Role of nitric oxide and cyclic guanosine 3',5'-monophosphate in the estrogen regulation of cervical epithelial permeability.

Treatment of cultured human cervical epithelia on filters with 17beta-estradiol increases paracellular permeability in a time- and dose-related manner (EC50, 1.1 nM). The objective of the present study was to understand the molecular mechanisms of estrogen action. In cultured human cervical epithelial cells the nitric oxide (NO) donors sodium nitroprusside (SNP) and N-[ethoxycarbonyl]-3-[4-morpholinyl]sydnoneimine (SIN-I) and the cell-permeable cGMP analog 8-bromo-cGMP (8-Br-cGMP) increased paracellular permeability. In estrogen-treated cells SNP and 8-Br-cGMP increased permeability to a lesser degree than in estrogen-deprived cells, suggesting that NO and cGMP mediate the effect of estrogen on permeability. Tamoxifen blocked the estrogen-induced increase in permeability, but it had no effect on increases in permeability that were induced by SNP or by 8-Br-cGMP. LY-83583 (blocker of guanylate cyclase) attenuated the effect of SNP, whereas KT-5823 (blocker of cGMP-dependent protein kinase) abrogated the effects of both SNP and 8-Br-cGMP. Treatment with 17beta-estradiol increased NO release and cellular cGMP in a dose-related manner (EC50, approximately 1 nM), and the effects were inhibited by tamoxifen. Treatment with SNP increased cGMP maximally, even in estrogen-deficient cells. LY-83583 blocked the estrogen-induced increase in cGMP, but neither LY-83583 nor KT-5823 had a significant effect on the estrogen-induced increases in NO release and cellular cGMP. The NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester decreased NO release, and pretreatment of cells with L-arginine reversed the effect. Cultured human cervical epithelial cells express messenger RNA for the NOS isoforms endothelial NOS (ecNOS), brain NOS, and inducible NOS. 17beta-Estradiol up-regulated ecNOS messenger RNA, and tamoxifen blocked the effect. Based on these results we suggest that the effect of estradiol on permeability involves four signaling steps: 1) activation of estrogen receptors, 2) increase in ecNOS transcription and up-regulation of NO activity, 3) NO activation of guanylate cyclase and increase in cGMP, and 4) cGMP activation of cGMP-dependent protein kinase.