Stress, alcohol and infection during early development: A brief review of common outcomes and mechanisms.

Although stress is an adaptive physiological response to deal with adverse conditions, its occurrence during the early stages of life, such as infancy or adolescence, can induce adaptations in multiple physiological systems, including the reproductive axis, the hypothalamic-pituitary-adrenal (HPA) axis, the limbic cortex and the immune system. These early changes have consequences in adult life, as seen in the physiological and behavioural responses to stress. This review highlights the impact of several stress challenges incurred at various stages of development (perinatal, juvenile, adolescent periods) and how the developmental timing of early-life stress confers unique physiological adaptations that may persist across the lifespan. In doing so, we emphasise how intrinsic sex differences in the stress response might contribute to sex-specific vulnerabilities, the molecular processes underlying stress in the adult, and potential therapeutic interventions to mitigate the effects of early stage stress, including the novel molecular mechanism of SUMOylation as a possible key target of HPA regulation during early-life stress.

Melanocortin 4 receptor activates ERK-cFos pathway to increase brain-derived neurotrophic factor expression in rat astrocytes and hypothalamus.

Melanocortins are neuropeptides with well recognized anti-inflammatory and anti-apoptotic effects in the brain. Of the five melanocortin receptors (MCR), MC4R is abundantly expressed in the brain and is the only MCR present in astrocytes. We have previously shown that MC4R activation by the α-melanocyte stimulating hormone (α-MSH) analog, NDP-MSH, increased brain-derived neurotrophic factor (BDNF) expression through the classic cAMP-Protein kinase A-cAMP responsive element binding protein pathway in rat astrocytes. Now, we examined the participation of the mitogen activated protein kinases pathway in MC4R signaling. Rat cultured astrocytes treated with NDP-MSH 1 µM for 1 h showed increased BDNF expression. Inhibition of extracellular signal-regulated kinase (ERK) and ribosomal p90 S6 kinase (RSK), an ERK substrate, but not of p38 or JNK, prevented the increase in BDNF expression induced by NDP-MSH. Activation of MC4R increased cFos expression, a target of both ERK and RSK. ERK activation by MC4R involves cAMP, phosphoinositide-3 kinase (PI3K) and the non receptor tyrosine kinase, Src. Both PI3K and Src inhibition abolished NDP-MSH-induced BDNF expression. Moreover, we found that intraperitoneal injection of α-MSH induces BDNF and MC4R expression and activates ERK and cFos in male rat hypothalamus. Our results show for the first time that MC4R-induced BDNF expression in astrocytes involves ERK-RSK-cFos pathway which is dependent on PI3K and Src, and that melanocortins induce BDNF expression and ERK-cFos activation in rat hypothalamus.

Molecular mechanisms involved in interleukin 1-beta (IL-1ß)-induced memory impairment. Modulation by alpha-melanocyte-stimulating hormone (α-MSH).

Pro-inflammatory cytokines can affect cognitive processes such as learning and memory. Particularly, interleukin-1ß (IL-1ß) influences the consolidation of hippocampus-dependent memories. We previously reported that administration of IL-1ß in dorsal hippocampus impaired contextual fear memory consolidation. Different mechanisms have been implicated in the action of IL-1ß on long-term potentiation (LTP), but the processes by which this inhibition occurs in vivo remain to be elucidated. We herein report that intrahippocampal injection of IL-1ß induced a significant increase in p38 phosphorylation after contextual fear conditioning. Also, treatment with SB203580, an inhibitor of p38, reversed impairment induced by IL-1ß on conditioned fear behavior, indicating that this MAPK would be involved in the effect of the cytokine. We also showed that IL-1ß administration produced a decrease in glutamate release from dorsal hippocampus synaptosomes and that treatment with SB203580 partially reversed this effect. Our results indicated that IL-1ß-induced impairment in memory consolidation could be mediated by a decrease in glutamate release. This hypothesis is sustained by the fact that treatment with d-cycloserine (DCS), a partial agonist of the NMDA receptor, reversed the effect of IL-1ß on contextual fear memory. Furthermore, we demonstrated that IL-1ß produced a temporal delay in ERK phosphorylation and that DCS administration reversed this effect. We also observed that intrahippocampal injection of IL-1ß decreased BDNF expression after contextual fear conditioning. We previously demonstrated that α-MSH reversed the detrimental effect of IL-1ß on memory consolidation. The present results demonstrate that α-MSH administration did not modify the decrease in glutamate release induced by IL-1ß. However, intrahippocampal injection of α-MSH prevented the effect on ERK phosphorylation and BDNF expression induced by IL-1ß after contextual fear conditioning. Therefore, in the present study we determine possible molecular mechanisms involved in the impairment induced by IL-1ß on fear memory consolidation. We also established how this effect could be modulated by α-MSH.

Seasonal variations of Substance P in the striatum of the female rat are affected by maternal and offspring pinealectomy.

The effect of pinealectomy (PIN-X) and PIN-X+melatonin treatment during pregnancy (PIN-X+MEL) 100µg/100g body weight on Substance P (SP) in the striatum was investigated in offspring female rats. Female offspring were divided into control and PIN-X at the neonatal period, and studied at days 31 and 60. PIN-X mother/control offspring showed a positive influence on striatal SP values in winter, at both ages and in spring at day 31. However, this effect of maternal PIN-X was not observed in summer or fall. The effect of PIN-X on the offspring showed a positive effect in spring at day 31 and summer at the two ages studied. This effect was not observed in fall or winter. Two generations, PIN-X mother/PIN-X offspring, altered the effect of mother or offspring PIN-X and decreased the SP values in winter, spring and summer. Only striatal SP at day 60 in fall was increased. In two generations PIN-X, the striatal SP values were similar to those observed in control mother/control offspring. The effect of PIN-X+MEL treatment on mothers during pregnancy was inhibitory for the intact offspring and stimulatory for PIN-X offspring. In conclusion, the results indicate that maternal and offspring PIN-X seem to exert a rotative and positive seasonal influence from winter to spring to summer. Two generations PIN-X disrupted this rotative circuit and in fall a compensatory discharge of SP was observed.

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.

Neurokinin A inhibits oxytocin and GABA release from the posterior pituitary by stimulating nitric oxide synthase.

Neurokinin A (NKA) is a tachykinin that participates in the control of neuroendocrine functions. The posterior pituitary lobe (PP) contains abundant nitric oxide synthase (NOS), suggesting that nitric oxide (NO) may play a role in controlling the release of neuropeptides and neurotransmitters. In the present project, we investigated the in vitro effect of NKA on oxytocin release from hypothalamic explants and PP of male rats and the possible involvement of NO in the action of NKA. Since NKA inhibits gamma-aminobutyric acid (GABA) release from PP, we also examined the role of NO in the effect of NKA on basal and K(+)-evoked GABA release. NKA (10(-7)-10(-5) M) significantly decreased oxytocin release from PP, whereas it did not affect its release from hypothalamic explants. The inhibitory effect of NKA on oxytocin release from PP was completely blocked by the NOS inhibitors N(G)-monomethyl-L-arginine (L-NMMA, 0.5 mM) or N(G)-nitro-L-arginine-methyl-ester (L-NAME, 1 mM). Sodium nitroprusside (0.5 mM), an NO releaser, had no effect on basal GABA release but significantly decreased K(+)-evoked GABA release. L-NMMA (0.3 mM) and L-NAME (0.5 mM) increased K(+)-evoked GABA release, indicating that NO plays an inhibitory role in GABA release from PP. The inhibition in both basal and K(+)-evoked GABA release induced by NKA (10(-7) M) was reduced by L-NAME (1 mM). Also, NKA (10(-7) M) increased NO synthesis as measured by [(14)C] citrulline production. Considered all together, our data indicate that NO may mediate the inhibitory effect of NKA on the release of both oxytocin and GABA from PP.

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.

Interaction between substance P and TRH in the control of prolactin release.

Substance P (SP) may participate as a paracrine and/or autocrine factor in the regulation of anterior pituitary function. This project studied the effect of TRH on SP content and release from anterior pituitary and the role of SP in TRH-induced prolactin release. TRH (10(-7) M), but not vasoactive intestinal polypeptide (VIP), increased immunoreactive-SP (ir-SP) content and release from male rat anterior pituitary in vitro. An anti-prolactin serum also increased ir-SP release and content. In order to determine whether intrapituitary SP participates in TRH-induced prolactin release, anterior pituitaries were incubated with TRH (10(-7) M) and either WIN 62,577, a specific antagonist of the NK1 receptor, or a specific anti-SP serum. Both WIN 62,577 (10(-8) and 10(-7) M) and the anti-SP serum (1:250) blocked TRH-induced prolactin release. In order to study the interaction between TRH and SP on prolactin release, anterior pituitaries were incubated with either TRH (10(-7) M) or SP, or with both peptides. SP (10(-7) and 10(-6) M) by itself stimulated prolactin release. While 10(-7) M SP did not modify the TRH effect, 10(-6) M SP reduced TRH-stimulated prolactin release. SP (10(-5) M) alone failed to stimulate prolactin release and markedly decreased TRH-induced prolactin release. The present study shows that TRH stimulates ir-SP release and increases ir-SP content in the anterior pituitary. Our data also suggest that SP may act as a modulator of TRH effect on prolactin secretion by a paracrine mechanism.

Role of phosphodiesterase and protein kinase G on nitric oxide-induced inhibition of prolactin release from the rat anterior pituitary.

OBJECTIVE: In order to determine the mechanism by which nitric oxide (NO) inhibits prolactin release, we investigated the participation of cGMP-dependent cAMP-phosphodiesterases (PDEs) and protein kinase G (PKG) in this effect of NO. METHODS: Anterior pituitary glands of male rats were incubated with inhibitors of PDE and PKG with or without sodium nitroprusside (NP). Prolactin release, and cAMP and cGMP concentrations were determined by RIA. RESULTS AND CONCLUSIONS: The inhibitory effect of NP (0.5 mmol/l) on prolactin release and cAMP concentration was blocked by EHNA (10(-4)mol/l) and HL-725 (10(-4)mol/l), inhibitors of cGMP-stimulated cAMP-PDE (PDE2). 8-Br-cGMP (10(-4) and 10(-3)mol/l), which mimics cGMP as a mediator of NP effects on prolactin release, also decreased cAMP concentration. Zaprinast (10(-4)mol/l), a selective inhibitor of specific cGMP-PDE (PDE5), potentiated the NP effect on cAMP concentration. Rp-8-[(4-chlorophenyl)thio]-cGMP triethylamine (Rp-8-cGMP, 10(-7)-10(-6)mol/l), an inhibitor of PKG, reversed the effect of NP on prolactin release. The present study suggests that several mechanisms are involved in the inhibitory effect of NO on prolactin release. The activation of PDE2 by cGMP may mediate the inhibitory effect of NO on cAMP concentration and therefore on prolactin release. NO-activated PKG may also be participating in the inhibitory effect of NO on prolactin release.

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.

Intracellular distribution of GABA in the rat anterior pituitary. An electron microscopic autoradiographic study.

We studied the internalization and intracellular distribution of [3H] GABA in rat anterior pituitary cells. Electron microscopic autoradiography of anterior pituitary fragments or dispersed pituitary cells incubated with [3H] GABA showed that lactotrophs and, to a lesser extent, somatotrophs were the only cells that contained radioactive grains. Grain density analysis performed on dispersed pituitary cells after a pulse-chase experiment (10 min pulse and then change to a medium without radioactive GABA for various periods up to 2 h) revealed that GABA internalized by lactotrophs was distributed in various intracellular membranous organelles. Of the cell compartments examined, plasma membrane, Golgi apparatus, mitochondria and secretory granules had different time-dependent labeling patterns. The highest grain density values were associated with plasma membrane (at the first chase time) and the Golgi apparatus. Mitochondria and secretory granules also showed significant grain density values. A similar pattern of distribution was observed when fragments of prolactin-secreting pituitary adenomas were incubated with [3H] GABA. These results provide morphological data on the cellular specificity and intracellular distribution of GABA in anterior pituitary cells.

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.

Modulation of the hypothalamo-pituitary-gonadal axis and the pineal gland by neurokinin A, neuropeptide K and neuropeptide gamma.

Modulation of the hypothalamo-pituitary-gonadal axis and the pineal gland by neurokinin A, neuropeptide K, and neuropeptide gamma. PEPTIDES 1999. Neurokinin A (NKA), neuropeptide K (NPK) and neuropeptide gamma (NPG) are members of the family of tachykinins, and act preferentially on NK-2 tachykinin receptors. These peptides are widely distributed and are potent stimulators of smooth muscle contraction, especially in the respiratory and gastrointestinal tract. They also induce vasodilatation and plasma extravasation. Through their effects on the vascular tone, they are also potential regulators of the blood flow and therefore of the function of many organs and tissues. Tachykinins have been demonstrated to influence the secretory activity of endocrine cells, and they may have a physiological role as regulators of endocrine functions. A number of reports have indicated that NPK, NKA and NPG act on the hypothalamo-pituitary gonadal axis to regulate functions related to reproduction. Therefore, we thought that, at this point, it was important to review the available evidence suggesting the role of these tachykinins on reproductive functions by effects exerted at 3 different levels of regulation: the hypothalamus, the anterior pituitary and the gonads. These 3 tachykinin peptides were reported to have effects on reproductive functions, acting on the control of the secretion of gonadotropin and prolactin at the level of the hypothalamo-pituitary axis, and on the steroid secretion by the testes and the ovaries. Acting on the hypothalamus, tachykinins, mainly NPK, were reported to inhibit LH secretion, but this effect is dependent on the presence of gonadal steroids. On the anterior pituitary gland, however, tachykinins were shown to stimulate LH and prolactin secretion, and this effect is also dependent on the presence of gonadal steroids. Tachykinin concentrations in the hypothalamus and pituitary are regulated by steroid hormones. In the hypothalamus, estrogens and testosterone increase tachykinin concentration. In the anterior pituitary gland, estradiol and thyroid hormones markedly depress tachykinin concentrations. Ovariectomy and exposure to short photoperiods significantly increase anterior pituitary tachykinins in the Siberian hamster. In the pineal gland, SP and NK-1 receptors are present and, more recently, the presence of NKA and probably also NPK was demonstrated. Castration and steroid replacement modified the content of tachykinins in the pineal gland. The removal of the superior cervical ganglia was followed by an increase in NKA content in the pineal gland. These results suggest that gonadal steroids may influence tachykinins in the pineal gland. In the gonads, tachykinins stimulated the secretory activity of Sertoli cells, but inhibited testosterone secretion by Leydig cells. There are very few reports on the role of tachykinins in the ovary, but some of them indicated that these peptides are present in some of the ovarian structures, and they may affect the secretion of ovarian steroids. Thus, NKA, NPK and NPG appear to have a modulatory role, mainly acting as paracrine factors, on the hypothalamo-pituitary-gonadal axis.

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.

Effect of lipopolysaccharide on tumor necrosis factor and prolactin release from rat anterior pituitary cells.

TNF-alpha plays a critical role in the cascade of neuroendocrine events during inflammation and septic shock. It also affects the release of pituitary hormones and acts as a growth factor in immune and nonimmune cells. The aim of the present study was to investigate the release of TNF-alpha from rat anterior pituitary cells and the effect of the steroid medium on its release. Cultured anterior pituitary cells from lactating rats spontaneously released TNF-alpha. The presence of lipopolysaccharide (LPS, 0.1 microg/mL) in the culture medium significantly increased TNF-alpha release and inhibited prolactin release. Chronic estrogenization of ovariectomized rats or the presence of 17 beta-estradiol in the culture medium also increased TNF-alpha release. LPS significantly stimulated TNF-alpha release in all groups and abrogated the estrogen-induced prolactin release. We also investigated the effect of TNF-alpha on prolactin release. The presence of TNF-alpha (50 ng/mL) in the culture medium inhibited prolactin release from anterior pituitary cells. These data show that anterior pituitary cells in culture release TNF-alpha and that this release is stimulated by estrogens. Our results also indicate that LPS inhibits prolactin release in an estrogenic environment, suggesting that TNF-alpha could affect pituitary hormone release during endotoxemia.

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.

Vasoactive intestinal peptide (VIP) mediates the effect of estrogens on the dopaminergic tone in the hypothalamic-pituitary axis of ovariectomized (OVX) rats.

The role of vasoactive intestinal peptide (VIP) in the regulation of dopamine (DA) concentration in mediobasal hypothalamus (MBH), posterior and anterior pituitary of ovariectomized (OVX) estrogenized rats was studied using passive immunization against VIP with a specific antiserum (a-VIP). Chronic estradiol administration decreased DA concentration in MBH, and in posterior and anterior pituitary, compared to OVX control rats. DA tissue concentration increased following a-VIP administration to control and estrogenized OVX rats. In vitro study of VIP and a-VIP on DA release from MBH in chronically estrogenized OVX rats showed that estrogens decreased DA evoked-release from MBH;a-VIP increased DA evoked-release from MBH of control OVX and estrogenized rats. VIP decreased DA evoked-release from MBH of OVX rats, but had no effect on estrogenized rats. VIP decreased DA tissue concentration in MBH of OVX control but not of estrogenized rats. It is suggested that VIP decreases DA synthesis and release from hypothalamic neurons in female rats, and that VIP partially mediates the inhibitory effect of long-term estrogen administration on DA release from MBH.

Role of nitric oxide/cyclic GMP pathway in the inhibitory effect of GABA and dopamine on prolactin release.

The anterior pituitary gland is a site of nitric oxide (NO) production and action, suggesting a local regulatory function. We recently reported that NO inhibits in vitro prolactin release. The aim of the present study was to establish the mechanism of action of NO on prolactin release and to determine whether NO is involved in the inhibitory effect of GABA on prolactin release. Since NO exerts its action through cGMP by activating guanylate cyclase in different tissues, we examined the effect of sodium nitroprusside (NP), a NO releaser, on intrapituitary cGMP levels. Incubation of anterior pituitary glands with 0.5 mM NP 4-fold increased intrapituitary cGMP content, but decreased intrapituitary cAMP levels. In addition, we studied the effect of NP on prolactin release in the presence of LY 83583, an inhibitor of guanylate cyclase activity and 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase activity. 10 microM LY 83583 and 0.5 mM IBMX blocked the inhibitory effect of NP on prolactin release. (10(-3) M) 8Br-cGMP, an analogue of cGMP, mimicked the effect of NP on prolactin release. On the other hand, NO seems to be involved in the inhibitory effect of GABA on prolactin release since hemoglobin, a scavenger of NO, and Nw-nitro-L-arginine methyl ester, an inhibitor of NO synthase (NOS), blocked the pituitary response to GABA. Moreover, GABA (10(-6) M) stimulated NOS activity by almost 50%. GABA increased intrapituitary cGMP levels and decreased cAMP. Dopamine stimulated NOS activity weakly. These observations suggest that NO, acting through the guanylate cyclase-cGMP pathway, inhibits prolactin secretion. In addition, NO may be involved in the inhibitory effect of GABA and dopamine on prolactin release.