Glutamate induces apoptosis in anterior pituitary cells through group II metabotropic glutamate receptor activation.

Glutamate can induce neuronal cell death by activating ionotropic glutamate receptors (iGluRs) as well as metabotropic glutamate receptors (mGluRs). In the present study, we investigated whether glutamate induces apoptosis of cultured anterior pituitary cells from female rats. Glutamate (1 mm) significantly reduced the metabolic activity of viable cells and increased the percentage of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells and caspase-3 activity in anterior pituitary cells. The inhibitory effect of glutamate on the viability of anterior pituitary cells was not observed in the presence of [2S]-alpha-ethylglutamic acid (0.75 mm), a specific group II mGluR antagonist. Also, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (LCCG-I; 0.75 mm), a specific group II mGluR agonist, reduced viability and increased the percentage of TUNEL-positive anterior pituitary cells. Group I and III mGluRs and iGluRs agonists failed to modify the metabolic activity of anterior pituitary cells. Glutamate and LCCG-I increased the percentage of TUNEL-positive lactotropes and somatotropes. The subunit mGluR2/3, belonging to group II mGluR, was localized in these cell types. Glutamate increased nitric oxide (NO) synthase (NOS) activity and inducible NOS expression in anterior pituitary cells. N-methyl-l-arginine (NMMA, 0.5 mm), a NOS inhibitor, potentiated the apoptotic effect of glutamate in anterior pituitary cells, indicating that NO may restrain glutamate-induced apoptosis. Incubation of anterior pituitary cells with a cAMP analog (N6, 2'-o-dibutyryladenosine 3', 5'-cyclic monophosphate; 1 mm) attenuated the apoptosis induced by glutamate. Glutamate and LCCG-I decreased prolactin release from anterior pituitary cells. N6, 2'-o-dibutyryladenosine 3', 5'-cyclic monophosphate reversed the inhibitory effect of glutamate on prolactin release, but NMMA failed to modify it. Our data show that glutamate induces apoptosis of lactotropes and somatotropes through group II mGluR activation, probably by decreasing cAMP synthesis.

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.

Differential effects of glutamate agonists and D-aspartate on oxytocin release from hypothalamus and posterior pituitary of male rats.

In order to determine whether ionotropic (iGluRs) and metabotropic (mGluRs) glutamate receptor activation modulates oxytocin release in male rats, we investigated the effect of agonists of both types of glutamate receptors on oxytocin release from hypothalamus and posterior pituitary. Kainate and quisqualate (1 mM) increased hypothalamic oxytocin release. Their effects were prevented by selective AMPA/kainate receptor antagonists. NMDA (0.01-1 mM) did not modify hypothalamic oxytocin release. Group I mGluR agonists, such as quisqualate and 3-HPG, significantly increased hypothalamic oxytocin release. These effects were blocked by AIDA (a selective antagonist of group I mGluRs). In the posterior pituitary, oxytocin release was not modified by kainate, quisqualate, trans-ACPD (a broad-spectrum mGluR agonist) and L-SOP (a group III mGluR agonist). However, NMDA (0.1 mM) significantly decreased oxytocin release from posterior pituitary. D-Aspartate significantly increased oxytocin release from the hypothalamus, while it decreased oxytocin release from posterior pituitary. AP-5 (a specific NMDA receptor antagonist) reduced the D-Aspartate effect in the hypothalamus, but not in the posterior pituitary. Our data indicate that the activation of non-NMDA receptors and group I mGluRs stimulates oxytocin release from hypothalamic nuclei, whereas NMDA inhibits oxytocinergic terminals in the posterior pituitary. D-Aspartate also has a dual effect on oxytocin release: stimulatory at the hypothalamus and inhibitory at the posterior pituitary. These results suggest that excitatory amino acids differentially modulate the secretion of oxytocin at the hypothalamic and posterior pituitary levels.

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.

NMDA receptor-mediated control of GABA release from neurointermediate lobes of female and male rats.

The effect of glutamate (GLUT) and its ionotropic receptor agonists on K(+)-evoked GABA release from the neurointermediate lobe (NIL) was investigated in diestrus, ovariectomized, ovariectomized-estrogenized female rats and intact male rats. GLUT and N-methyl-D-aspartate (NMDA) increased K(+)-evoked GABA release from the NIL in all the experimental groups. This stimulatory effect of NMDA was blocked by specific NMDA receptor antagonists but not by non-NMDA receptor antagonists. However, kainate did not modify evoked GABA release from the NIL in any of these groups. Neither GLUT nor NMDA modified nitric oxide synthase activity. These results indicate that GLUT, acting through NMDA receptors, stimulates evoked GABA release from the NIL of female and male rats. This effect is not influenced by gonadal status and does not appear to be mediated by nitric oxide production.

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.