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The role of oestradiol in the control of uterine responsiveness to oxytocin was investigated by measuring oxytocin-induced phospholipase C activation in [³H]inositol-labelled cultured human myometrial cells. Addition of oestradiol to steroid-free culture medium (10% (v/v) fetal calf serum treated with dextran-coated charcoal in phenol red-free medium) enhanced formation of inositol phosphates and this effect was completely abolished by the anti-oestrogen tamoxifen. The inhibitory effect of tamoxifen on oxytocin-induced phospholipase C activation occurred in both steroid-free and complete culture medium; it was time- and concentration-dependent and was only partly reversed by oestradiol. When phospholipase C was activated with PGF_2α or fluoroaluminate instead of oxytocin, oestradiol and tamoxifen had the same stimulatory and inhibitory effects, respectively. The inhibitory effect of tamoxifen could not be prevented by treating the cells with pertussis toxin. Moreover, the effect of tamoxifen was not mediated by inhibition of protein kinase C, since the use of staurosporine (a protein kinase inhibitor) resulted in potentiation of phospholipase C activation by oxytocin. Both oestradiol and tamoxifen increased [³H]inositol incorporation into cellular lipids and cell proliferation. These results suggest that oestradiol enhances myometrial responsiveness to oxytocin and other agonists by facilitating phospholipase C activation at a post-receptor level. This effect is antagonized by tamoxifen; however, tamoxifen also has oestrogen-independent inhibitory effects.

The expression of heterotrimeric (αβγ 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 α_s, α_i3 α_i1,2, α_q,11 and β subunits. Three antibodies against α_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 Gα_s. The α₂ 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 Gα_i2. Oxytocin provoked an increase in formation of inositol phosphates and intracellular calcium concentration, which was partly pertussis toxin resistant, providing evidence of Gα_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 α_i or βγ 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 Gα_s:Gα_i activation, whereas activation of the inositol phospholipid pathway and rises in intracellular calcium involved both G_q,11 and G_i pathways.