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Biomedical subjects

S R Vincent

Publications and source records attributed to S R Vincent.

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

Autoradiographic localization of [3H]-cyclic GMP binding sites in the rat brain.

Both the atriopeptides and nitric oxide act in the nervous system by activating guanylyl cyclases to stimulate the production of cyclic GMP. Thus a key to understanding the roles of these messengers is to understand the functions of cyclic GMP in the nervous system. Three potential targets for cyclic GMP have been identified, phosphodiesterases, protein kinases and ion channels. In this study we describe a method using autoradiography to localize specific [3H]-cGMP binding sites in the brain. The specific binding of [3H]-cGMP to rat brain sections was saturable (Bmax = 1.5 pmol/mg protein) and of high affinity (KD = 164 nM). The pharmacological characteristics were consistent with binding to the cGMP-dependent protein kinase. Highest densities of binding were seen in the medial habenula, basal ganglia, locus ceruleus and nucleus of the solitary tract. The CA1 pyramidal cells of the hippocampus, the neocortex, thalamus and cerebellum were also labelled. This method should prove useful in studies of potential targets for cyclic GMP in the brain.

Animals↗
PubMed1995

Effects of methylene blue and LY83583 on neuronal nitric oxide synthase and NADPH-diaphorase.

Methylene blue and 6-anilino-5,8-quinolinedione (LY83583) have often been used as 'selective' inhibitors of soluble guanylyl cyclase. We report that in in vitro assays, both these compounds were potent inhibitors of rat cerebellar nitric oxide synthase activity. Methylene blue had an apparent Ki of 2.7 microM, while for LY83583 the Ki was 15.8 microM. Furthermore, methylene blue, but not LY83583, inhibited the NADPH-diaphorase histochemical reaction associated with nitric oxide synthase. Our results indicate that many of the effects of these drugs which have been attributed to inhibition of guanylyl cyclase, may derive from their direct inhibition of nitric oxide synthase activity instead.

Aminoquinolines↗
PubMed1995

Expression of the olfactory cyclic nucleotide gated channel (CNG1) in the rat brain.

The expression of the olfactory cyclic nucleotide-gated channel (CNG1) was studied in the rat brain. Using RT-PCR, levels of CNG1 mRNA were determined relative to the expression of a constantly expressed gene, alpha-tubulin. RT-PCR showed that CNG1 mRNA was detectable in the pituitary gland, the olfactory bulb, and the cerebellum of adult and 5-day-old rats. A 3.4 kb mRNA was detected in the olfactory bulb by Northern blotting. In situ hybridization analysis showed that CNG1 mRNA expression is present in the olfactory bulb and in the Purkinje cells of the cerebellum. RT-PCR studies on Purkinje cell-enriched cultures obtained from the cerebellum of 16-day-old embryos (E16) confirmed the expression of CNG1 mRNA in these neurones. Our results show that CNG1 is not restricted to the olfactory epithelium but is also present in specific regions of the brain. These results suggest that cyclic nucleotides may act in the regions that possess CNG1 gene expression to affect the electrical activity of certain neurones directly.

Animals↗
PubMed1995

Molecular characterization of a type II cyclic GMP-dependent protein kinase expressed in the rat brain.

We applied reverse transcription-PCR to examine the gene expression of cyclic GMP (cGMP)-dependent protein kinase in the rat brain. A PCR product with the size predicted from the type II cGMP-dependent protein kinase (cGK II) cDNA was detected in various regions of the brain, with highest expression in the thalamus. The amplified product of this cDNA was subcloned, sequenced, and consequently shown to be cGK II. Northern analysis confirmed that this kinase was highly expressed in the thalamus. In situ hybridization with riboprobes derived from this cDNA indicated that cGK II mRNA was highly expressed in the outer layers of the cortex, the septum, amygdala, and olfactory bulb with highest levels in the thalamus. High amounts of cGK II mRNA were also found in specific brainstem loci, including the medial habenula, the subthalamic nucleus, the locus ceruleus, the pontine nucleus, the inferior olivary nuclei, and the nucleus of the solitary tract. Only low levels of cGK II mRNA were detected in the striatum, cerebellum, and hippocampus. These data suggest that the effects of guanylyl cyclase activators, such as nitric oxide and the atriopeptides, in various regions of the CNS may be mediated through cGK II.

Animals↗
PubMed1994

Localization and functional properties of a rat brain alpha 1A calcium channel reflect similarities to neuronal Q- and P-type channels.

Functional expression of the rat brain alpha 1A Ca channel was obtained by nuclear injection of an expression plasmid into Xenopus oocytes. The alpha 1A Ca current activated quickly, inactivated slowly, and showed a voltage dependence typical of high voltage-activated Ca channels. The alpha 1A current was partially blocked (approximately 23%) by omega-agatoxin IVA (200 nM) and substantially blocked by omega-conotoxin MVIIC (5 microM blocked approximately 70%). Bay K 8644 (10 microM) or omega-conotoxin GVIA (1 microM) had no significant effect on the alpha 1A current. Coexpression with rat brain Ca channel beta subunits increased the alpha 1A whole-cell current and shifted the current-voltage relation to more negative values. While the beta 1b and beta 3 subunits caused a significant acceleration of the alpha 1A inactivation kinetics, the beta 2a subunit dramatically slowed the inactivation of the alpha 1A current to that seen typically for P-type Ca currents. In situ localization with antisense deoxyoligonucleotide and RNA probes showed that alpha 1A was widely distributed throughout the rat central nervous system, with moderate to high levels in the olfactory bulb, in the cerebral cortex, and in the CA fields and dentate gyrus of the hippocampus. In the cerebellum, prominent alpha 1A expression was detected in Purkinje cells with some labeling also in granule cells. Overall, the results show that alpha 1A channels are widely expressed and share some properties with both Q- and P-type channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

D2 dopamine receptor mRNA distribution in cholinergic and somatostatinergic cells of the rat caudate-putamen and nucleus accumbens.

An in situ hybridization procedure that identifies cells expressing D2 dopamine receptor mRNA was combined in double-labelling studies with immunohistochemical procedures that identify cells expressing either choline acetyltransferase (ChAT) or somatostatin. D2 receptor mRNA was detected in almost all of the ChAT positive caudate-putamen cells, approximately half of the ChAT positive nucleus accumbens cells and none of the somatostatin-positive cells in either brain region.

Acetylcholine↗
PubMed1994

Characterization and localization of [3H] cyclosporin A binding sites in rat brain.

The immunosuppressant drug [3H]cyclosporin A binds specifically and with high affinity to rat brain membrane preparations. The highest density of binding sites was observed in the hippocampus, cerebellum, cortex and basal ganglia. A similar distribution pattern was seen using a quantitative autoradiographic analysis. This distribution agrees with the localizations of cyclophilin and calcineurin reported in immunohistochemical and in situ hybridization studies. Thus inhibition of calcineurin activity following cyclosporin A binding to cyclophilin may occur in neurones, as it does in T-cells. These results suggest that the neurological side-effects of cyclosporin A may be mediated through its interaction with these proteins in neurones.

Amino Acid Isomerases↗
PubMed1994

Inhibition of nitric oxide synthase by antineoplastic anthracyclines.

Nitric oxide synthase, the enzyme responsible for the synthesis of nitric oxide and citrulline from arginine, was potently inhibited by the anthracycline antibiotics doxorubicin (Ki = 24 microM) and aclarubicin (Ki = 50 microM). These drugs were non-competitive inhibitors with respect to arginine. This action on nitric oxide synthase may explain some of the cardiovascular and cytotoxic actions of these chemotherapeutic drugs.

Aclarubicin↗
PubMed1994

Metalloporphyrins inhibit nitric oxide-dependent cGMP formation in vivo.

Sodium nitroprusside produced a dose-dependent increase in extracellular levels of cGMP in the cerebellar cortex in vivo. This was independent of nitric oxide synthase activity. The metalloporphyrins zinc-protoporphyrin-IX, tin-protoporphyrin-IX and zinc-deuteroporphyrin-IX,2,4-bis glycol prevented the increase in cGMP in the cerebellar cortex produced by sodium nitroprusside. At high doses, tin-protoporphyrin-IX also decreased the basal extracellular levels of cGMP. These drugs had no effect on nitric oxide synthase activity. We conclude that the neuropharmacological effects of metalloporphyrins may result from their direct inhibition of soluble guanylyl cyclase, rather than from an effect on carbon monoxide synthesis.

Amino Acid Oxidoreductases↗
PubMed1994

NMDA-dependent nitric oxide release in the hippocampus in vivo: interactions with noradrenaline.

Nitric oxide appears to act as a novel intercellular messenger activating soluble guanylyl cyclase to cause an increase in cGMP in target cells. In the nervous system, NMDA receptor activation has often been shown to result in activation of NO synthase, and many of the actions of NMDA are thought to be mediated by NO. We have recently combined intracerebral microdialysis with a sensitive assay for the NO oxidation products nitrite and nitrate, to assess NO release directly in awake, freely-moving animals. In the present study, we have applied this method to the hippocampus, where we have found that local NMDA application increases NO release. This was prevented by prior administration of an NMDA receptor antagonist or a nitric oxide synthase inhibitor. These pretreatments also reduced the basal extracellular nitrite and nitrate levels, suggesting that there may be a tonic glutamate-induced NO production in the hippocampus in awake, freely-moving animals. Previous work on NMDA-induced increases in cGMP suggested a possible role for noradrenaline. We found that pretreatment with the alpha 1A antagonists WB4101 or 5-methyl-urapidil, reduced the basal levels of NO oxidation products, however, NMDA still induced an increase in extracellular NO in the presence of these alpha 1A antagonists. In contrast, prior lesion of the central noradrenergic system with DSP4 prevented the increase in hippocampal NO release seen following local NMDA application. These results indicate that in vivo microdialysis can be used to monitor NO release in the hippocampus in response to NMDA receptor activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1994

Brain heme oxygenase isoenzymes and nitric oxide synthase are co-localized in select neurons.

Two isoforms of the enzyme heme oxygenase are expressed in distinct populations of neurons in the brain. These enzymes catalyse the oxidative cleavage of heme to the cellular antioxidant biliverdin resulting in the release of carbon monoxide in the process. Both heme and carbon monoxide may play important roles in regulating the nitric oxide-cyclic guanosine monophosphate signal transduction system. Thus we have examined the distributions of both isoforms of heme oxygenase in the rat brain, and compared their localizations with that of nitric oxide synthase determined with the NADPH-diaphorase histochemical technique. Heme oxygenase-1 is highly expressed in a few select populations of neurons including cells in the hilus of the dentate gyrus, in the hypothalamus, cerebellum and brainstem. This enzyme appears to be coexpressed with nitric oxide synthase only in a few cells in the dentate gyrus. Heme oxygenase-2 is much more widely expressed. It is present in mitral cells in the olfactory bulb, pyramidal cells in the cortex and hippocampus, granule cells in the dentate gyrus, many neurons in the thalamus, hypothalamus, cerebellum and caudal brainstem. However, only some of these labelled neurons also displayed nitric oxide synthase. Instead, many neurons expressing heme oxygenase-2 correspond to those known to express high levels of the hemoprotein soluble guanylyl cyclase. These results suggest that heme oxygenase may play a role in modulating guanylyl cyclase independent of nitric oxide synthase. This may result from regulation of intracellular heme and carbon monoxide levels by the heme oxygenase system.

Amino Acid Oxidoreductases↗
PubMed1994

Projections of nitric oxide synthase-containing fibers from the sphenopalatine ganglion to cerebral arteries in the rat.

The origin and distribution of cerebral perivascular nerves containing nitric oxide, a short-acting messenger or neurotransmitter, have been studied in the rat by histochemistry for reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity, a specific marker for neuronal nitric oxide synthase. Positively stained nerve fibers were distributed throughout the major vessels of the cerebral arteries, though the fiber density was higher in the anterior circulation, including the circle of Willis, than in the posterior arteries. Examination using axonal transport methods indicated that nitric oxide-containing neurons in the sphenopalatine ganglion innervate the cerebral arteries bilaterally. Nitric oxide synthase in these ganglionic cells often co-existed with vasoactive intestinal polypeptide. The anatomical information obtained is discussed in terms of non-adrenergic, non-cholinergic neuronal transmission in the cerebral arteries.

Acetylcholine↗
PubMed1994

Nitric oxide-dependent efflux of cGMP in rat cerebellar cortex: an in vivo microdialysis study.

The stimulation of excitatory amino acid receptors in the cerebellar cortex results in the Ca2+/calmodulin-dependent activation of nitric oxide synthase. This leads to an increase in tissue levels of cGMP following the interaction of nitric oxide with soluble guanylyl cyclase. The cerebellar cortex has the highest levels of nitric oxide synthase and cGMP in the brain; however, the levels of guanylyl cyclase and cGMP-phosphodiesterase are remarkably low. Thus, the mechanisms regulating cGMP levels in cerebellar cells are unclear. One report has noted that cGMP can be released from cerebellar slices. We have therefore used intracerebellar microdialysis in awake, freely moving rats to test the hypothesis that activation of nitric oxide synthase in the cerebellar cortex results in the release of cGMP. Climbing fibers, which release excitatory amino acids in the cerebellum, were activated with systemic harmaline. This resulted in an immediate increase in extracellular cGMP, which was blocked by TTX or the removal of extracellular Ca2+, and attenuated by prior lesion of the climbing fibers. Blockade of N-type calcium channels with omega-conotoxin also antagonized the harmaline-induced increase. In contrast, blockade of L-type calcium channels, or inhibition of anion transport with probenecid or bromosulfophthalein, potentiated the increase in cGMP seen in response to harmaline. Inhibitors of nitric oxide synthase or guanylyl cyclase prevented the harmaline-induced increase in extracellular cGMP, while phosphodiesterase inhibitors potentiated the increase. Local application of the NMDA antagonist 2-amino-5-phosphonopentanoic acid or the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione attenuated the effect of harmaline.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1993

Structure and functional expression of a member of the low voltage-activated calcium channel family.

Oscillatory firing patterns are an intrinsic property of some neurons and have an important function in information processing. In some cells, low voltage-activated calcium channels have been proposed to underlie a depolarizing potential that regulates bursting. The sequence of a rat brain calcium channel alpha 1 subunit (rbE-II) was deduced. Although it is structurally related to high voltage-activated calcium channels, the rbE-II channel transiently activated at negative membrane potentials, required a strong hyperpolarization to deinactivate, and was highly sensitive to block by nickel. In situ hybridization showed that rbE-II messenger RNA is expressed in regions throughout the central nervous system. The electrophysiological properties of the rbE-II current are consistent with a type of low voltage-activated calcium channel that requires membrane hyperpolarization for maximal activity, which suggests that rbE-II may be involved in the modulation of firing patterns.

Amino Acid Sequence↗
PubMed1993

Altered histamine H3 binding in rat forebrain after reserpine treatment.

Groups of 6 rats were treated for 5 days with either reserpine hydrochloride (5 mg/kg i.p., per diem), or saline. Regional binding of the histamine H3 agonist N alpha-[3H]methyl-histamine ([3H]NAMH) was determined in forebrain sections by quantitative autoradiography and Scatchard analysis. Highest maximal binding was in nucleus accumbens (107 +/- 18 fmol/mg) and corpus striatum (58 +/- 9 fmol/mg), where the apparent affinity was close to 4 nM. Maximal binding of [3H]NAMH in the insular cortex (39 +/- 6 fmol/mg) was higher than in other cortical areas examined. Reserpine treatment produced a 50% decrease in both the Bmax and the apparent Kd in the corpus striatum and nucleus accumbens, but binding parameters in the cortex and septum were unaltered. Therefore, the response of H3 receptors in rat forebrain to reserpine treatment for 5 days was regionally heterogenous such that maximal [3H]NAMH binding was typically higher in insular cortex (36 +/- 6 fmol/mg) than in corpus striatum (24 +/- 3 fmol/mg) of reserpine-treated rats.

Animals↗
PubMed1993

N-methyl-D-aspartate-induced nitric oxide release: an in vivo microdialysis study.

Increasing evidence indicates that nitric oxide acts as an intercellular signal transduction molecule in the nervous system. In particular, in vitro studies have demonstrated that nitric oxide is produced in the cerebellar cortex and is responsible for the increases in cyclic GMP seen in response to glutamate receptor activation. In this study, we have combined the technique of intracerebellar microdialysis with a sensitive assay for nitric oxide oxidation products nitrate and nitrite, to assess nitric oxide release directly in awake, freely moving animals. We have found that infusion of N-methyl-D-aspartate via the microdialysis probe results in a dose-dependent increase in cerebellar nitric oxide release. This increase was prevented by prior administration of an N-methyl-D-aspartate receptor antagonist, or the nitric oxide synthase inhibitor NG-nitroarginine. Both these pretreatments also reduced the basal extracellular nitrite and nitrate levels, suggesting that there is a tonic glutamate-induced nitric oxide production in the cerebellum of awake, freely moving animals. These results provide direct evidence for nitric oxide release in response to N-methyl-D-aspartate receptor activation in the adult cerebellar cortex, in vivo. This new approach, coupling microdialysis with the azo dye detection method of Griess, should thus prove useful for the in vivo study of nitric oxide release from various brain regions in response to pharmacological, physiological or behavioral manipulations.

Amino Acid Oxidoreductases↗
PubMed1993

Histochemical localization of nitric oxide synthase in rat enteric nervous system.

The localization of nitric oxide synthase, the enzyme responsible for producing the short-acting messenger nitric oxide, has been determined in the digestive tract of the rat using histochemistry for reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity, a specific marker for neuronal nitric oxide synthase. Positively stained neurons were found throughout the entire digestive tract from the esophagus to the rectum. Positive neuronal somata were very common in the myenteric ganglia. Dense positive fibers were distributed in internodal strands, the secondary plexus, the tertiary plexus, and were particularly abundant in the deep muscular plexus, while very few were observed in the submucosal ganglia. The density of these positive structures was higher in the small and large intestine than in the esophagus and stomach. The pattern of distribution suggested that some of these positive cells innervate gut muscles. Double-staining revealed that in these enteric neurons, nitric oxide synthase does not co-localize with acetylcholinesterase. Instead, vasoactive intestinal polypeptide almost always coexists with nitric oxide synthase in the myenteric plexus. Thus, nitric oxide and vasoactive intestinal polypeptide may be co-transmitters in a population of non-adrenergic, non-cholinergic neurons in the enteric nervous system.

Acetylcholinesterase↗