Expression of nitric oxide synthase isoforms in pregnant human myometrium

1. Endogenous nitric oxide has been proposed to play a role in the control of myometrial contractility in pregnancy. In this study, the expression, lo calisation and regulation of nitric oxide synthase (NOS) isoforms have been examined in human pregnant myometrium and cultured human myometrial smooth muscle cells, by immunoblotting, immunohistochemistry and reverse transcri ption-polymerase chain reaction. 2. Immunoblotting of extracts from freshly isolated myometrial tissue, affi nity-enriched for NOX proteins by precipitation with ADP-sepharose, reveale d expression of endothelial NOS (eNOS or NOS3) in tissues from preterm, ter m non-labour and active labour at term. Inducible NOS (iNOS or NOS2) and ne uronal NOS (nNOS or NOS1) proteins were not detected at any stage of pregna ncy. 3. Immunohistochemical detection showed that expression of eNOS protein was restricted to the endothelium of the myometrial vasculature, with no stain ing detected in myometrial smooth muscle cells. 4. Messenger RNA for all three NOS isoforms was detected, although iNOS and nNOS mRNAs were detectable only with high cycle number, implying a low cop y number. 5. NOS isoforms were not detectable in human myometrial smooth muscle cells cultured from term non-labour pregnancies. Cytokine stimulation of culture d myometrial cells did not induce iNOS expression or nitrite accumulation i n the culture medium, although both iNOS protein and nitrite release were d etected in the human pulmonary epithelial cell line A549. 6. Levels of eNOS protein and of NOS mRNA expression were not correlated wi th gestational stage, suggesting that endogenously produced NO is not likel y to be a modulator of myometrial tone during human pregnancy.