l-Arginine ameliorates recirculation and metabolic derangement in brain ischemia in hypertensive rats

The effects of

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-arginine (a precursor of nitric oxide, NO) on cerebral blood flow (CBF), cerebrovascular resistance (CVR) and metabolites in the ischemic brain were examined in spontaneously hypertensive rats with bilateral carotid artery occlusion for 30 min followed by 60 min-recirculation. The administration of

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-arginine (300 mg/kg, i.v.) increased the CBF by an average of 11 ml·100 g⁻¹·min⁻¹ (P<0.05 vs. at rest), and N^ω-nitro-

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-arginine (

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-NNA, an inhibitor of NO synthase, 5 mg/kg, i.v.) reduced the CBF by 5–6 ml·100 g⁻¹·min⁻¹ with increase in the mean arterial pressure by 26 mmHg. During ischemia the CBF significantly decreased to below 8% of the resting values in all rats. The largest blood flow in postischemic hyperemia was 171±9% of the resting CBF in the rats with

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-arginine (P<0.05 vs.

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-NNA and saline), followed by 126±5 with saline and 109±3 with

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-NNA. The CVR at 60 min of recirculation was 3.291±0.144 mmHg·ml⁻¹·100 g⁻¹·min⁻¹ in the rats with saline, remained low level of 2.711±0.124 with

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-arginine (P<0.01 vs.

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-NNA and P<0.05 vs. saline) and in contrast, significantly increased to 5.732±0.184 with

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-NNA (P<0.01 vs.

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-arginine and saline, respectively). Tissue lactate with saline increased 2.3-fold at 60 min of recirculation, whereas the increase was inhibited to 1.4-fold after

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-arginine treatment (P<0.01 vs.

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-NNA) and in contrast, significantly increased 5.7-fold with

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-NNA. The ATP and glucose levels were better preserved in the rats with

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-arginine than in those with

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-NNA or saline. These findings support that the enhanced postischemic hyperemia is beneficial to the ischemic brain and the administration of

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-arginine may be potentially useful for the treatment of acute stroke.     

The Effect of μ-Opioid Receptor Antisense on Morphine Potency and Antagonist-Induced Supersensitivity and Receptor Upregulation

The present study examined the effect of in vivo antisense oligodeoxynucleotide treatment on naltrexone (NTX)-induced functional supersensitivity and

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-opioid receptor upregulation in mice. On day 1 mice were implanted SC with a NTX or placebo pellet and injected IT and ICV with dH₂O or oligodeoxynucleotides. The oligodeoxynucleotides were designed so that they were either perfectly complementary to the first 18 bases of the coding region of mouse

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-opioid receptor mRNA, or had one (Mismatch-1) or four (Mismatch-4) mismatches. On days 3, 5, 7, and 9, mice were again injected IT and ICV with dH₂O or one of the oligodeoxynucleotides. After the final injections on day 9, placebo and NTX pellets were removed, and 24 h later mice were tested for morphine analgesia or sacrificed for saturation binding studies ([³H]DAMGO). Naltrexone increased the analgesic potency of morphine in dH₂O treated mice by ≈ 70%. In binding studies, NTX significantly increased density of brain (≈ 60%) and spinal cord (≈ 140%)

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-opioid receptors without affecting affinity. The

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-opioid antisense and the oligodeoxynucleotide with one mismatch (Mismatch-1) significantly reduced the potency of morphine by ≈ twofold in placebo-treated mice. The oligodeoxynucleotide with four mismatches (Mismatch-4) did not significantly alter morphine potency. When placebo-treated mice were treated with either the antisense to the mouse

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-opioid receptor, Mismatch-4 or Mismatch-1 there were no significant changes in the density of

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-opioid receptors. Thus,

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-opioid antisense significantly reduced morphine potency without changing

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-opioid receptor density. When NTX and oligodeoxynucleotide treatments were combined, there was no change in NTX-induced supersensitivity and

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-opioid receptor upregulation. These data suggest that opioid antagonist-induced supersensitivity and upregulation of

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-opioid receptors does not involve changes in gene expression.     

Two novel types of l-glutamate receptors with affinities for NMDA and l-cysteine in the olfactory organ of the Caribbean spiny lobster Panulirus argus

A subset of olfactory receptor neurons of the Caribbean spiny lobster Panulirus argus possesses receptors for

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-glutamate that can mediate both excitatory and inhibitory responses (P.C. Daniel, M.F. Burgess, C.D. Derby, Responses of olfactory receptor neurons in the spiny lobster to binary mixtures are predictable using a non-competitive model that incorporates excitatory and inhibitory transduction pathways, J. Comp. Physiol. A 178 (1992) 523–536). In this study, we have used biochemical and electrophysiological techniques to understand the role of these receptors in olfactory transduction, and to compare these olfactory glutamate receptors with peripheral and central

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-glutamate receptors in other animals. Using a radioligand-binding assay with a membrane-rich preparation from the dendrites of olfactory receptor neurons, we have identified two types of binding sites for

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-glutamate. Both sites showed rapid, reversible, and saturable association with radiolabeled

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-glutamate, and their K_d values (1 nM and 3 μM) are effective in physiological studies of glutamate-sensitive olfactory neurons, suggesting these binding sites are receptors involved in olfactory transduction. Both sites were completely inhibited by high concentrations of NMDA and

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-cysteine, and only partially inhibited by other

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-glutamate analogs and odorants. Electrophysiological recordings from

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-glutamate-best olfactory receptor neurons showed that NMDA and

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-cysteine are both partial agonists and antagonists of glutamate receptors. Together, these results suggest the olfactory

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-glutamate receptors of spiny lobsters are novel types of

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-glutamate receptors that are functionally important in mediating olfactory responses.     

Activation of protein kinase C by phorbol dibutyrate potentiates [

In contrast to the pharmacological studies implicating δ-opioid receptor subtypes, cloning studies have identified only a single cDNA encoding a δ receptor, DOR-1. Antisense studies have established the importance of DOR-1 in δ analgesia in mice. Antisense mapping extends this approach to include oligodeoxynucleotides which are targeted against each of the exons of the gene. Five different antisense oligodeoxynucleotides based upon the three DOR-1 exons all block both spinal and supraspinal analgesic actions of the δ₂ ligand [

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-Ala²,Glu⁴]deltorphin, consistent with the suggestion that DOR-1 encodes the δ₂ receptor. At the spinal level, [

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-Pen²,

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-Pen⁵]enkephalin (DPDPE) acts also acts through δ₂ receptors and all the antisense probes block spinal DPDPE analgesia. However, supraspinally only the two antisense probes targeting exon 3 block DPDPE analgesia. The remaining three antisense probes based upon exons 1 and 2 are inactive. Thus, the δ receptors responsible for spinal and supraspinal DPDPE analgesia can be discriminated at the molecular level by antisense mapping. © 1997 Elsevier Science B.V. All rights reserved.     

Characterization of [

In contrast to the pharmacological studies implicating δ-opioid receptor subtypes, cloning studies have identified only a single cDNA encoding a δ receptor, DOR-1. Antisense studies have established the importance of DOR-1 in δ analgesia in mice. Antisense mapping extends this approach to include oligodeoxynucleotides which are targeted against each of the exons of the gene. Five different antisense oligodeoxynucleotides based upon the three DOR-1 exons all block both spinal and supraspinal analgesic actions of the δ₂ ligand [

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-Ala²,Glu⁴]deltorphin, consistent with the suggestion that DOR-1 encodes the δ₂ receptor. At the spinal level, [

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-Pen²,

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-Pen⁵]enkephalin (DPDPE) acts also acts through δ₂ receptors and all the antisense probes block spinal DPDPE analgesia. However, supraspinally only the two antisense probes targeting exon 3 block DPDPE analgesia. The remaining three antisense probes based upon exons 1 and 2 are inactive. Thus, the δ receptors responsible for spinal and supraspinal DPDPE analgesia can be discriminated at the molecular level by antisense mapping. © 1997 Elsevier Science B.V. All rights reserved.     

Antisense mapping DOR-1 in mice: further support for δ receptor subtypes

In contrast to the pharmacological studies implicating δ-opioid receptor subtypes, cloning studies have identified only a single cDNA encoding a δ receptor, DOR-1. Antisense studies have established the importance of DOR-1 in δ analgesia in mice. Antisense mapping extends this approach to include oligodeoxynucleotides which are targeted against each of the exons of the gene. Five different antisense oligodeoxynucleotides based upon the three DOR-1 exons all block both spinal and supraspinal analgesic actions of the δ₂ ligand [

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-Ala²,Glu⁴]deltorphin, consistent with the suggestion that DOR-1 encodes the δ₂ receptor. At the spinal level, [

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-Pen²,

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-Pen⁵]enkephalin (DPDPE) acts also acts through δ₂ receptors and all the antisense probes block spinal DPDPE analgesia. However, supraspinally only the two antisense probes targeting exon 3 block DPDPE analgesia. The remaining three antisense probes based upon exons 1 and 2 are inactive. Thus, the δ receptors responsible for spinal and supraspinal DPDPE analgesia can be discriminated at the molecular level by antisense mapping. © 1997 Elsevier Science B.V. All rights reserved.     

Characterization of α,β-Methylene ATP Binding Sites in Mouse Crude Synaptic Membranes

Since ATP has been reported to be a potent excitatory transmitter in the mammalian central nervous system (CNS), we studied the neurochemical characters of the binding sites of

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,

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-methylene ATP, an agonist of P_2X receptors, in mouse crude synaptic membranes. ATP and its related compounds inhibited [³H]

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,

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-methylene ATP binding in a concentration-dependent manner. The potency order in the inhibition of the binding was as follows;

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,

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-methylene

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>

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> ATP ≥ ADP >

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,

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-methylene ATP UTP > 2-methylthio ATP. And adenosine did not affect the binding. The order was different from those reported in peripheral tissues. And Sr²⁺, Ca²⁺, Mg²⁺, and Cd²⁺ enhanced the binding. These results suggest that

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,

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-methylene ATP binding sites in CNS have different characters from those in peripheral tissues.     

Selective metabotropic receptor agonists distinguish non-ionotropic glutamate binding sites

Metabotropic glutamate receptors (mGluRs) are thought to mediate diverse processes in brain including synaptic plasticity and excitotoxicity. These receptors are often divided into three groups by their pharmacological profiles. [³H]Glutamate binding in the presence of compounds selective for ionotropic glutamate receptors can be used as a general assay for these receptors; subtypes of this non-ionotropic [³H]glutamate binding differ in both pharmacology and anatomical distribution, and are differentially sensitive to quisqualate. The characteristics of these binding sites are consistent with those of group 1 (high-affinity quisqualate) and group 2 (low-affinity quisqualate) mGluRs. Under our assay conditions, no [³H]glutamate binding to group 3-like (

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-AP4 sensitive) sites could be demonstrated. We have attempted to characterize particular agents which may selectively measure [³H]glutamate binding to mGluR subtypes. We used two isomers of 2-(carboxycyclopropyl)glycine,

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-CCG-I and

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-CCG-II, and the (2S,1′R,2′R,3′R) isomer of 2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) as competitors of non-ionotropic [³H]glutamate binding sites. DCG-IV clearly distinguishes two binding sites. Quantitative levels of DCG-IV binding by anatomic region correlate with quisqualate-defined binding subtypes: high-affinity DCG-IV binding correlates with low-affinity quisqualate binding, whereas low-affinity DCG-IV binding correlates with high-affinity quisqualate binding.

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-CCG-II displaces only one type of non-ionotropic [³H]glutamate binding, corresponding to high-affinity quisqualate binding. Therefore DCG-IV and

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-CCG-II at appropriate concentrations appear to distinguish binding to putative group 2 vs. group 1 mGluRs.

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-CCG-I displaces both high- and low-affinity quisqualate binding sites, but unlike the other two compounds, does not clearly distinguish between them.     

NMDA receptor antagonist blocks the bradycardic but not the pressor response to l-glutamate microinjected into the nucleus tractus solitarius (NTS) of unanesthetized rats

In the present study we evaluated the role of NMDA receptors on the pressor and bradycardic responses to

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-glutamate (

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-Glu) microinjected into the nucleus tractus solitarius (NTS) of unanesthetized rats.

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-Glu (1 nmol/100 nl) was microinjected into the NTS before and 10 min after microinjection of phosponovaleric acid (AP-5), a selective NMDA receptor antagonist, into the NTS of three different groups of rats (0.5, 2.0 and 10.0 nmol/100 nl). Microinjection of AP-5 into the NTS produced a dose-dependent reduction in the bradycardic response to

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-Glu. However, no significant change in the pressor response to

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-Glu was observed. These results indicate that the activation of the cardiovagal component (bradycardia) by

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-Glu involves NMDA receptors and suggest that the activation of the sympatho-excitatory component (pressor response) by

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-Glu in the commissural NTS is mediated by non-NMDA receptors.© 1997 Elsevier Science B.V. All rights reserved.     

p-Chlorophenylalanine and fluoxetine inhibit

Cardiovascular effects elicited by microinjection of

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-S-nitrosocysteine in the nucleus tractus solitarii (NTS) were compared and contrasted with those produced by the dextroisomer, other nitric oxide donors and nitric oxide itself.

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-S-nitrosocysteine produced dose-related decreases of arterial pressure and heart rate. In contrast,

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-S-nitrosocysteine, S-nitrosoglutathione, glyceryl trinitrate, and sodium nitroprusside produced minimal responses that were not dose-related. Likewise, injection of cystine and nitric oxide, two products of S-nitrosocysteine breakdown, produced no significant response. Headspace analysis using chemiluminescence revealed that

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- and

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-S-nitrosocysteine released identical amounts of nitric oxide when exposed to homogenates of whole rat brain. Responses to

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-S-nitrosocysteine were not affected by local injection of oxyhemoglobin or the nitric oxide synthase inhibitor

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-nitroarginine methylester. Although injection of

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-cysteine into the NTS produced responses similar to those seen with injection of

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-S-nitrosocysteine, blockade of excitatory amino acid receptors with kynurenic acid inhibited responses to cysteine but not those to the nitrosothiol. The study demonstrates that S-nitrosocysteine is biologically active in the NTS. Its action is independent of release of nitric oxide from the nitrosothiol but may be mediated through stereoselective sites on target neurons.     

The neuronal NOS inhibitor

Cardiovascular effects elicited by microinjection of

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-S-nitrosocysteine in the nucleus tractus solitarii (NTS) were compared and contrasted with those produced by the dextroisomer, other nitric oxide donors and nitric oxide itself.

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-S-nitrosocysteine produced dose-related decreases of arterial pressure and heart rate. In contrast,

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-S-nitrosocysteine, S-nitrosoglutathione, glyceryl trinitrate, and sodium nitroprusside produced minimal responses that were not dose-related. Likewise, injection of cystine and nitric oxide, two products of S-nitrosocysteine breakdown, produced no significant response. Headspace analysis using chemiluminescence revealed that

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- and

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-S-nitrosocysteine released identical amounts of nitric oxide when exposed to homogenates of whole rat brain. Responses to

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-S-nitrosocysteine were not affected by local injection of oxyhemoglobin or the nitric oxide synthase inhibitor

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-nitroarginine methylester. Although injection of

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-cysteine into the NTS produced responses similar to those seen with injection of

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-S-nitrosocysteine, blockade of excitatory amino acid receptors with kynurenic acid inhibited responses to cysteine but not those to the nitrosothiol. The study demonstrates that S-nitrosocysteine is biologically active in the NTS. Its action is independent of release of nitric oxide from the nitrosothiol but may be mediated through stereoselective sites on target neurons.     

Actions of S-nitrosocysteine in the nucleus tractus solitarii are unrelated to release of nitric oxide

Cardiovascular effects elicited by microinjection of

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-S-nitrosocysteine in the nucleus tractus solitarii (NTS) were compared and contrasted with those produced by the dextroisomer, other nitric oxide donors and nitric oxide itself.

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-S-nitrosocysteine produced dose-related decreases of arterial pressure and heart rate. In contrast,

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-S-nitrosocysteine, S-nitrosoglutathione, glyceryl trinitrate, and sodium nitroprusside produced minimal responses that were not dose-related. Likewise, injection of cystine and nitric oxide, two products of S-nitrosocysteine breakdown, produced no significant response. Headspace analysis using chemiluminescence revealed that

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- and

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-S-nitrosocysteine released identical amounts of nitric oxide when exposed to homogenates of whole rat brain. Responses to

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-S-nitrosocysteine were not affected by local injection of oxyhemoglobin or the nitric oxide synthase inhibitor

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-nitroarginine methylester. Although injection of

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-cysteine into the NTS produced responses similar to those seen with injection of

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-S-nitrosocysteine, blockade of excitatory amino acid receptors with kynurenic acid inhibited responses to cysteine but not those to the nitrosothiol. The study demonstrates that S-nitrosocysteine is biologically active in the NTS. Its action is independent of release of nitric oxide from the nitrosothiol but may be mediated through stereoselective sites on target neurons.     

Direct evidence for the role of nitric oxide on the glutamate-induced neuronal death in cultured cortical neurons

It has been reported that glutamate-induced neurotoxicity is related to an increase in nitric oxide (NO) concentration. An NO-sensitive electrode has been developed to measure NO concentration directly. Using this electrode, we examined NO concentration and neuronal survival after glutamate application in rat cultured cortical neurons. We also examined the effects of NMDA receptor antagonists, MK-801 and ketamine, and the NO synthetase inhibitor,

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-NMMA on NO production and neuronal death. After 7 days in culture, application of glutamate (1 mM) or

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-arginine (0.3 mM) to the cultured medium increased NO concentration, and decreased the number of anti-microtubule-associated protein 2 positive neurons. Both pretreatment with MK-801 (300 μm) and ketamine (300 μm) prevented glutamate-, but not

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-arginine-induced increase in NO concentration and neuronal death.

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-NMMA prevented both glutamate- and

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-arginine-induced NO production and neuronal death. The nitric oxide donor, S-nitroso-N-acetyl-

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,

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-penicillamine (SNAP) also caused neuronal death, and MK-801, ketamine and

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-NMMA did not prevent SNAP-induced toxicity. We have demonstrated excitatory amino acid-induced changes of NO concentration and the parallel relationship between changes of NO concentration and neuronal death. In conclusion, an increase in NO concentration does induce neuronal death, and the inhibition of the production of NO prevents glutamate-induced neuronal death.     

Quinine suppression of single facial taste fiber responses in the channel catfish

Two groups of single facial taste fibers that were responsive to quinine hydrochloride (QHCl) and amino acids were identified in the channel catfish, Ictalurus punctatus. Group I fibers were significantly more excited by quinine hydrochloride (QHCl) than were group II fibers. QHCl (10⁻³ M), as one component in a binary mixture, suppressed taste responses of group II fibers to 10⁻⁴ M amino acids (other component) by 61%, but did not inhibit significantly the responses to

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-alanine of group I fibers. QHCl (10⁻² M) suppressed the response to 10⁻⁴ M

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-alanine of group I fibers by 58% and group II fibers to 10⁻⁴ M

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-alanine,

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-arginine and

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-proline by 89–100%. The suppression of amino acid responses of both groups of fibers by QHCl was reversible in subsequent testing of stimuli in the absence of QHCl. QHCl also suppressed the taste responses to other bitter stimuli [10⁻³ M caffeine and 10⁻² M denatonium benzoate(DB)]; however, neither caffeine nor DB suppressed amino acid taste responses. Possible mechanisms for the suppressive effect of QHCl on taste nerve activity are discussed.     

Modulatory effect of

Intraventricular administration of carbachol can induce phase shifts in wheel-running activity in rodents, which depend on circadian phase and are mediated via muscarinic cholinergic receptors in Syrian hamsters. We studied the circadian variation in binding of [

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]-N-methylscopolamine ([

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]NMS), a hydrophilic muscarinic receptor antagonist, in micropunches obtained from the anterior hypothalamus and occipital cortex of Syrian hamsters housed in a 14:10 light:dark cycle. Binding sites were characterized on cells contained within 1 mm punches (obtained from slices 300 μm thick), using a method to selectively detect cell surface (functional) receptors. Atropine sulphate was used to determine nonspecific binding. Cortex showed a significant daily rhythm in [

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]NMS binding with a peak occurring late in the light phase and a trough at lights on, while the hypothalamus showed no detectable rhythm. Following suprachiasmatic nucleus (SCN) ablation or maintenance in constant darkness, the rhythm in the cortex was abolished. These findings suggest that photic information conveyed via the SCN is responsible for the receptor binding rhythm in the cortex. Autoradiographic studies ([

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]NMS; 2 nM, 3 weeks exposure) clearly revealed both M1 and M2 subtypes of muscarinic receptors in the region of the SCN and the visual cortex.     

Daily variation of muscarinic receptors in visual cortex but not suprachiasmatic nucleus of Syrian hamsters

Intraventricular administration of carbachol can induce phase shifts in wheel-running activity in rodents, which depend on circadian phase and are mediated via muscarinic cholinergic receptors in Syrian hamsters. We studied the circadian variation in binding of [

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]-N-methylscopolamine ([

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]NMS), a hydrophilic muscarinic receptor antagonist, in micropunches obtained from the anterior hypothalamus and occipital cortex of Syrian hamsters housed in a 14:10 light:dark cycle. Binding sites were characterized on cells contained within 1 mm punches (obtained from slices 300 μm thick), using a method to selectively detect cell surface (functional) receptors. Atropine sulphate was used to determine nonspecific binding. Cortex showed a significant daily rhythm in [

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]NMS binding with a peak occurring late in the light phase and a trough at lights on, while the hypothalamus showed no detectable rhythm. Following suprachiasmatic nucleus (SCN) ablation or maintenance in constant darkness, the rhythm in the cortex was abolished. These findings suggest that photic information conveyed via the SCN is responsible for the receptor binding rhythm in the cortex. Autoradiographic studies ([

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]NMS; 2 nM, 3 weeks exposure) clearly revealed both M1 and M2 subtypes of muscarinic receptors in the region of the SCN and the visual cortex.     

Changes in noradrenergic sensitivity to tumor necrosis factor-α in brains of rats administered clonidine

Tumor necrosis factor-alpha (TNFα) and the imidazoline clonidine modulate norepinephrine (NE) release from noradrenergic nerve terminals in the central nervous system. The present study demonstrates an intrinsic association between presynaptic α₂-adrenergic receptor sensitivity and TNFα responsiveness in governing this NE release. Superfusion and electrical field stimulation were applied to a series of rat hippocampal brain slices in order to study the regulation of [

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]-NE release. The α₂-adrenergic agonist clonidine and the cytokine TNFα concentration-dependently inhibit [

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]-NE release; whereas, the α₂-adrenergic antagonist idazoxan potentiates [

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]-NE release. The fractional release of [

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]-NE during field stimulation of control hippocampal slices was decreased by the addition of TNFα in a concentration-dependent manner, an effect which was potentiated by the α₂-adrenergic antagonist idazoxan; whereas, TNFα attenuated the concentration-dependent potentiating effect of idazoxan. Furthermore, constitutive TNFα, demonstrated to be present in several brain areas, was significantly decreased following administration of the α₂-adrenergic agonist clonidine (0.6 mg/kg, i.p., twice daily) to rats for either 1 or 14 days, without a change in TNFα mRNA accumulation. We next investigated whether the presynaptic sensitivity to TNFα was changed after clonidine administration to rats. TNFα enhanced, rather than inhibited, [

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]-NE release after 1 day of clonidine administration, while a suppressed sensitivity to TNFα was observed in the hippocampus after 14 days of clonidine administration. In addition, in the presence of idazoxan, TNFα potentiation of [

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]-NE release after 1 day clonidine administration was reversed to a decreased inhibition as compared to control slices exposed to idazoxan. Therefore, the temporary reversal in the presynaptic TNFα response after 1 day of clonidine administration illustrates a mechanism of action for its persistent antihypertensive effect, its transient sedative and antihyperpathic effects, and its acute ability to promote antidepressants. These results demonstrate a novel role for an immune mediator in the central nervous system, and demonstrates that presynaptic TNFα responsiveness is intimately associated with adrenergic receptor sensitivity.     

Interferon-

The third intracellular loop of adrenergic receptors has been implicated in their interaction with guanine nucleotide-binding proteins (G proteins). One of the mechanisms involved in the modulation of receptor function is the phosphorylation of specific residues by intracellular kinases.

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-Adrenergic receptor is phosphorylated in vitro by cAMP-dependent protein kinase (PKA), although its physiological effect remains to be determined. We have produced fusion proteins formed by glutathione S-transferase and sequences of the third intracellular loop of mouse

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-,

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-, and

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-adrenergic receptor subtypes, and used them as substrates for PKA. Only the fusion protein containing the

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sequence was phosphorylated in vitro by this kinase. Site-directed mutagenesis of a serine (homologue to serine 278 of the rat sequence, RSS) to an alanine residue precluded phosphorylation by PKA.     

Harmaline competitively inhibits [

The third intracellular loop of adrenergic receptors has been implicated in their interaction with guanine nucleotide-binding proteins (G proteins). One of the mechanisms involved in the modulation of receptor function is the phosphorylation of specific residues by intracellular kinases.

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-Adrenergic receptor is phosphorylated in vitro by cAMP-dependent protein kinase (PKA), although its physiological effect remains to be determined. We have produced fusion proteins formed by glutathione S-transferase and sequences of the third intracellular loop of mouse

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-,

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-, and

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-adrenergic receptor subtypes, and used them as substrates for PKA. Only the fusion protein containing the

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sequence was phosphorylated in vitro by this kinase. Site-directed mutagenesis of a serine (homologue to serine 278 of the rat sequence, RSS) to an alanine residue precluded phosphorylation by PKA.     

Phosphorylation of the Third Intracellular Loop of the Mouse α1b-Adrenergic Receptor by cAMP-dependent Protein Kinase

The third intracellular loop of adrenergic receptors has been implicated in their interaction with guanine nucleotide-binding proteins (G proteins). One of the mechanisms involved in the modulation of receptor function is the phosphorylation of specific residues by intracellular kinases.

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-Adrenergic receptor is phosphorylated in vitro by cAMP-dependent protein kinase (PKA), although its physiological effect remains to be determined. We have produced fusion proteins formed by glutathione S-transferase and sequences of the third intracellular loop of mouse

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-,

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-, and

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-adrenergic receptor subtypes, and used them as substrates for PKA. Only the fusion protein containing the

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sequence was phosphorylated in vitro by this kinase. Site-directed mutagenesis of a serine (homologue to serine 278 of the rat sequence, RSS) to an alanine residue precluded phosphorylation by PKA.