E. Blanco

Coexistence of NADPH-diaphorase with vasopressin and oxytocin in the hypothalamic magnocellular neurosecretory nuclei of the rat

Coexistence of NADPH-diaphorase with vasopressin and oxytocin was studied in the magnocellular neurosecretory nuclei of the rat hypothalamus by use of sequential histochemical and immunocytochemical techniques in the same sections. Coexistence was found in all the nuclei examined (supraoptic, paraventricular, circular, fornical, and in some isolated neurons located in the hypothalamic area between the paraventricular and supraoptic nuclei). The ratios of neurons expressing both markers (NADPH-diaphorase and vasopressin, NADPH-diaphorase and oxytocin) in each of the nuclei were very similar. Although further studies must be carried out, the partial coexistence found in all nuclei suggests that NADPH-diaphorase is probably not related to general mechanisms involving vasopressin and oxytocin, but rather in specific functions shared by certain hypothalamic neuronal cell populations.

Immunohistochemical evidence of the presence of aromatase P450 in the rat hypophysis

Abstract In order to analyze whether aromatase is present in the hypophysis of adult rats, we have performed an immunohistochemical study in young adult male and female rats. Our study has revealed that the hypophysis of adult rats contains aromatase, although marked differences are found between the sexes. The hypophyses of male rats have cells immunoreactive for the enzyme, 34.40% of these hypophyseal cells showing reaction. By contrast, cells from female rats show very little reaction, only 0.84% of them being reactive. No significant differences in the percentage of immunoreactive cells between one phase and another are observed during the estrous cycle. Our results point to the immunohistochemical expression of aromatase in the hypophysis of adult rats and at the same time suggest that its expression is sex-dependent. The enzyme may therefore be involved in the regulation of adenohypophyseal cytology by androgens.

Expression of the μ-opioid receptor in the anterior pituitary gland is influenced by age and sex

Abstract To analyze whether opioids are able to modulate endocrine regulation by acting directly on rat pituitary cells, an immunohistochemical study of μ-opioid receptor expression in these cells was performed, with attention given to the analysis of potential age- and sex-related variations in receptor expression patterns. In both sexes, the μ-opioid receptor was detected in the pituitary pars distalis. However, significant age-related differences were observed. Both in male and female rats, the percentage of μ-opioid receptor-expressing cells decreased significantly from postnatal week one through the 24 months of our study. Interestingly, pituitary cells containing the μ-opioid receptor were significantly more numerous in male than in female, with exception of the pre-pubertal phase and old rats. According to two-way analysis of variance, the gender-related differences in μ-receptor expression were independent of age-related variations. Thus, without excluding hypothalamic actions, our results suggest that opioids may exert their endocrine function by acting directly on pituitary cells.

Localization of tyrosine hydroxylase mRNA in the axons of the hypothalamo-neurohypophysial system

With in situ hybridization we examined the localization of mRNA coding for tyrosine hydroxylase (TH) in the rat hypothalamo-neurohypophysial system (HNS) under conditions of acute osmotic stress. Fifteen min after salt loading, hybridization signal of TH mRNA could be located in the magnocellular hypothalamic nuclei and in the median eminence (ME). In untreated animals, TH mRNA was detected only in the ME. In osmotically challenged animals that had been pretreated with colchicine, signals for TH mRNA remained confined to the ME, while pretreatment of salt loaded rats with a polymerase II transcription inhibitor resulted in labelling of the magnocellular perikarya but a decrease of the hybridization signal in the ME. Our results suggest that also TH mRNA is among the RNAs which are axonally transported in the HNS. TH mRNA can probably be stored in axons of the hypothalamo-neurohypophysial tract, to be transported retrogradely and translated upon certain stimuli.

Coexistence of NADPH-diaphorase with tyrosine hydroxylase in hypothalamic magnocellular neurons of the rat

The presence and distribution of NADPH-diaphorase (ND) neurons as well as tyrosine hydroxylase-immunoreactive (TH) neurons in the hypothalamus are well established. Previous studies have shown the coexistence of ND with neuroactive substances such as calbindin, somatostatin, vasopressin and oxytocin in neurons of this region of the brain. As the tópographical patterns of distribution of ND and TH coincide in many cases, the aim of this study was to determine the possible coexistence of both substances in the main hypothalamic magnocellular nuclei of the albino rat. Histochemical-immunocytochemical double labelling was employed on the same sections as well as a morphometric study. NADPH-diaphorase and tyrosine hydroxylase neurons were observed in all the nuclei under study (supraoptic, paraventricular and accessory nuclei), although most neurons showing the coexistence of both substances were mainly located in the supraoptic nucleus, isolated neurons with double labelling being found in the magnocellular parts of the paraventricular nucleus and in some of the accessory nuclei. Although both substances have previously been shown to be modified in hypothalamic neurons after osmotic stimuli, the range of functions of ND in the CNS is only beginning to be understood. Further studies are needed to elucidate the functional role that ND/TH neurons play in the nervous system.

Local transformations of androgens into estradiol by aromatase P450 is involved in the regulation of prolactin and the proliferation of pituitary prolactin-positive cells.

In previous studies we demonstrated the immunohistochemical expression of aromatase in pituitary cells. In order to determine whether pituitary aromatase is involved in the paracrine regulation of prolactin-producing pituitary cells and the physiological relevance of pituitary aromatase in the control of these cells, an in vivo and in vitro immunocytochemical and morphometric study of prolactin-positive pituitary cells was carried out on the pituitary glands of adult male rats treated with the aromatase antagonist fadrozole. Moreover, we analyzed the expression of mRNA for the enzyme in pituitary cells of male adult rats by in situ hybridization. The aromatase-mRNA was seen to be located in the cytoplasm of 41% of pituitary cells and was well correlated with the immunocytochemical staining. After in vivo treatment with fadrozole, the size (cellular and nuclear areas) of prolactin cells, as well as the percentage of prolactin-positive cells and the percentage of proliferating-prolactin cells, was significantly decreased. Moreover, fadrozole decreased serum prolactin levels. In vitro, treatment with fadrozole plus testosterone induced similar effects on prolactin-positive cells, inhibiting their cellular proliferation. Our results suggest that under physiological conditions aromatase P450 exerts a relevant control over male pituitary prolactin-cells, probably transforming testosterone to estradiol in the pituitary gland.

Dopamine Modulates Insulin Release and Is Involved in the Survival of Rat Pancreatic Beta Cells

The local synthesis of dopamine and its effects on insulin release have been described in isolated islets. Thus, it may be accepted that dopamine exerts an auto-paracrine regulation of insulin secretion from pancreatic beta cells. The aim of the present study is to analyze whether dopamine is a regulator of the proliferation and apoptosis of rat pancreatic beta cells after glucose-stimulated insulin secretion. Glucose stimulated pancreatic islets obtained from male Wistar rats were cultured with 1 or 10 μM dopamine from 1 to 12 h. Insulin secretion was analyzed by RIA. The cellular proliferation rate of pancreatic islets and beta cells was studied with immunocytochemical double labelling for both insulin and PCNA (proliferating cell nuclear antigen), and active caspase-3 was detected to evaluate apoptosis. The secretion of insulin from isolated islets was significantly inhibited (p<0.01), by treatment with 1 and 10 μM dopamine, with no differences between either dose as early as 1 h after treatment. The percentage of insulin-positive cells in the islets decreased significantly (p<0.01) after 1 h of treatment up to 12 h. The proliferation rate of insulin-positive cells in the islets decreased significantly (p<0.01) following treatment with dopamine. Apoptosis in pancreatic islets and beta cells was increased by treatment with 1 and 10 μM dopamine along 12 h. In conclusion, these results suggest that dopamine could modulate the proliferation and apoptosis of pancreatic beta cells and that dopamine may be involved in the maintenance of pancreatic islets.

Morphometric changes of specific located vasopressin-reacting parvicellular neurons in the paraventricular nucleus of the rat after adrenalectomy.

The morphological-morphometric consequences of bilateral adrenalectomy on vasopressin-reacting neurons of the paraventricular nucleus of the rat hypothalamus were analyzed. Bilateral adrenalectomy led to a dramatic increase in the cellular area as well as the number of immunoreactive cells (when compared to those obtained in normal colchicine-treated animals) in the neurons located in the anterior, medial and periventricular parvicellular subdivisions of the paraventricular nucleus. By contrast, no changes were observed in either the dorsal or lateral parvicellular subdivisions or in any of the magnocellular subdivisions of the paraventricular nucleus.

Morphological and functional study of the GH-immunoreactive adenohypophyseal cells in ovariectomized rats

SummaryIn view of the existence of a different secretion pattern of growth hormone (GH) between male and female rats, the aim of the present study was to analyse the role played by ovarian steroid hormones in the modulation of such secretion. To do so, postpuberal female rats were ovariectomized and killed at 30 days after the operation. The basal serum levels of growth hormone, together with cell area, cytoplasmic area and nuclear area of the hypophyseal somatotropic cells of normal and ovariectomized rats were compared. The results obtained show that ovariectomy induces a significant decrease (p<0.05) in the basal serum levels of GH, accompanied by an increase in cellular and cytoplasmic areas, with no significant differences in nuclear area. Overiectomy was also accompanied by an increase in reaction intensity and the number of GH-immunoreactive cells (p<0.01). These findings point to the shift towards a masculine secretory and morphological pattern following ovariectomy and supports the hypothesis that ovarian steroids intervene in the establishment of a different pattern in females compared to males.

The expression of AIB1 correlates with cellular proliferation in human prolactinomas.

Estrogens as well as certain growth factors strongly influence the development and growth of prolactinomas. However, the molecular mechanisms by which extracellular factors trigger prolactinomas are not well known. Amplified in breast cancer 1 (AIB1), also known as steroid receptor co-activator 3 (SRC-3), belongs to the p160/SRC family of nuclear receptor co-activators and is a major co-activator of the estrogen receptor. Here, we report that the estrogen receptor coactivator AIB1 is overexpressed in human prolactinomas and correlates with the detection of aromatase and estrogen receptor α (ERα). Of the 87 pituitary tumors evaluated in women, 56%, corresponding to hyperoprolactinemic women, contained an enriched population of prolactin-positive cells and hence were further classified as prolactinomas. All prolactinomas stained positive for both ERα and AIB1. Moreover, AIB1 sub-cellular distribution was indicative of the cell-cycle status of tumors; the nuclear expression of AIB1 was correlated with proliferative markers whereas the cytoplasmic localization of AIB1 coincided with active caspase-3. Thus, our results demonstrate for the first time that AIB1 is expressed in prolactinomas and suggest its participation in the regulation of proliferation and apoptosis of tumoral cells. Because aromatase expression is also enhanced in these prolactinomas and it is involved in the local production of estradiol, both mechanisms, ER-AIB1 and aromatase could be related.