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G protein expression and second messenger formation in human granulosa cells

The expression of heterotrimeric (β 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 _s, _{i3 i1,2}, _q,11 and β subunits. Three antibodies against _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_s. The ₂ adrenoceptor agonist, clonidine, inhibited hCG-stimulated cAMP formation and this effect was blocked with pertussis toxin, thus involving a G_i-type protein, most likely G_i2. Oxytocin provoked an increase in formation of inositol phosphates and intracellular calcium concentration, which was partly pertussis toxin resistant, providing evidence of G_q,11 activation. However, a significant component of the response to oxytocin could be blocked by pertussis toxin, indicating G_i-mediated phospholipase C activation (by either _i or β 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_s:G_i activation, whereas activation of the inositol phospholipid pathway and rises in intracellular calcium involved both G_q,11 and G_i pathways.