G protein expression and second messenger formation in human granulosa cells

The expression of heterotrimeric (alpha beta gamma subunits) GTP-binding regulatory proteins (G proteins) and the activation of G protein-linked receptors in human granulosa cells were investigated. The cells were obtained from stimulated follicles in women undergoing in vitro fertilization and were cultured in serum-supplemented medium. Immunoblotting with specific antibodies showed that granulosa cell membranes express alpha s, alpha i3 alpha i1,2, alpha q,11 and beta subunits. Three antibodies against alpha o failed to detect this protein. The cells responded to hCG and to prostaglandin E2 with a dose-dependent increase in cAMP formation, confirming the functional activation of G alpha s. The alpha 2 adrenoceptor agonist, clonidine, inhibited hCG-stimulated cAMP formation and this effect was blocked with pertussis toxin, thus involving a Gi-type protein, most likely G alpha i2. Oxytocin provoked an increase in formation of inositol phosphates and intracellular calcium concentration, which was partly pertussis toxin resistant, providing evidence of G alpha q,11 activation. However, a significant component of the response to oxytocin could be blocked by pertussis toxin, indicating Gi-mediated phospholipase C activation (by either alpha i or beta gamma subunits). These data demonstrate the presence of G proteins in granulosa cells and suggest a complex regulation of hormonal signalling. The concentration of cAMP in these cells depended on the balance of G alpha s:G alpha i activation, whereas activation of the inositol phospholipid pathway and rises in intracellular calcium involved both Gq,11 and Gi pathways.