Estrogen downregulates neuronal nitric oxide synthase in rat anterior pituitary cells and GH(3) tumors

The anterior pituitary gland produces neuronal nitric oxide synthase (nNOS) and nitric oxide regulates secretion of various anterior pituitary hormone s, Estrogen has many functions in anterior pituitary cells including stimul ation of prolactin (PRL) cell proliferation and secretion of various anteri or pituitary hormones. However, the role of estradiol-17 beta (E-2) in regu lating pituitary nNOS expression has not been previously examined, We studi ed the regulation of nNOS in normal pituitaries, and neoplastic GH(3) pitui tary tumors in order to analyze the effects of E-2 on nNOS in pituitary cel ls, GH(3) tumors expressed higher levels of nNOS proteins compared to norma l pituitaries, Estrogen downregulated nNOS mRNA and protein in both estroge n-treated pituitaries with PRL cell hyperplasia and in GH(3) tumors implant ed into the flank of rats treated with E-2 in silastic tubing. Reverse tran scriptase-polymerase chain reaction (RT-PCR) analysis demonstrated three al ternatively spliced nNOS transcript isoforms - nNOSa, nNOSb, and nNOSc mRNA s - with distinct 5' untranslated first exons that arose from alternative s plicing to a common second exon, All three spliced isoforms were found in t he normal rat pituitary, whereas nNOSa and nNOSb, but not nNOSc, were expre ssed in GH(3) tumors implanted into Wistar-Furth rats. E-2 also downregulat ed the nNOSa alternative mRNA transcript isoform in vivo. These results indicate that the biological activity of nNOS in the normal r at anterior pituitary and in pituitary tumors is regulated by a complex pat tern of alternative splicing and that some of these mRNA isoforms as well a s nNOS protein are regulated by estrogen, Our results also indicate that th e levels of nNOS and the alternatively spliced nNOS transcript between norm al and GH(3) pituitary tumors are different.