. We purified the i nhibitory substance from the heart of spontaneously hypertensive rats by collecting active fractions after gel-filtration column chromatogra phy and two steps of reverse-phase high performance liquid chromatogra phy. The amino acid composition and automated gas-phase sequencing of the full-length substance and fragments cleaved by AspN indicated that the prostacyclin-inhibitory peptide was identical to coupling factor 6. Recombinant rat coupling factor 6, which was synthesized using a cl eavable fusion protein strategy, attenuated base-line and bradykinin ( 10(-6) M)-induced prostacyclin synthesis and [H-3]arachidonic acid (AA ) release in human umbilical vein endothelial cells in a dose-dependen t manner (10(-9)-10(-7) M). Exogenous AA- and prostaglandin H-2-induce d prostacyclin synthesis were unchanged even after treatment with 10(- 7) M recombinant coupling factor 6. Base-line and bradykinin-induced [ H-3]AA release were suppressed by arachidonyltrifluoromethyl ketone, a relatively specific inhibitor of cytosolic phospholipase A(2) at 40 m u M, and simultaneous administration of coupling factor 6 showed no fu rther effect. Neither oleyloxyethyl phosphorylcholine at 1 mu M nor br omoenol lactone at 1 mu M affected AA release. Preincubation (1 min) w ith 10(-7) M recombinant coupling factor 6 had no influence on adenosi ne diphosphate- and collagen-induced platelet aggregations. We conclud e that coupling factor 6 possesses a novel function of prostacyclin sy nthesis inhibition in endothelial cells via suppression of Ca2+-depend ent cytosolic phospholipase A(2), although it is unclear whether coupl ing factor 6 functions in normal conditions or only in pathophysiologi cal states.