ACTIVATION OF THE PROSTAGLANDIN FP RECEPTOR IN HUMAN GRANULOSA-CELLS

Prostaglandin F-2 alpha (PGF(2 alpha)) has regulatory (mainly luteolyt ic) effects in the ovary but the mechanism of action is not completely understood. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques were used to demonstrate the presence of mRNA encoding the PGF(2 alpha) receptor (FP receptor) in human granulosa-lutein cells. S pecific primers for the amplification of cDNA were designed and yielde d a single product of 696 bp corresponding to the FP receptor. The ide ntity of this product was verified by sequencing. Fluprostenol, a sele ctive FP receptor agonist, activated phospholipase C (PLC) and increas ed intracellular free calcium concentration, confirming the functional activation of the receptor. We have demonstrated by Western blotting that granulosa cells express PLC-beta and PLC-gamma isoforms. The cell s responded to pervanadate with increased PLC activity and increased t yrosine phosphorylation, demonstrating a functional PLC-gamma tyrosine kinase pathway. However, fluprostenol did not provoke any detectable tyrosine phosphorylation. Moreover, the effect of fluprostenol was inh ibited through protein kinase C stimulation by phorbol 12,13-dibutyrat e, and was not affected when cells were treated with phenylarsine oxid e, which blocks tyrosine phosphorylation. These results suggest that t he FP receptor activates PLC-beta rather than PLC-gamma isoforms. Flup rostenol-induced activation was pertussis toxin resistant. Granulosa c ells express G proteins of the G(q) family (resistant to pertussis tox in) and mRNA for both G alpha(q) and G alpha(11) has been identified b y RT-PCR. In conclusion, human granulosa cells have a functional FP re ceptor the effects of which are mediated through PLC-beta activation p robably via G(q/11).