Effect of ionotropic and metabotropic glutamate agonists and D-aspartate on prolactin release from anterior pituitary cells.

Although the presence of ionotropic (iGluRs) and metabotropic (mGluRs) glutamate receptors has been demonstrated in the anterior pituitary, recent reports on the direct effect of glutamate on prolactin (PRL) secretion by anterior pituitary cells have presented contradictory results. Hence, the aim of this study was to determine the effect of ionotropic (iGluRs) and metabotropic (mGluRs) glutamate receptor agonists on prolactin (PRL) release. In addition, since D-Aspartate (D-Asp) is found in the pituitary and is involved in neuroendocrine regulation, we also studied the direct action of D-Asp on PRL secretion. Finally, since the posterior pituitary participates in the regulation of PRL secretion, we examined the influence of the posterior pituitary on the effects of NMDA and D-Asp on PRL release. Glutamate (1000 microM) increased PRL secretion from cultured anterior pituitary cells. Both NMDA (100 microM) and kainate (100 microM) increased PRL secretion and these effects were blocked by a specific NMDA receptor antagonist. AMPA did not modify PRL release in these cultures. The group I and II mGluR agonist, trans-ACPD (1000 microM), and a specific group II mGluR agonist, L-CCG-I (100-1000 microM), inhibited whereas specific group I and III mGluR agonists, 3-HPG and L-AP4 respectively, had no effect on PRL release. Finally, D-Asp (100-1000 microM) stimulated PRL secretion and this effect was reduced by a NMDA receptor antagonist. When anterior pituitary cells were cultured in the presence of posterior pituitary cells, NMDA did not modify PRL or GABA release, while D-Asp increased PRL secretion and decreased GABA release in these cocultures. In conclusion, our results show that L-glutamate has a differential direct effect on PRL release: it exerts a stimulatory action via iGluRs and an inhibitory effect via mGluRs. D-Asp could directly stimulate PRL release through NMDA receptors. D-Asp may also stimulate PRL release by decreasing GABA release from the posterior pituitary.

Estrogenic status influences nitric oxide-regulated TNF-alpha release from human peripheral blood monocytes.

Cytokines and nitric oxide (NO) have been implicated in bone loss caused by estrogen deficiency. Here we evaluated the effect of nitric oxide synthase (NOS) inhibitors on the bone particle resorbing activity and TNF-alpha release of cultured peripheral blood monocytes (PBM) obtained from 10 premenopausal (PreM) and 10 postmenopausal (PostM) women. Gonadal status (menopause < 3 yr) was assessed by FSH and estradiol. Bone alkaline phosphatase and N-Telopeptide were significantly increased in PostM. Significant differences between PreM and PostM women were observed in bone mineral density of lumbar spine. The bone particle resorbing activity of PBM cultured in the presence of L-arginine-methyl ester (NAME) or aminoguanidine, NOS inhibitors, was determined by (45)Ca release from rat bone labeled particles. TNF-alpha release was assayed in supernatants by ELISA. (45)Ca release was higher in PostM (p < 0.01) and was enhanced by NAME (p < 0.02). Furthermore, TNF-alpha release from PBM was significantly higher in PostM (p < 0.01). Aminoguanidine significantly increased TNF-alpha release in PreM. Based on these findings and on the evidence that estrogen stimulates NOS, we suggest that estrogen withdrawal may reduce the inhibitory effect of NO on TNF-alpha release. Thus, this increased production of TNF-alpha could contribute to the increased postmenopausal bone turnover.

Nitric oxide mediates the inhibitory effect of tumor necrosis factor-alpha on prolactin release.

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine that markedly affects neuroendocrine functions. This cytokine is expressed in the anterior pituitary where its receptors are also present. Nitric oxide (NO) is synthesized in gonadotropes and folliculo-stellate cells of the anterior pituitary. Since NO directly inhibits prolactin secretion, we investigated the involvement of NO in the inhibitory effect of TNF-alpha on prolactin release from anterior pituitary cells of female rats. The presence of L-NAME (1 mM), an inhibitor of NO synthase (NOS), in the incubation medium significantly blunted the inhibition of prolactin release produced by TNF-alpha (50 ng/ml). TNF-alpha increased nitrite release to the incubation medium. The activity of NOS as measured by [(14)C]citrulline production was significantly enhanced when anterior pituitary cells were incubated with TNF-alpha for 8 h or more. Also, TNF-alpha induced iNOS gene expression in anterior pituitary cells as assessed by reverse transcriptase-polymerase chain reaction. The current results indicate that NO is involved in the inhibitory effect of TNF-alpha on prolactin secretion and that TNF-alpha induces iNOS transcription and stimulates NO synthesis in anterior pituitary cells.

Estrogens modulate the inhibitory effect of tumor necrosis factor-alpha on anterior pituitary cell proliferation and prolactin release.

Considering that tumor necrosis factor-alpha (TNF-alpha) is involved in normal tissue homeostasis and that its receptors are expressed in the anterior pituitary, we examined the effect of this cytokine on pituitary cell growth. Because anterior pituitary function depends on hormonal environment, we also investigated the influence of gonadal steroids in the effects of TNF-alpha on cell proliferation and the release of PRL from anterior pituitary cells. In addition, the release of TNF-alpha and its action on the release of PRL from anterior pituitary cells of rats at different stages of the estrous cycle was evaluated. In minimum essential medium D-valine, a medium that restricts fibroblastic proliferation, TNF-alpha (10 and 50 ng/mL) reduced 3H-Thymidine incorporation, DNA content, and active cell number. TNF-alpha failed to affect proliferation of cells from ovariectomized (OVX) rats. However, it significantly inhibited growth of cells from OVX rats cultured with 17beta-estradiol (E2) (10(-9) M) and from chronically estrogenized rats. TNF-alpha decreased the release of PRL from cells of intact rats, especially in proestrous, OVX rats cultured with E2 and chronically estrogenized rats. The release of anterior pituitary TNF-alpha was higher in proestrous rats. These results indicate that TNF-alpha plays an inhibitory role in anterior pituitary cell growth and the release of PRL in an estrogen-dependent manner.

Effect of interleukin-6 and tumor necrosis factor-alpha on GABA release from mediobasal hypothalamus and posterior pituitary.

The release of cytokines during infection, inflammation and stress induces brain-mediated responses, including alterations of neuroendocrine functions. We examined the effect of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on release of gamma-aminobutyric acid (GABA) from mediobasal hypothalamic (MBH) explants and posterior pituitaries (PP) of male rats. IL-6 (10 ng/ml) did not modify basal GABA release from MBH and PP, but significantly increased GABA release under depolarizing conditions (40 mM K(+)). This effect was abolished by incubation of the tissue with indomethacin, an inhibitor of cyclooxygenase activity, indicating that prostaglandins could mediate the stimulation of GABA release induced by IL-6. On the contrary, TNF-alpha (50 ng/ml) significantly decreased K(+)-evoked GABA release from both MBH and PP. This inhibitory effect was not modified by indomethacin. Neither IL-6 nor TNF-alpha affected nitric oxide synthesis, as measured by [(14)C]citrulline production. The current results indicate that IL-6 stimulates GABA release from both hypothalamus and posterior pituitary by a mechanism mediated by prostaglandins. On the contrary, TNF-alpha inhibits GABA release from both tissues. These results suggest the possibility that GABAergic activity in the hypothalamic-pituitary axis could be involved in neuroendocrine responses to cytokines.

The hormonal status modulates the effect of neurokinin A on prolactin secretion in female rats.

We have previously reported that neurokinin A (NKA), a tachykinin closely related to substance P, increases the release of prolactin (PRL) from the anterior pituitary gland of male rats, but not from pituitaries of ovariectomized (OVX) female rats. In this study, we evaluated the influence of estrogens in the action of NKA on PRL secretion in female rats. NKA stimulated the in vitro release of PRL from pituitary glands of OVX-chronically estrogenized rats, and of proestrus and estrus rats, but had no effect in anterior pituitaries of diestrus rats. In addition, we observed that cultured anterior pituitary cells of OVX rats responded to NKA only when they were incubated for 3 days in the presence of estradiol 10(-9) M. This effect was blocked by L-659,877, an NK-2 receptor antagonist. We also studied the action of NKA on PRL release during lactation. The response of anterior pituitary cells to NKA was variable over this period. The maximal sensitivity to NKA was observed at day 10 of lactation. Furthermore, the blockade of endogenous NKA by the administration of an anti-NKA serum to lactating rats reduced the PRL surge induced by the suckling stimulus. These results show that the responsiveness of the anterior pituitary gland of female rats to NKA is modulated by the endocrine environment, and suggest that NKA may participate in the control of PRL secretion during the estrus cycle and lactation.

The effect of excitatory aminoacids on GABA release from mediobasal hypothalamus of female rats.

The purpose of the present study was to examine the in vitro effect of L-glutamate and its agonists on basal and potassium-evoked GABA release from incubated mediobasal hypothalamus (MBH) of intact, ovariectomized (OVX) and OVX-estrogenized female rats. L-glutamate (100 microM) decreased evoked GABA release from MBH of intact female rats in diestrus. NMDA and quisqualate (10 and 100 microM) modified neither basal nor evoked hypothalamic GABA release of intact rats. However, kainate (10 and 100 microM) decreased hypothalamic basal and evoked GABA release of intact rats. Kainate induced no changes in basal or in evoked GABA release from hypothalami of OVX rats, but decreased GABA release in chronically estrogenized rats. DNQX (6,7-dinitroquinoxaline-2,3-dione), a non-NMDA receptor antagonist, failed to affect GABA release but blocked the inhibitory effect of kainate. The kainate effect was not Mg2+-sensitive and was not inhibited by D-AP5 (D(-)-2-amino-5-phosphonopentanoic acid), an NMDA-specific receptor antagonist. Kainate induced no changes in nitric oxide synthase activity in MBH of either intact or estrogenized rats. These data indicate that kainate decreases GABA release from MBH of female rats through a non-NMDA receptor subtype, and provide evidence to support the view that kainate-mediated decrease of the hypothalamic GABAergic tone is affected by estrogens.

Nitric oxide donors modify free intracellular calcium levels in rat anterior pituitary cells.

The effect of nitric oxide donors on intracellular calcium concentration [Ca2+]i was studied in anterior pituitary cells using ratiometric FURA 2 fluorescence measurements. Sodium nitroprusside (NP) induced a transient decrease in [Ca2+]i, after which [Ca2+]i returned to, or even increased over basal values. S-Nitroso glutathione (GSNO) induced a similar decrease. NP also inhibited high [Ca2+]i achieved by depolarization with 25 mM K+. The inhibitory effect of NP was partially blunted by pretreatment with methoxy-verapamil, and in calcium free buffer, and was not altered by thapsigargin. Interestingly, in calcium free buffer there was a significant stimulatory effect of NP, which was partially blunted by thapsigargin. We conclude that NO donors modify [Ca2+]i in anterior pituitary cells. The action is biphasic, with an initial decrease in [Ca2+]i probably related to a decrease of Ca2+ influx through VDCC, and an increase evidenced in calcium free buffer in which the inhibitory component is absent, and partially depends on thapsigargin sensitive calcium stores.

Nitric oxide inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing gamma-aminobutyric acid.

Nitric oxide synthase (NOS)-containing neurons, termed NOergic neurons, occur in various regions of the hypothalamus, including the median eminence-arcuate region, which plays an important role in controlling the release of luteinzing hormone-releasing hormone (LHRH). We examined the effect of NO on release of gamma-aminobutyric acid (GABA) from medial basal hypothalamic (MBH) explants incubated in vitro. Sodium nitroprusside (NP) (300 microM), a spontaneous releaser of NO, doubled the release of GABA. This release was significantly reduced by incubation of the tissue with hemoglobin, a scavenger of NO, whereas hemoglobin alone had no effect on the basal release of GABA. Elevation of the potassium concentration (40 mM) in the medium increased GABA release 15-fold; this release was further augmented by NP. Hemoglobin blocked the increase in GABA release induced by NP but had no effect on potassium-induced release, suggesting that the latter is not related to NO. As in the case of hemoglobin, NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, had no effect on basal release of GABA, which indicates again that NO is not significant to basal GABA release. However, NMMA markedly inhibited the release of GABA induced by high potassium, which indicates that NO plays a role in potassium-induced release of GABA. In conditions in which the release of GABA was substantially augmented, there was a reduction in GABA tissue stores as well, suggesting that synthesis of GABA in these conditions did not keep up with release of the amine. Although NO released GABA, there was no effect of the released GABA on NO production, for incubation of MBH explants with GABA had no effect on NO release as measured by [14C]citrulline production. To determine whether GABA had any effect on the release of LHRH from these MBH explants, GABA was incubated with the tissue and the effect on LHRH release was determined. GABA (10(-5) or 10(-6) M) induced a 70% decrease in the release of LHRH, indicating that in the male rat GABA inhibits the release of this hypothalamic peptide. This inhibition in LHRH release induced by GABA was blocked by NMMA (300 microM), which indicates that GABA converts the stimulatory effect of NO on LHRH release into an inhibitory one, presumably via GABA receptors, which activate chloride channels that hyperpolarize the cell. Previous results have indicated that norepinephrine stimulates release of NO from the NOergic neurons, which then stimulates the release of LHRH. The current results indicate that the NO released also induces release of GABA, which then inhibits further LHRH release. Thus, in vivo the norepinephrinergic-driven pulses of LHRH release may be terminated by GABA released from GABAergic neurons via NO.