Mechanism(s) by which activation of protein kinase C is coupled to prostacyclin synthesis in granulosa cells

We examined the mechanisms by which the phospholipid-sensitive, calcium-dependent protein kinase (protein kinase C) regulates prostacyclin synthesis by ovarian cells. In monolayer cultures of swine granulosa cells, specific phorbol esters significantly augmented production of the stable immunoreactive metabolite of prostacyclin, 6-keto-prostaglandin F_1α by 3- to 8-fold. These stimulatory actions were dose (0.03-30 ng/ml) and time (24-96 h) dependent, could be reproduced by non-diterpene activators of protein kinase C, and were corroborated by high performance liquid chromatography and mass spectrometry. The rank order of potency of phorbol esters was 12-O-tetradecanoylphorbol 13-acetate (TPA) > phorbol 12,13-dibenzoate > phorbol 12,13-dibutyrate > pure phorbol base. TPA enhanced de novo synthesis of prostacyclin, and synergized with the divalent cation ionophore, A23187. Although prostacyclin synthetase activity was not induced, microsomal cyclooxygenase activity was significantly increased by phorbol treatment. Moreover, TPA doubled the intracellular accumulation of free arachidonic acid. An inhibitor of phospholipase A₂ (quinacrine 100 μM) impeded, whereas melittin (0.01 μM), an activator of cellular phospholipase A₂, and purified bacterial phospholipase A₂ (5 and 50 mU/ml) both augmented prostacyclin production. RH 59022 (30 μM), an inhibitor of diacylglyceride lipase, also suppressed prostacyclin synthesis. We conclude that the protein kinase C effector pathway is functionally coupled to de novo prostacyclin production in the swine granulosa cell. Increased eicosanoid synthesis can be accounted for by enhanced phospholipase A₂ and diacylglyceride lipase-mediated availability of arachidonic acid substrate and an activated cyclooxygenase enzyme without a change in prostacyclin synthetase activity.