Estrogen downregulates neuronal nitric oxide synthase in rat anterior pituitary cells and GH3 tumors.

The anterior pituitary gland produces neuronal nitric oxide synthase (nNOS) and nitric oxide regulates secretion of various anterior pituitary hormones. Estrogen has many functions in anterior pituitary cells including stimulation of prolactin (PRL) cell proliferation and secretion of various anterior pituitary hormones. However, the role of estradiol-17beta (E2) in regulating pituitary nNOS expression has not been previously examined. We studied the regulation of nNOS in normal pituitaries, and neoplastic GH3 pituitary tumors in order to analyze the effects of E2 on nNOS in pituitary cells. GH3 tumors expressed higher levels of nNOS proteins compared to normal pituitaries. Estrogen downregulated nNOS mRNA and protein in both estrogen-treated pituitaries with PRL cell hyperplasia and in GH3 tumors implanted into the flank of rats treated with E2 in silastic tubing. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated three alternatively spliced nNOS transcript isoforms--nNOSa, nNOSb, and nNOSc mRNAs--with distinct 5' untranslated first exons that arose from alternative splicing to a common second exon. All three spliced isoforms were found in the normal rat pituitary, whereas nNOSa and nNOSb, but not nNOSc, were expressed in GH3 tumors implanted into Wistar-Furth rats. E2 also downregulated the nNOSa alternative mRNA transcript isoform in vivo. These results indicate that the biological activity of nNOS in the normal rat anterior pituitary and in pituitary tumors is regulated by a complex pattern of alternative splicing and that some of these mRNA isoforms as well as nNOS protein are regulated by estrogen. Our results also indicate that the levels of nNOS and the alternatively spliced nNOS transcript between normal and GH3 pituitary tumors are different.