Anti-stress effects of ONO-2952, a novel translocator protein 18 kDa antagonist, in rats.

Accumulating evidence has shown the pathophysiological significance of the translocator protein 18 kDa (TSPO) in the central nervous system. In this study, we evaluated the beneficial effects of ONO-2952, a novel TSPO antagonist in rat stress models. ONO-2952 potently bound both rat and human TSPO (Ki=0.330-9.30 nmol/L) with high selectivity over other receptors, transporters, ion channels and enzymes. ONO-2952 inhibited both neurosteroid accumulation and noradrenaline release in the brain of rats exposed to acute stress. The inhibitory effect of ONO-2952 on stress-induced noradrenaline release was attenuated by co-treatment with the TSPO agonist CB34 in a dose-dependent manner. ONO-2952, at 0.3 mg/kg or higher, dose-dependently suppressed restraint stress-induced defecation in rats with brain TSPO occupancy of more than 50%. In addition, ONO-2952, at 1 mg/kg or higher, suppressed conditioned fear stress-induced freezing behavior in rats with an efficacy equivalent to that of diazepam, given orally at 3 mg/kg. Results of the passive avoidance learning test revealed that ONO-2952, unlike diazepam, did not affect learning and memory even at doses 10 times higher than its effective doses in the stress models. The present findings indicate that ONO-2952 is a promising candidate for the treatment of stress-related disorders.

Effects of tanshinone VI on phosphorylation of ERK and Akt in isolated cardiomyocytes and cardiac fibroblasts.

Effects of tanshinone VI, a diterpene from Tan-Shen, on humoral factor-induced phosphorylation of ERK and Akt during hypertrophy of cardiomyocytes and fibrosis of cardiac fibroblasts isolated from neonatal rats were examined. Treatment of cultured cardiomyocytes with 10 nM insulin-like growth factor-1 (IGF-1) or 10 nM endothelin-1 resulted in an increase in leucine incorporation into acid-insoluble fraction. Treatment of cultured cardiac fibroblasts with 10 nM IGF-1 or 10 nM angiotensin II increased incorporation of proline. IGF-1 increased phosphorylated extracellular signal-regulated kinase (pERK) and protein kinase B (pAkt) of cardiomyocytes, whereas endothelin-1 increased pERK, but not pAkt. Treatment of cardiac fibroblasts with 10 nM IGF-1 or 10 nM angiotensin II also increased pERK, whereas pAkt was increased by treatment with IGF-1 alone. When the cardiomyocytes were incubated in the presence of 10 microM tanshinone VI, IGF-1- and endothelin-1-induced increases in pERK, but not pAkt, were partially attenuated. Treatment of cardiac fibroblasts with 10 microM tanshinone VI also attenuated IGF-1-induced increases in pERK and pAkt. Tanshinone VI also partially attenuated angiotensin II-induced increase in proline incorporation into cardiac fibroblasts. PD98059, an inhibitor for phosphorylation of extracellular signal-regulated kinase (ERK), but not wortmannin, that for protein kinase B phosphorylation, attenuated an increase in leucine incorporation into cardiomyocytes in the presence of either IGF-1 or endothelin-1. These results suggest that tanshinone VI is a possible agent that can attenuate the humoral factor-induced hypertrophy of cardiomyocytes and fibrosis of cardiac fibroblasts via an attenuation of ERK phosphorylation in these cells.

V1a vasopressin receptors maintain normal blood pressure by regulating circulating blood volume and baroreflex sensitivity.

Arginine-vasopressin (AVP) is a hormone that is essential for both osmotic and cardiovascular homeostasis, and exerts important physiological regulation through three distinct receptors, V1a, V1b, and V2. Although AVP is used clinically as a potent vasoconstrictor (V1a receptor-mediated) in patients with circulatory shock, the physiological role of vasopressin V1a receptors in blood pressure (BP) homeostasis is ill-defined. In this study, we investigated the functional roles of the V1a receptor in cardiovascular homeostasis using gene targeting. The basal BP of conscious mutant mice lacking the V1a receptor gene (V1a-/-) was significantly (P < 0.001) lower compared to the wild-type mice (V1a+/+) without a notable change in heart rate. There was no significant alteration in cardiac functions as assessed by echocardiogram in the mutant mice. AVP-induced vasopressor responses were abolished in the mutant mice; rather, AVP caused a decrease in BP, which occurred in part through V2 receptor-mediated release of nitric oxide from the vascular endothelium. Arterial baroreceptor reflexes were markedly impaired in mutant mice, consistent with a loss of V1a receptors in the central area of baroreflex control. Notably, mutant mice showed a significant 9% reduction in circulating blood volume. Furthermore, mutant mice had normal plasma AVP levels and a normal AVP secretory response, but had significantly lower adrenocortical responsiveness to adrenocorticotropic hormone. Taken together, these results indicate that the V1a receptor plays an important role in normal resting arterial BP regulation mainly by its regulation of circulating blood volume and baroreflex sensitivity.

Resistin concentrations in murine adipose tissue and serum measured by a new enzyme immunoassay.

OBJECTIVE: In an attempt to clarify the conflicting data on resistin mRNA expression and protein analysis by western blotting in adipose tissue and serum, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for direct measurement of mouse resistin. RESEARCH METHODS AND PROCEDURES: We developed polyclonal antibodies directed to the N (21 to 40) and C (79 to 91) termini of mouse resistin. Then, affinity-purified anti-C-terminal resistin immunoglobin G (IgG) was biotinylated. ELISA was based on the sandwiching of antigen between antibody IgG coated on polystyrene plates and biotinylated antibody IgG. The bound biotinylated antibody was quantified with streptavidin-linked horseradish peroxidase. RESULTS: New ELISA can measure a concentration as low as 0.5 ng/mL of recombinant mouse resistin and is sensitive and specific enough to measure resistin protein in various adipose tissues and in sera. In normal mice, decreases in resistin concentrations in both white adipose tissue and serum were age dependent during 6 to 24 weeks of development. Resistin concentrations were significantly higher in omental adipose tissue in comparison with perirenal and abdominal adipose tissues and were 2- to 5-fold higher in females than males during the growth period. ob/ob mice had significantly lower resistin concentrations than the control mice in both sera and the white adipose tissues, particularly in the omental fat. The treatment by testosterone, but not progesterone or beta-estradiol, in cultured adipocytes reduces resistin protein levels in a dose-dependent manner. DISCUSSION: New sensitive ELISA for mouse resistin clarified that the resistin concentrations in normal mice were markedly elevated in the omental adipose depots as compared with the perirenal and abdominal adipocyte depots and significantly elevated compared with adipose tissues in genetically obese mice.

Preferable anesthetic conditions for echocardiographic determination of murine cardiac function.

Ketamine and xylazine are routinely used for measurement of hemodynamics of mice and rats by echocardiography. The anesthetic agents produce low heart rate (HR) in the animals, which may result in misleading data in the hemodynamic profiles of the small animals. The purpose of the present study was to select an appropriate anesthetic condition in the evaluation of mouse and rat cardiac function by echocardiography. Echocardiographic measurement was performed in male C57BL6 mice anesthetized with an intraperitoneal injection of 30 or 40 mg/kg pentobarbital (P30 or P40) or a combination of 60 mg/kg ketamine and 6 mg/kg xylazine (KX) and in male Wistar rats with an intraperitoneal injection of 40 or 50 mg/kg pentobarbital (P40 or P50) or a combination of 100 mg/kg ketamine and 10 mg/kg xylazine (KX). Basal HR of P30-anesthetized mice and P40-anesthetized were comparable to those in the conscious state, whereas KX-anesthetized mice and rats were 38% and 74% of those of the conscious animals, respectively. Fractional shortening (FS) and cardiac output index (COI) of the P30-anesthetized mice or the P40-anesthetized rats were greater than those of KX-anesthetized animals. Intraperitoneal injection of dobutamine at 0.3 and 1 mg/kg increased HR, FS, and COI of the P30-anesthetized mice and the P40-anesthetized rats, respectively, whereas the percent responses of these parameters in KX animals were greater than those in pentobarbital-anesthetized ones due to the lower basal values for the cardiac functional parameters. Anesthesia with P30 for the mouse and P40 for the rat rather than ketamine/xylazine may be relevant to the evaluation of cardiac function using echocardiography.

Cabergoline stimulates synthesis and secretion of nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor by mouse astrocytes in primary culture.

Neuroprotection is the primary concern in patients with newly diagnosed Parkinson's disease. The D2/weak D1 dopamine agonist cabergoline elicits neuroprotection by antioxidation and scavenging free radicals, and may protect neurons by up-regulating endogenous neurotrophic factors synthesis in the brain. In primary cultured mouse astrocytes, cabergoline 37 micromol/l immediately elevated concentrations of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor (GDNF) in culture medium, reaching 9.9-, 2.6- and 30-fold, respectively, of control levels at 16 h. Relative mRNA levels were 3.0-, 1.5- and 1.9-fold, respectively, of controls at 3 h. These effects may be mediated partly by the dopamine D2 receptor. Cabergoline may be a good candidate for an inducer of GDNF, which may have neuroprotective and neurorestorative properties in dopaminergic nigral neurons.

Enzyme-linked immunosorbent assay for circulating human resistin: resistin concentrations in normal subjects and patients with type 2 diabetes.

BACKGROUND: Resistin is a recently identified adipocyte-secreted hormone in rodents, and has been proposed to serve as a link between obesity and insulin resistance. The aim of this study was to develop a sensitive enzyme-linked immunosorbent assay (ELISA) for human resistin and evaluate serum resistin concentrations in normal subjects and patients with type 2 diabetes. METHODS: Using ELISA developed by two polyclonal antibodies, resistin concentrations were measured in 90 patients with type 2 diabetes and compared to 74 healthy control subjects. RESULTS: This ELISA has high specificity and sensitivity over the concentration of range 0.5-100 ng/ml with good percentage recovery (97.1 +/- 4.7%) and reproducibility (within-day assay, CV = 4.8-8.6%; between-day assay, CV = 5.6-9.7%). The mean concentration of resistin in sera from type 2 diabetic patients was significantly higher than that in normal subjects (mean +/- S.E.: 20.8 +/- 0.7 vs. 14.9 +/- 0.5 ng/ml, p < 0.001). A moderate positive correlation was observed between serum resistin levels and body mass indices in both normal subjects (r = 0.412, p < 0.0003) and patients with type 2 diabetes (r = 0.395, p < 0.0001). CONCLUSIONS: Our ELISA will be useful to confirm the physiological and pathophysiological role of resistin in humans.

Effects of tanshinone VI on insulin-like growth factor-1-induced hypertrophy of isolated cardiomyocytes from neonatal rats.

The effects of tanshinone VI (Tan), a diterpene extracted from Salvia miltiorrhiza, on insulin-like growth factor-1 (IGF-1)-induced hypertrophy of cardiomyocytes were examined. Cultured cardiomyocytes were isolated from neonatal rat hearts. The incorporation of [(3)H]-leucine into the trichloroacetic acid (TCA)-insoluble fraction was measured as a marker of protein synthesis, which revealed cardiomyocyte hypertrophy. Various concentrations of IGF-1, ranging from 0.1 nM to 10 nM, increased [(3)H]-leucine incorporation into the TCA-insoluble fraction of cardiomyocytes in a dose-dependent manner. IGF-1 induced an increase in phosphorylated extracellular signal-regulated kinase 1/2 (ERK), but did not change ERK protein content in cardiomyocytes. When the cells were incubated in the presence of Tan (0.1 muM to 10 muM), [(3)H]-leucine incorporation into IGF-1-untreated cells was unaltered. When the cells were incubated with 10 muM Tan, IGF-1-induced increases in [(3)H]-leucine incorporation into the TCA-insoluble fraction and phosphorylated ERK were attenuated. These results suggest that Tan is a possible agent for the suppression of IGF-1-induced hypertrophy of cardiomyocytes via an attenuation of ERK activation.

Effects of tanshinone VI on the hypertrophy of cardiac myocytes and fibrosis of cardiac fibroblasts of neonatal rats.

The possible effects of tanshinone VI (tsh), a diterpene from the root of Tan-Shen (Salvia miltiorrhiza, Bunge (Labiatae)) on hypertrophy and fibrosis in cultured neonatal rat cardiac myocytes and fibroblasts were examined. Tsh had no significant effect on protein synthesis, which was evaluated by [3H]-leucine incorporation into the acid insoluble fraction in the cells, in the absence of stimulatory factors in cardiac myocytes. The amount of protein produced in cardiac myocytes was increased by 10(-8) M endothelin-1 (ET-1), 10(-6) M phenylephrine (PE), or 10(-8) M insulin-like growth factor-1 (IGF-1), suggesting that hypertrophy of cardiac myocytes in vitro was induced by these factors. The ET-1-, PE-, or IGF-1-induced increase in protein synthesis was attenuated by treatment with 10(-5) M tsh. Treatment with 10(-5) M tsh significantly decreased the synthesis of collagen by cardiac fibroblasts, which was evaluated by [3H]-proline incorpolation into acid-insoluble fraction of the fiblobrasts, in the absence of stimulatory factors for the production. Fetal bovine serum (FBS) or IGF-1 increased collagen synthesis in a concentration-dependent manner. The increase at 5% FBS or 10(-8) M IGF-1 was inhibited by 10(-5) M tsh. Fibroblast-conditioned medium (FB-CM) increased protein synthesis in cardiac myocytes in a concentration-dependent manner (10; - 100 %). Tsh attenuated the FB-CM-induced increase in protein synthesis by cardiac myocytes. These results show that tsh may attenuate the humoral factor-induced hypertrophy of cardiac myocytes and fibrosis of cardiac fibroblasts. The findings suggest that tsh may improve the development of cardiac remodeling under pathophysiological conditions. Abbreviations. ANP:atrial natriuretic peptide DMEM:Dulbecco-modified Eagle's medium ET-1:endothelin-1 FB-CM:fibroblast-conditioned medium FBS:fetal bovine serum IGF-1:insulin-like growth factor-1 PE:phenylephrine tsh:tanshinone VI