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17beta-Estradiol (17beta-E(2)) is known to exert neuroprotective activity against beta-amyloid, but its exact target and mechanism of action in this effect have not been elucidated. The involvement of astroglia in neuroprotection of 17beta-E(2) against the beta-amyloid fragment [betaAP((25-35))] has been evaluated using an experimental paradigm in which medium conditioned from rat astroglia pretreated with 17beta-E2 was transferred to pure rat cortical neurons challenged with 25 microm betaAP((25-35)) for 24 h. The toxicity of betaAP((25-35)) was assessed by flow cytometry, evaluating the ability of the peptide to induce an aberrant mitotic cell cycle in neurons. The results obtained indicate that conditioned medium from astrocytes preexposed to 17beta-E(2) for 4 h increased the viability of cortical neurons treated with betaAP((25-35)). This effect was not modified by treatment with the estrogen receptor antagonist ICI 182,780, added directly to neurons, nor was it mimicked by direct addition of 17beta-E(2) to neuronal cultures during exposure to betaAP((25-35)). A soluble factor stimulated by 17beta-E(2) seemed to be involved, and accordingly, the intracellular and released levels of TGF-beta1 were increased by 17beta-E(2) treatment, as established by Western blot analysis. In addition, the intracellular content of TGF-beta1 in immunopositive cells, as detected by flow cytometry, was reduced, suggesting that 17beta-E(2) stimulated mainly the release of the cytokine. In support of a role for TGF-beta1 in astrocyte-mediated 17beta-E(2) neuroprotective activity, incubation with a neutralizing anti-TGF-beta1 antibody significantly modified the reduction of neuronal death induced by 17beta-E(2)-treated astrocyte-conditioned medium.

To investigate possible effects that may contribute, together with a direct action on neurohormone secretion, to the impairment of gonadal axis function during inflammation, we evaluated the effect of TNF alpha on the growth and viability of GT1-7 hypothalamic neurons and the intracellular transduction pathways involved in these effects. TNF alpha caused a reduction of cell number and an induction of apoptotic death. These effects were mimicked by cell-permeable analogs of ceramide and by neutral or acidic sphingomyelinase. Exposure to acidic sphingomyelinase induced a persistent (up to 48 h) reduction of cell growth and apoptosis, whereas the effect of neutral sphingomyelinase was time limited. The involvement of acidic sphingomyelinase in TNF alpha action was demonstrated by the partial prevention of ceramide generation, apoptosis, and reduced cell growth by the inhibitor of the acidic sphingomyelinase-generating pathway, D609, whereas the involvement of ceramide was proved by complete prevention of TNF alpha-induced effects by treatment with okadaic acid at concentrations inhibiting ceramide-dependent protein phosphatase. The present data indicate that TNF alpha, through activation of ceramide-generating pathways, is able to affect GT1-7 cell viability, suggesting an additional effect that may contribute to the global action of this cytokine on neuroendocrine activities.

A role for nitric oxide (NO) in the regulation of hypothalamic neurohormone secretion has been suggested. The aim of the present study was to establish a direct involvement of this novel intracellular regulatory molecule in the control of GnRH release. For this purpose, the GT1-1 GnRH-secreting continuous cell line was treated with various agents that can modify the endogenous NO synthase activity or, alternatively, with substances that can liberate NO, mimicking an increased concentration of this molecule in the cell. Treatment of GT1-1 cells with increasing concentrations of L-arginine, the direct precursor of NO, produced a marked reduction of norepinephrine-stimulated GnRH release despite a lack of effect on basal secretion. Similarly, the NO donors SIN-1 and acidified NaNO2 potently reduced basal as well as KCl-stimulated GnRH secretion. Conversely, sodium nitroprusside caused a significant inhibition of KCl-stimulated, but not basal, GnRH secretion. Addition of these agents to GT1-1 cells resulted in a marked increase in intracellular cGMP accumulation. Addition of the NO synthase inhibitors N-nitro-L-arginine and N-nitro-L-arginine methyl ester stimulated basal GnRH secretion without modifying norepinephrine- or KCl-stimulated release. In addition, treatment of GT1-1 cells with both L-arginine analogs produced a significant inhibition of the basal cGMP concentration. Together, these data suggest an inhibitory role for NO in the control of GnRH secretion from GT1-1 cells.

We investigated the involvement of arachidonate in the PRL secretory process using three experimental systems: hemipituitary glands incubated in vitro, cultured pituitary cells, and dispersed anterior pituitary cells perifused in columns. Arachidonate (100 microM) significantly (P less than 0.05) stimulated PRL release in the former system and stimulated PRL secretion in a dose-related manner in cultured cells. In hemipituitary glands, indomethacin, a cyclooxygenase inhibitor, potentiated the arachidonate-mediated stimulation, whereas nordihydroguaiaretic acid or BW755c abolished it. The latter two agents, but not indomethacin, abolished the effect of phospholipase A2 on PRL release in vitro. BW755c also inhibited the stimulatory effect of TRH on PRL release in both experimental systems. Conversely, the stimulation of PRL release by phorbol myristate acetate (PMA), although significantly reduced, was not abolished by either nordihydroguaiaretic acid or BW755c. Quinacrine, a phospholipase A2 inhibitor, also abolished the stimulatory effect of phospholipase A2 or TRH on PRL release. In cultured cells, quinacrine inhibits basal PRL release, but does not affect PRL release induced by arachidonate or (Bu)2 cAMP. These results more firmly establish a role for arachidonate as an intracellular mediator of PRL release and suggest the involvement of an arachidonate metabolic pathway(s) (lipoxygenase and epoxygenase) other than prostaglandin or thromboxane formation. The effect of PMA on PRL release may be attributable only in part to an increase in the production of arachidonate metabolites, and most of PMA's effect on PRL release may relate to its activation of protein kinase C.

Dopamine reduces the stimulation of intracellular [3H]arachidonate release produced by the two PRL-stimulating peptides angiotensin-II and TRH. This effect is concentration dependent and is mediated by stimulation of D-2 dopamine receptors. D-2 receptor agonists (bromocriptine, dihydroergocryptine, and dihydroergocristine) inhibit the release of fatty acid induced by angiotensin-II with a potency that parallels their ability to inhibit PRL release in vitro. Conversely, the selective D-2 receptor antagonist L-sulpiride completely prevents dopamine's effect, whereas SCH 23390 (a D-1 receptor antagonist) is ineffective. The inhibitory action of dopamine does not seem to be consequent to an action on the adenylate cyclase-cAMP system, as 8-bromo-cAMP (1 mM) does not affect either basal or dopamine-inhibited [3H]arachidonate release. However, a 24-h pertussis toxin pretreatment significantly reduces the action of dopamine on fatty acid release. Collectively, these results suggest that D-2 dopamine receptor-mediated inhibition of intracellular [3H]arachidonate release requires the action of a GTP-binding protein, but is not a consequence of an inhibitory action on cAMP levels.

We investigated the effects of angiotensin peptides on the breakdown of specific membrane phospholipids, the inositol lipids, in anterior pituitary cells in culture, measuring the water-soluble products (inositol phosphates) produced during the cleavage of phosphoinositides by phospholipase C. Both angiotensin II and angiotensin I in the presence of 10 mM LiCl potently increased, in a concentration-dependent manner, total [3H]inositol phosphate and PRL release in cultured rat anterior pituitary cells. The release of LH, TSH, or GH was not significantly enhanced by the peptides. The effect on inositol phosphate accumulation was significant at 0.01 nM, and maximal stimulation (approximately 5-fold increase) occurred at 10 nM, with an ED50 of about 0.3 nM. The stimulatory effects of both angiotensin II and angiotensin I were antagonized by the receptor antagonists saralasin and Sar1,Ile8-angiotensin II. Moreover, 1 microM captopril, an inhibitor of angiotensin-converting enzyme, antagonized the effects of 0.1 and 1 nM angiotensin I, suggesting that the effect of angiotensin I on phosphoinositide breakdown and PRL release is dependent on prior conversion of angiotensin I to angiotensin II. The effect of angiotensin II was very rapid. Fractionation of the water-soluble inositol phosphates showed that angiotensin II significantly increased inositol bisphosphate and inositol triphosphate at 10 sec, whereas inositol monophosphate was increased only after 40 sec. These data indicate that in the pituitary, and presumably in the lactotroph, the binding of angiotensin II to specific membrane receptors provokes increased polyphosphoinositide hydrolysis, leading to increased production of intracellular messengers, i.e. inositol triphosphate and 1,2-diacylglycerol, responsible for the stimulation of PRL release.

We studied the association between the inhibition of phosphatidylinositol (PI) turnover and the inhibition of PRL secretion in the presence of dopamine. The incorporation of radiolabeled phosphate into anterior pituitary gland PI as well as serum PRL levels were significantly (P less than 0.01) greater in female than in male rats. No significant sex-related difference was found in the incorporation by pituitary tissue of 32P into phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Dopamine decreased the incorporation of 32P into PI, but not into PC or PE, by female rat pituitary glands; this effect was reversed by two dopamine receptor-blocking agents, haloperidol and pimozide. After dopamine was removed from the incubation medium, basal 32P incorporation into PI was restored within 10 min. The administration of bromocriptine (500 micrograms/kg, ip, 4 h earlier) significantly reduced pituitary PI turnover. Conversely, in vivo injection of alpha-methyl-p-tyrosine (alpha MpT; 200 mg, ip, 2.5 h before death), an inhibitor of catecholamine biosynthesis, dramatically increased serum PRL levels. In vitro incorporation of 32P into PI, but not into PC or PE, increased (+130%) when these glands were incubated for 30 min with radiolabeled phosphate. The in vitro addition of 0.5 microM dopamine to glands from alpha MpT-treated rats counteracted the stimulation of 32P incorporation into PI produced by alph MpT treatment. In rats bearing the transplantable PRL-secreting tumor MtTW15, the hyperprolactinemia produced by the tumor stimulates hypothalamic turnover of dopamine, with a consequent inhibition of pituitary gland PRL secretion. 32P incorporation into PI, but not into PC or PE, was significantly (P less than 0.01) inhibited (-41%) in pituitary glands from these rats. The injection of alpha MpT (200 mg/kg, ip) or haloperidol (2 mg/kg, ip) 12 and 3 h before death into MtTW15 tumor-bearing rats abolished the inhibition of 32P incorporation into pituitary PI. Dopamine also decreased PI turnover in the 7315a PRL-secreting pituitary tumor. Our data indicate that the PI cycle may be an intracellular mechanism controlling PRL release in the rat and that the changes in its cleavage and turnover may be an early postreceptor event responsible for the inhibition of PRL secretion produced by factors such as dopamine.

The in vitro effect of dopamine, haloperidol and pimozide on 32Pi incorporation into phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine by anterior pituitary glands was studied. Dopamine decreased the incorporation of 32Pi into phosphatidylinositol in a dose-related manner without affecting phosphatidylcholine and phosphatidylethanolamine. Haloperidol and pimozide did not modify 32Pi incorporation into these phospholipids by themselves but completely reversed the inhibitory effect of dopamine on phosphatidylinositol. The inhibition of 32Pi incorporation into phosphatidylinositol synthesis in the presence of 500 nM dopamine was significant at 20 min of incubation and maximal at 30 min. The possibility that a decrease in phosphatidylinositol cleavage and turnover may be involved in the inhibitory regulation of prolactin secretion by DA is suggested.