NO increases permeability of cultured human cervical epithelia by cGMP-mediated increase in G-actin

Human cervical epithelial cells express mRNA for the nitric oxide (NO) synt hase (NOS) isoforms ecNOS, bNOS, and iNOS and release NO into the extracell ular medium. N-G-nitro-L-arginine methyl ester (L-NAME), an NOS inhibitor, and Hb, an NO scavenger, decreased paracellular permeability; in contrast, the NO donors sodium nitroprusside (SNP) and N-(ethoxycarbonyl)-3-(4-morpho linyl)sydnonimine increased paracellular permeability across cultured human cervical epithelia on filters, suggesting that NO increases cervical parac ellular permeability. The objective of the study was to understand the mech anisms of NO action on cervical paracellular permeability. 8-Bromo-cGMP (8- BrcGMP) also increased permeability, and the effect was blocked by KT-5823 (a blocker of cGMP-dependent protein kinase), but not by LY-83583 (a blocke r of guanylate cyclase). In contrast, LY-83583 and KT-5823 blocked the SNP- induced increase in permeability. Treatment with SNP increased cellular cGM P, and the effect was blocked by Hb and LY-83583, but not by KT-5823. Neith er SNP nor 8-BrcGMP had modulated cervical cation selectivity. In contrast, both agents increased fluorescence from fura a-loaded cells in the Ca2+-in sensitive wavelengths, indicating that SNP and 8-BrcGMP stimulate a decreas e in cell size and in the resistance of the lateral intercellular space. Ne ither SNP nor 8-BrcGMP had an effect on total cellular actin, but both agen ts increased the fraction of G-actin. Hb blocked the SNP-induced increase i n G-actin, and KT-5823 blocked the 8-BrcGMP-induced increase in G-actin. On the basis of these results, it is suggested that NO acts on guanylate cycl ase and stimulates an increase in cGMP; cGMP, acting via cGMP-dependent pro tein kinase, shifts actin steady-state toward G-actin: this fragments the c ytoskeleton and renders cells more sensitive to decreases in cell size and resistance of the lateral intercellular space and, hence, to increases in p ermeability. These results may be important for understanding NO regulation of transcervical paracellular permeability and secretion of cervical mucus in the woman.