V. L. Raghavendra Rao

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.

Increased Nitric Oxide Synthase Activities and l‐[3H]Arginine Uptake in Brain Following Portacaval Anastomosis

Abstract: 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 ∼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.

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

Abstract: 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.

Increased activities of MAOA and MAOB in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

Using radioenzymatic assays, activities of MAOA and MAOB were measured in autopsied brain tissue from cirrhotic patients who died in hepatic coma and in material from an equal number of age-matched subjects who were free from hepatic, neurological or psychiatric disorders. Activities of both MAOA and MAOB were significantly increased in frontal cortex and caudate nucleus, two brain regions shown previously to be the site of functional and morphological alterations of astrocytes and increased concentrations of the acid metabolites of dopamine and serotonin. These findings suggest that increased monoamine metabolism and subsequent modifications of monoaminergic synaptic function could contribute to the pathogenesis of hepatic encephalopathy.

Alterations of thiamine phosphorylation and of thiamine-dependent enzymes in Alzheimer's disease

There is a growing body of evidence to suggest that thiamine neurochemistry is disrupted in Alzheimers Disease (AD). Studies in autopsied brain tissue from neuropathologically proven AD patients reveal significantly reduced activities of the thiamine phosphate dephosphorylating enzymes thiamine diphosphatase (TDPase) and thiamine monophosphatase (TMPase) as well as the thiamine diphosphate-dependent enzymes, pyruvate dehydrogenase complex, α-ketoglutarate dehydrogenase (αKGDH) and transketolase. Reductions in enzyme activities are present both in affected areas of AD brain as well as in relatively well conserved tissue. Decreased TDP concentrations and concomitantly increased TMP in autopsied brain tissue from AD patients and in CSF from patients with Dementia of the Alzheimer Type suggests that CNS thiamine phosphorylation-dephosphorylation mechanisms are disrupted in AD. αKGDH is a rate-limiting enzyme for cerebral glucose utilization and decreases in its activity are associated with lactic acidosis, cerebral energy failure and neuronal cell loss. Deficiencies of TDP-related metabolic processes could therefore participate in neuronal cell death mechanisms in AD.

Portacaval Shunting and Hyperammonemia Stimulate the Uptake of l‐[3H]Arginine but Not of l‐[3H]Nitroarginine into Rat Brain Synaptosomes

Abstract: 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.

Characterization of binding sites for the ω3 receptor ligands [3H]PK11195 and [3H]RO5-4864 in human brain

The kinetics and pharmacology of the isoquinoline and benzodiazepine binding sites of the omega3 or peripheral-type benzodiazepine receptors were studied using the specific ligands [3H] 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin -2-one ([3H]PK11195) and [3H]1-(2-Chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarb oxamide ([3H]RO5-4864), respectively. Binding of both ligands was saturable, reversible, displayed nanomolar affinity, and best fit to a single site model. Occipital cortex and cerebellum displayed highest and lowest densities of binding sites respectively; for both ligands. Bmax values of [3H]PK11195 were several-fold higher than that of [3H]RO5-4864 in all regions studied consistent with their binding to distinct subunits of the human peripheral-type benzodiazepine receptor heteromeric complex. However, the isoquinoline and benzodiazepine ligands were found to be mutually competitive at nanomolar concentrations suggesting allosteric interactions between these two sites. Competition binding experiments showed that the binding of both ligands was displaced by diazepam with Ki values in the nM range, and by clonazepam in the microM range. The novel peripheral-type benzodiazepine receptor ligand 2-(4-fluorophenyl)-N,N-di-n-hexyl-1H-indole-3-acetamide (FGIN1-27) displaced only [3H]PK11195 binding with high potency. Heterogeneity of the two sites is observed, manifested by their differential susceptibility towards detergents and alcohols. Histidine residue modification by diethylpyrocarbonate treatment abolished only [3H]PK11195 binding but had no effect on [3H]RO5-4864 binding. These studies demonstrate that the isoquinoline and benzodiazepine sites on the peripheral-type benzodiazepine receptor in human brain manifest many pharmacological characteristics that are distinct from each other and from rodent brain peripheral-type benzodiazepine receptors.

Increased expression of the peripheral-type benzodiazepine receptor-isoquinoline carboxamide binding protein mRNA in brain following portacaval anastomosis

Using RT-PCR, gene expression of the peripheral-type benzodiazepine receptor isoquinoline carboxamide-binding protein (PTBR-IBP) was studied in the frontal cortex of rats four weeks following end-to-side portacaval anastomosis, an experimental animal model of hepatic encephalopathy, or sham operation. Portacaval anastomosis resulted in increased expression of PTBR-IBP in frontal cortex and in a concomitant increase in densities (Bmax) of binding sites for the PTBR ligand [3H]PK11195. In view of the findings that the PTBR modulates the synthesis of neurosteroids with high affinity for excitatory and inhibitory neurotransmitter systems in brain, increased expression of these receptors could be implicated in the pathogenesis of hepatic encephalopathy.

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.

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 shamoperated 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.