Higinio Uría

Neurohormone melatonin prevents cell damage: effect on gene expression for antioxidant enzymes.

It is well known that porphyrins cause a toxic light‐mediated effect due to their capability to generate free radicals. Several reports have proved that melatonin is a potent free radical scavenger. The aim of this work has been to study the ability of melatonin to prevent the cell damage caused by porphyrins in the Harderian gland of female Syrian hamsters. Cell injury was evaluated estimating the percentage of damaged cells found in the gland and analyzing the degree of this damage at ultrastructural level. To explain the mechanism by which this hormone could prevent the cell damage caused by porphyrins, its capability to both decrease porphyrin synthesis and increase the mRNA levels for antioxidant enzymes was evaluated. Our results demonstrate that melatonin administration decreases the percentage of damaged cells, porphyrin synthesis, and aminolevulinate synthase (ALA‐S) mRNA levels and increases the mRNA levels for manganese superoxide‐dismutase and copper‐zinc superoxide dismutase. When observed under an electron microscope, the lesions in the clear cells of the treated females were much less severe than in the corresponding cells of the control animals. Melatonin exerts a cytoprotective effect by inhibiting the ALA‐S gene expression (and so porphyrin synthesis) and by raising the mRNA levels for several antioxidant enzymes.—Antolín, I., Rodríguez, C., Sáinz, R. M., Mayo, J. C., Uría, H., Kotler, M. L., Rodríguez‐Col‐ unga, M. J., Tolivia, D., Menéndez‐Peláez, A. Neurohormone melatonin prevents cell damage: effect on gene expression for antioxidant enzymes. FASEB J. 10, 882‐890 (1996)

Melatonin prevents apoptosis induced by 6-hydroxydopamine in neuronal cells: Implications for Parkinson's disease

Abstract: It was recently reported that low doses of 6‐hydroxydopamine (6‐OHDA) induce apoptosis of naive (undifferentiated) and neuronal (differentiated) PC 12 cells, and this system has been proposed as an adequate experimental model for the study of Parkinsons disease. The mechanism by which this neurotoxin damages cells is via the production of free radicals. Given that the neurohormone melatonin has been reported 1) to be a highly effective endogenous free radical scavenger, 2) to increase the mRNA levels and the activity of several antioxidant enzymes, and 3) to inhibit apoptosis in other tissues, we have studied the ability of melatonin to prevent the programmed cell death induced by 6‐OHDA in PC12 cells. We found that melatonin prevents the apoptosis caused by 6‐OHDA in naive and neuronal PC12 cells as estimated by 1) cell viability assays, 2) counting of the number of apoptotic cells, and 3) analysis and quantification of DNA fragmentation. Exploration of the mechanisms used by melatonin to reduce programmed cell death revealed that this chemical mediator prevents the 6‐OHDA induced reduction of mRNAs for several antioxidant enzymes. The possibility that melatonin utilized additional mechanisms to prevent apoptosis of these cells is also discussed. Since this endogenous agent has no known side effects and readily crosses the blood‐brain‐barrier, we consider melatonin to have a high clinical potential in the treatment of Parkinsons disease and possibly other neurodegenerative diseases, although more research on the mechanisms is yet to be done.

The pineal neurohormone melatonin prevents in vivo and in vitro apoptosis in thymocytes

Abstract: Recently, melatonin was found to be the most potent physiological free radical scavenger known to date. In this work, we attempted to define the role this neurohormone plays in the regulation of apoptosis, since the effect of bcl‐2, the main gene implicated in its inhibition, acts via an antioxidant mechanism. We investigated the role of melatonin in cell death of thymus, a well known model for the study of apoptosis. Two sets of experiments were carried out: in vivo experiments, performed with Wistar rats, and in vitro experiments, performed with primary cultures of young Wistar rat thymocytes treated with glucocorticoids in order to induce apoptosis. Morphometrical studies in semithin sections of thymus and analysis of DNA fragmentation by gel electrophoresis show that physiological apoptosis occurring in thymus of 65 days old rats, is prevented by the daily administration of melatonin beginning when the rats were 25 days old. Also, we found that at a concentration of 10−7 M, melatonin decreases by 35% the percentage of apoptotic cells induced by glucocorticoids in cultured thymocytes of 25 day old rats. 10−9 M melatonin decreases cell death by 20%. Finally, melatonin at 10−11 Mdid not have any effect. Several hypothesis are discussed to explain this effect: direct interaction of melatonin with glucocorticoid receptors in the thymus; induction of interleukin‐4 release; direct genomic action modulating the expression of apoptosis‐inhibiting genes; an effect on nitric oxide synthase; and finally, the antioxidant action of melatonin. Since apoptosis is a possible mechanism involved in neuronal death shown in several neurodegenerative diseases such as Parkinson or Alzheimers diseases, investigative efforts should be directed to the possible role of melatonin in inhibiting cell death in tissues other that the thymus. Melatonin might be a potent therapeutic agent in some of these conditions.

Androgen regulation of gene expression in the Syrian hamster Harderian gland

The androgenic control of sexual dimorphism has been studied in the Harderian gland from Syrian hamster and compared to rat Harderian gland, a system without dimorphism. Hybridization in situ with a rat cDNA clone has revealed the presence of androgen receptor mRNA in all secretory cells from male and female hamster glands. Testosterone or 5-alpha-dihydrotestosterone administration to females both caused a 60% decrease in the levels of 5-aminolevulinate synthase mRNA after 1 day of treatment, but the resulting patterns of in vitro translation using RNA from glands treated with the two androgens are different. Testosterone alters the mRNA levels for androgen receptor and 5-aminolevulinate synthase in the glands only 6 h after its implantation in females, and the action is maintained up to 10 days of treatment. Finally, androgen administration to females or deprivation in males alter androgen receptor but not 5-aminolevulinate synthase mRNA levels in rat Harderian glands. Our results suggest that the androgen receptor from Harderian glands is responsible for the sexual dimorphism found in Syrian hamsters, whereas the lack of sexual dimorphism in rat seems to be due to a restricted effect of androgens in the glands.

Development and androgen regulation of the secretory cell types of the Syrian hamster (Mesocricetus auratus) Harderian gland

The secretory cell types of the hamster Harderian glands were studied in both male and female Syrian hamsters. As previously demonstrated, female hamsters showed a single secretory cell type (type I), while male hamsters displayed two secretory cell types (type I and type II). Type-II cells were observed after the first month of age correlating with the increase in testosterone levels. The administration of testosterone to adult female hamsters resulted in a marked increase in the percentage of type-II cells without a significant increase in the number of mitotic figures. Very low levels of serum testosterone were able to maintain the percentage of type-II cells. Castration of male hamsters produced a decrease in the percentage of type-II cells. This drop correlated with the reduction in serum testosterone levels. The chronic administration of a luteinizing hormone-releasing hormone agonist to male Syrian hamsters induced a significant reduction in both serum luteinizing hormone and testosterone. However, the percentage of type-II cells was similar to that of control hamsters suggesting that very low levels of circulating testosterone are able to maintain the percentage of type-II cells. In a final experiment male Syrian hamsters were treated with the antiadrogen cyproterone acetate. No changes were observed in the percentage of type-II cells, whereas serum luteinizing hormone and testosterone levels were significantly modified. We concluded that (1) type-II cells differentiate from type-I cells; (2) gonadal androgens are the major factor controlling this differentiation; and (3) the disappearance of type-II cells after androgen deprivation occurs through holocrine and apocrine mechanisms. The possible implication of 5α-reductase in the regulation of secretory cell types in the Harderian glands of hamsters is discussed.

The harderian gland of the rodent octodon degus: A structural and ultrastructural study

Harderian glands from male and female Octodon degus were examined by light and transmission electron microscopy. Two types of secretory units, designated as type I and type II, were observed. Type I secretory units comprise three types of epithelial cells: Cells packed with numerous lipid droplets (Type a), cells with few lipid droplets (Type b), and cells with numerous mitochondria and a very well developed Golgi complex (Type c). Type II secretory units were found exclusively in female Octodon degus and comprised a type of secretory cells which contained numerous basophilic granules in their apical cytoplasm. In addition, in female Octodon degus, clusters of lymphocyte-like cells and plasmatic cells were also observed. The vascularization of the gland appeared very well developed. The most unique feature of the blood supply was the existence of large sinusoidal vessels extremely variable in shape. In the medullar region, the sinsoidal wall adapts its contour to that of the tubuloalveolar surface. Unmyelinated and myelinated nerve fibers were found in the connective stroma of the gland.

Photoperiod and the pineal gland regulate the male phenotype of the Harderian glands of male Syrian hamsters after androgen withdrawal

Coto‐Montes AM, Rodriguez‐Colunga MJ, Una H, Antolin I, Tolivia D, Buzzell GR, Menendez‐Pelaez A. Photoperiod and the pineal gland regulate the male phenotype of the Haderian glands of male Syrian hamsters after androgen withdrawal. J. Pineal Res. 1994: 17: 48–54.

Androgen-dependent mast cell degranulation in the Harderian gland of female Syrian hamsters: in vivo and organ culture evidence

Abstract In previous articles we have reported the ’’disappearance’’ of Harderian gland mast cells (HGMC) after treatment with testosterone. In the present work we study: (a) if the apparent decrease in the number of mast cells caused by this androgen is real or is due to the fact that testosterone induces mast cell degranulation that avoids its recognition by toluidine blue staining; (b) if testosterone acts through its receptor directly on the Harderian gland (HG). In order to give an answer to the first question, we observed HG of female Syrian hamsters treated with testosterone under the electron microscope to find the possible degranulated mast cells not recognizable with the aid of the toluidine blue staining. We also studied in vivo and in vitro the effects of the β-agonists isoproterenol and salbutamol, given that they increase cAMP and can therefore prevent degranulation of mast cells. Finally we have used cytocalasin B, which inhibits degranulation by blocking actin depolimerization. Both the β-agonists and cytochalasin B were able to prevent the decrease of mast cells, as recognized by staining with toluidine blue after treatment with testosterone. Indeed, when observed under the electron microscope, abundant degranulated mast cells were found after treatment with testosterone. For solving the second issue we analyzed the effect of the antiandrogen cyproterone acetate in vivo and in vitro. Our results demonstrate that testosterone is able to induce degranulation of HGMC in the Syrian hamster Mesocricetus auratus and that this effect is achieved directly through its receptor on the Harderian gland.

Melatonin decreases mRNA for histone h4 in thymus of young rats

The antiproliferative properties of melatonin have been previously demonstrated for several normal and tumoral tissues. In a recent report we have shown that melatonin is able to inhibit programmed cell death in thymus both, in vivo and in vitro. Given that other authors have related programmed cell death and cell proliferation and that no previous reports on melatonin and cell division exist on thymus, we decide to study the possible antiproliferative effect of melatonin in this organ measured as the levels of mRNA for the histone H4. We found that melatonin inhibits cell division on thymus when administered chronically both, at high (500 microg/body weight) and low (50 microg/body weight) dose. We also found a circadian rhythm of the mRNA for histone H4, opposed to the one previously described for melatonin, supporting the negative regulation by this hormone of cell division on thymus. A single dose of melatonin (50 microg/body weight) was not able to decrease the levels of mRNA for H4 in the time-points studied but after two hours of its administration. Finally, we report the inhibitory effect of melatonin in the cell proliferation of Harderian gland, brain, lung and kidney.

INVASIVE PROCESSES IN THE NORMAL HARDERIAN GLAND OF SYRIAN HAMSTER

In this contribution we will pay special attention to several morphological findings that we can observe, under some circumstances, in the normal Harderian gland of the Syrian hamster. The accumulation of porphyrins in this gland results in mitochondrial damage and extensive cell death. Many damaged cells are secreted into the lumen of the tubule‐alveoli, but most of them seem to produce an invasive process that even affects the vascular components of the gland. In this way, many blood vessels are invaded and appear partially filled with the invasive mass, which sometimes totally occludes the lumen of the vessels. We have also observed other surprising features related to a special kind of activity in certain secretory cells. Such activity results in a peculiar “segregation” of a cytoplasmic fragment, containing the nucleus. The affected cells seem to gather up their cytoplasm and nucleus towards the basal zone, while the rest of the cell, including practically the whole amount of lipid droplets, is relegated to the vicinity of the lumen. All these phenomena seem finally to result in the detachment of some clusters, composed of a limited number of cells, which display a basophilic cytoplasm practically free of lipid droplets.