Biomedical subjects

V L Rao

Publications and source records attributed to V L Rao.

At least 37 records · Page 2Linked to original sources

PubMed1997

Increased neuronal nitric oxide synthase expression in brain following portacaval anastomosis.

It has previously been suggested that increases of L-arginine uptake into brain following portacaval shunting may result in increased activities of constitutive neuronal nitric oxide synthase (nNOS). In order to further address this issue, nNOS protein and gene expression were studied by Western blot analysis using a monoclonal nNOS antibody and RT-PCR respectively in the brains of rats following portacaval shunting or sham operation. Portacaval shunting resulted in a 2-fold increase (P < 0.01) in nNOS protein and a concomitant 2.4-fold increase (P < 0.01) in nNOS mRNA. Increased nNOS activity in brain and the resulting increase in nitric oxide production could contribute to the increased cerebral blood flow and to the pathogenesis of hepatic encephalopathy in chronic liver disease.

Animals↗

PubMed1997

Portacaval shunting and hyperammonemia stimulate the uptake of L-[3H] arginine but not of L-[3H]nitroarginine into rat brain synaptosomes.

Elevated activities of nitric oxide synthase (NOS) have been reported previously in the brains of portacaval-shunted (PCS) rats, a model of chronic hepatic encephalopathy (HE). As L-arginine availability for nitric oxide synthesis depends on a specific uptake mechanism in neurons, we studied the kinetics of L-[3H]-arginine uptake into synaptosomes prepared from the brains of PCS rats. Results demonstrate that L-arginine uptake is significantly increased in cerebellum (60%; p < 0.01), cerebral cortex (42%; p < 0.01), hippocampus (56%; p < 0.01), and striatum (51%; p < 0.01) of PCS rats compared with sham-operated controls. Hyperammonemia in the absence of portacaval shunting also stimulated the transport of L-[3H]arginine; kinetic analysis revealed that the elevated uptake was due to increased uptake capacity (Vmax) without any change in affinity (Km). Incubation of cerebellar synaptosomes with ammonium acetate for 10 min caused a dose-dependent stimulation of L-[3H]arginine uptake. Neither portacaval shunting nor hyperammonemia had any significant effect on the synaptosomal uptake of NG-nitro-L-[3H]arginine. These studies demonstrate that increased NOS activity observed in experimental HE may result from increased availability of L-arginine resulting from a direct stimulatory effect of ammonia on L-arginine transport.

Acetates↗

PubMed1997

Effects of ifenprodil, a polyamine site NMDA receptor antagonist, on reperfusion injury after transient focal cerebral ischemia.

Polyamines and N-methyl-D-aspartate (NMDA) receptors are both thought to play an important role in secondary neuronal injury after cerebral ischemia. Ifenprodil, known as a noncompetitive inhibitor of polyamine sites at the NMDA receptor, was studied after transient focal cerebral ischemia occurred. Spontaneously hypertensive male rats, each weighing between 250 and 350 g, underwent 3 hours of tandem middle cerebral artery (MCA) and common carotid artery occlusion followed by reperfusion for a period of 3 hours or 21 hours. Intravenous ifenprodil (10 microg/kg/minute) or saline infusion was started immediately after the onset of MCA occlusion and continued throughout the ischemic period. Physiological parameters including blood pressure, blood gas levels, blood glucose, hemoglobin, and rectal and temporal muscle temperatures were monitored. Six rats from each group were evaluated at 6 hours postocclusion for brain water content, an indicator of brain edema, and Evans blue dye extravasation for blood-brain barrier breakdown. Infarct volume was also measured in six rats from each group at 6 and 24 hours postocclusion. Ifenprodil treatment significantly reduced brain edema (82.5 +/- 0.4% vs. 83.5 +/- 0.4%, p < 0.05) and infarct volume (132 +/- 14 mm3 vs. 168 +/- 25 mm3, p < 0.05) compared with saline treatment, with no alterations in temporal muscle (brain) or rectal (body) temperature (35.9 +/- 0.4 degrees C vs. 36.2 +/- 0.2 degrees C; 37.7 +/- 0.4 degrees C vs. 37.6 +/- 0.6 degrees C; not significant). These results demonstrate that ifenprodil has neuroprotective properties after ischemia/reperfusion injury in the absence of hypothermia. This indicates that antagonists selective for the polyamine site of the NMDA receptors may be a viable treatment option and helps to explain some of the pathophysiological mechanisms involved in secondary injury after transient focal cerebral ischemia has occurred.

Adrenergic alpha-Antagonists↗

PubMed1996

Vesicular dysfunction during experimental thiamine deficiency is indicated by alterations in dopamine metabolism.

Experimental and clinical studies indicate that catecholamines play an important role in the neurobehavioural symptomatology of thiamine deficiency. Given the cerebral region-selective vulnerability and the behavioural impairment commonly encountered in thiamine deficiency, we undertook to investigate regional catecholamine metabolism in the brains of pyrithiamine-induced thiamine-deficient rats. Dopamine metabolism was unaffected in the striatum. In contrast, other regions also known to be involved in sensory processing and intellectual function (e.g., frontal cortex, hypothalamus, thalamus), but having a greater noradrenergic input, had increased levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and decreased levels of other dopaminergic metabolites including noradrenaline. In these regions levels of the vesicular amine transporter, defined by tetrabenazine-sensitive [3H]ketanserin binding, were also decreased. Our data suggest a region-selective vesicular dysfunction resulting in intraneuronal release, and subsequent degradation, of dopamine. These disruptions of dopamine and consequently noradrenaline metabolism may account for certain neurobehavioural deficits commonly encountered in thiamine deficiency.

Adrenergic Uptake Inhibitors↗

PubMed1996

Nitric oxide synthase activities are selectively decreased in vulnerable brain regions in thiamine deficiency.

Pyrithiamine-induced thiamine deficiency in the rat exhibits many neuropathological and biochemical similarities to Wernicke's Encephalopathy in human. Activities of constitutive nitric oxide synthase (NOS) were measured in vulnerable (thalamus and cerebellum) and non-vulnerable (hippocampus and striatum) brain regions of pyrithiamine-induced thiamine-deficient rats. NOS activities were significantly decreased in the thalamus (by 26%, P < 0.05) of presymptomatic thiamine-deficient rats compared to pair-fed controls. Following onset of symptoms, in addition to thalamus (-38%, P < 0.01), cerebellum (-50%, P < 0.01) also manifested significantly decreased activities of NOS. Hippocampal and striatal activities of NOS were unchanged at both presymptomatic and symptomatic stages of thiamine deficiency. Selectively decreased activities of neuronal NOS in the thalamus and the cerebellum extends the previous observations of region-selective metabolic changes and, ultimately, neuronal cell loss observed in thiamine deficiency.

Animals↗

PubMed1996

Na+,K(+)-ATPase activity is selectively increased in thalamus in thiamine deficiency prior to the appearance of neurological symptoms.

The relationship between progression of neurological status and the activities of both Na+,K(+)- and Mg(2+)-dependent-ATPase (adenosine 5'-triphosphate phosphohydrolase) was investigated in brain regions of pyrithiamine-induced thiamine deficient rats. Thalamic Na+,K(+)-ATPase activity was selectively increased by 200% (P < 0.01) prior to the appearance of symptoms of thiamine deficiency and normalized in symptomatic rats. This selective transitory activation precludes a mediation by brain soluble fraction Na+,K(+)-ATPase modifiers as does the unaltered distribution in regional high-affinity [3H]ouabain binding densities observed throughout the time-course used in these experiments. Na+,K(+)-ATPase maintains cellular ionic gradients and has been implicated in neurotransmitter uptake and release mechanisms. The fact that the increased thalamic Na+,K(+)-ATPase activity coincides with the early alterations in serotonin metabolism observed in similarly treated animals and the concomitantly early increase in glucose utilization previously observed in the thalamus of thiamine-deficient rats is discussed.

Animals↗

PubMed1996

Regional distribution of binding sites for the nitric oxide synthase inhibitor L-[3H]nitroarginine in rat brain.

The regional distribution of NG-nitro-L-[(3)H]arginine (L-[(3)H]NOARG) binding to different regions of rat brain was studied by quantitative autoradiography. These studies revealed highest density of binding sites in cerebellum, anterior olfactory nucleus, islands of Calleja and substantia nigra with appreciable binding site densities in inferior colliculus, superior colliculus, olfactory tubercle and dorsal tegmental nucleus. The regional distribution of L-[(3)H]NOARG binding, is in good agreement with the distribution of nitric oxide synthase studied previously by NADPH-diaphorase staining and immunohistochemistry using antibodies against neuronal nitric oxide synthase. The kinetics of L-[(3)H]NOARG binding to the cytosolic preparations of cerebral cortex, cerebellum, hippocampus and striatum was studied using an in vitro binding technique. Specific L-[(3)H]NOARG binding was of nanomolar affinity, saturable, and best fit to a single-site model in all four brain regions. These studies support the potential use of L-[(3)H]NOARG binding as a tool for further elucidation of the regional distribution and functional properties of NOS in the central nervous system.

Animals↗

PubMed1996

Kinetics, pharmacology, and autoradiographic distribution of L-[3H]nitroarginine binding sites in rat cerebellum.

The kinetics and pharmacology of NG-nitro-L-[2,3,4,5-3H]arginine (L-[3H]NOARG) binding to rat cerebellum were investigated using in vitro radioligand binding. Specific L-[3H]NOARG binding in cerebellum was of nanomolar affinity, reversible, saturable, and best fit to a single-site model. Specific binding was Ca2+ dependent and sensitive to pH (with an optimum of 5.5-7.0). Added calmodulin (1.5-40 micrograms/ml) had no influence on specific L-[3H]NOARG binding. However, the calmodulin antagonists W-5, W-13, and calmidazolium inhibited L-[3H]-NOARG binding with IC50 values in the micromolar range, and calmodulin (10 micrograms/ml) competitively reversed this inhibition. Nitric oxide synthase (NOS) inhibitors (NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine acetate) and L-arginine displaced L-[3H]NOARG binding with IC50 values in the nanomolar range, whereas D-arginine and basic amino acids (L-lysine and L-histidine) displaced L-[3H]NOARG binding with IC50 values in the millimolar range. A comparison of the NOS functional assay with L-[3H]NOARG binding in rat cerebellum showed similar profiles of Ca2+ dependency and inhibitory kinetics. Quantitative autoradiographic distribution of L-[3H]NOARG binding sites was significantly higher in the molecular layer than in the granular layer of cerebellum. These studies confirm the potential use of L-[3H]NOARG binding to study the regional distribution and functional properties of NOS.

Animals↗

PubMed1996

Alterations in serotonin parameters in brain of thiamine-deficient rats are evident prior to the appearance of neurological symptoms.

Biochemical alterations of serotoninergic parameters have been demonstrated in experimental thiamine deficiency. In addition, hypophagia and hypothermia, two physiological processes associated with changes in the serotonin [5-hydroxytryptamine (5-HT)] system, are manifest early during the progression of thiamine deficiency. The binding of selected 5-HT radioligands was therefore investigated in discrete brain regions of pyrithiamine-induced thiamine-deficient rats. Using quantitative receptor autoradiography, the binding of 8-hydroxy-2-(di-n-[3H]propylamino) tetralin, a ligand used to label the somatodendritic 5-HT1A autoreceptor of the dorsal raphe nucleus, was found to be unaffected in this region, suggesting that the structural integrity of the 5-HT cell bodies is maintained throughout the course of pyrithiamine treatment. Increased binding of [3H]-ketanserin was observed in regions considered vulnerable as well as in some considered to be nonvulnerable during the course of thiamine deficiency. These binding changes, which appear to represent changes in the density of the postsynaptic 5-HT2A receptor population rather than the "tetrabenazine-sensitive" vesicular monoamine transporter, are evident before the appearance of histopathologic lesions and coincide with altered tissue concentrations of 5-HT. These data suggest that 5-HT neurons, although structurally intact, are functionally affected early during the progression of thiamine deficiency. These alterations, which are likely a part of adaptive neuronal change consequent to thiamine dysfunction, may be important in the physiological manifestations and the learning deficits commonly encountered in experimental thiamine deficiency.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1996

L-[3H]nitroarginine and L-[3H]arginine uptake into rat cerebellar synaptosomes: kinetics and pharmacology.

Characteristics of the transport of the nitric oxide synthase substrate L-arginine and its inhibitor, NG-nitro-L-arginine (L-NOARG), into rat cerebellar synaptosomes were studied. Uptake of both L-arginine and L-NOARG was linear with increasing amount of protein (up to 40 micrograms) and time of incubation (up to 5 min) at 37 degrees C. Uptake of both compounds reached a steady state by 20 min. Maximal uptake of L-NOARG (650 pmol/mg of protein) was three to four times higher than that of L-arginine (170 pmol/mg of protein). L-NOARG uptake showed biphasic kinetics (Km1 = 0.72 mM, Vmax 1 = 0.98 nmol/min/mg of protein; Km2 = 2.57 mM, Vmax2 = 16.25 nmol/min/mg of protein). L-Arginine uptake was monophasic with a Km of 106 microM and a Vmax of 0.33 nmol/min/mg of protein. L-NOARG uptake was selectively inhibited by L-NOARG, NG-nitro-L-arginine methyl ester, and branched-chain and aromatic amino acids. L-Alanine and L-serine also inhibited L-NOARG uptake but with less potency. Uptake of L-arginine was selectively inhibited by NG-monomethyl-L-arginine acetate and basic amino acids. These studies suggest that in rat cerebellar synaptosomes, L-NOARG is transported by the neutral amino acid carrier systems T and L with high affinity, whereas L-arginine is transported by the basic amino acid carrier system y+ with high affinity. These data indicate that the concentration of competing amino acids is an important factor in determining the rates of uptake of L-NOARG and L-arginine into synaptosomes and, in this way, may control the activity of nitric oxide synthase.

Amino Acids↗

PubMed1995

A quantitative autoradiographic study of muscarinic cholinergic receptor subtypes in the brains of pyrithiamine-treated rats.

Previous studies describe decreased acetylcholine synthesis in brain as well as neurobehavioral evidence for a central muscarinic cholinergic deficit in pyrithiamine-induced thiamine-deficient rats. In order to further evaluate this possibility, quantitative autoradiographic procedures using [3H]quinuclidinyl benzilate (for total muscarinic binding sites), [3H]pirenzepine (for muscarinic M1 sites) and [3H]AF-DX 384 (for muscarinic M2 sites) were performed at early (presymptomatic) and late (symptomatic) stages of thiamine deficiency induced in rats by administration of the central thiamine antagonist, pyrithiamine. No significant alterations in densities of M1, M2 or total muscarinic binding sites were observed in any brain structure evaluated at either early or late stages of thiamine deficiency. These findings do not support a major role for modifications of muscarinic cholinergic function in the pathogenesis of the neurological symptoms of thiamine deficiency.

Animals↗

PubMed1995

Increased nitric oxide synthase activities and L-[3H]arginine uptake in brain following portacaval anastomosis.

Glutamatergic synaptic dysfunction has been proposed as a causal factor in portal-systemic encephalopathy. Increased in vitro and in vivo glutamate release and decreased glutamate binding to NMDA receptors were previously reported in the brains of portacaval-shunted rats. Such changes could lead to alterations in the second messenger systems coupled to glutamate receptors. As NMDA receptors have been shown to act via the nitric oxide/cyclic GMP second messenger system, we studied the activities of constitutive nitric oxide synthase (NOS) in the brains of rats following portacaval shunting. Results demonstrate that NOS activities are significantly increased in cerebellum (by 54%, p < 0.01), cerebral cortex (by 65%, p < 0.01), hippocampus (by 88%, p < 0.01), and striatum (by 64%, p < 0.01) of shunted rats compared with sham-operated controls. As L-arginine transport is a prerequisite for nitric oxide production, we also studied L-[3H]arginine transport into cerebellar and cerebral cortical synaptosomes prepared from the brains of portacaval-shunted and sham-operated rats. L-[3H]Arginine uptake was significantly increased (by approximately 50%, p < 0.01) in both cerebellum and cortex. Increased NOS activities of neuronal and/or astrocytic origin and the resultant increased production of nitric oxide in brain could be the consequence of increased NMDA receptor activation following portacaval shunting. Furthermore, increased nitric oxide production could contribute to the increased cerebral blood flow consistently observed following portacaval shunting.

Amino Acid Oxidoreductases↗

PubMed1995

Selective alterations of extracellular brain amino acids in relation to function in experimental portal-systemic encephalopathy: results of an in vivo microdialysis study.

Portal-systemic encephalopathy (PSE) is characterized by neuropsychiatric symptoms progressing through stupor and coma. Previous studies in human autopsy tissue and in experimental animal models of PSE suggest that alterations in levels of brain amino acids may play a role in the pathogenesis of PSE. To assess this possibility, levels of amino acids were measured using in vivo cerebral microdialysis in frontal cortex of portacaval-shunted rats administered ammonium acetate (3.85 mmol/kg, i.p.) to precipitate severe PSE. Sham-operated rats served as controls. Portacaval shunting resulted in significant increases of levels of extracellular glutamine (threefold, p < 0.001), alanine (38%, p < 0.01), aspartate (44%, p < 0.05), phenylalanine (170%, p < 0.001), tyrosine (140%, p < 0.001), tryptophan (63%, p < 0.001), leucine (75%, p < 0.001), and serine (60%, p < 0.001). Administration of ammonium acetate to sham-operated animals led to a significant increase in extracellular glutamine and taurine content, but this response was absent in shunted rats. The lack of taurine release into extracellular fluid following ammonium acetate administration in portacaval-shunted rats could relate to the phenomenon of brain edema in these animals. Ammonium acetate administration resulted in significant increases in the extracellular concentrations of phenylalanine and tyrosine in both sham-operated and portacaval-shunted rats. Severe PSE was not accompanied by significant increases in extracellular fluid concentrations of glutamate, aspartate, GABA, tryptophan, leucine, or serine, suggesting that increased spontaneous release of these amino acids in cerebral cortex is not implicated in the pathogenesis of hepatic coma.

Acetates↗

PubMed1995

Thiamine phosphatases in human brain: regional alterations in patients with alcoholic cirrhosis.

Activities of thiamine monophosphatase (TMPase) and thiamine diphosphatase (TDPase) were measured in homogenates of brain tissue obtained at autopsy from eight alcoholic cirrhotic patients who died in hepatic coma and nine controls matched for age and for postmortem delay interval and free from neurological or psychiatric disorders, hepatic disease, or other conditions of grossly impaired nutritional status. Enzyme activities were measured by standard spectrophotometric techniques. Both TMPase and TDPase were distributed unevenly in brain with highest activities being recorded in temporal cortex. Regional correlations between TMPase and TDPase, however, were poor. TDPase activities in brain tissue from alcoholic cirrhotic patients were significantly increased in 5 of 6 brain regions, by 26 to 153% (p < 0.05). TMPase activities in alcoholic cirrhotics, on the other hand, were unchanged in all brain regions, with the exception of caudate nucleus where they were increased by 70% (p < 0.05). These findings add to the substantial body of evidence suggesting that alcoholic liver disease is associated with abnormal thiamine status and with altered thiamine neurochemistry. Increased TDP degradation resulting from increased activities of TDPase could contribute to the pathophysiological mechanisms involved in alcohol-related brain dysfunction.

Acid Anhydride Hydrolases↗

PubMed1994

Alterations of [3H]8-OH-DPAT and [3H]ketanserin binding sites in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy.

Affinities and densities of binding sites for the 5HT1A receptor ligand [3H]8-hydroxy(di-n-propylamino)tetralin ([3H]8-OH-DPAT) and the 5HT2 receptor ligand [3H]ketanserin were measured using a rapid filtration assay in crude membrane preparations from frontal cortex and hippocampus of nine cirrhotic patients who died in hepatic encephalopathy and from an equal number of age-matched subjects free from hepatic, neurological or psychiatric disorders. Binding site densities (Bmax) obtained by Scatchard analysis of saturation binding isotherms for [3H]8-OH-DPAT were decreased in frontal cortex (by 56%, P < 0.05) and hippocampus (by 30%, P < 0.05). [3H]ketanserin binding sites were concomitantly increased (by 55%, P < 0.05) in hippocampus of cirrhotic patients. Ligand binding affinities were within normal ranges in all cases. Previous reports have described the accumulation of the 5HT metabolite 5-hydroxyindoleacetic acid and increased activities of the 5HT-metabolizing enzyme MAOA in this same material from patients with hepatic encephalopathy. Taken together, these findings suggest that alterations of serotoninergic function in brain could be responsible for some of the neuropsychiatric symptoms of hepatic encephalopathy observed in humans with chronic liver disease.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1994

Activities of monoamine oxidase-A and -B are altered in the brains of congenitally hyperammonemic sparse-fur (spf) mice.

Activities of monoamine oxidases, MAOA and MAOB, were measured using radiometric assays in different brain regions of the sparse-fur (spf/Y) mouse, a model of congenital hyperammonemia resulting from an X-chromosomal defect of ornithine transcarbamylase. MAOA activities were decreased in cerebellum (by 23%, P < 0.05) and brainstem (by 16%, P < 0.05) of spf mice; activities of MAOB were concomitantly increased in cerebellum (by 22%, P < 0.05), brainstem (by 20%, P < 0.05) and cerebral cortex (by 22%, P < 0.05). These findings offer a rational explanation for previous findings of increased acidic metabolites of monoamines in the brain of spf mice. Altered monoaminergic function could be a key factor in the pathogenesis of neurological dysfunction in congenital hyperammonemias.

Ammonia↗

PubMed1994

Tissue-specific alterations of binding sites for peripheral-type benzodiazepine receptor ligand [3H]PK11195 in rats following portacaval anastomosis.

Kinetics of binding of [3H]PK11195, an antagonist ligand with high selectivity for the peripheral-type (mitochondrial) benzodiazepine receptor (PTBR), was studied in homogenates of cerebral cortex, kidney, heart, and testis of portacaval shunted rats and sham-operated controls. Portacaval anastomosis resulted in a significant two- to threefold increase in the number of [3H]PK11195 binding sites in cerebral cortex and kidney. A reduction in the number of [3H]PK11195 binding sites was observed in testis preparations, while the number of binding sites in the heart remained unaltered. These differences in the response of PTBRs to portacaval anastomosis, in different organs suggest that the physiological function of these receptors and the factors regulating them are modulated by distinct mechanisms. The finding of increased densities of [3H]PK11195 binding sites in brain and kidney following portacaval anastomosis parallels the cellular hypertrophy in these tissues and, together with previous observations of similar increases of these binding sites in brain and kidney in congenital hyperammonemia, suggest a pathophysiologic role for ammonia in these changes. In contrast, the significant loss of [3H]PK11195 binding sites in testicular preparations following portacaval anastomosis together with the known effects of steroid hormones on these sites suggests a role for PTBRs in the pathogenesis of testicular atrophy in chronic liver disease.

Animals↗

PubMed1994

Choline acetyltransferase and acetylcholinesterase activities are unchanged in brain in human and experimental portal-systemic encephalopathy.

Activities of choline acetyltransferase, acetylcholinesterase and butyrylcholinesterase were studied in the frontal cortex, temporal cortex, cerebellum and caudate nucleus obtained at autopsy from eight alcoholic cirrhotic patients who died in hepatic coma and from an equal number of age-matched subjects free from hepatic, neurological or psychiatric disorders. Activities of these enzymes were unaltered in the brains of cirrhotics compared to controls. Choline acetyltransferase and cholinesterase activities were also studied in the cerebral cortex, cerebellum, brain stem and striatum of rats four weeks following portacaval anastomosis and their sham-operated controls. Portacaval-shunting did not cause any statistically significant differences in the activities of choline acetyltransferase, acetyl or butyrylcholinesterases. These results argue against a presynaptic cholinergic lesion in human and experimental portal-systemic encephalopathy.

Acetylcholinesterase↗