Rosa Maria Tolón

Corticosterone Modulates Growth Hormone-Releasing Factor and Somatostatin in Fetal Rat Hypothalamic Cultures

It is well known that chronic supraphysiological doses of glucocorticoids (GC) inhibit GH secretion in vivo, and stimulate GH secretion from the somatotropes in vitro. It has been suggested that GC exert an inhibitory role in the hypothalamus surpassing the GC-positive effect at the somatotrope level. To test the hypothesis that GC can affect growth hormone-releasing releasing factor (GRF) and somatostatin (SS) at the hypothalamic level, we studied the effect of corticosterone on the immunoreactive content of GRF (IR-GRF) and SS (IR-SS) in cells and media of fetal hypothalamic cells in culture. After 20 days in culture, cells were incubated with serum-free medium containing corticosterone (from 0.3 to 300 nM) for 48 h. Corticosterone had a dual effect on IR-GRF. Concentrations in the range of the glucocorticoid receptor Kd (3 nM) increased peptide content, whereas higher concentrations (30 and 300 nM) decreased IR-GRF content in cells and media. Conversely, corticosterone increased SS cell content, only at a concentration of 3 nM, inducing a 2- to 3-fold increment in media content with the highest doses (30 and 300 nM). These results demonstrated that both GRF and SS are modulated by corticosterone in primary fetal rat hypothalamic cultures. Whereas GRF exhibited a dual response, stimulatory and inhibitory, at low and high corticosterone doses, respectively, SS showed a parallel increase with the corticosterone concentrations.

Regulation of somatostatin and growth hormone-releasing factor by gonadal steroids in fetal rat hypothalamic cells in culture

The mechanism underlying the sexually dimorphic pattern of growth hormone (GH) secretion in the rat has not been clearly elucidated. In the present study, we assayed the possible direct effect of gonadal steroids on both somatostatin (SS) and growth hormone-releasing factor (GRF) in fetal rat hypothalamic cells in culture. Hypothalamic cells, obtained by mechanical dispersion, were maintained as monolayer cultures in serum-supplemented medium. After 20 days in culture, cells were incubated with serum free medium containing testosterone (T, 10, 20, 40 ng/dl) or estradiol (E, 0.1, 1, 10 ng/dl) for 48 h. At the end of the experiments, immunoreactive SS (IR-SS) and immunoreactive GRF (IR-GRF) were measured by specific radioimmunoassays (RIAs) in media and cell extracts. After 48 h of incubation with testosterone, somatostatin in both media and cells was significantly reduced. On the contrary, this treatment lead to a dose-dependent increase in media and cell GRF content. When cells were incubated with estradiol for 48 h, a significant inhibition in medium SS release was observed, whereas intracellular SS slightly increased at the highest concentration of 10 ng/dl. Estradiol treatment resulted in an inconsistent decrease in media and cells IR-GRF. Our results indicate that both SS and GRF are under the influence of testosterone and estradiol acting at the hypothalamic level, and furthermore suggest that at this stage of brain development, gonadal steroids may regulate GH secretion through their ability to modulate hypothalamic SS and GRF.

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.

Neurotransmitter regulation of somatostatin secretion by fetal rat cerebral cortical cells in culture

Extensive studies exploring the regulation of hypothalamic somatostatin GHRIH release have been reported, but the factors regulating GHRIH release in the cerebral cortex have not been well defined. We have studied the effects of central neurotransmitters on GHRIH secretion by cultured fetal rat cerebral cortical cells and on intracellular GHRIH levels. Cells maintained in vitro for 15–20 days were incubated with dopamine (DA), acetylcholine (ACh), gamma-aminobutyric acid (GABA), norepinephrine (NE), serotonin (SE) or histamine (His) (10−11 M to 103 M) for 30 minutes. Following incubation, immunoreactive GHRIH was measured by RIA in cell extracts and incubation media. DA increased intracellular GHRIH content but have no effect on GHRIH in the media. Both media and intracellular GHRIH content were significantly reduced by GABA and SE. The effect of NE was stimulatory at low (10−9 M) and inhibitory at high (10−5 M to 10−3 M) concentrations. ACh was found to increase media GHRIH and to decrease intracellular GHRIH content; 30 min exposure to His did not significantly modify either media or intracellular GHRH. Our findings with fetal rat cerebral cortical cells in culture demonstrate that endogenous neurotransmitters do have the capacity to directly influence GHRIH regulation.

Role of Locally Produced Growth Hormone-Releasing Factor in Somatostatin Regulation by Fetal Rat Brain Cells in Culture

To determine the possible physiological role of endogenous growth hormone-releasing factor (GRF) in the neuronal content and release of cerebral somatostatin (SS), we studied the effect of endogenous GRF blockade on the immunoreactive SS (IR-SS) content of cells and media in fetal rat cerebral cortical and hypothalamic cells in culture. Cells were cultured in minimum essential medium (MEM) with 10% fetal calf serum and 10% horse serum. After 7-10 days in vitro, media were replaced with MEM without sera containing anti-GRF immunoglobulins G (IgG) for 1, 5 or 24 h. Controls were incubated with equal amounts of IgG from normal rabbit serum (NRS). In another group of experiments, cells were incubated with GRF (10(-11) to 10(-7) M) for 1 or 24 h. Long-term exposure (24 h) to anti-GRF IgG resulted in decreased media and intracellular IR-SS content, in both cerebral cortical and hypothalamic cells. 24 h treatment with GRF caused a dose-dependent increase in the IR-SS content of cells and media, the stimulatory action being abolished by the addition of anti-GRF to plates containing 10(-7) M GRF. On the contrary, when cells were exposed to anti-GRF IgG for 1 h, IR-SS increased in the media as compared to the control group. Short-term incubation (1 h) with GRF (10(-9) to 10(-7) M) resulted in a dose-dependent inhibition of IR-SS content in the cells and media. This inhibitory action was partially prevented by the addition of anti-GRF to plates containing 10(-7) M GRF.(ABSTRACT TRUNCATED AT 250 WORDS)

Potassium depolarization-induced cAMP stimulates somatostatin mRNA levels in cultured diencephalic neurons.

In the nervous system, signals transmitted across synapses are known to regulate gene expression in the postsynaptic cells. This process often involves membrane depolarization and subsequent elevation of intracellular Ca(2+). We have previously demonstrated in fetal cerebrocortical cells, that somatostatin (SS) mRNA levels can be induced by depolarizing agents such as high potassium concentrations and veratridine (VTD), and that these effects are calcium dependent. SS expression is regulated by cAMP, and in the cerebral cortex adenylate cyclase activity is regulated through fluctuations in intracellular Ca(2+) concentrations. The present experiments were undertaken to determine the mechanism by which calcium upregulates the levels of SS mRNA. Cerebrocortical cells from 17-day-old fetuses were exposed to the different agents for 24 h and the levels of SS mRNA were measured by Northern blot. Incubation of cells with the calcium channel antagonist nifedipine (Nf), the calcium chelating agent EGTA, calcium free KRB and the calcium calmodulin inhibitors trifluoroperazine (TFP) and the napthelene sulfonamide, W7, resulted in the inhibition of K(+)-induced SS mRNA levels. K(+)-evoked depolarization increased the intracellular concentration of cAMP and this effect was antagonized by verapamil (VPM). Forskolin (Fk) provoked a higher increment in cAMP concentration than potassium, although the induction of SS mRNA was more evident following K(+) depolarization indicating a lack of correlation between levels of cAMP and induction of SS mRNA. The role of K(+)-induced cAMP on the increment of SS mRNA that occurred upon membrane depolarization was further explored with the inhibitor of protein kinase A (PKA), Rp cAMP whose presence significantly reduced depolarization-induced SS mRNA levels. This study confirms that Ca(2+) influx is required for K(+)depolarization-induced stimulation of cAMP whereby the increment of SS mRNA is partly produced.

Growth Hormone-Releasing Factor Regulation by Somatostatin, Growth Hormone and Insulin-Like Growth Factor I in Fetal Rat Hypothalamic-Brain Stem Cell Cocultures

Information about growth hormone-releasing factor (GRF) regulation by somatostatin, GH and IGF-I is scarce and controversial. This could be due to the in vivo interactions among these signals and the lack of models for individualizing the action of one of them from the others upon GRF regulation. The aim of the present work was to study GRF regulation by these signals, using primary fetal rat hypothalamic-brain stem cell cocultures. Coculturing of these two cytotypes increases hypothalamic immunoreactive rat GRF (IR-rGRF) content in cells by 45% and in media by 36%. The effect of SS on GRF in cocultures was examined by using a multiple approach: (1) depleting endogenous SS by adding 1 mM cysteamine (CSH); (2) blocking endogenous SS by incubation with SS antiserum, and (3) incubating with synthetic SS14 at different concentrations and exposure periods. 1 mM CSH depleted IR-SS content (pg/plate, mean +/- SE) in cells (CSH-treated: 68 +/- 8 vs. control: 322 +/- 10, p < 0.01) and media (CSH-treated: 211 +/- 15 vs. control: 880 +/- 70; p < 0.01). In the CSH-induced SS-depleted cultures, a slight reduction in the IR-rGRF content in cells was observed (CSH-treated: 93.5 +/- 4.5 vs. control: 111 +/- 6; p < 0.05), with no effect on media content. When SS antiserum was added to plates, there was a slight reduction in the IR-rGRF content in cells and media, but it was not significantly different from the controls. However, SS14 (10(-10)-10(-8) M) could not modify IR-rGRF content in media and cells. The GH effect on IR-rGRF was studied in the absence of CSH and in CSH-induced SS-depleted cultures. GH (5 microM, 24 h) decreased (52%) the IR-rGRF content in media (GH-treated: 28.7 +/- 4.6 vs. control: 60.2 +/- 7; p < 0.01) without causing changes in cell content. In SS-depleted cultures, the inhibitory action of GH on media IR-rGRF was greater (62% decrease) (GH-treated: not detected, control 56 +/- 10; p < 0.01) and also affected IR-rGRF cell content (GH-treated: 64.3 +/- 7.3 vs. control: 160 +/- 9.6; p < 0.01). In the same experiments, GH increased IR-SS content in cells (GH-treated: 31.8 +/- 4.6 vs. control 20.9 +/- 0.5; p < 0.01) and in media (GH-treated: 413 +/- 7 vs. control: 286 +/- 9; p < 0.01). 1 mM CSH again depleted IR-SS content and abolished the GH stimulatory effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Thyroid Hormones Regulate Release and Content of Vasoactive Intestinal Peptide in Cultured Fetal Cerebral Cortical Cells

The effects of thyroid hormones (TH) on brain immunoreactive-vasoactive intestinal peptide (IR-VIP) secretion and content in cultured fetal rat cortical cells were studied. Cerebral cortical cells were maintained as monolayer cultures for 14-18 days. T3 or T4 (10(-7) M) caused a time-dependent decrease in total IR-VIP. Significant suppression was observed following treatment periods of 6 h or longer (24 and 48 h). Depending on the length of time cells had been deprived of TH prior to the addition of exogenous T3 or T4, these two thyroid hormones had different effects on IR-VIP accumulation. Both T3 and T4 caused a dose-dependent suppression or IR-VIP accumulation when there was no deprivation period or when it lasted 4 h. However, a biphasic effect was observed when cells were deprived of TH for 17 and 24 h: low doses of T3 or T4 (from 10(-12) to 10(-10) M) significantly increased (p less than 0.05) total IR-VIP, while high T3 or T4 doses (10(-8) and 10(-7) M) caused a significant decrease (p less than 0.01). The TH action was furthermore shown to be reversible. After T3 (10(-7) M) removal and subsequent incubation in serum-free medium for 6, 24 and 48 h, T3-treated and control cells exhibited similar levels of IR-VIP release and content. At this time, a new exposure to T3 (10(-7) M) again had a suppressive effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Triiodothyronine regulates somatostatin gene expression in cultured fetal rat cerebrocortical cells

The effect of triiodothyronine (T3) on somatostatin (SS) mRNA levels in cultured fetal rat cerebrocortical cells was studied. Two different experimental approaches were sought. They differed in the length of time in which cells were deprived of thyroid hormones prior to the addition of exogenous T3. When the cells were not deprived of thyroid hormones, T3 caused a dose-related decrease in SS mRNA content at all doses tested. However, when the cells were deprived of T3 for 24 h, a biphasic effect was observed. These findings suggest that T3 regulates SS gene expression in fetal cultured cerebrocortical cells.

Influence of thyroid hormones on somatostatin processing in cultured cerebro-cortical cells

Previous data from our laboratory showed that prolonged exposure of cultural cerebral cortical cells to high potassium concentrations and veratridine resulted in the stimulation of immunoreactive somatostatin (IR-SRIF) synthesis and caused a major increase in its high molecular weight forms. Somatostatin (SRIF) synthesis by cortical and hypothalamic cells was also affected by thyroid hormone (TH). In the present work we have examined to what extent TH might also affect SRIF processing. Cerebral cortical cells maintained as monolayer culture for 7-10 days received triiodothyronine (T3) in concentrations of 10(-11) M and 10(-7) M for 48 h. We found that the total amount of IR-SRIF was increased by high T3 concentrations as reported previously. When the IR-SRIF was characterized by high pressure liquid chromatography (HPLC) or gel filtration, it was evident that thyroid hormone treatment modified the elution profile of IR-SRIF in cells and medium on Bio-Gel P-10 and HPLC, increasing somatostatin 28 (S-28) and decreasing somatostatin 14 (S-14). The results indicate that thyroid hormones affect SRIF processing, leading to a major increase in the synthesis of its high molecular weight forms.