Mode of action of prostaglandin F2 alpha in human luteinized granulosa cells: role of protein kinase C.

It is well documented that prostaglandin F2 alpha (PGF2 alpha) inhibits progesterone production in luteal cells, but its mode of action is uncertain. It has recently been suggested that PGF2 alpha acts by activating the calcium and phospholipid-dependent protein kinase, protein kinase C (PKC). This hypothesis has been tested by comparing the site and mode of action of PGF2 alpha, a PGF2 alpha analogue (cloprostenol) and the PKC activator phorbol myristate acetate (4 beta PMA) in human granulosa-lutein cells. PGF2 alpha and cloprostenol exerted similar concentration-dependent inhibitory actions on gonadotrophin-stimulated cyclic AMP (cAMP) accumulation and progesterone production by human granulosa-lutein cells. The similarity in the actions of PGF2 alpha and cloprostenol in human granulosa-lutein cells suggests that they can be used interchangeably to study the role of PGF2 alpha in the regulation of steroidogenesis in the human ovary. Gonadotrophin-stimulated cAMP accumulation and progesterone production was also concentration-dependently inhibited by 4 beta PMA. In addition, cloprostenol and 4 beta PMA also inhibited dibutyryl cAMP-stimulated progesterone production, suggesting that these compounds inhibit LH action at sites before and after the generation of cAMP. The pre-cAMP site of action can be localised to the stimulatory G-protein (Gs) as both compounds inhibited cholera toxin-stimulated cAMP accumulation without affecting forskolin-stimulated cAMP accumulation. The post cAMP site of action can be localised to actions on cholesterol side chain cleavage enzyme, as both cloprostenol and 4 beta PMA inhibited 22R hydroxycholesterol-supported progesterone production without affecting pregnenolone-supported progesterone production. The finding that cloprostenol and 4 beta PMA interact with the steroidogenic cascade in a similar manner is indicative of a shared common mediator of their actions in human granulosa-lutein cells, i.e. PKC. The inhibitory actions of PGF2 alpha and 4 beta PMA on hLH-stimulated progesterone production were abolished in the presence of the PKC inhibitor, staurosporine. In addition, in PKC-depleted cells (achieved by exposure to 4 beta PMA for 20 h) the inhibitory actions of PGF2 alpha and 4 beta PMA were abolished. These results support the hypothesis that the inhibitory actions of PGF2 alpha are mediated by PKC in human granulosa-lutein cells.