Manuel Prieto Rubio

Infrequent cellular coexistence of NADPH-diaphorase and calretinin in the neurosecretory nuclei and adjacent areas of the rat hypothalamus

Colocalization of the calcium-binding protein calretinin and NADPH-diaphorase activity at the cellular level was studied in the magnocellular secretory nuclei of the rat hypothalamus using sequential immunocytochemical and histochemical staining of the same sections. A low degree of colocalization of these markers was observed in certain cellular subpopulations within all the areas considered (supra-optic, paraventricular, circular and both fornicals nuclei and in the hypothalamic area located between the supraoptic and paraventricular nuclei). However, since in the paraventricular nucleus both markers were expressed by different neuronal populations, the coexistence was almost non-existent in some subdivisions of this nucleus. This rare coexistence strongly suggests that NADPH-diaphorase and calretinin are related to different functions shared by restricted hypothalamic neuronal populations.

Expression of Aromatase P450 is Increased in Spontaneous Prolactinomas of Aged Rats

We have recently reported the presence of aromatase P450 in the rat hypophysis. This enzyme is responsible for the aromatization of testosterone to estradiol. Since the induction of prolactinomas has been demonstrated in the rat following chronic treatment with estradiol, the aim of the present study was to analyze whether a relationship exists between the presence of pituitary aromatase and the appearance of spontaneous prolactinomas in aged rats. Of a series of 90 adenomas studied, 53% showed prolactin immunoreactive cells and were classified as prolactinomas; only 33% of the adenomas were pure prolactinomas and the other 20% were multi-hormonal protactinomas. Moreover, 60% of the adenomas were aromatase-positive tumors. Interestingly, 100% of the pure prolactinomas were aromatase-positive while only 60% of the multi-hormonal prolactinomas expressed the enzyme. Western blotting with anti-aromatase antibodies revealed a 3.8-fold increase in expression of aromatase in pituitary tumors as compared to normal rat pituitary gland. Double immunohistochemical labeling detected the coexistence of prolactin and aromatase P450 in prolactinoma cells. ACTH- and LH-positive adenomas were considered as controls; only multi-hormonal ACTH and LH tumors display aromatase-positive cells and all of these also contained prolactin-positive cells. Our results demonstrate for the first time that aromatase is expressed in pituitary adenomas and that it is related to the functional nature of the tumor, especially in the case of pure prolactinomas, suggesting the possibility that an abnormally high conversion of testosterone into estradiol in pituitary cells may contribute to the genesis of spontaneous prolactinomas in aged rats.

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.

IN VITRO MORPHOMETRIC AND PROLIFERATIVE VARIATIONS IN VIP-IMMUNOREACTIVE PITUITARY CELLS INDUCED BY ESTRADIOL

Using double labelling for proliferating cell nuclear antigen (PCNA) and vasoactive intestinal peptide (VIP), a study was conducted to elucidate the repercussions of the addition of estradiol to monolayer pituitary cultures on the morphology, size, number, mitotic index and PCNA labelling index of VIP-immunoreactive cells, comparing the results with those obtained in control cultures. The addition of estradiol (100 nM) over 3 h of incubation led to a significant increase (p < 0.01) in the cellular, cytoplasmic and nuclear areas of VIP-immunoreactive cells and in the number of these cells. The same treatment elicited an even greater increase in the mitotic index (p < 0.01) and PCNA labelling index (p < 0.01) of VIP-immunoreactive cells. The results obtained suggest that estrogens induce proliferation and hyperplasia of in vitro VIP-immunoreactive pituitary cells.

NADPH-diaphorase activity and vasopressin in the paraventricular nucleus of the hypothalamus following adrenalectomy

In order to investigate novel neuroendocrine functions of the nitric oxide synthesizing enzyme a combined histochemical and immunocytochemical study focused on the paraventricular nucleus of the hypothalamus was conducted to check a possible influence of bilateral adrenalectomy on three different neuronal populations, NADPH diaphorase (ND)-positive, vasopressin (VP)-immunoreactive and neurons expressing both markers. In the adrenalectomized animals, a slight increase (P > 0.05) of the number of ND magnocellular neurons was detected, whereas no changes were observed in the ND-parvicellular population and in the neurons showing coexistence (magno- and parvicellular) (P > 0.05). By contrast, following bilateral adrenalectomy, a significant increase (P < 0.05) in the VP-parvicellular population (anterior, medial and periventricular subdivisions) was detected, which was reversed when the animals received daily doses of corticosterone. These results suggest that nitric oxide is not closely related to the hypothalamic regulation of the adenocorticotropin secretion exerted by the paraventricular nucleus.

In vitro and in vivo evidence for direct dopaminergic inhibition of VIP-immunoreactive pituitary cells

In order to establish whether dopamine is involved in the regulation of immunocytochemical expression of VIP in pituitary cells, in vivo and in vitro studies were carried out on male rats after treatment with haloperidol and dopamine, respectively. In the in vivo studies, following intraventricular (third ventricle) administration of haloperidol, an increase in the total number of VIP-immunoreactive cells (p < 0.05) was observed; additionally, the cellular, cytoplasmic and nuclear areas were increased (p < 0.01). The in vitro studies demonstrated that dopamine induces a significant decrease (p < 0.05) in the total number of VIP-immunoreactive cells, which were smaller than the cells from the control dishes, because dopamine induces a significant decrease in their cellular, cytoplasmic and nuclear areas (p < 0.01). These results support the hypothesis that dopamine is a physiological inhibitor of pituitary VIP expression and suggest that dopamine modulates the auto- or paracrine effects of VIP in the rat pituitary.

Dopamine inhibits in vitro release of VIP and proliferation of VIP-immunoreactive pituitary cells.

A double immunohistochemical study for VIP and proliferating cell nuclear antigen (PCNA) was carried out on monolayer cultures from adult male rats pituitary glands treated with dopamine (ranging from 10(-9) to 10(-5) M), in order to establish whether or not dopamine is involved in the regulation of the proliferation rate of pituitary VIP-immunoreactive cells. For all doses of dopamine assayed, the release of VIP to the culture medium, the numerical density of VIP-immunoreactive cells and the percentages of VIP- and PCNA-immunoreactive cells decreased significantly after dopamine treatment. These results suggest that dopamine could be a physiological inhibitor involved in the modulation of pituitary VIP proliferation rate.

Stereo Video Surveillance Multi-agent System: New Solutions for Human Motion Analysis

This article presents a distributed agent-based system that can process the visual information obtained by stereoscopic cameras. The system is embedded within a global project whose objective is to develop an intelligent environment for location and identification within dependent environments that merges with other types of technologies. In this kind of environments, vision algorithms are very costly and require a lot of time to produce a response, which is highly inconvenient since many applications can require action to be taken in real time. A multi-agent system (MAS) can automate the process of analyzing images obtained by cameras, and optimize the procedure. This study presents a MAS that can process stereoscopic images to detect and classify people by combining a series of novel techniques.The article shows in detail the combination of techniques used to perform the detection process. The process can be subdivided into human detection, human tracking, and human behavior understanding. With the addition of a case-based reasoning (CBR) model, the system can also incorporate reasoning capabilities. The system was tested under different conditions and environments.

Corticosterone induces hypoactivity of prolactin-immunoreactive cells.

In order to determine whether corticosterone regulates activity of rat lactotrophs by acting directly at the pituitary level, immunohistochemical studies were carried out in adrenalectomized rats, subjected or not to treatment with corticosterone or colchicine, and in monolayer cultures after incubation with corticosterone. Adrenalectomy increased cellular and nuclear areas (p<0.01) of prolactin-immunoreactive cells without affecting their cytoplasmic area. Similar results were found in adrenalectomized and colchicine-treated animals. Corticosterone reversed the effects of adrenalectomy, although normal values were partially reversed. In cultured pituitary cells, exposure to corticosterone reduced numerical density and cellular, cytoplasmic and nuclear areas with respect to control dishes. Morphological differences in shape, arrangement and nuclear features were observed after treatment with corticosterone. These results demonstrate an inhibitory effect of corticosterone on the activity of rat pituitary prolactin cells and suggest that corticosterone induces hypoactivity by acting on the pituitary prolactin cells of male rats.

Decreases in the Size and Proliferation Rate of VIP-lmmunoreactive Cells Induced in vitro by Testosterone Are Associated with Decreases in VIP Release

As in vitro study was carried out to determine whether testosterone directly inhibits the secretion of pituitary vasoactive intestinal peptide (VIP). A study was also conducted to elucidate the repercussions of treatment with 10(-6) M testosterone of monolayer cultures in the size and proliferation rate of pituitary VIP-immunoreactive cells, using double immunocytochemical labelling for proliferating cell nuclear antigen (PCNA) and VIP. Testosterone decreased the in vitro release of VIP from pituitary cells. It also decreased the percentage of pituitary VIP-immunoreactive and PCNA- and VIP-immunoreactive cells and decreased the size of the cellular and nuclear areas of these cells. Our results suggest that the inhibition of pituitary VIP secretion induced by testosterone could be exerted at pituitary level and that testosterone could act as a modulator of the proliferation rate of rat pituitary VIP-immunoreactive cells.