Enhanced dopamine D2 receptor function in hypothalamus and corpus striatum: their role in liver, plasma and in vitro hepatocyte ALDH regulation in ethahol treated rats

Dopamine D2 receptors are involved in ethanol self- administration behavior and also suggested to mediate the onset and offset of ethanol drinking. In the present study, we investigated dopamine (DA) content and Dopamine D2 (DA D2) receptors in the hypothalamus and corpus striatum of ethanol treated rats and aldehyde dehydrogenase (ALDH) activity in the liver and plasma of ethanol treated rats and in vitro hepatocyte cultures. Hypothalamic and corpus striatal DA content decreased significantly (P\0.05, P\0.001 respectively) and homovanillic acid/ dopamine (HVA/DA) ratio increased significantly (P\0.001) in ethanol treated rats when compared to control. Scatchard analysis of [3H] YM-09151-2 binding to DA D2 receptors in hypothalamus showed a significant increase (P\0.001) in Bmax without any change in Kd in ethanol treated rats compared to control. The Kd of DA D2 receptors significantly decreased (P\0.05) in the corpus striatum of ethanol treated rats when compared to control. DA D2 receptor affinity in the hypothalamus and corpus striatum of control and ethanol treated rats fitted to a single site model with unity as Hill slope value. The in vitro studies on hepatocyte cultures showed that 10-5 M and 10-7 M DA can reverse the increased ALDH activity in 10% ethanol treated cells to near control level. Sulpiride, an antagonist of DA D2, reversed the effect of dopamine on 10% ethanol induced ALDH activity in hepatocytes. Our results showed a decreased dopamine concentration with enhanced DA D2 receptors in the hypothalamus and corpus striatum of ethanol treated rats. Also, increased ALDH was observed in the plasma and liver of ethanol treated rats and in vitro hepatocyte cultures with 10% ethanol as a compensatory mechanism for increased aldehyde production due to increased dopamine metabolism. A decrease in dopamine concentration in major brain regions is coupled with an increase in ALDH activity in liver and plasma, which contributes to the tendency for alcoholism. Since the administration of 10-5 M and 10-7 M DA can reverse the increased ALDH activity in ethanol treated cells to near control level, this has therapeutic application to correct ethanol addicts from addiction due to allergic reaction observed in aldehyde accumulation.

DOPAMINE D2 RECEPTOR FUNCTIONAL REGULATION : cAMP AND IP3 ROLE IN PANCREATIC REGENERATION

The present study deals with the differential regulation of Dopamine content in pancreas and functional regulation of Dopamine D2 receptor in brain regions such as hypothalamus, brain stem, cerebral cortex and corpus striatum play an important role during pancreatic islets cell proliferation and insulin secretion. Though may reports are there implicating the functional interaction between DA receptor and pancreatic islets cell insulin secretion, the involvement of specific DA D2 receptors and changes in second messenger system during insulin secretion and pancreatic islets cell proliferation were not given emphasis. Down regulation of DA content in brain regions and pancreatic islets were observed during pancreatic regeneration. Up regulation of DA content in plasma and adrenals down regulated sympathetic activity in pancreas which cause an increase in insulin secretion and pancreatic islets cell proliferation during pancreatic regeneration. There was a differential regulation of DA D2 receptor in brain regions. The pancreatic islets DA D2 receptors were lip regulated during pancreatic regeneration. DA D2 receptor activation at specific concentration has accounted for increased pancreatic islets cell proliferation. In vitro experiments have proved the differential regulation of DA on insulin synthesis and pancreatic islets cell proliferation. Inhibitory effect of DA on cAMP and stimulatory effect of DA on IP3 through DA D2 receptors were observed in in vitro cell culture system. These effects are correlating with the DA, cAMP and IP3 content during pancreatic regeneration and islets cell proliferation. Up regulation of intracellular Ca2+ was also observed at 10-8 M DA, a specific concentration of DA which showed maximum increase of IP3 content in pancreatic islets through DA D2 receptor activation in in vitro culture. These in vitro data was highly correlating with the changes in DA, cAMP and IP3 content in pancreas during pancreatic regeneration and insulin secretion. Thus we conclude that there is a differential functional regulation of DA and DA D2 receptors in brain and pancreas during pancreatic regeneration. In vitro studies confirmed a concentration depend functional regulation of DA through DA D2 receptors on pancreatic islets cell proliferation and insulin secretion mediated through increased cAMP, IP3 and intracellular Ca2+ level. This will have immense clinical significance in the management in diabetes mellitus.

Dopamine D1 and D2 Receptor Functional Regulation:Glutamate Receptor Gene Expression in the Brain of Hypoglycaemic and Hyperglycaemic Rats

In the present study a detailed investigation on the alterations of dopamine and its receptors in the brain regions of streptozotocin induced diabetic and insulin induced hypoglycaemic rats were carried out. Glutamate receptor, NMDARI gene expression in the hypoglycaemic and hyperglycaemic brain was also studied. EEG recording in hypoglycaemic and hyperglycaemic will be carried out to measure brain activity. in vitro studies on glucose uptake and insulin secretion, with and without specific antagonists were carried out to confirm the specific receptor subtypes - DA D1, DA D2 and NMDA involved in the functional regulation during hyperglycaemic and hypoglycaemic brain damage. The molecular studies on the brain damage through dopaminergic and glutamergic receptors will elucidate the therapeutic role in the corrective measures of the damage to the brain during hypoglycaemia and hyperglycaemia. This has importance in the management of diabetes and antidiabetic treatment for better intellectual functioning of the individual.

Dopamine D2 and 5-HT2A receptor gene expression

Neuronal dopamine and serotonin receptors are widely distributed in the central and the peripheral nervous systems at different levels. Dopaminergic and serotonergic systems have crucial role in aldehyde dehydrogenase regulation Stimulation of autonomic nervous system during ethanol treatment is suggested to be an important factor in regulating the ALDH function. The ALDH enzyme activity was increased in plasma, cerebral cortex, and liver but decreased in cerebellum. The ALDH enzyme affinity was decreased in plasma, brainstem and liver and increased in cerebral cortex and cerebellum. Dopamine and serotonin content decreased in liver and brain regions - cerebral cortex, corpus striatum of ethanol treated rats with an increased HVA/DA, 5-HIAA/5-HT tumover rate. Dopamine content decreased in brainstem with an increased HVA/DA turnover rate and serotonin content decreased with an increased 5-HIAA/5-HT turnover rate in the brainstem of ethanol treated rats compared to control. Serotonin content increased in hypothalamus with a decreased 5-HIAA/5—HT turnover rate where as dopamine content decreased in hypothalamus with an increased HVA/DA tumover rate of ethanol treated rats compared to control.alterations of DA D2 and 5-HTQA receptor function and gene expression in the cerebellum, hypothalamus, corpus striatum, cerebral cortex play an important role in the sympathetic regulation of ALDH enzyme in ethanol addiction. There is a serotonergic and dopaminergic functional regulation of ALDH activity in the brain regions and liver of ethanol treated rats. Gene expression studies of DA D2 and 5'HT2A studies confirm these observations. Perfusion studies using DA, 5-HT and glucose showed ALDH regulatory function. Brain activity measeurement using EEG showed a prominentfrontal brain wave difference. This will have immense clinical significance in the management of ethanol addiction.

Molecular, cellular and physiological investigation of myostatin propeptide-mediated muscle growth in adult mice

Inhibition of myostatin signalling or its biological activity has recently emerged as a potential remedial approach against muscle wasting and degenerative diseases such as muscular dystrophies. In the present study we systemically administered a recombinant AAV8 vector expressing a mutated myostatin propeptide (AAV8ProMyo) to healthy mice in order to assess its impact on the histological, cellular and physiological properties of the skeletal muscle, exploiting the fact that myostatin is naturally inhibited by its own propeptide. We report that a single intravenous administration of AAV8ProMyo leads to increases in muscle mass of tibialis anterior, extensor digitorum longus and gastrocnemius muscles 8 weeks post-injection and tibialis anterior, gastrocnemius and rectus femoris muscles 17 weeks post-injection. Moreover, treatment resulted in muscle fibre hypertrophy but not hyperplasia, with IIB myofibres responding to the greatest extent following propeptide-induced myostatin inhibition. Additionally, myofibre nuclear: cytoplasmic ratio was decreased in the AAV8ProMyo treated animals. Importantly, the hypertrophic EDL muscle 8 weeks after AAV8ProMyo treatment did not show the dramatic decrease in specific force displayed by the germline myostatin null mice. (C) 2009 Elsevier B.V. All rights reserved.

Functional coupling of the human dopamine D-2 receptor with G alpha(i1), G alpha(i2), G alpha(i3) and G alpha(o) G proteins: evidence for agonist regulation of G protein selectivity

1 The human dopamine D-2long (D-2L) receptor was expressed with four different G proteins in Sf9 cells using the baculovirus expression system. When co-expressed with G(i)/G(o) G proteins (G(i1)alpha, G(i2)alpha, G(i3)alpha, or G(o)alpha, plus Gbeta(1) and Ggamma(2)) the receptor displayed a high-affinity binding site for the agonists (dopamine and NPA), which was sensitive to GTP (100 mum), demonstrating interaction between the receptor and the different G proteins. 2 The receptor to G protein ratio (R: G ratio) was evaluated using [H-3]-spiperone saturation binding (R) and [S-35]-GTPgammaS saturation binding (G). R: G ratios of 1: 12, 1: 3, 1: 14 and 1: 5 were found for G(i1), G(i2), G(i3), and Go preparations, respectively. However, when R:G ratios of 1:2 and 1: 12 were compared for G(i2) and G(o), no difference was found for the stimulation of [S-35]-GTPgammaS binding. 3 Several agonists were tested for their ability to stimulate [S-35]-GTPgammaS binding to membranes co-expressing the receptor and various G proteins. All the compounds tested showed agonist activity in preparations expressing G(i3) and G(o). However, for G(i2) and G(i1) preparations, compounds such as S-(-)-3-PPP and p-tyramine were unable to stimulate [S-35]-GTPyS binding. 4 Most of the compounds showed higher relative efficacies (compared to dopamine) and higher potencies in the preparation expressing G(o). Comparison of the effects of different agonists in the different preparations showed that each agonist differentially activates the four G proteins. 5 We conclude that the degree of selectivity of G protein activation by the D-2L receptor can depend on the conformation of the receptor stabilised by an agonist.

Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). In vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.

Effect of apoE genotype and vitamin E on biomarkers of oxidative stress in cultured neuronal cells and the brain of targeted replacement mice

The aetiology of apoE4 genotype-Alzheimer's disease (AD) association are complex. The current study emphasizes the impact of apoE genotype and potential beneficial effects of vitamin E (VE) in relation to oxidative stress. Agonist induced neuronal cell death was examined 1) in the presence of conditioned media containing equal amounts of apoE3 or apoE4 obtained from stably transfected macrophages, and 2) after pretreatment with alpha- and gamma-tocopherol, and -tocotrienol. ApoE3 and apoE4 transgenic mice were fed a diet poor or rich in VE to study the interplay of both apoE genotype and VE status, on membrane lipid peroxidation, antioxidative enzyme activity and glutathione levels in the brain. Cytotoxicity of hydrogen peroxide and glutamate was higher in neuronal cells cultured with apoE4 than apoE3 conditioned media. VE pre-treatment of neurons counteracted the cytotoxicity of a peroxide challenge but not of nitric oxide. No significant effects of apoE genotype or VE supplementation were observed on lipid peroxidation or antioxidative status in the brain of apoE3 and apoE4 mice. VE protects against oxidative insults in vitro, however, no differences in brain oxidative status were observed in mice. Unlike in cultured cells, apoE4 may not contribute to higher neuronal oxidative stress in the brain of young targeted replacement mice.

Antitumour activity of [1,2-di(cyclopentadienyl)-1,2-di(p-N,N-dimethylaminophenyl)-ethanediyl] titanium dichloride in xenografted Ehrlich's ascites tumour

The effects of a new titanocene compound with an ansa ligand in the cyclopentadienyl rings, the 1,2-di(cyclopentadienyl)-1,2-di(p-NNdimethylaminophenyl)-ethanediyl] titanium dichloride (TITANOCENE X), on the growth and differentiation of granulocyte-macrophage progenitor cells [colony-forming unit-granulocyte-macrophage (CFU-GM)] and Natural killer (NK) cell activity in Ehrlich's ascites tumour (EAT)-bearing mice were studied. Myelosuppression concomitant with increased numbers of spleen CFU-GM was observed in tumour-bearing mice. Treatment of these animals with TITANOCENE X (2.5-50mg/kg/day) produced an increase in myelopoicsis, in a dose-dependent manner, and reduced spleen colony formation. In addition, the treatment of EAT-bearing mice with 3 doses of 20 or 50 mg/kg TITANOCENE X restored to normal values the reduced Natural killer cell function observed during tumour growth. In parallel, TITANOCENE X prolonged, in a dose-dependent manner, the survival of mice inoculated with Ehrlich's ascites tumour. The highest dose of 50 mg/kg prolonged in 50% the survival time of EAT-bearing mice, compared to non-treated tumour-bearing controls. In comparison with previous results from our laboratory addressing the effects of titanocenes on haematopoiesis, we observed with TITANOCENE X a similar effective profile as for bis(cyclopentadienyl) dithiocyanate titanium(IV), being both less effective than di(cyclopentadienyl) dichloro titanium(IV), since the latter not only prolonged, but also increased the rate of survival. These differences in efficacy may be due to the nature of the ansa-cyclopentadienyl ligand used in TITANOCENE X, since the C, bridge between the two cyclopentadienyl groups will increase the hydrolytic stability by an organometallic chelate effect. Also, the introduction of two dimethylamino substituents increases the water solubility of TITANOCENE X when compared to titanocene dichloride itself (c) 2006 Elsevier B.V. All rights reserved.

Sulforaphane protects cortical neurons against 5-S-cysteinyl-dopamine-induced toxicity through the activation of ERK1/2, Nrf-2 and the upregulation of detoxification enzymes

The degeneration of dopaminergic neurons in the substantia nigra has been linked to the formation of the endogenous neurotoxin 5-S-cysteinyl-dopamine. Sulforaphane (SFN), an isothiocyanate derived from the corresponding precursor glucosinolate found in cruciferous vegetables has been observed to exert a range of biological activities in various cell populations. In this study, we show that SFN protects primary cortical neurons against 5-S-cysteinyl-dopamine induced neuronal injury. Pre-treatment of cortical neurons with SFN (0.01-1 microM) resulted in protection against 5-S-cysteinyl-dopamine-induced neurotoxicity, which peaked at 100 nM. This protection was observed to be mediated by the ability of SFN to modulate the extracellular signal-regulated kinase 1 and 2 and the activation of Kelch-like ECH-associated protein 1/NF-E2-related factor-2 leading to the increased expression and activity of glutathione-S-transferase (M1, M3 and M5), glutathione reductase, thioredoxin reductase and NAD(P)H oxidoreductase 1. These data suggest that SFN stimulates the NF-E2-related factor-2 pathway of antioxidant gene expression in neurons and may protect against neuronal injury relevant to the aetiology of Parkinson's disease.

Synthesis and biological evaluation of a polyglutamic acid-dopamine conjugate: a new antiangiogenic agent

Dopamine has previously been shown to inhibit angiogenesis in vitro and in vivo, but its clinical applications in this context are severely limited by its short half-life. Here we report the synthesis of a polyglutamic acid-dopamine conjugate and show that conjugation significantly extends (from 1 to 24 h) dopamine’s antiangiogenic activity in vitro and in vivo. These findings form the basis for the development of a new class of agents for the treatment of angiogenesis-dependent diseases.

Role for protease activity in visceral pain in irritable bowel syndrome

Mediators involved in the generation of symptoms in patients with irritable bowel syndrome (IBS) are poorly understood. Here we show that colonic biopsy samples from IBS patients release increased levels of proteolytic activity (arginine cleavage) compared to asymptomatic controls. This was dependent on the activation of NF-kappaB. In addition, increased proteolytic activity was measured in vivo, in colonic washes from IBS compared with control patients. Trypsin and tryptase expression and release were increased in colonic biopsies from IBS patients compared with control subjects. Biopsies from IBS patients (but not controls) released mediators that sensitized murine sensory neurons in culture. Sensitization was prevented by a serine protease inhibitor and was absent in neurons lacking functional protease-activated receptor-2 (PAR2). Supernatants from colonic biopsies of IBS patients, but not controls, also caused somatic and visceral hyperalgesia and allodynia in mice, when administered into the colon. These pronociceptive effects were inhibited by serine protease inhibitors and a PAR2 antagonist and were absent in PAR2-deficient mice. Our study establishes that proteases are released in IBS and that they can directly stimulate sensory neurons and generate hypersensitivity symptoms through the activation of PAR2.

Fermentable carbohydrate alters hypothalamic neuronal activity and protects against the obesogenic environment

Obesity has become a major global health problem. Recently, attention has focused on the benefits of fermentable carbohydrates on modulating metabolism. Here, we take a system approach to investigate the physiological effects of supplementation with oligofructose-enriched inulin (In). We hypothesize that supplementation with this fermentable carbohydrate will not only lead to changes in body weight and composition, but also to modulation in neuronal activation in the hypothalamus. Male C57BL/6 mice were maintained on a normal chow diet (control) or a high fat (HF) diet supplemented with either oligofructose-enriched In or corn starch (Cs) for 9 weeks. Compared to HF+Cs diet, In supplementation led to significant reduction in average daily weight gain (mean ± s.e.m.: 0.19 ± 0.01 g vs. 0.26 ± 0.02 g, P < 0.01), total body adiposity (24.9 ± 1.2% vs. 30.7 ± 1.4%, P < 0.01), and lowered liver fat content (11.7 ± 1.7% vs. 23.8 ± 3.4%, P < 0.01). Significant changes were also observed in fecal bacterial distribution, with increases in both Bifidobacteria and Lactobacillius and a significant increase in short chain fatty acids (SCFA). Using manganese-enhanced MRI (MEMRI), we observed a significant increase in neuronal activation within the arcuate nucleus (ARC) of animals that received In supplementation compared to those fed HF+Cs diet. In conclusion, we have demonstrated for the first time, in the same animal, a wide range of beneficial metabolic effects following supplementation of a HF diet with oligofructose-enriched In, as well as significant changes in hypothalamic neuronal activity.

Platelet protein disulfide isomerase is required for thrombus formation but not essential for hemostasis in mice

Protein disulfide isomerase (PDI) derived from intravascular cells is required for thrombus formation. However, it remains unclear whether platelet PDI contributes to the process. Using platelet-specific PDI-deficient mice, we demonstrate that PDI-null platelets have defects in aggregation and ATP secretion induced by thrombin, collagen, and ADP. Such defects were rescued by exogenously-added wild-type but not mutant PDI, indicating that the isomerase activity of platelet surface PDI is critical for the regulatory effect. PDI-deficient platelets expressed increased levels of intracellular ERp57 and ERp72. Platelet PDI regulated αIIbβ3 integrin activation but not P-selectin exposure, Ca2+ mobilization, β3-talin interaction, and platelet spreading on immobilized fibrinogen. Inhibition of ERp57 further diminished αIIbβ3 integrin activation, aggregation and ATP secretion of activated PDI-deficient platelets, suggesting distinct roles of PDI and ERp57 in platelet functions. We found that platelet PDI is important for thrombus formation on collagen-coated surfaces under arteriolar shear. Intravital microscopy demonstrates that platelet PDI is important for platelet accumulation but not initial adhesion and fibrin generation following laser-induced arteriolar injury. Tail bleeding time and blood loss in platelet-specific PDI-deficient mice were not significantly increased. Our results provide important evidence that platelet PDI is essential for thrombus formation but not for hemostasis in mice.

Genetic evidence for a predominant role of PI3Kβ catalytic activity in ITAM- and integrin-mediated signaling in platelets.

Phosphatidylinositol 3-kinase (PI3K) isoforms PI3Kbeta and PI3Kgamma are implicated in platelet adhesion, activation, and aggregation, but their relative contribution is still unclear or controversial. Here, we report the first comparative functional analysis of platelets from mice expressing a catalytically inactive form of PI3Kbeta or PI3Kgamma. We demonstrate that both isoforms were similarly required for maximal activation of the small GTPase Rap1b and for complete platelet aggregation upon stimulation of G protein-coupled receptors for adenosine 5'-diphosphate (ADP) or U46619. Their contribution to these events, however, was largely redundant and dispensable. However, PI3Kbeta, but not PI3Kgamma, enzymatic activity was absolutely required for Akt phosphorylation, Rap1 activation, and platelet aggregation downstream of the immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptor glycoprotein VI (GPVI). Moreover, PI3Kbeta was a major essential regulator of platelet adhesion to fibrinogen and of integrin alpha(IIb)beta(3)-mediated spreading. These results provide genetic evidence for a crucial and selective role of PI3Kbeta in signaling through GPVI and integrin alpha(IIb)beta(3).