Arturo Cardounel

Anti-stress effects of dehydroepiandrosterone: protection of rats against repeated immobilization stress-induced weight loss, glucocorticoid receptor production, and lipid peroxidation.

In the present study, we have (i) examined the biological effects of repeated immobilization stress, and (ii) tested the hypothesis that the adrenal steroid hormone dehydroepiandrosterone (DHEA) is an anti-stress hormone, using male Sprague-Dawley rats. Rats (N = 6) were divided into the following four groups: (i) control, (ii) repeated immobilization stress (2 hr daily, for 60 days), (iii) repeated immobilization stress (2 hr daily, for 60 days) plus daily i.p. administration of 5 mg DHEA/0.1 mL DMSO, and (i.v.) daily i.p. administration of 5 mg DHEA/0.1 mL DMSO alone. Results obtained showed that repeated immobilization stress resulted in a significant (25%) inhibition in body weight gain, a significant increase in adrenal weight, an increase in glucocorticoid receptor (GR) in the liver, thymus, and spleen, decreased plasma triglyceride levels, and increased lipid peroxidation in the liver and heart as compared with control unstressed animals. Interestingly, DHEA administration resulted in a significant reversal in stress-induced inhibition in body weight gain, adrenal weight, GR levels in liver, thymus, and spleen, and lipid peroxidation levels in the liver and heart. In addition, animals treated with DHEA alone without stress showed a significant (15%) inhibition in body weight gain and an almost 60% decrease in plasma triglyceride levels as compared with control unstressed animals. It is concluded that DHEA acts as an anti-stress hormone in rats, as shown in its antagonizing the effects of repeated immobilization stress on total body weight, adrenal weight, GR levels, and free radical generation.

Pregnenolone Protects Mouse Hippocampal (HT-22) Cells against Glutamate and Amyloid Beta Protein Toxicity

In the present work we have examined whether the neurosteroid pregnenolone has any neuroprotective effects against glutamate and amyloid beta protein neurotoxicity using immortalized clonal mouse hippocampal cell line (HT-22). The neurosteroid pregnenolone protects HT-22 cells against both 5 mM glutamate and 2 μM amyloid beta protein induced cell death in a concentration dependent manner. Optimum protection was attained at 500 nM pregnenolone, against both 5 mM glutamate as well as 2 μM amyloid beta protein induced HT-22 cell death. Furthermore, using confocal immunoflourescence microscopy we observed that 20 hours of treatment with 5 mM glutamate resulted in intense nuclear localization of the glucocorticoid receptor (GR) in HT-22 cells as compared to control untreated cells. Interestingly, 500 nM pregnenolone treatment for 24 hours, followed by 20 hours treatment with 5 mM glutamate resulted in dramatic reduction in GR nuclear localization. These results show that (i) pregnenolone has neuroprotective effects against both glutamate and amyloid beta protein neuropathology and (ii) prevention of glucocorticoid receptor (GR) localization to the nucleus may be involved in the observed neuroprotective effects of pregnenolone against glutamate neurotoxicity.

Biological Effects of Long-Term Caloric Restriction: Adaptation with Simultaneous Administration of Caloric Stress Plus Repeated Immobilization Stress in Rats

In the present study, we have established the biological effects during 8 weeks of (i) caloric restriction (Cal) and (ii) simultaneous administration of Cal plus 2 hr daily immobilization stress using male Sprague-Dawley rats. Animals were divided into three equal groups: (i) ad libitum fed, (ii) 30% restriction of food intake of the ad libitum diet, and (iii) 30% restriction of food intake plus 2 hr daily immobilization stress. Caloric-restricted animals gained only 30% of the total body weight of the unrestricted animals but received 70% of the food of those rats. Cal animals showed a significant loss in their relative liver and thymus weight and a significant gain in their relative adrenal and testis weight as compared to the control animals. Cal animals had almost 2-fold higher levels of plasma corticosterone levels with a dramatic decrease in the total glucocorticoid receptor (GR) levels in the liver, thymus, heart, and testis as compared to ad libitum fed control animals. Interestingly, Cal animals showed higher levels of lipid peroxidation in both the liver and heart, indicating increased oxidative activities in these tissues when compared with the control animals. In addition, Cal animals had increased heat shock protein 70 (HSP 70) content in the testis. Surprisingly, hardly any significant differences were observed in either total body weight gain, organ weights, Plasma corticosterone levels, or lipid peroxidation between Cal animals and Cal plus immobilization-stressed animals. The results obtained suggest that (i) several stress-related responses such as inhibition of total body weight gain, increased adrenal weight, decreased thymus weight, increased plasma corticosterone, and lipid peroxidation levels in the liver and heart are associated with Cal, but (ii) no additional effects were observed on the parameters that were measured when two stress regimens were given simultaneously, suggesting that animals subjected to two stress regimens can protect themselves by controlling their stress-related thresholds of response through adaptation.

Repeated immobilization stress increases total cytosolic glucocorticoid receptor in rat liver

Glucocorticoids are well-known mediators of stress-related endocrine, autonomic, and behavioral responses in mammals and human beings. However, our understanding of the mechanisms of glucocorticoid action in response to stress remains elusive. Therefore, in the present study, an effort has been made to systematically examine glucocorticoid action during acute (2 h) and repeated (2 h daily for 7, 15, and 30 days) immobilization stress in male Sprague-Dawley rats. Prolonged 30-day stress resulted in reduced total body weight gain. There was a dramatic 3- to 4-fold increase in plasma corticosterone levels after single acute stress paradigm, which remained augmented 2- to 3-fold higher than basal control levels during the repeated 30-day immobilization conditions. There was good relationship between increased plasma corticosterone levels and elevation of tyrosine aminotransferase activity in the liver during 30 days of stress. Because repeated immobilization stress animals showed increased levels of both plasma corticosterone and tyrosine aminotransferase activity, the regulation of cytosolic glucocorticoid receptor (GR) in rat liver, a major target tissue for glucocorticoid, was carried out during repeated stress by using GR binding assay, exchange assay, and Western blotting techniques. Exposure of animals to acute and repeated stress resulted in decreased free cytosolic GR. Interestingly, the bound cytosolic GR increased remarkably in liver during prolonged stress of 7-30 days. Overall, results obtained by using both binding assays and Western blotting for the first time showed that repeated stress animals had higher levels of total hepatic cytosolic GR as compared to control animals. These novel results suggest that repeated stress influences the hypothalamic-pituitary-adrenal axis in rats by elevating both the level of plasma corticosterone and total hepatic cytosolic GR.

The environmental estrogenic compound bisphenol A exerts estrogenic effects on mouse hippocampal (HT-22) cells: neuroprotection against glutamate and amyloid beta protein toxicity.

We have examined using immortalized clonal mouse hippocampal cell line (HT-22) whether the environmental estrogenic compound bisphenol A (BPA), like estrogen, has any neuroprotective effect against glutamate and amyloid beta protein-induced neurotoxicity. BPA protects HT-cells against both 5 mM glutamate and 2 microM amyloid beta protein-induced cell death in a dose dependent manner. Optimum protection was attained at 1 microM and 500 nM BPA against 5 mM glutamate and 2 microM amyloid beta protein-induced HT-22 cell death, respectively. Using confocal immunoflourescence microscopy technique, we observed that 20 h of treatment with 5 mM glutamate resulted in intense nuclear localization of the glucocorticoid receptors (GR) in HT-22 cells as compared to control untreated cells. Interestingly, 1 microM BPA treatment for 24 h, followed by 20-h treatment with 5 mM glutamate, resulted in dramatic reduction in GR nuclear localization. We conclude that: (i) BPA mimics estrogen and exerts neuroprotective effects against both neurotoxins used; (ii) BPA inhibits enhanced nuclear localization of GR induced by glutamate; and (iii) HT-22 cells provide a good in vitro model system for screening the potencies of various environmental compounds for their estrogenic activity.

Biological effects of single and repeated swimming stress in male rats: beneficial effects of glucocorticoids.

We have examined the biological effects of single (45 min at 22°C) and repeated swimming stress (45 min at 22°C for 7 d) using male Sprague-Dawley rats. Repeated swimming for a week resulted in a significant inhibition in total body weight (25%) as compared to control unstressed animals. There was significant increase in adrenal and kidney relative weight and decreases in relative thymus weight in repeated swimming-stressed animals as compared to control animals. Repeated swimming stress resulted in almost threefold increase in plasma corticosterone levels with concomitant dramatic decrease in total glucocoticoid receptor (GR) levels in liver, thymus, and heart as compared to control unstressed animals. Interestingly, single swimming stress resulted in a significant elevation in lipid peroxidation levels in the liver and heart. In contrast, there was no change in the lipid per oxidation levels in the liver and heart between chronic stressed and control unstressed animals. Finally, both single and repeated swimming-stress animals had almost 50% reduction in plasma triglyceride levels as compared to control unstressed animals. It is concluded that elevated plasma corticosterone levels by downregulating GR during repeated swimming stress exerts beneficial effects in rats by retarding the total body weight gain and lowering plasma triglyceride levels without affecting free-radicals-induced oxidative stress.

Tamoxifen protects clonal mouse hippocampal (HT-22) cells against neurotoxins-induced cell death

In the present work using an established clonal mouse hippocampal (HT-22) cell line, we have examined whether the estrogen antagonist tamoxifen antagonizes the observed neuroprotective effects of estrogen against glutamate and amyloid beta protein neurotoxicity. Results obtained suggest that like estrogen, tamoxifen protects HT-22 cells against both 5mM glutamate and 2 microM amyloid beta protein induced cell death in a concentration dependent manner. Optimum protection was obtained at 500 nM tamoxifen. Tamoxifen was found to offer more potent protection at this dose against amyloid beta protein induced neurotoxicity when compared with glutamate neurotoxicity. We were unable to detect either estrogen receptor (ER)--ER alpha or ER beta presence in HT-22 cells using western blot technique. However, amyloid beta protein treatment significantly increases total glucocorticoid receptors (GRs) as determined by western blot technique, while prior treatment with estrogen or tamoxifen followed by amyloid beta protein resulted in the reduction of total GRs to the levels comparable to that observed for the control untreated cells. In addition, using confocal immunoflourescence microscopy technique, we observed that 20 h of treatment with 2 microM amyloid beta protein resulted in enhanced nuclear localization of GRs in HT-22 cells as compared to control untreated cells or 500 nM tamoxifen alone treated cells. Interestingly, 500 nM tamoxifen treatments for 24h, followed by 20 h treatment with 2 microM amyloid beta protein resulted in dramatic reduction in GRs nuclear localization. In conclusion, tamoxifen (i) protects HT-22 cells against amyloid beta protein neurotoxicity and (ii) neuroprotective effect is independent of ERs.

Heat shock preconditioning and pretreatment with glucocorticoid antagonist RU 486 protect rat myogenic cells H9c2 against glutamate-induced cell death

We have observed that the treatment of rat-heart derived H9c2 myoblasts for 20 h with the excitatory amino acid glutamate resulted in cell death in a dose dependent manner as determined by LDH release. The optimum cardiotoxicity was seen at 25 mM glutamate. Preconditioning with either sublethal heat shock (42°C for 30 min) or pretreatment with 500 nM of the glucocorticoid antagonist RU 486 for 24 h almost completely protected H9c2 cells against subsequent 20 h treatment with 25 mM lethal glutamate. In addition, we have observed that glutamate treatment resulted in intense nuclear localization of glucocorticoid receptors (GR) in H9c2 cells as judged by the confocal immunofluorescence microscopy. Furthermore, pretreatment with either heat shock or RU 486 followed by glutamate treatment resulted in dramatic decrease in GR nuclear localization which was almost comparable to that observed with control untreated cells. In conclusion, we have shown for the first time using H9c2 cells that (i) protection from glutamate cardiotoxicity occurs with prior treatment with sub lethal heat shock or RU 486 and (ii) these measures down regulate the intense nuclear localization of GR induced by glutamate. The block to GR nuclear localization is likely to be involved in cardioprotective effects offered against glutamate toxicity by pretreatment with heat shock or RU 486.

Identification and characterization of a novel synthetic cannabinoid CP 55,940 binder in rat brain cytosol

We have detected the presence of a specific [3H] CP 55,940 binder in the cytosol of rat cerebral cortex. Competition studies showed that only cold CP 55,940 and to a lesser extent D9THC was able to compete with [3H] CP 55,940; little competition was observed with either D8;THC or anandamide. Scatchard analysis of the data indicate the presence of two distinct binding components having affinity constants (Kd) of 0.97 ± 0.03 nM, 5.83 ± 0.08 nM, and Bmax of 3.31 ± 0.06 pmol/mg protein, 22.2 ± 1.2 pmol/mg protein respectively. The cytosolic CP 55,940 binder was heat stable up to 30øC. Besides the brain cytosol, lesser amounts of binding were also detected in the spleen, and testis. Liver, kidney and muscle cytosol preparations were found to be devoid of this binder. Unlike the previously characterized brain membrane cannabinoid receptor, this binder was found to be salt, sulfhydryl blocking reagents and nucleotide resistant. Interestingly, dithiothreitol (DTT), a protein-disulfide group reducing agent, inhibited the binding of [3H] CP-55,940 to the receptor and approximately 80% binding inhibition was obtained at a 5 mM concentration. Western blot analysis using anti-receptor antibody reveal the presence of a 95-110, 50 and 38 kDa band in the brain, spleen and testis cytosolic preparations. In conclusion, we have identified the presence of a novel CP 55,940 binder in rat cerebral cortex cytosol possessing biochemical properties distinct from those previously observed using rat cerebral cortex membrane cannabinoid receptor.