L. Cacicedo

Regulation of Gonadal and Somatotropic Axis by Chronic Intraventricular Infusion of Insulin-Like Growth Factor 1 Antibody at the Initiation of Puberty in Male Rats

There has been increasing experimental evidence to suggest that insulin-like growth factor 1 (IGF-I) may be one of the essential regulators in the reproductive system of the rat. IGF-I is synthesized in the hypothalamus and IGF-I immunoreactivity increases during puberty. Consequently we hypothesized that centrally located IGF-I might contribute to the initiation of puberty. Centrally located IGF-I was immunoneutralized to assess this hypothesis. Male Wistar rats, 28 days old, were infused intracerebroventricularly with specific purified IgGs from rabbit IGF-I antiserum (IGF-I-Ab). The intracerebroventricular administration of IGF-I-Ab resulted in a reduction in testicular weight and consequently in delayed pubertal development. There was also a reduction in serum testosterone, pituitary immunoreactive (IR) luteinizing hormone (LH) and serum IR follicle-stimulating hormone (FSH). The accumulation of βLH mRNA was not modified, whereas βFSH mRNA was increased. An increment in the serum growth hormone (GH) levels was also observed. There were no significant alterations in hypothalamic IR growth hormone releasing factor content, although IR somatostatin (SRIH) content was increased by IGF-I-Ab. The body weight gain remained unaltered. As a whole, our study suggests that centrally located IGF-I influences pubertal development, production and release of gonadotropins and supports the finding that endogenous centrally located IGF-I plays a role at the initiation of puberty in the male rat. It also gives support to the physiological role of centrally located IGF-I in the release of GH mediated by hypothalamic SRIH at the initiation of puberty.

Sex differences in growth hormone response to growth hormone-releasing hormone

The aim of the present study was to ascertain whether sex differences exist in GH response to GHRH, and the influence that menstrual cycle have on this response. A GHRH test was performed on nine healthy men and ten women on days one and twelve of the menstrual cycle. Basal GH levels, (mean ± SE) (7.98 ± 3.09 ng/ml in women and 0.13 ± 0.07 ng/ml in men, p < 0.05) as well as maximal GH response (40.17 ± 11.96 ng/ml in women and 9.63 ± 2.32 ng/ml in males, p < 0.01) were significantly higher in women than in men. In spite of a significant increase in estradiol levels during the menstrual cycle (75.88 ± 2.48 pg/ml on day one and 198.40 ± 28.65 pg/ml on day twelve, p < 0.01) neither basal plasma GH levels, nor GH response to GHRH were significantly different. In conclusion, these results confirm that GH response to GHRH is higher in women than in men, and that this difference is not modified during the first phase of the menstrual cycle.

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.

Differential regulation of gonadotropins and glycoprotein hormone α-subunit by IGF-I in anterior pituitary cells from male rats

IGF-I has been demonstrated to stimulate basal and GnRH-induced gonadotropin release. IGF-I also elicites a-subunit secretion in human pituitary tumor cells. The aims of this study were to evaluate both the effect of IGF-I on gonadotropin LH-gB and FSH-gB mRNA levels and glycoprotein a-subunit gene expression in cultured rat anterior pituitary cells. The exposure of pituitary cells to recombinant human IGF-I (rhIGF-I; 2 gMg/ml) for 72 h markedly stimulated basal LH and FSH release whereas their mRNA levels remained unmodified. IGF-I elicited a-subunit release from pituitary cells (p<0.01) and augmented its mRNA levels. Exposure to IGF-I consistently reduced GH release from pituitary cells. This study shows that the gonadotropin-releasing effects of IGF-I are not paralleled by changes in their mRNAs whereas IGF-I stimulates not only α-subunit release but also its mRNA levels. This study provides the first observation of a-subunit regulation by IGF-I in normal pituitary cells, where a differential regulation between release and synthesis for gonadotropin a-and gB-subunits is also shown.

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.

Nutrition and iodine versus genetic factors in endemic goiter

Consanguinity has been considered to be the major factor responsible for the high incidence of goiter in the area of Las Hurdes in Spain. However, iodine deficiency was later found to be severe enough to account for endemic goiter, and the presence of cretins in this area. Children from very similar family and socioeconomic backgrounds were found to be on three different nutritional programs, depending on the schools they were attending, and it appearedof interest to determine the effect of nutrition on the goiter incidence in children from the same population. Total iodine, nitrogen and creatinine concentrations were measured in casual urine samples. All three were found to change in parallel in the different subpopulations. They were low in schoolchildren receiving most of their meals at home. Such meals were composed of locally grown food. They were normal in children living in a boarding school, where all of the food is provided from outside the area. They were intermediate in children from a day care center, who received only some meals from an outside source. In the boarding school, goiter incidence was 21%, as opposed to 87% for schoolchildren fed at home. Such results indicate that in this area consanguinity plays a minor role, if any, in the high incidence of goiter.

Influence of Chronic Depolarization on Synthesis and the Relative Amounts of Different Forms of Somatostatin in Cultured Fetal Cerebrocortical Cells

The neuropeptide somatostatin is widely distributed throughout the nervous system. In addition to its well known role as a hypophysiotropic hormone, somatostatin also appears to serve as a neurotransmitter or neuromodulator (1,2). Although the effects of conventional neural depolarizing agents on the release of somatostatin (SRIF) have been extensively studied (3), little is known about the influence of these agents on SRIF synthesis and processing. We and others previously reported that in using cultured fetal cerebral cortical cells, immunoreactive (ir)-SRIF release occurred in response to acute depolarization induced by high potassium concentrations or veratridine (VTD) (3,4). In the present work, we sought to investigate to what extent SRIF synthesis is regulated by factors that influence its secretion, by studying the effect of chronic exposure to depolarizing agents on ir-SRIF synthesized by cultured cortical cells. As an index of synthesis, we followed the accumulation of ir-SRIF (cell content + media). We examined the response of the cultured neurons to high concentrations of K+ and found that K+ increased total ir-SRIF and that this effect is calcium-dependent.

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.

Potassium-induced depolarization stimulates somatostatin gene expression in cultured fetal rat cerebrocortical cells.

The stimulatory effect of potassium depolarization upon somatostatin mRNA (SS mRNA) levels in primary cultures of fetal cerebrocortical cells was analyzed. Depolarizing stimuli, such as 56 mM K+ concentration for 24 hours, elicited an increase in immunoreactive somatostatin (IR-SS) release to the media and SS mRNA levels, suggesting that somatostatin secretion can be coupled to SS mRNA accumulation. These changes were inhibited by the Ca2+ channel antagonist verapamil (VPM). In contrast, Na+ channel blockade by tetrodotoxin (TTX) did not modify the 24 h potassium-induced increase in SS mRNA. These results suggest that the induction of SS mRNA expression by K+ involves the modulation of calcium ion channels.