Nitric oxide synthase-containing cells were visualized in the anterior pituitary gland by immunocytochemistry. Consequently, we began an evaluation of the possible role of NO in the control of anterior pituitary function.Prolactin is normally under inhibitory hypothalamic control, and in vitro the gland secretes large quantities of the hormone. When hemipituitaries were incubated for 30 min in the presence of sodium nitroprusside, a releaser of NO, prolactin release was inhibited. This suppression was completely blocked by the scavenger of NO, hemoglobin. Analogs of arginine, such as NG-monomethyl-L-arginine (NMMA, where NG is the terminal guanidino nitrogen) and nitroarginine methyl ester, inhibit NO synthase. Incubation of hemipituitaries with either of these compounds significantly increased prolactin release. Since in other tissues most of the actions of NO are mediated by activation of soluble guanylate cyclase with the formation of cyclic GMP, we evaluated the effects of cyclic GMP on prolactin release. Cyclic GMP (10 mM) produced an "40%o reduction in prolactin release. Prolactin release in vivo and in vitro can be stimulated by several peptides, which include vasoactive intestinal polypeptide and substance P. Consequently, we evaluated the possible role of NO in these stimulations by incubating the glands in the presence of either of these peptides alone or in combination with NMMA. In the case of vasoactive intestinal polypeptide, the significant stimulation of prolactin release was augmented by NMMA to give an additive effect. In the case of substance P, there was a smaller but significant release of prolactin that was not significantly augmented by NMMA. We conclude that NO has little effect on the stimulatory action of these two peptides on prolactin release. Dopamine (0.1 ,uM), an inhibitor of prolactin release, reduced prolactin release, and this inhibitory action was significantly blocked by either hemoglobin (20 jig/ml) or NMMA and was completely blocked by 1 mM nitroarginine methyl ester. Atrial natriuretic factor at 1 ,uM also reduced prolactin release, and its action was completely blocked by NMMA. In contrast to these results with prolactin, luteinizing hormone (LH) was measured in the same medium in which the effect of nitroprusside was tested on prolactin release, there was no effect of nitroprusside, hemoglobin, or the combination of nitroprusside and hemoglobin on luteinizing hormone release. Therefore, in contrast to its inhibitory action on prolactin release NO had no effect on luteinizing hormone release. Immunocytochemical studies by others have shown that NO synthase is present in the folliculostellate cells and also the gonadotrophs of the pituitary gland. We conclude that NO produced by either of these cell types may diffuse to the lactotropes, where it can inhibit prolactin release. NO appears to play little role in the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accorda...

TNF-alpha is involved in the regulation of normal tissue homeostasis affecting cell proliferation, differentiation, and death. We previously reported that TNF-alpha reduces anterior pituitary cell proliferation and PRL release in an estrogen-dependent manner. In the present project we studied the induction of apoptosis by TNF-alpha in anterior pituitary cells from female rats. TNF-alpha (50 ng/ml) decreased the viability of anterior pituitary cells. Incubation with TNF-alpha for 24 h increased the percentage of terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling-positive cells. TNF-alpha increased the percentage of somatotropes and lactotropes with apoptotic nuclear morphology without affecting the proportion of apoptotic corticotropes or gonadotropes. TNF-alpha increased the percentage of apoptotic lactotropes in cultured cells from rats killed in proestrus and estrus, but not in diestrus. This effect was significantly higher in cells from rats in proestrus than in estrus. In anterior pituitary cells from ovariectomized rats, TNF-alpha significantly increased the percentage of apoptotic lactotropes only when the cells were incubated in the presence of 17beta-estradiol. These results indicate that TNF-alpha induces apoptosis in somatotropes and lactotropes from female rats. The apoptotic effect of TNF-alpha on lactotropes is dependent on estrogens and could be involved in the regulation of anterior pituitary cell renewal during the estrous cycle.

The Fas/FasL system provides the major apoptotic mechanism for many cell types, participating in cell turnover in hormone-dependent tissues. In the present study, we localized both Fas and FasL in anterior pituitary cells, mainly in lactotropes and somatotropes. The percentage of anterior pituitary cells showing immunoreactivity for Fas or FasL was higher in cells from rats killed in proestrus than in diestrus. Also, the proportion of pituitary cells from ovariectomized (OVX) rats expressing Fas or FasL increased in the presence of 17beta-estradiol (10(-9) M). This steroid increased the percentage of lactotropes with immunoreactivity for Fas or FasL and the percentage of somatotropes expressing Fas. Activation of Fas by an agonist anti-Fas antibody (Mab-Fas) decreased the vi-ability-3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT assay)-of anterior pituitary cells from OVX rats cultured in the presence of 17beta-estradiol. Also, membrane-bound FasL decreased cell viability-[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (MTS assay)-only when anterior pituitary cells from OVX rats were incubated with 17beta-estradiol. Moreover, FasL increased the percentage of hypodiploid anterior pituitary cells (flow cytometry). Mab-Fas increased the percentage of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive pituitary cells and lactotropes from OVX rats only when cells were incubated in the presence of 17beta-estradiol. Also, Mab-Fas triggered apoptosis of anterior pituitary cells from rats killed at proestrus but not at diestrus. Our results show that 17beta-estradiol up-regulates the expression of the Fas/FasL system in anterior pituitary cells and increases Fas-induced apoptosis in lactotropes, suggesting that Fas-induced apoptosis could be involved in the pituitary cell renewal during the estrous cycle.

It is now accepted that estrogens not only stimulate lactotrope proliferation but also sensitize anterior pituitary cells to proapoptotic stimuli. In addition to their classical mechanism of action through binding to intracellular estrogen receptors (ERs), there is increasing evidence that estrogens exert rapid actions mediated by cell membrane-localized ERs (mERs). In the present study, we examined the involvement of membrane-initiated steroid signaling in the proapoptotic action of estradiol in primary cultures of anterior pituitary cells from ovariectomized rats by using estren, a synthetic estrogen with no effect on classical transcription and a cell-impermeable 17beta-estradiol conjugate (E2-BSA). Both compounds induced cell death of anterior pituitary cells after 60 min of incubation as assessed by flow cytometry and the [3-(4,5-dimethylthiazol-2-yl)]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Estren, E2, and E2-BSA induced apoptosis of lactotropes and somatotropes as evaluated by the deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and immunodetection of prolactin (PRL) and growth hormone (GH). The proapoptotic effect of E2-BSA was abrogated by ICI-182,780, an antagonist of ERs. The expression of membrane-associated ERalpha was observed in PRL- and GH-bearing cells. Our results indicate that estradiol is able to exert a rapid apoptotic action in anterior pituitary cells, especially lactotropes and somatotropes, by a mechanism triggered by mERs. This mechanism could be involved in anterior pituitary cell turnover.