N-甲基-D-天冬氨酸受体激活在脑缺血中的神经保护及神经毒性作用研究进展

N-甲基-D-天冬氨酸受体(NMDAR)介导的神经元兴奋毒性损伤与脑缺血发生密切相关,但生理水平的NMDAR却具有神经保护、抵抗损伤的功能,并且在突触可塑性及突触传递方面发挥重要作用。这种功能的双面性正是使用NMDAR拮抗剂治疗脑缺血、卒中等疾病临床效果欠佳的原因之一。深入了解NMDAR及其介导的促神经元存活或死亡信号通路在缺血性脑损伤中的作用,在不影响促神经元存活以及突触可塑性通路前提下,选择性地阻断NMDAR介导的神经元死亡信号通路,是临床治疗缺血/缺氧性脑损伤、脑卒中等疾病的发展方向。本文就NMDAR激活介导的信号通路在缺血性脑损伤中的作用作一综述。     

Tweaking agonist efficacy at N-methyl-D-aspartate receptors by site-directed mutagenesis

The structural basis for partial agonism at N-methyl-D-aspartate (NMDA) receptors is currently unresolved. We have characterized several partial agonists at the NR1/NR2B receptor and investigated the mechanisms underlying their reduced efficacy by introducing mutations in the glutamate binding site. Key residues were selected for mutation based on ligand-protein docking studies using a homology model of NR2B-S1S2 built from the X-ray structure of NR1-S1S2 in complex with glycine. Wild-type and mutant forms of NR2B were coexpressed with NR1 in Xenopus laevis oocytes and characterized by two-electrode voltage-clamp electrophysiology. By combining mutagenesis of residues His486 or Val686 with activation by differently substituted partial agonists, we introduce varying degrees of steric clash between the ligand and the two binding domains S1 and S2. In cases where ligand-protein docking predicts increased steric clashes between agonists and the residues forming the S1-S2 interface, the agonists clearly show decreased relative efficacy. Furthermore, we demonstrate that the mutation S690A affects both potency and efficacy in an agonist-specific manner. The results indicate that essential residues in the ligand binding pocket of NR2B may adopt different conformations depending on the agonist bound. Together, these data indicate that agonist efficacy at the NR2B subunit can be controlled by the extent of steric clashes between the agonist and the ligand binding domains and by ligand-dependent arrangements of residues within the binding pocket.     

Ncm-D-aspartate: a novel caged D-aspartate suitable for activation of glutamate transporters and N-methyl-D-aspartate (NMDA) receptors in brain tissue

The D-isomer of aspartate is both a substrate for glutamate transporters and an agonist of N-methyl-D-aspartate (NMDA) receptors. To monitor the behavior of these receptors and transporters in intact tissue we synthesized a new photo-labile analogue of D-aspartate, N-[(6-nitrocoumarin-7-yl)methyl]-D-aspartic acid (Ncm-D-aspartate). This compound was photolyzed rapidly (t(1/2)=0.11 micros) by UV light with a quantum efficiency of 0.041 at pH 7.4. In acute hippocampal slices, photolysis of Ncm-D-aspartate by brief (1 ms) exposure to UV light elicited rapidly activating inward currents in astrocytes that were sensitive to inhibition by the glutamate transporter antagonist DL-threo-beta-benzyloxyaspartic acid (TBOA). Neither Ncm-D-aspartate nor the photo-released caging group exhibited agonist or antagonist activity at glutamate transporters, and Ncm-D-aspartate did not induce transporter currents prior to photolysis. Glutamate transporter currents were also elicited in cerebellar Purkinje cells in response to photolysis of Ncm-D-aspartate. Photo-release of D-aspartate from Ncm-D-aspartate did not induce alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor or metabotropic glutamate receptor (mGluR) currents, but triggered robust NMDA receptor currents in neurons; Ncm-D-aspartate and the photolzyed caging group were similarly inert at NMDA receptors. These results indicate that Ncm-D-aspartate can be used to study NMDA receptors at excitatory synapses and interactions between transporters and receptors in brain tissue.     

Suramin inhibits beta-bungarotoxin-induced activation of N-methyl-D-aspartate receptors and cytotoxicity in primary neurons

We demonstrated that beta-bungarotoxin (beta-BuTX), a snake presynaptic neurotoxin, exhibited a potent cytotoxic effect on cultured cerebellar granule neurons. The mechanism of action of beta-BuTX and the cytoprotective agents against beta-BuTX were studied. The neuronal death of cerebellar granule neurons induced by beta-BuTX was manifested with apoptosis and necrosis processes as revealed by neurite fragmentation, morphological alterations, and staining apoptotic bodies with the fluorescent dye Hoechst 33258. By means of microspectrofluorimetry and fura-2, we measured intracellular Ca2+ concentration, [Ca2+]i and found that [Ca2+]i was increased markedly prior to the morphological changes and cytotoxicity. The downstream pathway of the increased [Ca2+]i was investigated: there was increased production of free radicals, decreased mitochondrial membrane potential, and depleted cellular ATP content. MK801 and suramin effectively suppressed these detrimental effects of beta-BuTX. Furthermore, the [3H]MK801 binding was reduced by unlabeled MK801, beta-BuTX, and suramin. Thus, activation of N-methyl-D-aspartate (NMDA) receptors appeared to play a crucial role in the cytotoxic effects following betaBuTX exposure. In conclusion, the novel finding of this study was that a polypeptide beta-BuTX exerted a potent cytotoxic effect through sequential events, including activating NMDA receptors followed by increasing [Ca2+]i, ROS production, and impaired mitochondrial energy metabolism. Suramin, clinically used as a trypanocidal agent, was an effective antagonist against beta-BuTX. Data suggest that suramin might have value to detect the possible pathway of certain neuropathological disorders.     

Putative partial agonist 1-aminocyclopropanecarboxylic acid acts concurrently as a glycine-site agonist and a glutamate-site antagonist at N-methyl-D-aspartate receptors

1-Aminocyclopropanecarboxylic acid (ACPC) has been shown to protect against neuronal cell death after ischemic insult in vivo. Such results can be correlated with in vitro assays in which ACPC protected neurons against glutamate-induced neurotoxicity by reducing the activity of N-methyl-D-aspartate (NMDA) channel activation. Electrophysiological studies have determined that ACPC inhibits NMDA receptor activity by acting as a glycine-binding site partial agonist. In this study, rapid drug perfusion combined with whole-cell voltage-clamp was used to elicit and measure the effects of ACPC on NMDA receptor-mediated responses from cultured hippocampal neurons and cerebellar granule cells. The ACPC steady-state dose-response curve had both stimulatory and inhibitory phases. Half-maximal activation by ACPC as a glycine-site agonist was 0.7 to 0.9 microM. Half-maximal inhibition by ACPC was dependent on NMDA concentration. Peak responses to a >100 microM ACPC pulse in the presence of 1 microM glutamate were similar to those of glycine but decayed to a steady-state amplitude below that of glycine. The removal of ACPC initially caused an increase in inward current followed by a subsequent decrease to baseline levels. This suggests that relief of low-affinity antagonism occurs before high-affinity agonist dissociation. Simulations of ACPC action by a two glutamate-binding site/two glycine-binding site model for NMDA channel activation in conjunction with the concurrent role of ACPC as a glycine-site full agonist and glutamate-site competitive antagonist were able to successfully approximate experimental results.     

Characterization of organophosphate interactions at N-methyl-D-aspartate receptors in brain synaptic membranes.

Several competitive antagonists of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors are phosphonate analogs of L-glutamic acid. The position of the phosphonate has been shown to be important in the structure-activity relationships of these analogs. To investigate whether other phosphorous-containing compounds had activity at the NMDA receptor, several organophosphates were tested for the ability to inhibit the specific binding to brain synaptic membranes of 3-((+-)-2-carboxypiperazin-4-yl)-[1,2-3H]propyl-1-phosphonic acid ([3H]CPP), a selective antagonist of NMDA receptors. Diisopropylfluorophosphate (DFP), dichlorvos, cyanophos, mipafox, and o-ethyl o-4-nitrophenyl phenylphosphonothioate are relatively potent inhibitors of [3H]CPP binding to synaptic membranes. The inhibition produced by DFP is selective for the NMDA subtype of excitatory amino acid receptors, is irreversible, and can be prevented by preincubation with excess CPP, 2-amino-7-phosphonoheptanoic acid, or L-glutamate. Rat brain synaptic membranes have a population of phosphonate-sensitive [3H]DFP binding sites that are covalently labeled by [3H]DFP. Two protein bands of synaptic membrane proteins subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis are labeled by [3H]DFP in a 2-amino-5-phosphonopentanoic acid-sensitive manner. These proteins have an average molecular size of 47-50 and 32 kDa. Proteins of nearly identical molecular sizes have been shown in other studies to be components of an NMDA receptor complex. These observations are indicative of an interaction between the organophosphates and the NMDA receptor protein complex and suggest that DFP may be another important pharmacological tool that can be used in the elucidation of the molecular structure of the NMDA receptor complex.     

Possible endogenous agonist mechanism for the activation of secretin family G protein-coupled receptors

The class B family of G protein-coupled receptors contains several potentially important drug targets, yet our understanding of the molecular basis of ligand binding and receptor activation remains incomplete. Although a key role is recognized for the cysteine-rich, disulfide-bonded amino-terminal domain of these receptors, detailed insights into ligand docking and resultant conformational changes are not clear. We postulate that binding natural ligands to this domain results in a conformational change that exposes an endogenous ligand which interacts with the body of the receptor to activate it. In this work, we examined whether a synthetic peptide corresponding to a candidate region between the first and third conserved cysteines could act as an agonist. Indeed, this peptide was a weakly potent but fully efficacious agonist, stimulating a concentration-dependent cAMP response in secretin receptor-bearing cells. This effect was maintained as the peptide length was reduced from 30 to 5, and ultimately, three residues focused on the conserved residue Asp49. The agonist potency was enhanced by cyclization through a diaminopropionic acid linker and by amino-terminal fatty acid acylation. Both ends of the cyclic peptide were shown to interact with the top of transmembrane segment 6 of the receptor, using probes with a photolabile benzoyl-phenylalanine on each end. Analogous observations were also made for two other members of this family, the vasoactive intestinal polypeptide type 1 and calcitonin receptors. These data may provide a unique molecular mechanism and novel leads for the development of small-molecule agonists acting at potential drug targets within this physiologically important receptor family.     

Structural requirements for activation of the glycine coagonist site of N-methyl-D-aspartate receptors expressed in Xenopus oocytes

Five structural features important for activation of the glycine recognition site on N-methyl-D-aspartate (NMDA) receptors were identified by structure-activity studies of more than 60 glycine analogues in voltage-clamped Xenopus oocytes injected with rat brain mRNA. First, sterically unhindered and ionized carboxyl and amino termini were essential for action at this site. Second, an increase in the interterminal separation by greater than one carbon (e.g., beta-alanine) markedly attenuated activity at this site. Third, activity at the glycine site was stereoselective. The D-isomers of alanine and serine were approximately 20 and 30 times more potent than the L-isomers. Fourth, only small sterically unobtrusive substitutions at the alpha-carbon could be tolerated. alpha-Methyl (D-alanine) and alpha-cyclopropyl (1-amino-cyclopropane carboxylic acid) (ACC) substitutions were effective as agonists but most larger aliphatic and aromatic alpha-carbon substitutions were simply inactive. Glycine, D-alanine, and ACC probably have only a two-point attachment to the receptor. Finally the alpha-carbon substituent of D-serine is envisioned as binding to a third site on the receptor probably via hydrogen bonding of the omega-terminal hydroxyl group. Thus, serine, an hydroxymethyl substitution of glycine, permitted activation of NMDA receptor-mediated currents, whereas isosteric substitutions incapable of hydrogen bonding (e.g., 2-aminobutyric acid) were inactive. Additionally, the position and size of the hydroxyl-containing group is critical for agonist action; D-threonine, DL-homoserine, and hydroxyphenolic substitutions at the alpha-carbon were all inactive. Halogenated analogs of a size comparable to D-serine but capable only of proton acceptance at the omega-terminus (beta-fluoro-D-alanine and beta-chloro-D-alanine) possessed agonist action, whereas an analog capable of only proton donation (1,2-diaminopropionic acid) was inactive. Full concentration-response curves were constructed for those analogs displaying greater than 25% of the effect of glycine when tested at 3 microM. With the exception of (R)-(+)cycloserine and beta-fluoro-D-alanine, all compounds were nearly full agonists and had Hill coefficients not significantly different from unity. The order of relative potency of the active analogs was ACC greater than glycine greater than D-serine greater than D-alanine greater than beta-fluoro-D-alanine greater than (R)-(+)-cycloserine greater than L-serine greater than L-alanine. Molecular modelling of a series of active and inactive analogs with close structural relation to glycine was undertaken. These results were complementary to those data obtained from the electrophysiological investigation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Targeting N-methyl-D-aspartate receptors for treatment of neuropathic pain

Neuropathic pain remains a major clinical problem and a therapeutic challenge because existing analgesics are often ineffective and can cause serious side effects. Increased N-methyl-d-aspartate receptor (NMDAR) activity contributes to central sensitization in certain types of neuropathic pain. NMDAR antagonists can reduce hyperalgesia and allodynia in animal models of neuropathic pain induced by nerve injury and diabetic neuropathy. Clinically used NMDAR antagonists, such as ketamine and dextromethorphan, are generally effective in patients with neuropathic pain, such as complex regional pain syndrome and painful diabetic neuropathy. However, patients with postherpetic neuralgia respond poorly to NMDAR antagonists. Recent studies on identifying NMDAR-interacting proteins and molecular mechanisms of increased NMDAR activity in neuropathic pain could facilitate the development of new drugs to attenuate abnormal NMDAR activity with minimal impairment of the physiological function of NMDARs. Combining NMDAR antagonists with other analgesics could also lead to better management of neuropathic pain without causing serious side effects.     

Targeting N-methyl-D-aspartate receptors for treatment of neuropathic pain

Neuropathic pain remains a major clinical problem and a therapeutic challenge because existing analgesics are often ineffective and can cause serious side effects. Increased N-methyl-D-aspartate receptor (NMDAR) activity contributes to central sensitization in certain types of neuropathic pain. NMDAR antagonists can reduce hyperalgesia and allodynia in animal models of neuropathic pain induced by nerve injury and diabetic neuropathy. Clinically used NMDAR antagonists, such as ketamine and dextromethorphan, are generally effective in patients with neuropathic pain, such as complex regional pain syndrome and painful diabetic neuropathy. However, patients with postherpetic neuralgia respond poorly to NMDAR antagonists. Recent studies on identifying NMDAR-interacting proteins and molecular mechanisms of increased NMDAR activity in neuropathic pain could facilitate the development of new drugs to attenuate abnormal NMDAR activity with minimal impairment of the physiological function of NMDARs. Combining NMDAR antagonists with other analgesics could also lead to better management of neuropathic pain without causing serious side effects.     

Some properties of brain specific benzodiazepine receptors: new evidence for multiple receptors.

Several new lines of evidence suggest the existence of two or more distinct types of benzodiazepine receptors, in contrast to earlier results suggesting the presence of only one class of receptors. Appropriate thermoinactivation experiments indicate two receptors with different thermostabilities. Several triazolopyridazines, with some of the pharmacological properties of anxiolytics have recently been shown to displace 3H-diazepam and 3H-flunitrazepam with Ki values in the 6 to 100 nanomolar range. These new substances are active in conflict tests in rats and monkeys and prevent metrazol induced seizures in vivo, but strikingly lack the ataxia and sedative properties of the benzodiazepines. Hill analyses of dose-response curves for some of these substances yields Hill coefficients in the range of 0.4--0.6, suggesting that these compounds may be able to discriminate between several types of benzodiazepine receptors.     

Effect of tributyltin on the N-methyl-D-aspartate (NMDA) receptors in the mouse brain

The purpose of this study was to determine the effect of tributyltin chloride (TBTCI) on the NMDA receptor by in vitro and in vivo experiments. In the first in vitro experiment, the binding of [3H]MK-801 and of [3H]-CGP39653 were studied in membrane preparations from the cerebral cortex of intact mice to obtain control values. Saturation experiments for [3H]MK-801 and [3H]CGP39653 revealed single binding sites with Kd values of 10.27 and 37.8 nM, and receptor densities of 1.75 and 2.20 pmol/mg of protein, respectively. In the second in vitro experiment, displacement studies were carried out with TBTCI over a concentration range of 0.1 microM to 2 mM. TBTCI inhibited [3H]MK-801 binding but did not affect [3H]CGP39653 binding. In the in vivo experiments, the mice received 1-125 ppm TBTCI in the diet ad libitum for 30 days. Ligand binding to cortical membrane preparations from each mouse was measured by a one-concentration point (2 nM) binding assay. [3H]MK-801 binding was significantly lowered (P < 0.05) in the 5 and 125 ppm TBTCl-exposed animals compared with the controls. [3H]CGP39653 binding was also significantly lowered (P<0.05) in the 1 and 125 ppm TBTCI-exposed animals compared with the controls. These results suggest that the NMDA receptors in the mouse brain are sensitive to relatively low level exposure to TBTCl.     

Multiple signaling pathways involved in stimulation of osteoblast differentiation by N-methyl-D-aspartate receptors activation in vitro

Aim:

Glutamate receptors are expressed in osteoblastic cells. The present study was undertaken to investigate the mechanisms underlying the stimulation of osteoblast differentiation by N-methyl-D-aspartate (NMDA) receptor activation in vitro.

Methods:

Primary culture of osteoblasts was prepared from SD rats. Microarray was used to detect the changes of gene expression. The effect of NMDA receptor agonist or antagonist on individual gene was examined using RT-PCR. The activity of alkaloid phosphotase (ALP) was assessed using a commercial ALP staining kit.

Results:

Microarray analyses revealed that 10 genes were up-regulated by NMDA (0.5 mmol/L) and down-regulated by MK801 (100 μmol/L), while 13 genes down-regulated by NMDA (0.5 mmol/L) and up-regulated by MK801 (100 μmol/L). Pretreatment of osteoblasts with the specific PKC inhibitor Calphostin C (0.05 μmol/L), the PKA inhibitor H-89 (20 nmol/L), or the PI3K inhibitor wortmannin (100 nmol/L) blocked the ALP activity increase caused by NMDA (0.5 mmol/L). Furthermore, NMDA (0.5 mmol/L) rapidly increased PI3K phosphorylation, which could be blocked by pretreatment of wortmannin (100 nmol/L).

Conclusion:

The results suggest that activation of NMDA receptors stimulates osteoblasts differentiation through PKA, PKC, and PI3K signaling pathways, which is a new role for glutamate in regulating bone remodeling.     

Subunit-specific roles of glycine-binding domains in activation of NR1/NR3 N-methyl-D-aspartate receptors

N-Methyl-D-aspartate receptors (NMDARs) composed of NR1 and NR3 subunits differ from other NMDAR subtypes in that they require glycine alone for activation. However, little else is known about the activation mechanism of these receptors. Using NMDAR glycine-site agonists/antagonists in conjunction with functional mutagenesis of the NR1 and NR3 ligand-binding cores, we demonstrate quite surprisingly that agonist binding to NR3 alone is sufficient to activate a significant component of NR1/NR3 receptor currents. Thus, the apo conformation of NR1 in NR1/NR3 receptors is permissive for receptor activation. Agonist-bound NR1 may also contribute to peak NR1/NR3 receptor currents but specifically enables significant NR1/NR3 receptor current decay under the conditions studied here, pre-sumably via a slow component of desensitization. Ligand studies of NR1/NR3 receptors also suggest differential agonist selectivity between NR3 and NR1, as some high-affinity NR1 agonists only minimally activate NR1/NR3 receptors, whereas other NR1 agonists are as potent as glycine. Furthermore, liganded NR3 subunits seem necessary for effective engagement of NR1 in NR1/NR3 receptor activation, suggesting significant interactivity between the two subunits. NR3 subunits thus induce plasticity in NR1 with respect to subunit assembly and ligand binding/channel coupling that is unique among ligand-gated ion channel subunits.     

Bradykinin analogues: differential agonist and antagonist activities suggesting multiple receptors.

Bradykinin analogues with specific antagonist activity in several bioassays were evaluated for effects on [3H]-bradykinin receptor binding sites and inositol phosphate production in neuroblastoma N1E-115 cells. The analogues varied in their affinities for bradykinin receptors in guinea-pig ileum and N1E-115 cell membranes, in their effects on uterine and ileal contractions and in their agonist or antagonist activity on phosphoinositide turnover in N1E-115 cells. These tissue specific effects suggest the presence of multiple bradykinin receptor subtypes.     

Differential activation and trafficking of micro-opioid receptors in brain slices

The activation of G protein-coupled receptors results in a cascade of events that include acute signaling, desensitization, and internalization, and it is thought that not all agonists affect each process to the same extent. The early steps in opioid receptor signaling, including desensitization, have been characterized electrophysiologically using brain slice preparations, whereas most previous studies of opioid receptor trafficking have been conducted in heterologous cell models. This study used transgenic mice that express an epitope-tagged (FLAG) micro-opioid receptor (FLAGMOR) targeted to catecholamine neurons by regulatory elements from the tyrosine hydroxylase gene. Brain slices from these mice were used to study tagged MOR receptors in neurons of the locus ceruleus. Activation of the FLAGMOR with [Met5]enkephalin (ME) produced a hyperpolarization that desensitized acutely to the same extent as native MOR in slices from wild-type mice. A series of opioid agonists were then used to study desensitization and receptor trafficking in brain slices, which was monitored with a monoclonal antibody against the FLAG epitope (M1) conjugated to Alexa 594. Three patterns of receptor trafficking and desensitization were observed: 1) ME, etorphine, and methadone resulted in both receptor desensitization and internalization; 2) morphine and oxymorphone caused significant desensitization without evidence for internalization; and 3) oxycodone was ineffective in both processes. These results show that two distinct forms of signaling were differentially engaged depending on the agonist used to activate the receptor, and they support the hypothesis that ligand-specific regulation of opioid receptors occurs in neurons maintained in brain slices from adult animals.     

An endogenous modulator of N-methyl-D-aspartate receptor-coupled glycine receptors

Extensive washing of a membrane preparation from rat brain resulted in a progressive enhancement of strychnine-insensitive [3H]glycine binding, which was due to an increase in the number of binding sites with no changes in the apparent affinity of this radioligand, precluding an explanation based solely on the elimination of endogenous glycine. Moreover, after extensive washing a population of [3H]glycine binding sites with very high affinity for L-serine was observed in addition to the sites with low affinity for L-serine present in less extensively washed tissue. The observed changes in [3H]glycine binding were attributable to the elimination of a low molecular weight, heat-stable compound which was readily detected in the wash supernatant. Extensive washing also altered [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohept-5,10-i min e maleate ([3H]MK-801) binding to N-methyl-D-aspartate (NMDA) receptor-associated channels, decreasing basal binding at equilibrium and producing slower association rates in the presence of either glycine or L-glutamate. Moreover, in well-washed membranes both glycine and glutamate enhanced [3H]MK-801 binding acting at high- and low-affinity sites. These findings suggest that the NMDA receptor complex can assume interconverting conformational states regulated by an endogenous substance(s).     

Modulation of solitarial deglutitive N-methyl-D-aspartate receptors by dihydropyridines

The ability of the dihydropyridine calcium channel activators, (-)-S-BAY K 8644 and (+)-S-202-791 and the calcium channel inhibitor, (+)-R-BAY K 8644, to modify the differential deglutitive actions of glutamate and muscarine at premotor loci in the nucleus tractus solitarii was investigated in urethane-anaesthetised rats. At subnuclei ventralis and intermedialis loci, pneumophoretic application (20-100 pl) from multibarrelled glass micropipettes (tip diameter 2-5 microns) of glutamate (10-20 pmol) evoked aminophosphonovaleric acid (APV)-insensitive pharyngeal swallows; at sites in the subnucleus centralis of the nucleus tractus solitarii glutamate evoked an APV-sensitive single-wave oesophageal response, whereas muscarine (5-10 pmol) evoked rhythmic oesophageal contractions. Both (-)-S-BAY K 8644 and (+)-S-202-791, applied in prepulses of 10-20 fmol and 100-200 fmol, respectively, either had no effect or selectively and reversibly enhanced or inhibited the glutamate-evoked responses. Identical results were obtained by intravenous administration of (-)-S-BAY K 8644 (10-50 micrograms/kg). Micropneumophoretic (20-50 fmol) or intravenous (10-50 micrograms/kg) administration of (+)-R-BAY K 8644 suppressed the N-methyl-D-aspartate (NMDA)-mediated oesophageal responses in a reversible and selective manner. The dihydropyridine vehicle produced a transient depression of all types of deglutitive responses. It is concluded that, within the deglutitive subnuclei of the nucleus tractus solitarii, "L"-type voltage-operated calcium channels are associated with NMDA-receptor-mediated deglutitive mechanisms. The inhibition or a lack of effect produced by the dihydropyridine calcium channel activators is explained in part by their actions at other sites e.g. release of inhibitory transmitters.     

Antagonism of stimulation-produced analgesia by naloxone and N-methyl-D-aspartate: role of opioid and N-methyl-D-aspartate receptors

The present study aims to investigate the influence of electrical stimulation of periaqueductal gray (PAG) following peripheral nerve injury and its modulation by naloxone and N-methyl-D-aspartate (NMDA). Chronic neuropathic pain was induced by chronic constriction injury of the sciatic nerve, and subsequently a cannula was implanted in the PAG area for the purpose of electrical stimulation and intra-PAG drug administration. Intra-PAG administration of morphine, ketamine, and their combination were found to elicit antinociceptive response on hot-plate test. Electrical stimulation of PAG was also observed to demonstrate decreased pain response on hot-plate test, and this effect was reversed by the administration of naloxone, NMDA, and their combination, when injected into the PAG area. These findings suggest that apart from the opioid receptors, probably NMDA receptors also have a role to play in stimulation-produced analgesia.