PubMed Health⌕ Search

Biomedical subjects

V Ravindranath

Publications and source records attributed to V Ravindranath.

At least 37 records · Page 2Linked to original sources
PubMed1996

alpha-Lipoic acid protects against reperfusion injury following cerebral ischemia in rats.

Ischemic-reperfusion injury in humans occurs in conditions such as stroke, cardiac arrest, subarachnoid hemorrhage or head trauma. Maximal tissue damage is observed during reperfusion, which is primarily attributed to oxidative injury resulting from production of oxygen free radicals. One of the major consequences of such damage is the depletion of the cellular antioxidant, glutathione (GSH) leading to oxidation of protein thiols to disulfides and the loss of activity of critical enzymes having active thiol group(s). Thus, the maintenance of thiol homeostasis is an important factor in cell survival. The effect of thiol antioxidants like alpha-lipoic acid and the isopropyl ester of GSH was examined on the morbidity and mortality of rats subjected to reperfusion following cerebral ischemia induced by bilateral carotid artery occlusion and hypotension. While the GSH isopropyl ester had no significant protective effect; after pretreatment of rats, alpha-lipoic acid was detected in the rat brain and it dramatically reduced the mortality rate from 78% to 26% during 24 h of reperfusion. The natural thiol antioxidant, alpha-lipoic acid is effective in improving survival and protecting the rat brain against reperfusion injury following cerebral ischemia.

Animals↗
PubMed1996

Cerebral metabolism of imipramine and a purified flavin-containing monooxygenase from human brain.

Flavin-containing monooxygenase (FMO), previously reported both from hepatic and extrahepatic tissues, including brain, catalyze the oxidation of certain xenobiotics and drugs that contain a nucleophilic heteroatom. Psychoactive drugs, including the antidepressant imipramine, are substrates for the brain FMO. Since FMO-mediated metabolism of these drugs might contribute to local pharmacodynamic modulation within the human brain, the metabolism of imipramine by human brain FMO was studied in further detail. In the present study, the FMO activity was determined in human brain microsomes by estimating the actual amount of imipramine N-oxide formed. It was then compared with the corresponding activity measured using substrate (imipramine)-stimulated rates of nicotinamide adenine dinucleotide phosphate (NADPH) oxidation, which was significantly higher than the activity estimated as the amount of N-oxide assayed using high-pressure liquid chromatography (HPLC). The brain FMO activity was measurable only in the presence of detergents (sodium cholate or Lubrol PX) or in microsomes that were freeze-thawed several times. The activity was inhibited by an antibody to rabbit pulmonary FMO, but an antiserum to the rat liver NADPH cytochrome P-450 reductase had no effect indicating that cytochrome P-450 was not involved in the above metabolic pathway. The optimum pH for N-oxidation of imipramine was found to be 8.5; thermolability experiments indicated that the FMO activity was completely lost only after the incubation of brain microsomes at 45 degrees C for 20 minutes. An FMO purified to apparent homogeneity from a human brain had a molecular weight of 71,000 Da. The purified enzyme cross-reacted with the antibody to rabbit pulmonary FMO and efficiently catalyzed the metabolism of imipramine to its N-oxide. The human brain clearly contains an active FMO system, and it is conceivable that such enzymes are significantly involved in the local metabolism and modulation of pharmacological and/or toxic effects of certain xenobiotics, including psychoactive drugs.

Antidepressive Agents, Tricyclic↗
PubMed1995

Brain mitochondrial cytochromes P450: xenobiotic metabolism, presence of multiple forms and their selective inducibility.

The capability of rat brain mitochondria to metabolize a variety of xenobiotics was examined. The presence of cytochrome P450 (P450) and associated monooxygenase activities were estimated in isolated rat brain mitochondria and compared with the corresponding activities in microsomes. Total P450 content in brain mitochondria from naive rats was twice that of the corresponding microsomal level. The ability of brain mitochondria to metabolize the potent carcinogen N-nitrosodimethylamine was more than twofold that of the corresponding microsomal activity, while the 7-ethoxycoumarin-O-deethylase activity was significantly lower in mitochondria. Immunoblot experiments using antisera to purified rat liver microsomal P450s, namely P450 (2B1/2B2), P4501A1, and P4502E1, and purified phenobarbital-inducible rat brain P450, revealed the presence of immunoreactive bands in isolated brain mitochondria. These various antibodies to P450 inhibited the brain mitochondrial monooxygenase activities to significant, though varying extent. The addition of antiserum to microsomal NADPH cytochrome P450 reductase did not affect the mitochondrial P450 associated monooxygenase activities, although it completely inhibited the corresponding microsomal activities. Chronic ethanol administration resulted in twofold induction of total P450 content and the monooxygenase activities known to be mediated by P4502E1, such as N-nitrosodimethylamine-N-demethylase and p-nitrophenol hydroxylase in brain mitochondria. Pretreatment of animals with phenobarbital resulted in the induction of aminopyrine N-demethylase activity in brain mitochondria. The study demonstrates the presence of multiple forms of P450 in the rat brain mitochondria, their inducibility, and their capability to metabolize xenobiotics.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1995

Flavin-containing monooxygenase mediated metabolism of psychoactive drugs by human brain microsomes.

Flavin-containing monooxygenases (FMO) catalyze the oxidation of certain xenobiotics and drugs which contain a nucleophilic heteroatom. Here we report the first assessment of human brain flavin-containing monooxygenase from tissues obtained at autopsy from seven traffic accident victims. Human brain microsomes catalyzed the S-oxidation or N-oxidation of model substrates methimazole and N,N-dimethylaniline, respectively. The psychoactive drugs chlorpromazine, imipramine and fluoxetine, were also metabolized by human brain FMO. 'Western' immunoblot analyses revealed immunological cross-reactivity of the human brain FMO with rabbit pulmonary FMO. Immunocytochemistry further revealed the localization of the FMO predominantly in the neuronal cell bodies in the magnocellular reticular nuclei, colliculi and substantia nigra. Human brain clearly contains an active FMO system, and it is conceivable that such enzyme(s) are significantly involved in the local metabolism and modulation of pharmacological effects of psychoactive drugs.

Adult↗
PubMed1995

Cytochrome P450 and associated monooxygenase activities in the rat and human spinal cord: induction, immunological characterization and immunocytochemical localization.

We have discovered cytochrome P450 and associated monooxygenase activities in microsomes prepared from spinal cord tissues from rats and a human. Cytochrome P450 levels and nicotinamide adenine dinucleotide phosphate cytochrome c reductase activities in microsomes from rat spinal cord were similar to those observed from the whole brain. However, certain monooxygenase activities were significantly lower in the rat spinal cord microsomes as compared to the corresponding activities observed in the whole brain. Cytochrome P450-mediated monooxygenase activities were also detectable in microsomes prepared from human spinal cord. Immunoblot analyses of rat and human spinal cord microsomes using antisera to various forms of hepatic cytochrome P450 namely (2B1 + 2B2), 1A1, 1A2 and 2E1 revealed the presence of immunologically similar forms. The spinal cord microsomes also cross-reacted with the antiserum to the phenobarbital-inducible form of rat brain cytochrome P450. Immunocytochemical stain was predominant in the gray horns of the rat spinal cord. At the cervical level, lamina 1 and 2 representing the substantia gelatinosa were intensely stained. In the ventral horns, lamina 7, 8 and 9 containing the large motor neurons were strongly labelled, while small neurons revealed variable staining. In the white matter, the glial cells were stained but the axons remained non-reactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Xenobiotic metabolism in brain.

Recent hypothesis suggesting a role for environmental toxins in the pathogenesis of neurodegenerative disorders has stimulated interest in research on xenobiotic metabolizing capability of the brain. In addition to possible irreversible loss of neurons through bioactivation in situ in the nervous tissue, the metabolism of psychoactive drugs in the target tissue can lead to local pharmacological modulation at the site of action. The major drug metabolizing enzymes, cytochromes P-450 (P450) and flavin-containing monooxygenase (FMO) have been detected in rodent brain and human brain tissue obtained at autopsy. The brain microsomal and mitochondrial P450 systems are capable of metabolizing a variety of xenobiotics, while the brain FMO efficiently metabolizes a variety of psychoactive drugs to their respective N-oxides. Immunocytochemical studies have revealed the regional heterogeneity in the distribution of multiple forms of P450 in the brain and the co-localization of P450 and FMO predominantly in the neuronal cells. Although the brain P450 and FMO share many common features with similar enzymes present in other tissues such as liver and lung, there are some distinctive differences. It is evident from the studies carried out so far that the brain can metabolize a variety of lipophilic xenobiotics that enter by way of the blood stream.

Animals↗
PubMed1995

Protection and potentiation of 1-methyl-4-phenylpyridinium-induced toxicity by cytochrome P450 inhibitors and inducer may be due to the altered uptake of the toxin.

Earlier studies from our laboratory have demonstrated that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity could be modulated by inhibitors and inducer of cytochrome P450 (P450) in an in vitro model consisting of sagittal slices of mouse brain. To understand the molecular mechanisms underlying the role of P450 on MPTP toxicity, it was undertaken to study the effect of the modulators of P450 on the toxicity of the metabolite of MPTP, namely, 1-methyl-4-phenylpyridinium ion (MPP+). Incubation of mouse brain slices with various concentrations of MPP+ (1-100 microM) resulted in dose-dependent inhibition of mitochondrial enzyme NADH-dehydrogenase (NADH-DH) and leakage of the cytosolic enzyme lactate dehydrogenase from the slice into the medium. MPP(+)-induced toxicity was abolished by pretreatment of the slices with inhibitors of monoamine oxidase (MAO; pargyline and deprenyl) or inhibitors of P450 (piperonyl butoxide or SKF-525A) or dopamine uptake blocker (GBR-12909), as measured by the activity of NADH-DH in slices and leakage of lactate dehydrogenase from the slice into the medium. Slices prepared from mice pretreated with phenobarbital (an inducer of P450) potentiated the toxic effects of MPP+. Pretreatment of slices with MAO-inhibitor, P450 inhibitors, or dopamine uptake blocker attenuated the uptake of MPP+ into the slices. In contrast, MPP+ uptake was significantly increased in slices prepared from phenobarbital-pretreated mice. Thus, both MAO and P450 inhibitors abolish the toxicity of MPP+ in the sagittal slices of mouse brain by altering the uptake of the toxin into the slices.

1-Methyl-4-phenylpyridinium↗
PubMed1995

Glutathione and protein thiol homeostasis in brain during reperfusion after cerebral ischemia.

The status of glutathione (GSH) and protein thiol homeostasis was examined in rat brain regions during reperfusion after moderate and severe cerebral ischemia. GSH levels were decreased in brain regions during reperfusion for 1 hr after moderate or severe ischemia for 0.5 hr. Maximal loss of GSH (50-66%) was observed in the striatum and hippocampus. The GSH lost from the brain regions was essentially recovered as protein-glutathione mixed disulfide (PrSSG) with concomitant loss of protein thiols (PrSH). The activities of enzymes such as Na+K+ ATPase, NADH dehydrogenase and glutathione reductase were also inhibited but were restored after incubation of the brain homogenate with dithiothreitol. The depletion of GSH was also accompanied by an increase in the levels of malondialdehyde and reactive oxygen species. The total GSH recovered as sum of GSH and PrSSG was significantly higher than the sham-operated controls in the hippocampus and striatum after 1 hr of reperfusion, after moderate ischemia for 0.5 hr, and at the end of 24 hr of reperfusion the GSH-protein thiol homeostasis was restored. In contrast after 1 hr of reperfusion after severe ischemia, the GSH recovered as sum of GSH and PrSSG was not significantly different from sham-operated controls and at the end of 24 hr, 7 of 9 animals died. The recuperation of the brain from oxidative stress during reperfusion after moderate ischemia was thus preceded by increased recovery of total GSH essentially in the form of PrSSG. Thus, rapid restoration of thiol homeostasis in the brain during reperfusion may help the brain recover from reperfusion injury.

Animals↗
PubMed1995

Rat brain cytochrome P450. Reassessment of monooxygenase activities and cytochrome P450 levels.

There have been considerable interlaboratory variations in the reported levels of rat brain microsomal cytochrome P450 and associated monooxygenase activities. To ascertain if the variability could be accountable, at least in part, to different methodologies used for microsome preparation, cytochrome P450 monooxygenase components and activities were directly compared herein using brain microsome prepared by various methods. Rat brain microsome isolated using a calcium aggregation method in the presence of dithiothreitol and glycerol contained approximately 100 pmol of cytochrome P450/mg protein. Considerably lower cytochrome P450 levels (e.g. 20-40 pmol/mg protein) were found in brain microsome prepared in a more conventional manner using Tris or phosphate buffers without glycerol and dithiothreitol. The NADPH cytochrome c reductase activity was consistently approximately 23-25 nmol of cytochrome c reduced/min/mg protein, whatever the method of preparation of the brain microsome. Cytochrome P450-associated monooxygenase activities, namely morphine N-demethylase and ethoxycoumarin O-deethylase, were dependent on the amount of protein in the incubation medium, the length of incubation, and the ratio of the concentration of the substrate to the amount of protein in the incubation mixture. The specific activity of morphine N-demethylase was constant over a range of protein concentration, if the ratio of the concentration of the substrate to the protein was kept constant.

Animals↗
PubMed1993

L-BOAA induces selective inhibition of brain mitochondrial enzyme, NADH-dehydrogenase.

Lathyrism, a human neurological disorder has been linked to the excessive consumption of a plant toxin, beta-oxalylamino-L-alanine (L-BOAA) present in Lathyrus sativus. The present study was carried out to elucidate the biochemical mechanisms underlying L-BOAA-induced toxic insult. Incubation of sagittal slices of mouse brain with L-BOAA resulted in dose and time-dependent inhibition of mitochondrial NADH-dehydrogenase (NADH-DH). Significant inhibition of NADH-DH was seen following incubation of brain slices with very low concentration of L-BOAA (0.1 pM). L-BOAA also induced lactate dehydrogenase (LDH) leakage from the slice into the medium in dose-dependent manner. The inhibition of NADH-DH preceded LDH leakage from the slices into the medium. L-BOAA had no effect on other mitochondrial enzymes, namely, isocitrate dehydrogenase or cytochrome c oxidase. Incubation of isolated mouse brain mitochondria with L-BOAA also resulted in inhibition of NADH-DH. L-BOAA-induced inhibition of NADH-DH was prevented by non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonists in general and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist (NBQX) in particular. Other glutamate agonists examined namely, N-methyl-D-aspartate, beta-N-methylamino-L-alanine (L-BMAA), L-glutamic acid, N-acetylaspartylglutamate (NAAG), quisqualic acid, kainic acid or AMPA did not have any effect on NADH-DH activity in slices although they induced LDH leakage from the slice into the medium. Incubation of brain slices with L-BOAA did not induce lipid peroxidation or changes in glutathione levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids, Diamino↗
PubMed1993

Induction of brain cytochrome P-450IIE1 by chronic ethanol treatment.

Cytochrome P-450 mediated metabolism is potentially involved in the expression of the pharmacological and/or toxicological effects of a wide variety of drugs and environmental chemicals upon tissues which contain this metabolic system. In the present investigation, the presence of cytochrome P-450IIE1 and associated mono-oxygenase activities in brain and the effect of chronic ethanol treatment on brain cytochrome P-450 (P-450) were studied. Aniline hydroxylase, N-nitroso-dimethylamine N-demethylase and p-nitrophenol hydroxylase activities (known to be mediated by P-450IIE1) were detectable in brain microsomes from untreated rats and were about 5%, 125% and 8.3%, respectively, of the corresponding hepatic levels. Chronic ethanol treatment resulted in induction of the above enzyme activities in brain microsomes by 243%, 496% and 155%, respectively. Intake of ethanol for a prolonged period also resulted in the induction of total P-450 in the brain (150% of the control). Addition of the antisera raised against rat liver cytochrome P-450IIE1 markedly inhibited brain microsomal p-nitrophenol hydroxylase activity. Immunoblot analysis of rat brain microsomes using the above antisera also revealed the induction of brain cytochrome P-450IIE1 following chronic ethanol administration. Immunocytochemical localization of cytochrome P-450IIE1 using the above antisera, revealed the preferential localization of the enzyme in the neuronal cell bodies in the cortex, hippocampus, basal ganglia, hypothalamic nuclei and reticular nuclei in the brainstem of rats treated chronically with ethanol. Based upon these studies, it is conceivable that chronic alcohol ingestion could enhance the sensitivity of certain regions of the brain to environmental chemicals that are metabolized to more toxic derivatives by the P-450 system.

Animals↗
PubMed1993

Purification of multiple forms of cytochrome P450 from a human brain and reconstitution of catalytic activities.

The present study demonstrates the presence of multiple forms of cytochrome P450 (P450) in human brain obtained at autopsy, the purification of various isoforms to apparent homogeneity, and the monooxygenase activities in reconstituted systems. Sequential chromatography on octylamino-Sepharose 4B, DEAE-Sephacel, and DEAE-cellulose yielded four isoforms of P450 (A, B, C, and D) with specific contents of 11.0, 9.4, 12.5, and 8.3 nmol of P450/mg protein, respectively. While the forms A, B, and C were apparently homogeneous as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the P450D was not homogeneous. The apparent molecular masses of the four forms of P450 were 60,200 Da (P450A), 60,900 Da (P450B), 60,200 Da (P450C), and 61,000 Da (P450D), respectively. NADPH cytochrome P450 reductase (reductase) was also partially purified from the brain microsomes. Immunoblot analysis of the four forms of human purified P450, using antisera to purified rat liver P450 (IIB1 + IIB2), rat liver P450 (1A1 + 1A2), phenobarbital-inducible rat brain P450, human liver P450 IIE1, P450 1A2, P450 IIC, and P450 IIIA4, indicated differential immunological cross-reactivity. The monooxygenase activities of the purified human brain P450s were demonstrated with various substrates (aminopyrine, morphine, aniline, 7-ethoxycoumarin, and nifedipine) as examined in reconstituted systems consisting of purified human brain P450, purified rat brain NADPH cytochrome P450 reductase, deoxycholate, phospholipid, and NADPH.

Brain↗
PubMed1993

Catalytic activity and immunohistochemical localization of flavin-containing monooxygenase in rat kidney.

The presence of flavin-containing monooxygenase activity was examined in rat kidney microsomes using N,N-dimethylaniline and methimazole as substrates. Western immunoblot analysis using antisera to porcine liver and rabbit lung flavin-containing monooxygenase indicated immunological cross-reactivity between rat kidney, porcine liver and rabbit lung flavin-containing monooxygenase. Immunohistochemical studies using antisera to rabbit lung flavin-containing monooxygenase demonstrated localization of this enzyme in the proximal and distal convoluted tubules of the renal cortex, the collecting ducts in the renal medulla, but not the glomeruli. This observation indicates the colocalization of flavin-containing monooxygenase and cytochrome P-450 in the metabolically active and absorptive compartment of the renal parenchyma.

Aniline Compounds↗
PubMed1993

Billion-fold difference in the toxic potencies of two excitatory plant amino acids, L-BOAA and L-BMAA: biochemical and morphological studies using mouse brain slices.

Plant amino acids beta-N-oxalylamino-L-alanine (L-BOAA, present in Lathyrus sativus) and beta-N-methylamino-L-alanine (L-BMAA, present in Cycas circinalis) have been implicated in the pathogenesis of human neurological disorders lathyrism and amyotrophic lateral sclerosis-Parkinson's dementia complex of Guam (ALS-PD), respectively. In view of the conflicting reports that have emerged on the role of L-BMAA in ALS-PD, we reinvestigated the comparative toxicity of L-BMAA and L-BOAA. We report here the potent toxicity of L-BOAA as examined in an in vitro model consisting of sagittal slices of mouse brain. Incubation of sagittal slices of mouse brain with L-BOAA (1 pM) resulted in significant leakage of lactate dehydrogenase (LDH) and potassium from the slices into the medium. Under similar conditions, L-BMAA-induced LDH leakage from the slices into the medium was observed only at very high concentration of the toxin, namely 1 mM. N-Methyl-D-aspartate (NMDA) receptor antagonists ameliorated the toxic effects of L-BMAA, while non-NMDA receptor antagonists (quinoxalinediones) protected against the toxicity of L-BOAA. Incubation of slices with L-BOAA for 1 h resulted in extensive vacuolation and degeneration of neurons in the thalamus and brain stem, and to a lesser extent in the hippocampus and cerebellar nuclei. The large sized neurons appeared to be affected to a greater extent than the smaller ones. The neurons in other areas of the brain also revealed variable degree of degeneration with swelling of axons and dendrites.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1993

Cerebral flavin-containing monooxygenase-mediated metabolism of antidepressants in brain: immunochemical properties and immunocytochemical localization.

Flavin-containing monooxygenase (FMO)-mediated oxidation of the model substrates N,N-dimethylaniline and methimazole, and the antidepressants imipramine and fluoxetine, was determined in rat brain microsomes. No sex-related difference was observed in the activity of the FMO-mediated metabolism of the four substrates examined. The Km values for flavin-containing monooxygenase-mediated metabolism of N,N-dimethylaniline and methimazole were 2.8 and 0.8 mM, respectively, and the Km values for the oxidation of the antidepressants imipramine and fluoxetine were 20.9 and 9.8 microM, respectively. The Vmax values for oxidation of N,N-dimethylaniline, methimazole, imipramine and fluoxetine were 340, 31, 182 and 470 nmol nicotinamide adenine dinucleotide phosphate oxidized/min/mg protein, respectively. Western immunoblot analysis using antisera to purified porcine liver FMO did not reveal any immunological cross-reactivity with male or female brain microsomal protein. Antibody to rabbit lung flavin-containing monooxygenase cross-reacted with brain microsomes as examined by Western immunoblot studies. Addition of the antibody raised against rabbit lung FMO resulted in inhibition (43% inhibition) of the FMO-mediated metabolism of imipramine. Immunocytochemical examination of rat brain sections using the above antibody revealed the preferential localization of flavin-containing monooxygenase in the neuronal cell body. The flavin-containing monooxygenase-mediated metabolism of antidepressant drugs by brain microsomes is of profound pharmacological significance.

Animals↗