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.     

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.     

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.     

Nitric Oxide Inhibits Neuronal Activity in the Supraoptic Nucleus of the Rat Hypothalamic Slices

The presence of abundant nitric oxide synthase (NOS) in magnocellular neurons of the rat hypothalamus suggests that nitric oxide (NO) may be involved in controlling the release of oxytocin and vasopressin. To test this possibility, we examined the effect of NO-related drugs on extracellular discharges of 124 supraoptic nucleus (SON) neurons from slices of rat hypothalamus in vitro. Twenty-three (43%) of 53 neurons were inhibited by sodium nitroprusside (SNP), a spontaneous releaser of NO, at 1–3 mM. This inhibition was prevented by preincubation of the slices with 1

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hemoglobin, an inactivator of NO (

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), whereas hemoglobin alone enhanced neuronal activity in seven (35%) of 20 neurons.

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-Arginine (1 mM), a precursor of NO, inhibited neuronal activity in five (36%) of 14 neurons, while

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-arginine (1 mM), the inactive counterpart of

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-arginine, was ineffective (

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). N-

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-nitro-

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-arginine methyl ester (

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-NAME, 10

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), an inhibitor of NOS, also enhanced neuronal activity in five (29%) of 17 neurons, while N-

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-nitro-

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-arginine methyl ester (DNAME, 10

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), the inactive enantiomer of

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-NAME, was without effect (

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). Together, our data show that NO exerts predominantly an inhibitory effect on SON neurons and may serve as a negative feedback loop in controlling release of oxytocin and vasopressin.     

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.     

The effects of

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.     

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.     

Activation of protein kinase C by phorbol dibutyrate potentiates [

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.     

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.     

Maternal Exposure to Δ-Tetrahydrocannabinol (Δ-THC) Alters Indolamine Levels and Turnover in Adult Male and Female Rat Brain Regions

Perinatal exposure to

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-THC has been shown to produce effects on brain development. In this study we evaluated the changes induced by maternal exposure to

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-THC (5 mg/kg per day) from gestational day 5 to postnatal day 24 in eight discrete brain areas on the central serotoninergic system in both adult male and female rats. These result show that maternal exposure to

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-THC from gestational day 5 to postnatal day 24 affects development of the various central indoleaminergic system of the offsprings brain. Perinatal exposure to

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-THC decreased the levels of 5-HT in hypothalamus and rostral neostriatum in exposed males, and also decreased the levels of 5-HT in ventral hippocampus, septum, and midbrain raphe nuclei in both exposed males and females. Perinatal exposure to

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-THC increased the levels of 5-HIAA in dorsal hippocampus, hypothalamus, septum, midbrain raphe nuclei, and rostral neostriatum in exposed males and females. We have also found differences between nonexposed males and females in several brain regions. Our results confirm a regional and sexual specificity in endogenous levels of indoleamine after perinatal

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-THC treatment, being the midbrain raphe nuclei the most affected area.     

Monoamine oxidase-dependent metabolism of dopamine in the striatum and substantia nigra of

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.     

p-Chlorophenylalanine and fluoxetine inhibit

The exposure of cultured rat striatal neurons to

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-DOPA caused marked cell death. The

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-DOPA cytotoxicity was inhibited by the addition of Mg²⁺ to and by the removal of Ca²⁺ from the culture medium, and also by the application of tetrodotoxin. Moreover, prolonged application of

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-DOPA increased the glutamate content in the culture medium. These results indicate that

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-DOPA produces neurotoxicity by facilitating glutamate release.     

l-DOPA neurotoxicity is mediated by glutamate release in cultured rat striatal neurons

The exposure of cultured rat striatal neurons to

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-DOPA caused marked cell death. The

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-DOPA cytotoxicity was inhibited by the addition of Mg²⁺ to and by the removal of Ca²⁺ from the culture medium, and also by the application of tetrodotoxin. Moreover, prolonged application of

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-DOPA increased the glutamate content in the culture medium. These results indicate that

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-DOPA produces neurotoxicity by facilitating glutamate release.     

Evidence for involvement of the lateral parabrachial nucleus in mediation of cholinergic inputs to neurons in the rostral ventrolateral medulla of the rat

We examined whether sites in the lateral parabrachial nucleus (PBN) where

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-glutamate produced increases in arterial pressure were involved in mediation of cholinergic inputs to neurons in the rostral ventrolateral medulla (RVLM). Male Wistar rats were anesthetized, paralyzed and artificially ventilated. Unilateral microinjection of

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-glutamate into the lateral PBN produced a pressor response. Microinjection of the muscarinic receptor antagonist scopolamine into the unilateral RVLM inhibited the pressor response to

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-glutamate injected ipsilaterally into the lateral PBN, whereas microinjection of the cholinesterase inhibitor physostigmine into the RVLM enhanced it. PBN microinjection of

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-glutamate also enhanced the firing rate of RVLM sympathoexcitatory neurons and the enhancement of the firing rate was inhibited by scopolamine iontophoretically applied on neurons. PBN injection of

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-glutamate produced a tetrodotoxin (TTX)-sensitive release of ACh in the RVLM. Unilateral microinjection of TTX into the lateral PBN inhibited the pressor response induced by RVLM microinjection of physostigmine. These results provide evidence that neurons in the pressor sites of the lateral PBN are involved in mediation of cholinergic inputs responsible for pressor responses in the RVLM.     

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.     

Characterization of [

Previous studies in our laboratory have demonstrated that microinjection of N-methyl-

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-aspartate (NMDA) agonist into the nucleus magnocellularis (NMC) of the medial medulla increases muscle tone and/or produces locomotion, while injection of corticotropin-releasing factor (CRF) and non-NMDA agonists into the same or nearby sites suppresses muscle tone. In the first paper of this series, we report that myoclonic twitches or coordinated rhythmic leg movement (locomotion) can be induced by either NMDA or hemorrhagic bilateral lesion of the ventral mesopontine junction (vMPJ). In this paper, we report that microinjection of CRF (10 nM) or non-NMDA agonists, kainic acid (0.1–0.2 mM) and quisqualic acid (1–10 mM), into the NMC block locomotion and myoclonic twitches. The latency and duration of CRF and non-NMDA agonist-induced blockade of motor activity were short, at 34 s and 3.6 min, respectively. However, microinjection of the NMDA antagonists

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-2-amino-5-phosphonovaleric acid (APV; 50 mM) or

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-2-amino-5-phosphonopentanoic acid (AP5, 20 mM) block myoclonus at a latency of 0.6–3 min with the block lasting for a mean of 7 h. Thus, activation of non-NMDA receptors or inactivation of NMDA receptors in NMC can block myoclonus. An imbalance between the inputs to these receptor systems may contribute to the generation of abnormal motor activation in waking and sleep.     

Brainstem-mediated locomotion and myoclonic jerks. II. Pharmacological effects

Previous studies in our laboratory have demonstrated that microinjection of N-methyl-

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-aspartate (NMDA) agonist into the nucleus magnocellularis (NMC) of the medial medulla increases muscle tone and/or produces locomotion, while injection of corticotropin-releasing factor (CRF) and non-NMDA agonists into the same or nearby sites suppresses muscle tone. In the first paper of this series, we report that myoclonic twitches or coordinated rhythmic leg movement (locomotion) can be induced by either NMDA or hemorrhagic bilateral lesion of the ventral mesopontine junction (vMPJ). In this paper, we report that microinjection of CRF (10 nM) or non-NMDA agonists, kainic acid (0.1–0.2 mM) and quisqualic acid (1–10 mM), into the NMC block locomotion and myoclonic twitches. The latency and duration of CRF and non-NMDA agonist-induced blockade of motor activity were short, at 34 s and 3.6 min, respectively. However, microinjection of the NMDA antagonists

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-2-amino-5-phosphonovaleric acid (APV; 50 mM) or

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-2-amino-5-phosphonopentanoic acid (AP5, 20 mM) block myoclonus at a latency of 0.6–3 min with the block lasting for a mean of 7 h. Thus, activation of non-NMDA receptors or inactivation of NMDA receptors in NMC can block myoclonus. An imbalance between the inputs to these receptor systems may contribute to the generation of abnormal motor activation in waking and sleep.