IGF-I and vasoactive intestinal peptide (VIP) regulate cAMP-response element-binding protein (CREB)-dependent transcription via the mitogen-activated protein kinase (MAPK) pathway in pituitary cells: requirement of Rap1.

In previous studies we demonstrated that vasoactive intestinal peptide (VIP) mediation, and interactions between mitogen-activated protein kinase (MAPK) and cAMP/protein kinase A (PKA) signaling pathways are implicated in insulin-like growth factor I (IGF-I)- and VIP-induced lactotroph proliferation. These facts led us to investigate the intracellular mechanisms involved in IGF-I- and VIP-induced lactotroph proliferation. Exposure of cultured male rat pituitary cells to IGF-I (10(-7) M) or VIP (10(-7) M) stimulated the MAPK cascade. Studies in GH4C1 cells, with an expression vector for Rap1 GTPase-activating protein (Rap1 GAP1), demonstrated reduced VIP-induced MAPK activation, indicating that VIP-dependent activation of the extracellular signal-regulated kinase (ERK) pathway requires PKA-Rap1 signaling. IGF-I induced cAMP-response element (CRE)-binding protein (CREB) phosphorylation through the Ras-MAPK pathway, whereas VIP phosphorylated CREB directly via PKA. The mechanisms that regulate IGF-I-and VIP-CREB-dependent gene transcription were examined using GH4C1 cells transiently transfected with a CRE reporter gene. IGF-I and VIP stimulation of CRE-mediated transcription required activation of both Ras-MAPK and cAMP/PKA signaling. This activation was blocked in the presence of Rap1 GAP1. In summary, we showed that IGF-I and VIP stimulated MAPK activity and the phosphorylation of CREB in pituitary cells. Furthermore, VIP-dependent activation of PKA-Rap1-ERK pathways mediated VIP and IGF-I effects on CREB-dependent transcription in GH4C1 cells. Thus, it is possible that VIP- and IGF-I-induced lactotroph proliferation may involve Rap1.

Effects of thyroid hormones and thyrotropin-releasing hormone on thyrotropin biosynthesis by mouse pituitary tumor cells in vitro.

Mouse thyrotropic tumor cells grown in primary culture were shown to synthesize TSH and proteins, as determined by the incorporation of radioactive proline into immunoprecipitable TSH and trichloroacetic acid-precipitable proteins. The net TSH content of the cells and medium determined by RIA is also increased during 24 h of incubation, and newly formed hormone is detected in the medium within 1 h after the addition of proline tracer. To study the effect of T4 and T3 on TSH synthesis, cultures were pulse-labeled with [3H]proline after they had been exposed to either T3 or T4. After 48 but not 24 h, exposure to either T3 or T4 was followed by inhibition. When studied after 48 h of incubation, T4, (10(-13) M) or T3 (10(-11) M) at the lowest concentration tested, was inhibitory to TSH synthesis. At concentrations of T4 and T3 greater than 10(-9) M, the inhibitory effects on TSH synthesis were partially reversed, suggesting a biphasic response. Incubation in TRH (10(-7) M) for 24 h led to a significant increase in TSH synthesis, total protein, acid-precipitable protein, and total DNA. The effect of TRH on TSH biosynthesis was a function of the logarithm of its concentration over the range of 10(-11)-10(-7) M. The inhibitory action of 10(-6) M T3 on TSH synthesis was reversed by exposure to 10(-10) or 10(-7) M TRH.

Prolactin gene expression and secretion during pregnancy and lactation in the rat: role of dopamine and vasoactive intestinal peptide.

It is known that dopamine (DA) is the major PRL-inhibiting factor, and vasoactive intestinal peptide (VIP) is one of the most potent and physiological PRL-releasing factors. We have investigated the implication of DA and VIP in PRL gene expression and peptide secretion regulation during the physiological hyperprolactinemic states of pregnancy and lactation. Pregnant rats were studied on days 8, 15, and 20 of pregnancy. Lactating rats suckled by eight pups were studied on days 3 and 8 of postpartum, and nonsuckling postpartum rats were used as controls. Plasma estradiol, progesterone, and PRL were measured by RIA, as well as pituitary immunoreactive (IR-) PRL, pituitary IR-VIP, and hypothalamic IR-VIP. DA was studied by measuring changes in gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. TH, PRL, and VIP messenger RNA (mRNA) were assessed by Northern blot hybridization. The results showed very high plasma PRL levels in early pregnancy and during lactation, whereas plasma PRL concentrations were normalized at the end of gestation and in nonsuckling control rats. The physiological hyperprolactinemia of both early pregnancy and lactation correlated with higher pituitary PRL mRNA levels and lower pituitary IR-PRL content. Moreover, hypothalamic TH mRNA levels were lower in early pregnancy and lactation than at the end of gestation and in nonsuckling rats, respectively. The hypothalamic IR-VIP content was lower on day 8 of pregnancy than on days 15 and 20. However, VIP gene expression in the hypothalamus did not change throughout pregnancy. During lactation, neither hypothalamic IR-VIP content nor VIP mRNA was significantly altered. In the pituitary, IR-VIP content did not significantly change, and VIP mRNA levels were higher on day 15 of pregnancy than on the other days. During lactation, the pituitary IR-VIP content was very low on day 8 compared with those on day 3 of lactation and in nonsuckling control rats. VIP mRNA 1.0-kilobase transcript levels were higher in the lactating rats than in the control animals. These data show that both early pregnancy and lactation are physiological hyperprolactinemic states in which increased PRL mRNA accumulation coincides with decreased IR-PRL content in the pituitary and higher plasma IR-PRL, indicating regulation at the gene expression level and of PRL secretion. Low TH gene expression also occurs during hyperprolactinemia, suggesting that the diminution of DA activity that occurs during early pregnancy and lactation might be the major regulator of PRL alterations. If hypothalamic VIP plays a role as a neuroendocrine PRL-releasing factor during pregnancy and lactation, this may occur at the secretory level, as suggested by the alterations in IR-VIP, with no modifications in VIP mRNA accumulation, in the hypothalamus. Pituitary VIP does not seem to be a major regulator of PRL secretion during pregnancy, whereas during lactation, it regulates PRL secretion in a paracrine and/or autocrine manner.

Regulation of growth hormone (GH) gene expression and secretion during pregnancy and lactation in the rat: role of insulin-like growth factor-I, somatostatin, and GH-releasing hormone.

GH appears to play an important metabolic role during late pregnancy and in lactation maintenance. In this study, pregnant (days 8, 15, and 20 of gestation) and postpartum (days 3 and 8 postpartum, including lactating and nonlactating dams) Wistar rats were used to investigate pituitary GH gene expression and hormone secretion, and the potential alterations of the major signals regulating GH secretion and action [somatostatin (SS) and GH-releasing hormone (GHRH), GH receptor (GH-R), and insulin-like growth factor-I (IGF-I)]. GH and SS messenger RNA (mRNA) were quantitated by Northern blot, and both IGF-I and GH-R mRNA were analyzed by the ribonuclease protection assay technique. Pituitary IR-GH content and GH mRNA increased at midpregnancy. IR-GH content was decreased in lactating rats. Plasma GH levels progressively increased during pregnancy, whereas no significant alterations were shown during lactation. Elevated GH levels persisted during lactation. Levels at this time were higher in nonsuckling compared with suckling dams. Liver GH-R mRNA progressively decreased during pregnancy, but it remained unchanged during lactation. Plasma IGF-I and liver IR-IGF-I constantly decreased during pregnancy, and no significant modifications were seen either in suckling or in nonsuckling animals. IGF-I mRNA accumulation in the liver decreased during pregnancy. After delivery, a progressive decrease of liver IGF-I mRNA occurred. At the hypothalamic level, a progressive increase in the IR-SS content was found during pregnancy, with no SS mRNA modification. After delivery, a higher hypothalamic IR-SS content was found in lactating than in nonlactating rats, with no changes in SS mRNA levels. Hypothalamic IR-IGF-I also showed a progressive increase during pregnancy with no significant alterations during lactation. Hypothalamic IR-GHRH presented a nonsignificant mild increase during pregnancy with no modifications during lactation. In the pituitary, IR-IGF-I content progressively increased during gestation, reaching its highest concentration at day 20. During lactation, pituitary IGF-I did not change. In summary, our data show that the mechanisms of the increase in plasma GH levels occurring during pregnancy include an increase in GH gene expression in the pituitary, a decrease in SS secretion from the hypothalamus, an increase in IR-IGF-I content in the hypothalamus and in the pituitary, and a significant decrease in circulating IGF-I. Plasma and liver IR-IGF-I and IGF-I mRNA in the liver decreased throughout gestation due to a lower GH-R gene expression in the liver. This state of GH resistance with a higher GH/IGF-I ratio could be important in providing supplementary nutrients to the fetus. During lactation, GH and its regulatory machinery did not show important modifications.

Autocrine and/or paracrine action of vasoactive intestinal peptide on thyrotropin-releasing hormone induced prolactin release.

Several in vitro studies have demonstrated that vasoactive intestinal peptide (VIP) modulates basal PRL release in normal and hypothyroid anterior pituitary (AP) cells through an autocrine or paracrine action. As thyroid hormone is an important factor in the regulation of pituitary VIP synthesis and secretion, we analyzed the influence of the absence of thyroid hormone on basal PRL release in vitro to study whether the release of PRL induced by TRH might be mediated by a local action of pituitary VIP. When normal AP cells were cultured in a medium supplemented with a near-physiological concentration of free T2 (0.5 nM), basal PRL and VIP release decreased and PRL secretion was not altered by the blockade of VIP action. This finding allowed us to establish the culture conditions in which basal PRL secretion is apparently not under VIP influence. Consequently, we were able to study whether pituitary VIP may be implicated in TRH-induced PRL release. TRH (100 nM) increased PRL and VIP release in a parallel manner and decreased PRL and VIP intracellular content in incubations from 15-180 min. When AP cells wee incubated simultaneously with TRH and a VIP receptor antagonist, TRH-induced PRL release decreased when incubation lasted more than 30 min, whereas the depletion of PRL intracellular content induced by TRH was unchanged. TRH also slightly increased VIP messenger RNA levels at 3 and 24 h, but PRL messenger RNA levels were not modified. These data demonstrate that pituitary VIP participates in in TRH-induced PRL release and that the effect of thyroid hormone on basal pituitary VIP secretion should be borne in mind when studies on its effect, through autocrine and/or paracrine mechanisms, on PRL release stimulated by PRL-releasing factors are conducted.

Autoparacrine action of vasoactive intestinal peptide on dopaminergic control of prolactin secretion.

We have previously reported that pituitary vasoactive intestinal peptide (VIP) mediates through autoparacrine mechanisms insulin-like growth factor-I and TRH-stimulated PRL release. In the present study, we have investigated whether VIP might also be implicated in the PRL increase that follows dopamine (DA) withdrawal. The experiments were carried out in defined medium supplemented or not with T3, as in a previous study we had found that PRL release increases under low T3 culture conditions due to mediation by concomittant stimulus of VIP. Anterior pituitary (AP) cells from adult male rats were incubated for 1 h in the presence of DA (10 microM), a VIP-receptor antagonist (VIP-At) (200 nM), or DA plus VIP-At. Then media were removed and the cells were washed with PBS and reincubated under the same conditions but without the addition of DA. In the first incubation, DA inhibited PRL release by 80%, and VIP release by 20% in both T3-free and T3 medium. In the second period of incubation, PRL and VIP release were increased in AP cells previously exposed to DA. These effects were greater when the cells were cultured in T3-free medium than in T3-medium (225% and 150%, respectively for PRL release; and 155 and 135%, respectively for VIP release). PRL response to DA withdrawal was inhibited by the simultaneous presence of VIP-At. This inhibition was again greater when the cells were incubated in medium supplemented with T2. Thus, the stimulus of DA withdrawal was inhibited by 88% in T2-free medium and by 37% in T3-medium. To investigate whether pituitary VIP messenger RNA (mRNA) expression is under dopaminergic control, AP cells were incubated in the presence or absence of bromocriptine (BC) (10 nM) for 2 and 24 h. After 24 h of incubation, BC decreased mRNA levels of PRL and of the two transcripts which VIP expresses in AP. As with DA, BC also inhibited PRL and VIP release both at 2 and 24 h. These data demonstrate that VIP, at least partially, mediates DA withdrawal-induced PRL release through an autoparacrine action. They also suggest that simultaneous inhibition of pituitary VIP mRNA expression and VIP release may be a necessary mechanism for the dopaminergic inhibition of PRL mRNA expression and PRL release.

Thyroid hormone action on biosynthesis of somatostatin by fetal rat brain cells in culture.

Thyroid hormone (TH) action on somatostatin (SRIF) secretion and synthesis by fetal rat brain cells in culture was studied. Cortical and hypothalamic brain cells were maintained as monolayer cultures for 7-10 days. T3, T4, and [3H] phenylalanine [( 3H]Phe) (40 microCi/plate) were added simultaneously for 48 h. Alternately, cultures were pulse labeled with [3H] Phe for only the last 3 h, after being exposed to TH for 45 h. 3H-Labeled SRIF-like material [( 3H]IR-SRIF) was purified by immunoaffinity chromatography and further characterized by gel filtration in Bio-Gel P-10. Total protein synthesis was determined by the incorporation of [3H]Phe into trichloroacetic acid precipitable proteins. Forty eight-hour T3 treatment had a biphasic effect on secretion of IR-SRIF by both cortical and hypothalamic cells. In cortical cells, low doses of T3 (10(-11) M) significantly increased (P less than 0.01) and high T3 doses (10(-7) M) significantly decreased (P less than 0.05) total IR-SRIF (nanograms per plate); control: 2 +/- 0.25; T3 (10(-11) M): 3 +/- 0.3; T3 (10(-7) M): 1.3 +/- 0.1. Similarly, T4 had a significant stimulatory action at 10(-9) M, being inhibitory at 10(-7) M (picograms/plate); control: 290 +/- 20 T4 (10(-9) M): 510 +/- 40; T4 (10(-7) M): 201 +/- 10. When [3H]Phe was added during the 48 h of the experiment, [3H]IR-SRIF synthesis in response to T3 by cortical cells significantly increased after exposure to 10(-11) M (P less than 0.05) and decreased with 10(-7) M (P less than 0.05). When [3H]Phe was added for only the last 3 h or incubation with T3, the action was inhibitory at both 10(-11) M and 10(-7) M. Trichloroacetic acid precipitable material decreased in a dose response manner between T3, 10(-11) M and 10(-7) M. These findings suggest that at this time of brain development, SRIF synthesis by cortical and hypothalamic cells is affected by TH.

Growth hormone-releasing factor production by fetal rat cerebrocortical and hypothalamic cells in primary culture.

Recent data from our laboratory and others have shown radioimmunoassayable GRF (IR-GRF) in the rat brain cortex. In the present study the ontogenesis of immunoreactive rat(r) GRF (IR-GRF) in long term dissociated fetal rat cerebrocortical and hypothalamic cell cultures and the regulation of its secretion by potassium depolarization and calcium channel-blocking agents were investigated. The chromatographic profiles of IR-rGRF from cell extracts were determined and compared with those from in vivo cerebrocortical and hypothalamic tissues. Mechanically dispersed cultured telencephalic and diencephalic cells from 17- to 21-day-old fetal rats showed a progressive increase in IR-rGRF, reaching maximum values (media plus cells) between 775-1020 (pg/mg protein) in hypothalamic cells on days 10-20 and between 450 and 950 pg/mg protein in cortical cells on days 25-30. IR-rGRF from acidic extracts of cells and adult cortical and hypothalamic tissues adsorbed onto octadecylsilyl-silica columns corresponds primarily to rGRF-(1-43)OH on HPLC. In gel filtration chromatography, almost all IR-rGRF from cultured cerebrocortical cells and fetal and adult cortical tissues coeluted with rGRF-(1-43)OH. IR-rGRF from cultured hypothalamic cells showed an additional component with a higher mol wt eluting in the void volume. To determine the influence of membrane depolarization of rGRF release, potassium (K+) concentrations in the medium were increased to 30 and 56 mM, inducing a marked increase in medium rGRF concentrations. Verapamil, a Ca2+ channel blocker (20 microns) reverses the effect of 56 mM potassium depolarization, suggesting that it is at least partially dependent upon Ca2+ transport. These data indicate that fetal rat cerebrocortical and hypothalamic cells in culture produce and release rGRF in response to depolarizing agents. The presence of rGRF in cortical tissue suggests that there are other physiological roles besides those implicated in the stimulation of GH secretion.

Growth hormone gene expression and secretion in aging rats is age dependent and not age-associated weight increase related.

GH secretion declines with age in rats and humans and a reduction in GH gene expression has been demonstrated in aging rats. GH secretion also diminishes in obesity; thus, the aim of this study was to determine whether GH decrease in aging rats is due to body weight gain or to aging. Three groups of male Wistar rats of different ages were studied (young, 3 months; middle-aged, 11 months; old, 27 months). The middle-aged group was established on a statistical analysis and corresponded to the youngest age at which body weight was not significantly different from the old (27 month) group. Thus, by using this group as control for comparison with animals with the same weight and an older age, the effects due to aging itself could be determined. Body weight (g, mean +/- sD) 3 months: 361 +/- 5.6; 11 months: 713 +/- 39; 27 months: 635 +/- 38. In comparison with 3-month-old rats, the 11-month-old animals showed no difference in pituitary GH messenger RNA (mRNA) accumulation and pituitary and serum IR-GH levels. Similarly IGF-I.a, IGF-I.b mRNA transcripts and IG-FBP-3 mRNA accumulation in the liver showed no significant differences between the two groups. On the contrary, when the 27-month-old rats were compared with the 11-month-old animals, lower levels of pituitary GH mRNA and serum and pituitary IR-GH were found. Pituitary GH mRNA decreased 37.5 +/- 7.7% P < 0.001, pituitary IR-GH content diminished (5.2 +/- 3.4 vs. 55 +/- 10.7 ng/mg of protein, P < 0.001) and serum IR-GH decreased (3.5 +/- 1.8 vs. 12.5 +/- 4.2 ng/ml, P < 0.01). Liver IGF-I.a and IGF-I.b mRNA transcripts accumulation and serum IGF-I were significantly diminished. IGF-I.b mRNA accumulation decreased 35.8 +/- 1.2% P < 0.05 and IGF-I.a 36 +/- 5.6% P < 0.05; serum IR-IGF-I levels diminished (759 +/- 152 vs. 1327 +/- 67 ng/ml, P < 0.05). Liver IGFBP-3 mRNA accumulation decreased 79 +/- 4.2% P < 0.001. These results indicate that the decrease in GH gene expression and secretion, as well as the expression of genes induced by GH such as IGF-I and IGFBP-3, is due to aging and not to the increase in body weight that takes place with aging.

Growth hormone induces somatostatin and insulin-like growth factor I gene expression in the cerebral hemispheres of aging rats.

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.

Thyroid hormone regulates vasoactive intestinal peptide (VIP) mRNA levels in the rat anterior pituitary gland.

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.

Induction of vasoactive intestinal peptide gene expression and prolactin secretion by insulin-like growth factor I in rat pituitary cells: evidence for an autoparacrine regulatory system.

The effects of recombinant human insulin-like growth factor I (IGF-I) on both vasoactive intestinal peptide (VIP) and PRL production and gene expression were studied using rat anterior pituitary cell cultures grown in serum-free defined medium. We also examined whether pituitary VIP could be involved in the PRL response to IGF-I and hence in a paracrine regulatory system. Exposure of cultured anterior pituitary cells to IGF-I (2.6 nM) for 3 h caused a significant decrease in both VIP content and media PRL. Treatment with IGF-I (from 0.65-5.2 nM) for 48 h increased VIP production and VIP messenger RNA (mRNA) accumulation, whereas only an increase in media and intracellular PRL content without changes in mRNA was observed. In all these experiments, IGF-I led to a decrease in both GH secretion and expression. Immunoglobulins G purified from VIP antiserum inhibited IGF-I-induced PRL release without affecting intracellular and mRNA levels. The inhibition of both GH secretion and gene expression induced by IGF-I was not blocked by VIP antiserum. In conclusion, these results indicate that IGF-I induces VIP gene expression, and its secretion and also increases PRL secretion. The effect of IGF-I on PRL release is specifically mediated by VIP through a paracrine or autocrine mechanism.

Synthesis and secretion of vasoactive intestinal peptide by rat fetal cerebral cortical and hypothalamic cells in culture.

To establish the neurosecretory activity of brain vasoactive intestinal peptide (VIP)ergic neurons and to characterize the molecular forms of secretion of these cells, fetal cerebrocortical and hypothalamic cells were grown in primary cultures for periods up to 4 weeks, their regulation by depolarization and calcium and sodium channel active agents was studied, and the chromatographic patterns of cell and medium VIP were determined. Mechanically dispersed cultured fetal telencephalic and diencephalic cells showed a progressive increase in immunoreactive VIP in both cells and media, reaching maximum values between 110-290 pg/mg protein.culture plate on days 15-20. Immunoreactive VIP in both cells and media corresponded almost exclusively to VIP-28 on exclusion chromatography and HPLC, and was identical to the form extracted from whole fetal brain. This indicates that both the stored and secreted forms of VIP are the mature 28-amino acid-containing peptide. To determine the influence of membrane depolarization on VIP release, the potassium concentration in the medium was increased to 30 and 56 mM, inducing a marked increase in medium VIP concentrations. The effects of K+ were dependent on Ca2+ transport, since release was blocked by the addition of verapamil, a Ca2+ channel blocker (20 microM). VIP release was stimulated by Na+ channel activation using the drug veratridine (100 microM); this effect was blocked by tetrodoxin. The influence of GH-releasing factor (GRF) on VIP release was studied by adding GRF (10(-9)-10(-7) M), alone or in the presence of anti-rGRF immunoglobulins (antirat GRF immunoglobulin G) to the incubation medium. Rat GRF stimulated VIP release and the simultaneous addition of antirat GRF immunoglobulin G blocked this effect. These findings confirm that VIP regulation by brain cells corresponds to the well defined patterns of membrane activation observed in other neuronal systems. They, furthermore, demonstrate that VIP release is under GRF influence.

Distribution of insulin-like growth factor-I receptor mRNA in rat brain. Regulation in the hypothalamo-neurohypophysial system.

The distribution and regulation of mRNA for the IGF-I receptor (IGF-I-R) in the adult rat brain were studied by in-situ hybridization with a 35S-labelled cRNA probe. The pituitary gland showed a strong hybridization signal in the pars tuberalis (the surface of the median eminence), pars distalis and pars intermedia. Within the brain, a strong hybridization signal was found in the circumventricular organs, olfactory bulb, hippocampus, cerebellum and hypothalamus. IGF-I-R mRNA was consistently found in cell bodies of the hypothalamo-neurohypophysial system. Six days of intermittent salt-loading resulted in an increase in IGF-I-R gene expression in the supraoptic nucleus. The increase in IGF-I-R mRNA was accompanied by a high expression of c-Fos immunoreactivity in the same cells. The presence and regulation of IGF-I-R mRNA in the hypothalamus suggest that IGF-I may affect the local plasticity or modulation of activated magnocellular neurones by an autocrine or paracrine action through specific receptors in the hypothalamo-neurohypophysial system.

IGF-I inhibits apoptosis through the activation of the phosphatidylinositol 3-kinase/Akt pathway in pituitary cells.

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-immunoreactive peptide in adult human cerebellar Purkinje cells: co-localization with low-affinity nerve growth factor receptor.

It has been proposed that Insulin-like growth factor I is involved in the development, growth and maintenance of the central nervous system possibly interacting with other trophic factors. High levels of insulin-like growth factor I have been detected in the cerebellum during development and adulthood suggesting a specific role for insulin-like growth factor I in this brain area. While there is ever increasing data regarding the cell types containing endogenous insulin-like growth factor I in the rat brain, no information on the human brain is yet available. In the present study we sought to analyse the precise location of insulin-like growth factor I peptide in the adult human cerebellum using a specific antiserum against recombinant human insulin-like growth factor I. After immunocytochemistry, numerous Purkinje cells exhibited intense positive staining occupying the cell soma, dendrites and dendritic spines as well as axons. Occasionally, immunoreactive Purkinje cell axons were arciform and exhibited bulbous dilatations along their proximal length. Putative recurrent collaterals of Purkinje cell axons were also insulin-like growth factor I reactive. Double-staining immunocytochemistry in the same sections consistently showed, as expected, co-expression of insulin-like growth factor I and calbindin, although a few calbindin containing Purkinje cells lacked insulin-like growth factor I immunostaining suggesting there are insulin-like growth factor I positive Purkinje cell subsets in the human cerebellum. In addition, co-expression of insulin-like growth factor I and low-affinity nerve growth factor receptor-immunoreactive protein was found in a subpopulation of insulin-like growth factor I positive Purkinje cells. The results of this study prove the presence of insulin-like growth factor I immunoreactivity in a Purkinje cell subpopulation of the adult human cerebellum suggesting that insulin-like growth factor I may participate in paracrine or autocrine regulatory systems in the adult human brain.

Sensitivity to exogenous GH and reversibility of the reduced IGF-I gene expression in aging rats.

BACKGROUND: IGF-I gene expression and IGF-I plasma concentration decline with age. A decreased sensitivity to GH has been suggested to be a contributory mechanism to this, in addition to attenuated GH secretion. OBJECTIVE: This study focuses on the sensitivity to exogenous GH and the reversibility of the reduced IGF-I gene expression in aging male rats. DESIGN: Three groups of male Wistar rats aged 3 months (young adult), 11 months (middle-aged) and 27 months (old), received recombinant human GH (rhGH) (150 microg/12 h s.c.) for seven consecutive days. RESULTS: This rhGH treatment completely reversed plasma immunoreactive IGF-I (IR-IGF-I) and hepatic IGF-I mRNA levels in 11-month-old and 27-month-old animals to the levels of the young group of animals. The sensitivity in the old group (percentage of increment after the same or lower dose of rhGH per body weight) was increased for both parameters; serum IGF-I increment: 15% in 3-month-old, 42.6% in 11-month-old and 119.1% in 27-month-old rats; and hepatic IGF-Ib mRNA increase: 45% in 3-month-old, 27.8% in 11-month-old and 64.3% in 27-month-old rats. IGF binding protein-3 (IGFBP-3) mRNA level in the liver was significantly decreased in the old group and only a partial reversion occurred in this group after rhGH treatment; the percentage of increment was also higher in the old group of rats. In extrahepatic tissues IGF-I mRNA was not significantly different in the kidney and the testis of the three groups, and the rhGH treatment produced a significant and similar increase of IGF-I mRNA level in the kidney of the three groups of rats and in the testis of the 27-month-old animals. The GHr/GHBP mRNA remained unchanged in the liver and in the kidney or the testis of the three groups, and was not influenced by the rhGH treatment. Exogenous rhGH decreased pituitary GH mRNA accumulation in a more intense manner in the old group versus control of each group: young adult, 25%; middle-aged, 41.2%; and old rats, 55%. The action of rhGH on pituitary immunoreactive GH (IR-GH) content was only significantly evident in the young group. CONCLUSIONS: These results establish that exogenous rhGH recovers the attenuated liver IGF-I gene expression and the diminished plasma IR-IGF-I in old rats to the levels of young adult animals. They also indicate that the hepatic and extrahepatic (kidney and testis) sensitivity to one established dose per weight of exogenous rhGH is not altered in old animals, or could be potentially increased in some tissues.

Involvement of VIP on BDNF-induced somatostatin gene expression in cultured fetal rat cerebral cortical cells.

Previous studies have shown that BDNF promotes expression of SS. In earlier studies we demonstrated the stimulatory effect of locally produced VIP upon SS secretion. These facts led us to explore the peptidergic action of BDNF on VIP, and to determine if BDNF-induced SS might be mediated by the induction of VIP. Cultured fetal rat cerebrocortical cells were incubated with BDNF (50 ng/ml) and/or VIP (10(-11) M) for 2 and 5 days. In other experiments IgGs from BDNF or VIP antisera were also added. BDNF increased VIP and SS gene expression and peptide production. After 2 days of incubation with both BDNF and VIP the induction of SS mRNA was similar to that obtained with BDNF alone. However when the treatment was extended to 5 days the increase in SS mRNA was higher than that obtained with BDNF alone. This finding suggests the possibility that both factors acted synergistically. To define the potential role of VIP in the response of SS gene expression to BDNF, endogenous VIP was blocked with IgGs from VIP antiserum. Under these experimental conditions BDNF-induced SS decreased. Our study provides the first evidence that BDNF up-regulates VIP gene expression and concentration of the peptide. The involvement of VIP on BDNF-induced SS gene expression is also demonstrated.

Regulation of somatostatin gene expression by veratridine-induced depolarization in cultured fetal cerebrocortical cells.

The stimulatory effect of veratridine (VTD) depolarization upon somatostatin mRNA (SS mRNA) levels in primary cultures of fetal cerebrocortical cells was analyzed. Depolarizing stimuli, such as 100 microM VTD exposure for 30 min, elicited an increase in immunoreactive somatostatin (IR-SS) release to the media without affecting SS mRNA levels. These levels increased when exposure to depolarization stimuli was prolonged up to 3 or more hours. At this time, veratridine acted as a secretagogue, stimulating somatostatin secretion, but was also effective in stimulating somatostatin mRNA levels. These changes were blunted by the Na+ channel blockade tetrodotoxin (TTX), and partially abolished by the Ca2+ channel antagonist, verapamil (VPM). To study whether VTD may affect mRNA stability we determine the rate of disappearance of SS mRNA after inhibition of transcription by actinomycin D and demonstrated that VTD stimulation did not stabilize the SS mRNA. These results indicate that the induction of SS mRNA expression by VTD involves the modulation of Ca2+ and Na+ channels. The time course study confirmed that the VTD-induced SS mRNA accumulation is time-dependent, and requires a prolonged exposure to stimulate SS gene expression. VTD stimulation does not modify the SS mRNA rate of degradation.

Pineal vasoactive intestinal peptide is reduced during the proestrous stage of the rat estrous cycle.

Immunoreactive levels of vasoactive intestinal peptide (IR-VIP) in the rat pineal gland were examined during the estrous cycle. IR-VIP was shown to be identical to the synthetic porcine material by its similarity in competitive binding studies, and by both gel filtration and high pressure liquid chromatography. Pineal IR-VIP decreased at early proestrus (0300 h, dark), partially as a function of the rise in serum estradiol levels. Although the functional role of VIP in the pineal remains to be clearly elucidated, these results suggest that it might be involved in the reproductive function of the female rat, and that estrogens could partially modulate its pineal concentration.