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.

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 degrees 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.

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 microM 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 microM 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.

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 degrees C) and repeated swimming stress (45 min at 22 degrees 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 glucocorticoid 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.

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.

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.

Dehydroepiandrosterone protects hippocampal neurons against neurotoxin-induced cell death: mechanism of action.

Dehydroepiandrosterone (DHEA), an adrenal cortex hormone secreted in large quantities in humans, protects cells of the clonal mouse hippocampal cell line HT-22 against the excitatory amino acid glutamate (5 mM), and amyloid beta-protein (2 microM) toxicity in a dose-dependent manner with optimum protection obtained at 5 microM concentration of DHEA. The protective effects of DHEA appear to be specific in that other related steroids and metabolites of DHEA, such as 5-androstene-3beta,17beta-diol, etiocholan-3alpha-ol-17-one, etiocholan-3beta-ol-17-one, testosterone, and 5alpha-androstane-3, 17-dione, offered no protection even at 50 microM concentrations. In addition, using immunocytochemical techniques, we observed that 20 hr of treatment with 5 mM glutamate remarkably increased glucocorticoid receptor (GR) nuclear localization in neuronal cells. Interestingly, 5 microM DHEA treatment for 24 hr, followed by 5 mM glutamate treatment for 20 hr almost completely reversed the copious nuclear localization of GR observed by glutamate treatment alone. Results obtained suggest that DHEA protects hippocampal neurons, at least in part, by its antiglucocorticoid action via decreasing hippocampal cells nuclear GR levels.

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 delta9THC was able to compete with [3H] CP 55,940; little competition was observed with either delta8THC 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 30 degrees C. 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.

Glucocorticoids enhance the potency of Schwann cell mitogens.

Previous studies have documented that cultured Schwann cells require serum-containing medium to respond maximally to mitogens. We now report that Schwann cells are able to proliferate to a mitogenic response in a serum-free defined medium termed oligodendrocyte defined media (ODM). Glucocorticoids are the essential component of ODM which allow Schwann cell proliferation in the serum-free medium. Charcoal treatment of the fetal calf serum decreases the mitogenic potency of the axolemma-enriched fraction (AEF) by 50%. The addition of 2 microM hydrocortisone to charcoal-treated fetal calf serum restores 75% of the lost mitogenicity. These observations are consistent with the view that glucocorticoids present in fetal calf serum are potent co-mitogens essential for AEF-induced Schwann cell proliferation. The synthetic glucocorticoid, dexamethasone, is a more potent co-mitogen than hydrocortisone, with a maximal effect at concentrations less than 10 nM. In contrast, other steroids including aldosterone, progesterone, testosterone, and 17 beta-estradiol have no effect on enhancing the mitogenic response of Schwann cells to the AEF. The glucocorticoid antagonists RU 486 and dehydroepiandrosterone (DHEA), but not the antiestrogenic compound tamoxifen, block AEF-induced Schwann cell proliferation. These results suggest that glucocorticoid-induced Schwann cell proliferation is mediated through a glucocorticoid receptor (GR) mechanism. We detected immunoreactivity to the GR in the cytoplasm, but not in the nuclei of Schwann cells grown in ODM lacking dexamethasone. The addition of 100 nM dexamethasone to these cultures resulted in immunoreactivity in the nucleus. This data suggests that glucocorticoids working through the GR are potent co-mitogens for Schwann cell proliferation.

Intracranial dehydroepiandrosterone blocks the activation of tryptophan hydroxylase in response to acute sound stress.

Bilateral infusion of dehydroepiandrosterone (DHEA) given intracerebroventricularly blocked the sound stress-induced increase in tryptophan hydroxylase activity observed ex vivo in midbrain and cortex but had no effect on the level of tryptophan hydroxylase activity from sham-stressed rats. DHEA (20 micrograms total dose) given bilaterally into the region of the central nucleus of the amygdala, 30 min prior to 1 h sound stress, also blocked the increase in enzyme activity in a dose-dependent manner. The DHEA treatment did not alter the activation of the enzyme seen in vitro in the presence of phosphorylating conditions. The effect of DHEA was steroid specific in that other sex steroids, such as estrogen, androgens, or progesterone, were without any effect. Coadministration, 20 micrograms each, of the potent glucocorticoid agonist, RU 28362, with DHEA 30 min prior to 1 h sound stress completely blocked the DHEA suppressive effect on sound stress-induced increases in tryptophan hydroxylase activity. The results obtained suggest that DHEA blocks this increase in tryptophan hydroxylase activity by antagonizing the effects of glucocorticoid.

Roles of sulfhydryl and disulfide groups in the binding of CP-55,940 to rat brain cannabinoid receptor.

The roles of sulfhydryl and disulfide groups in the specific binding of synthetic cannabinoid CP-55,940 to the cannabinoid receptor in membrane preparations from the rat cerebral cortex have been examined. Various sulfhydryl blocking reagents including p-chloromercuribenzoic acid (p-CMB), N-ethylmaleimide (NEM), o-iodosobenzoic acid (o-ISB), and methyl methanethiosulfonate (MMTS) inhibited the specific binding of [3H]CP-55,940 to the cannabinoid receptor in a dose-dependent manner. About 80-95% inhibition was obtained at a 0.1 mM concentration of these reagents. Scatchard analysis of saturation experiments indicates that most of these sulfhydryl modifying reagents reduce both the binding affinity (Kd) and capacity (Bmax). On the other hand, DL-dithiothreitol (DTT), a disulfide reducing agent, also irreversibly inhibited the specific binding of [3H]CP-55,940 to the receptor and about 50% inhibition was obtained at a 5 mM concentration. Furthermore, 5 mM DTT was abelt to dissociate 50% of the bound ligand from the ligand-receptor complex. The marked inhibition of [3H]CP-55,940 binding by sulfhydryl reagents suggests that at least one free sulfhydryl group is essential to the binding of the ligand to the receptor. In addition, the inhibition of the binding by DTT implies that besides free sulfhydryl group(s), the integrity of a disulfide bridge is also important for [3H]CP-55,940 binding to the cannabinoid receptor.

Role of the antiglucocorticoid RU 486 in the prevention of steroid-induced hypertension.

In the present study, we determined the effect of RU 486 on two experimental models of hypertension in the rat, deoxycorticosterone acetate (DOCA)-salt in nephrectomized rats and spontaneously hypertensive rats. Uni-nephrectomized saline-drinking male Sprague-Dawley rats were divided into three groups and each animal was given either 0.2 ml olive oil (control), 1 mg DOCA, or 1 mg DOCA + 10 mg RU 486 dissolved in 0.2 ml olive oil every third day for a period of three weeks. Within a week of steroid administration, there was a significant increase in the systolic blood pressure (SBP) in the DOCA-salt (157 +/- 3.8 mmHg) and DOCA + RU 486 (155 +/- 2.1 mmHg) treated rats over the control (116 +/- 2.6 mmHg) rats, which remained elevated throughout the experimental period. There was significant increase in the water intake and urine output in DOCA or DOCA + RU 486 treated rats as compared to the control untreated rats. In the experiment involving the spontaneously hypertensive rats, the rats were divided into three groups and each animal given 0.2 ml olive oil (control), 1 mg RU 486, or 5 mg RU 486 dissolved in 0.2 ml olive oil for six weeks. Instead of the expected decrease in the blood pressure, RU 486 significantly elevated blood pressure during the six weeks of drug administration. Water intake, urine output, and weights remained comparable in both groups. We conclude that RU 486 has no effect on the DOCA-salt model of hypertension but, surprisingly, elevates hypertension in the spontaneously hypertensive rats.

Dehydroepiandrosterone prevents dexamethasone-induced hypertension in rats.

Dehydroepiandrosterone (DHEA) is an endogenous steroid having a wide variety of biological and biochemical effects. In the present study, we have examined the role of DHEA on various rodent models of experimental hypertension. Sprague-Dawley rats were given subcutaneous injections of 1.5 mg dexamethasone every alternate day, resulting in an increase in systolic blood pressure within 1 wk. Interestingly, administration of a pharmacological dose of 1.5, 3, or 7.5 mg DHEA along with dexamethasone prevented dexamethasone-induced hypertension in a dose-dependent manner. DHEA had no effect on the hypertension induced by deoxycorticosterone acetate (DOCA)-salt administration using uninephrectomized rats or on the genetic model of spontaneously hypertensive rats. Dexamethasone administration resulted in a significant weight loss in rats, which was not prevented by simultaneous administration of DHEA. These results indicate that dexamethasone-mediated weight loss may involve mechanisms separate from its hypertensive action. Dexamethasone treatment resulted in a significant decrease in food consumption that was not reversed by DHEA. It is concluded that DHEA at doses above physiological levels when given subcutaneously has no effect on DOCA-salt or a genetic model of hypertension but has a beneficial effect on dexamethasone-induced hypertension.

Effect of a new mineralocorticoid antagonist mespirenone on aldosterone-induced hypertension.

The effects of the mineralocorticoid antagonist mespirenone on the development and maintenance of aldosterone-induced hypertension in Sprague-Dawley rats has been studied. Uninephrectomized saline-drinking male Sprague-Dawley rats were injected with either 0.2 ml olive oil, 50 g aldosterone, 1 mg mespirenone, 50 g aldosterone plus 500 g mespirenone, or 50 g aldosterone plus 1 mg mespirenone, each dissolved in 0.2 ml olive oil. Administration of aldosterone alone significantly increased the systolic blood pressure (SBP) from a control value of 114 +/- 3.6 to 162 +/- 4 mmHg by the end of the 3-wk experimental period. Mespirenone given alone had no effect on SBP. However, mespirenone given in combination with aldosterone reversed the hypertension caused by aldosterone in a dose-dependent manner. Saline consumption and urinary output were slightly increased in aldosterone-treated rats compared with the other groups, but the body and organ weights were comparable in all groups. Microscopic examination of kidney and heart showed no abnormalities due to mespirenone. These results suggest that in vivo administration of mespirenone to Sprague-Dawley rats effectively prevents the aldosterone-induced hypertension.