To define the role of GH during central nervous system development, we performed studies in cultured rat cerebral cortical cells from 14- (E14) and 17-d-old embryos (E17). The expression of GH receptor, IGF-I receptor, and IGF-I mRNAs was confirmed. In E17, GH increased total cell number (3.9-fold), [(3)H]-thymidine incorporation (3.5-fold), proliferating cell nuclear antigen levels (2.5-fold), and bromodeoxyuridine (BrdU)-positive cells (2.5-fold). GH action on nestin/BrdU-positive cells was increased in E14 cells at 3 d in vitro (80-fold) but not at 7 d in vitro. In E14 cells, GH increased (9.5-fold) beta-tubulin/BrdU cells. In E17 cells, GH induced neuronal differentiation, as indicated by the absence of beta-tubulin/BrdU-positive cells and the 5.9-fold increment of beta-tubulin protein, and increased glial fibrillary acidic protein/BrdU-positive cells (2.5-fold) and glial fibrillary acidic protein expression (4.5-fold). GH-induced proliferation and differentiation was blocked by IGF-I antiserum. GH increased IGF-binding protein-3 (IGFBP-3), IGF-I receptor protein and its phosphorylation. This study shows that GH promotes proliferation of neural precursors, neurogenesis, and gliogenesis during brain development. These responses are mediated by locally produced IGF-I. GH-induced IGFBP-3 may also have a role in these responses. Therefore, GH is able to activate the IGF-I/IGFBP-3 system in these cerebral cells and induce a physiological action of IGF-I.

There is extensive evidence that serotonin (5-HT) is implicated in the neuroendocrine control regulating the secretion of several anterior pituitary hormones. It has also been reported that the posterior pituitary is necessary for prolactin (PRL) response to 5-HT as well as to suckling, in which 5-HT implication has been demonstrated. As we have previously shown that vasoactive intestinal peptide (VIP) mediates through an autocrine or paracrine action the PRL release induced by insulin-like growth factor I, thyrotropin-releasing hormone (TRH) and dopamine withdrawal, the aim of the present work was to determine whether 5-HT has a direct action on pituitary secretion and to study the possible role of pituitary VIP in this situation. Cells from the anterior pituitary lobe (AP) were cultured either alone or together with cells from the posterior pituitary lobe (PP). As melanotropes from PP express glucocorticoid receptors in vitro, both AP cultures and cocultures of AP/PP cells were incubated in the presence or absence of corticosterone (0.1 µg/ml), thus designing four experimental conditions. Then both AP and mixed cultures were incubated with 5-HT (100 nM) for 20, 45 and 180. The release of PRL, growth hormone (GH), corticotropin (ACTH) and luteinizing hormone (LH) was stimulated by 5-HT, but only in cocultures of AP/PP cells preincubated with corticosterone, whereas follicle-stimulating hormone and thyroid-stimulating hormone release was not modified. As AP cultures did not show any response to 5-HT, both in the presence or absence of corticosterone, and as melanotropes are the main cellular type present in the PP cultures, we studied the response of α-melanocyte-stimulating hormone (αMSH) to 5-HT in PP cells cultured with or without corticosterone. Serotonin did not modify αMSH release either in the absence or the presence of corticosterone. VIP release was also stimulated by 5-HT in the cocultures, and the time response profile was only similar to that of PRL. In order to study whether pituitary VIP is implicated in 5-HT action, cocultures preincubated with corticosterone were incubated in the presence of 5-HT, a VIP-receptor antagonist (VIP-At) or simultaneously with 5-HT plus VIP-At. PRL response to 5-HT was abolished by the simultaneous presence of VIP-At, whereas GH, ACTH and LH response remained unchanged. These data demostrate that: (1) 5-HT stimulates the secretion of PRL, GH, ACTH, LH and VIP acting directly at pituitary level on PP, probably by releasing an unidentified mediator from melanotropes; (2) glucocorticoids make the response of AP cells to 5-HT possible due to the presence of PP cells in the coculture; (3) PRL response to 5-HT is mediated through an autocrine and/or paracrine action of VIP.

Vasoactive intestinal peptide (VIP) is a secretagogue for pituitary prolactin, but the importance of this peptide in the normal control of prolactin secretion is unclear. Recent studies suggest VIP synthesis within the rat anterior pituitary. We have shown (Endocrinology 124:1077) that the content of rat pituitary VIP increases in hypothyroidism. To confirm in situ pituitary synthesis of VIP and determine whether thyroid hormone effects on pituitary VIP relate to changes in VIP mRNA, Northern and in situ hybridization analyses of VIP mRNA in rat pituitaries were performed. Northern hybridization demonstrated an RNA species from rat pituitary consistent with rat VIP mRNA. Hypothyroidism increased the content of pituitary VIP mRNA, and replacement with 1-thyroxine prevented this increase. In situ hybridization showed multiple, widely-distributed hybridizing cells in pituitaries from hypothyroid animals. A distinct population of VIP-producing pituitary cells exists which may serve to modulate prolactin secretion in a paracrine or autocrine fashion.

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In previous studies we demonstrated that IGF-I induces proliferation of pituitary lactotrophs. In addition to its mitotrophic actions, IGF-I is known to prevent apoptosis induced by diverse stimuli in several cell types. In this study, we investigated the action of IGF-I on pituitary cell survival and the intracellular signaling transduction pathway implicated in this effect. Treatment of cultured male rat pituitary cells with IGF-I (10 −7 M) for 24 h prevented pituitary cell death induced by serum deprivation. The protective effect of IGF-I was blocked by phosphoinositide 3-kinase (PI3-kinase) inhibitor, LY294002, but was unaffected by PD98059, which inhibits MAP/ERK kinase (MEK1). IGF-I activation of PI3-kinase induced the phosphorylation and activation of the serine/threonine kinase Akt. Moreover, IGF-I increased the phosphorylation of the pro-apoptotic factor Bad and the levels of the anti-apoptotic protein Bcl-2 through the PI3-kinase pathway in primary pituitary cells.

Insulin-like growth factor I (IGF-I) is a potent inducer of oligodendrocyte development and myelination. Although IGF-I intracellular signaling has been well described in several cell types, intracellular mechanisms for IGF-I-induced oligodendrocyte development have not been defined. By using specific inhibitors of intracellular signaling pathways, we report here that the MAPK and phosphatidylinositol 3-kinase signaling pathways are required for the full effect of IGF-I on oligodendrocyte development in primary mixed rat cerebrocortical cell cultures. The MAPK activation, but not the phosphatidylinositol 3-kinase activation, leads to phosphorylation of the cAMP response element-binding protein, which is necessary for IGF-I to induce oligodendrocyte development. cAMP, although it does not show any effect on oligodendrocyte development, has an inhibitory effect on IGF-I-induced oligodendrocyte development that is mediated by the cAMPdependent protein kinase. Furthermore, cAMP also has an inhibitory effect on IGF-I-dependent MAPK activation. This is a cAMP-dependent protein kinase-independent effect and probably contributes to the cAMP action on IGF-I-induced oligodendrocyte development.

The neuropeptide somatostatin (SS) plays a role as a modulator of cognitive functions and as a potential tropic factor in the central nervous system. A reduction in SS levels has been demonstrated in the aging brain and in dementia. In addition, insulin-like growth factor I (IGF-I) acts as a paracrine factor in multiple GH actions and is also found in the cerebral hemispheres, where it exerts neurotropic effects. We used aging rats as an in vivo model of GH deficiency to study the possible participation of exogenous GH in the modulation of the cerebral hemispheric SS and IGF-I. Two sets of experiments were carried out. In the first set, the age-related patterns of GH, IGF-I, and SS in the serum, pituitary, and cerebral hemispheres were established. In the second experimental set, 90-day-old (adult) and 2-yr-old (aging) male rats received recombinant human GH (200 micrograms/ sc-day) or vehicle for 7 consecutive days. The serum levels of rat GH and IGF-I as well as pituitary GH messenger RNA decreased in 2-yr-old rats compared with those in adult rats. After GH treatment, pituitary GH messenger RNA levels decreased markedly in the 90-day-old and 2-yr-old rats. Serum immunoreactive GH decreased in the adult animals, whereas it remained unaffected in the aging ones, whereas serum IGF-I levels were not altered by GH treatment in either group. Immunoreactive levels and messenger RNA of both SS and IGF-I were low in the cerebral hemispheres of aging rats, but were restored to the levels found in adult rats after GH treatment. As treatment did not induce changes in the serum IGF-I levels, these results provide evidence of a stimulatory action of peripherally administered GH on the regulation of SS and IGF-I genes in the aging rat in the central nervous system. These data also show a new target action for GH and could provide a molecular basis for the improvement of some symptoms of GH deficiency that occurs after recombinant human GH treatment.

The stimulatory effect of potassium depolarization upon somatostatin (SS) mRNA levels in primary cultures of fetal cerebrocortical cells was analyzed. Depolarizing stimuli, such as 56 mM K+ exposure for 30 min, elicited an increase in immunoreactive somatostatin (IR-SS) release to the media and decreased SS mRNA levels. These were increased when exposure to depolarization stimuli was prolonged up to 3 or more hr. At this time, potassium (30 and 56 mM) acted as a secretagogue, stimulating SS secretion, but was also effective in stimulating SS mRNA levels, suggesting that SS secretion can be coupled to SS mRNA accumulation. These changes were inhibited by the Ca2+ channel antagonist verapamil. In contrast, Na+ channel blockade by TTX did not modify the 24 hr potassium-induced increase in SS mRNA, although it partially abolished potassium-induced SS secretion. Examination of the rate of disappearance of SS mRNA levels after inhibition of mRNA transcription by actinomycin-D revealed that K+ stimulation of cerebrocortical cells stabilized the SS mRNA. These results suggest that the induction of SS mRNA expression by K+ is dose dependent, and involves the modulation of ion channels. The time-course study confirmed that the K(+)-induced SS mRNA accumulation is time dependent, chronic activation of the Ca2+ channels being necessary to stimulate SS gene expression. K+ stimulation may also increase the level of SS mRNA in cerebrocortical cells by reducing its rate of degradation.