N-methyl-D-aspartate Receptor-ionophore Complex Research Articles

Human retina contains polyamine sensitive [3H]-ifenprodil binding sites: implications for neuroprotection?

AIMS This study characterised the pharmacology of [3H]-ifenprodil binding to the polyamine binding sites (PBS) on the N-methyl-d-aspartate (NMDA) receptor channel complex on human retinas. These data were correlated with...

Ethanol inhibition of N-methyl-D-aspartate-activated current in mouse hippocampal neurones: whole-cell patch-clamp analysis.

1. The action of ethanol on N-methyl-D-aspartate (NMDA)-activated ion current was studied in mouse hippocampal neurones in culture using whole-cell patch-clamp recording. 2. Ethanol inhibited NMDA-activated current in a voltage-independent manner, and did not alter the reversal potential of NMDA-activated current. 3. Concentration-response analysis of NMDA- and glycine-activated current revealed that ethanol decreased the maximal response to both agonists without affecting their EC50 values. 4. The polyamine spermine (1 microM) increased amplitude of NMDA-activated current but did not alter the percentage inhibition of ethanol. 5. Compared to an extracellular pH of 7.0, pH 6.0 decreased and pH 8.0 increased the amplitude of NMDA-activated current, but these changes in pH did not significantly alter the percentage inhibition by ethanol. 6. The sulphydryl reducing agent dithiothreitol (2 mM) increased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. 7. Mg2+ (10, 100, 500 microM), (5, 20 microM) or ketamine (2, 10 microM) decreased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. 8. The observations are consistent with ethanol inhibiting the function of NMDA receptors by a non-competitive mechanism that does not involve several modulatory sites on the NMDA receptor-ionophore complex.

Glutathione is an endogenous ligand of rat brain N-methyl-D-aspartate (NMDA) and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors.

A study was made of the effects of reduced (GSH) and oxidized (GSSG) glutathione on the Na(+)-independent and N-methyl-D-aspartate (NMDA) displaceable bindings of glutamate, on the binding of kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ligand of the brain NMDA receptor-ionophore complex: glycine, dizocilpine (MK-801) and (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP). GSH and GSSG strongly inhibited the binding of glutamate, CPP and AMPA, kainate and glycine binding being less affected. Both peptides enhanced the binding of dizocilpine in a time- and concentration-dependent manner. This activatory effect was not additive to that of saturating concentrations of glutamate or glutamate plus glycine. The activation of dizocilpine binding by GSH and GSSG was prevented by the competitive NMDA and glycine antagonists DL-2-amino-5-phosphonovalerate and 7-chlorokynurenate. GSH and GSSG may be endogenous ligands of AMPA and NMDA receptors, binding preferably to the glutamate recognition site via their gamma-glutamyl moieties. In addition to this, at millimolar concentrations they may regulate the redox state of the NMDA receptor-ionophore complex.

Supporting evidence for negative modulation by protons of an ion channel associated with the N-methyl- D-aspartate receptor complex in rat brain using ligand binding techniques

The addition of L-glutamic acid (Glu) alone, both Glu and glycine (Gly) or Glu/Gly/spermidine (SPD) was effective in potentiating[ 3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding before equilibrium to an ion channel associated with the N-methyl- D-aspartate (NMDA) receptor complex in brain synaptic membranes extensively washed and treated with Triton X-100. The binding dependent on Glu almost linearly increased in proportion to decreasing proton concentrations at a pH range of 6.0 to 9.0 in external incubation medium, while a Gly-dependent portion of the binding increased with decreasing proton concentrations up to a pH of 7.5 with a plateau thereafter. In contrast, the SPD-dependent binding increased in proportion to decreasing proton concentrations up to a pH of 7.0 with a gradual decline thereafter. Similar profiles were also obtained with [ 3H]MK-801 binding at equilibrium, with an exception that significant binding of [ 3H]MK-801 was detected in the absence of any added agonists. The potency of SPD to potentiate [ 3H]MK-801 binding before equilibrium increased in proportion to decreasing proton concentrations, with those of both Glu and Gly being unchanged. In contrast, the ability of (+)MK-801 to displace [ 3H]MK-801 binding at equilibrium was not significantly affected by a decrement of external proton concentrations from pH 7.5 to pH 8.5 in the presence of Glu/Gly and Glu/Gly/SPD added. However, similar changes in external proton concentrations did not similarly affect binding of several radioligands for the NMDA and Gly domains on the receptor complex. Decreasing proton concentrations were effective in exponentially potentiating binding of [ 3H]SPD at a pH range of 6.0 to 9.0 without virtually altering [ 3H] D, L-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid binding. In addition, [ 3H]kainic acid binding markedly decreased with decreasing proton concentrations only in the presence of Ca 2+ ions. These results suggest that protons negatively modulate neuronal responses mediated by the NMDA receptor ionophore complex through interference with opening mechanisms of the channel domain without disturbing association processes of the endogenous agonists with the respective recognition domains in rat brain. Moreover, possible modulation by protons of responses mediated by the kainate receptor in the presence of Ca 2+ ions at concentrations that occur in vivo is also suggested.

The effects of BTS 54,505, a metabolite of sibutramine, on monoamine and excitatory amino acid-evoked responses in the rat dorsolateral geniculate nucleus in vivo.

1. The effects of BTS 54,505, the primary amine metabolite of the non-tricylic putative antidepressant sibutramine, on the responses evoked by visual stimulation and ionophoretic application of noradrenaline (NA), 5-hydroxytryptamine (5-HT) and excitatory amino acids (EAAs) in the rat dorsolateral geniculate nucleus (dLGN) have been investigated. 2. Ionophoretic application of 5-HT to dLGN neurones attenuated visually-evoked (n = 46), NMDA-evoked (n = 21) and AMPA-evoked responses (n = 21), while ionophoretic application of NA potentiated visually-evoked activity in these cells (n = 27). 3. Simultaneous application of BTS 54,505 with 5-HT (over 120 s) resulted in a prolongation of the recovery time (i.e. the period required by a neurone to recover by 50%, RT50) from the 5-HT-mediated suppression of discharge activity (approximately 275% increase in RT50). BTS 54,505 also prolonged the recovery time from a NA-mediated potentiation of firing (approximately 450% increase in RT50). These effects on recovery time are attributed to the inhibition of uptake of both 5-HT and NA by BTS 54,505. The amplitude of the response to 5-HT or NA was unaffected by co-ejection of BTS 54,505. 4. Ionophoretic application of N-methyl-D-aspartate (NMDA) produced a current-dependent increase in neuronal firing, as did application of the non-NMDA receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). A simultaneous 120 s application of BTS 54,505 inhibited the NMDA response in all cells studied (mean ED50 = 16 +/- 5 nA) but had no effect on AMPA-evoked activity in the majority of the same cells (n = 15/21).5. Short 10 s applications of BTS 54 505, at ejecting currents (>30 nA) that attenuated NMDA-evoked activity in all cells studied, had no effect on either response amplitude or recovery time from ionophoretic application of 5-HT, suggesting that inhibition of NMDA-evoked activity by BTS 54 505 was not mediated by 5-HT uptake blockade.6. These results suggest that BTS 54 505 inhibits NMDA-evoked activity, and the observation that this effect is unlikely to be due to raised levels of endogenous 5-HT following monoamine uptake blockade indicate that BTS 54 505 may interact directly with the NMDA receptor ionophore complex.

Excitatory amino acid receptor binding in hippocampus of gerbils with transient global brain ischemia

Binding of a variety of ligands for brain excitatory amino acid receptors was examined in membrane preparations extensively washed and treated with Triton X-100 that were obtained from the hippocampus and cerebral cortex of gerbils that survived for different periods after transient global brain ischemia. Bilateral occlusion of the carotid arteries for 5 min did not affect the binding of [ 3H]( + )-5-methyl-10,11-dihydro-5 H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) to an open ion channel associated with the N-methyl- d-aspartate (NMDA)-sensitive subclass in both central structures of gerbils that survived for 1 to 4 weeks after the injury when determined at equilibrium in the presence of 3 different endogenous agonists including l-glutamic acid (Glu), glycine (Gly) and spermidine at maximally effective concentrations. In contrast, the ischemic occlusion significantly diminished [ 3H]MK-801 binding when determined before equilibrium in the presence of the 3 stimulants in hippocampal membranes without altering that in cortical membranes 2 weeks after the insult, so that the initial association rates were invariably reduced by more than 60%. Moreover, the occlusion not only reduced the binding of both [ 3H]Glu and [ 3H] d,l-( E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid to the NMDA domain on the NMDA receptor ionophore complex, but also decreased the binding of both [ 3H]Gly and [ 3H]5,7-dichlorokynurenic acid to the Gly domain. However, the insult did not induce any detectable changes under the experimental conditions employed in either the binding of [ 3H] dl-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) to the AMPA-sensitive subclass or the binding of kainic acid (KA) to the KA-sensitive subclass in both central regions of animals that survived for 2 weeks. These results suggest that transient global brain ischemia may predominantly impair neuronal and/or glial cells enriched of the NMDA receptor ionophore complex in gerbil hippocampus.

Comparative studies on binding of 3 different ligands to the N-methyl- d-aspartate recognition domain in brain synaptic membranes treated with Triton X-100

Treatment with a low concentration of Triton X-100 almost tripled the binding of [ 3H] d,l-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653), a novel competetive antagonist at an N-methyl- d-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, in synaptic membranes of the rat brain. The binding linearly increased with increasing protein concentrations of up to 0.4 mg/ml and also increased in proportion to incubation time with a plateau within 60 min after the initiation of incubation at 2°C in Triton-treated membranes. Elevation of incubation temperature from 2°C to 30°C resulted in a marked decrease in the binding at equilibrium by 80%, and a maximal level was obtained within 1 min after the initiation of incubation at 30°C with a gradual decline of up to 10 min. Bound [ 3H]CGP 39653 was rapidly dissociated by the addition of excess unlabeled l-glutamic acid (Glu), and the time required to attain complete dissociation was 60 min at 2°C and 1 min at 30°C, respectively. Among several agonists and antagonists tested, Glu was the most potent displacer of [ 3H]CGP 39653 binding with progressively less potent displacement by d-2-amino-5-phosphonovaleric,(±)-3-(2-carboxypiperain-4-yl)propyl-1-phosphonic (CPP), d-2-amino-7-phosphonoheptanoic, N-methyl- d-aspartic and N-methyl- l-aspartic acids. Agonists at the glycine (Gly) domain on the NMDA receptor ionophore complex invariably inhibited the binding of [ 3H]CGP 39653 without virtually affecting the binding of [ 3H]CPP or [ 3H]Glu in a manner that was sensitive to antagonism by 4 different antagonists at the Gly domain. In contrast, the Gly antagonist 1-hydroxy-3-aminopyrrolidone-2 markedly potentiated [ 3H]CPP binding with the binding of both [ 3H]CGP 39653 and [ 3H]Glu being unaltered. These results suggest that [ 3H]CGP 39653 may predominantly label an antagonist-preferring form of the NMDA domain in a state different from that favorable to labeling by [ 3H]CPP.

Arcaine and magnesium inhibition of the NMDA receptor ionophore complex: evidence for distinct voltage-dependent sites.

The inhibitory effects of the polyamine antagonist, arcaine, and magnesium on N-methyl-D-aspartate (NMDA) induced hippocampal [3H]norepinephrine release and [piperidyl-3,4-3H(N)]-[N-1-(2- thienyl)cyclohexyl]-3,4-piperidine (TCP) binding were studied. We report that the inhibitory effect of arcaine and magnesium on NMDA-induced [3H]norepinephrine release is diminished by increasing the extracellular K+ concentration, presumably reflecting a voltage-dependent block for both. However, unlike MK-801, the block by arcaine shows no evidence of use dependence. Further, the IC50 value for magnesium inhibition of [piperidyl-3,4-3H(N)]TCP binding varies with the state of activation of the channel, being the lowest when the channel is maximally activated and the highest when the channel is least activated. On the other hand, the apparent affinity of arcaine is not significantly affected by the activation of the channel by glutamate and glycine, but is decreased by the polyamine agonist, spermidine. These data suggest that the polyamine antagonist binding site is distinct from either the phencyclidine/MK-801 site or the voltage-dependent channel site for magnesium. Nonetheless, these data suggest that the site must be located in a region of the NMDA receptor ionophore complex capable of sensing transmembrane potential.

Identification and characterization of specific binding sites of [ 3H]spermidine in synaptic membranes of rat brain

Synaptic membranes of rat brain contained specific binding sites of [ 3H]spermidine (SPD) that exhibited an inverse temperature dependency, structure selectivity, reversibility, saturability, low affinity and high density with an uneven distribution profile. The affinities were not significantly different from each other in the rodent brain, while the highest density was found in the medulla-pons among the central structures examined with progressively lower densities in the midbrain, striatum, cerebellum, hypothalamus, hippocampus and cerebral cortex. The binding was insensitive to digestion by various proteases and glycosidases but sensitive to potentiation by phospholipases. A clear correlation was seen between the abilities of several natural and synthetic polyamines to displace [ 3H]SPD binding and to potentiate [ 3H] (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine binding to open cation channels associated with an N- methyl- d-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors. Treatment of brain membranes with deoxycholic acid resulted in a significant solubilization of [ 3H]SPD binding sites. Furthermore, [ 3H]SPD markedly associated with the acidic phospholipid phosphatidylserine irrespective of the presence of synaptic membranes in a manner sensitive to inhibition by a variety of calmodulin antagonists. These results suggest that endogenous polyamines may play a stimulatory role in neuronal responses mediated by the NMDA receptor ionophore complex through an interaction between their positive charges and negative charges of membranous phosphatidylserine in rat brain.

Alcohol-induced inhibition of [formula omitted] release of [ 3H]norepinephrine from brain is related to lipophilicity

A series of short chain alkanols were studied for their effects on N- methyl- d-aspartate (NMDA)-evoked release [ 3H]norepinephrine (NE) from slices of cortex of the rat. Methanol, ethanol, isopropanol, n-propanol, n-butanol and isoamyl alcohol inhibited NMDA-stimulated release of [ 3H]NE in a concentration-dependent manner, while having no effect on non-stimulated release of [ 3H]NE. The inhibitory potencies of the alkanols varied with the shorter chain alkanols, such as methanol, being less potent than the longer chain alkanols, such as isoamyl alcohol. Direct comparison of the effects of 100 mM methanol, ethanol, isopropanol, n-propanol and n-butanol indicated that increasing the chain length led to a greater efficacy for producing inhibition of NMDA-stimulated release of [ 3H]NE. A plot of the log IC 50 values, versus the log of the membrane/buffer partition coefficients for the various alkanols was linear, indicating that lipophilicity played some role in the inhibitory effect. The alkanols did not significantly depress the release of [ 3H]NE stimulated by 25 mM KCl, in the presence of 300 μM 2-amino-5-phosphonopentanoic acid (AP-5), suggesting that the alkanols have a selective effect at the NMDA receptor, as opposed to altering the release of neurotransmitter at the nerve terminal. The inhibitory effects of the alcohols were reversible, which suggests that the alcohols were not causing non-specific toxic effects on the slices. It is concluded that the inhibitory effect of ethanol and related short chain alkanols on NMDA-stimulated release of [ 3H]NE involves an interaction with a lipophilic target, at or near the NMDA receptor-ionophore complex.

Solubilization of Spermidine‐Sensitive (+)‐[3H]5‐Methyl‐10,11 ‐Dihydro‐5H‐Dibenzo[a,d]cyclohepten‐5,10‐Imine ([3H]MK‐801) Binding Activity from Rat Brain

The receptor-ionophore complex of the N-methyl-D-aspartate (NMDA)-sensitive receptor was solubilized by deoxycholic acid from rat brain using (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne ([3H]MK-801) binding as a marker for the receptor. Gel filtration of the solubilized preparations on a Sephadex G-25 column revealed significant [3H]MK-801 binding sensitive to potentiation by glutamate and glutamate/glycine, which was prevented by competitive antagonists for the NMDA and strychnine-insensitive glycine (GlyB) sites. In contrast to NMDA and glycine, spermidine markedly potentiated the amount of [3H]MK-801 binding in solubilized preparations by increasing the apparent affinity of the ligand. In the presence of all three stimulants, the solubilized preparations exhibited pharmacological profiles similar to those in the membrane preparations. These results clearly indicate that the whole macromolecular NMDA receptor-ionophore complex is solubilized under the experimental conditions used.

A comparison of the anticonvulsant effects of competitive and non-competitive antagonist of the [formula omitted] receptor

The anticonvulsant activity of two competitive antagonist of the N- methyl- d-aspartate (NMDA) receptor, 2-amino-7-hosphonoheptanoic acid (APH) and 3-[2-carboxypiperazin-4-yl]-propyl-1-phosphonate (CPP), and two non-competitive NMDA antagonists, phencyclidine (PCP) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), were compared in 4 models of induced seizures in mice. All 4 drugs protected against tonic extensor seizures induced by pentylenetetrazol (PTZ), by submaximal (15 mA) electroconvulsive shock (ECS) and by maximal (50 mA) ECS. Similar orders of potency (i.e., MK-801 > PCP ≥ CPP > APH) were seen in each of the 3 seizure models. All 4 drugs failed to block clonic seizures induced by picrotoxin in the dose ranges that protected from tonic seizures. These data are consistent with other data demonstrating that competitive and non-competitive NMDA antagonists have similar pharmacologic effects. These results also support the suggestion that the anticonvulsant effects of competitive and non-competitive NMDA antagonists are mediated by the NMDA receptor-ionophore complex.

Inhibition of N-methyl-D-aspartate- and kainic acid-induced neurotransmitter release by omega-conotoxin GVIA.

1. The role of voltage-sensitive calcium channels (VSCC) in N-methyl-D-aspartate (NMDA)- and kainic acid (KA)-evoked neurotransmitter release from rat cortical and hippocampal brain slices was evaluated by determining the effects of omega-conotoxin GVIA, an inhibitor of neuronal L- and N-type VSCC, and PN 200-110, a selective inhibitor of L-type VSCC. 2. Selective antagonists of the NMDA receptor ionophore complex, Mg2+, CPP and MK-801, inhibited NMDA- but not KA-evoked release of [3H]-noradrenaline from hippocampal and cortical brain slices. This suggests that cortical and hippocampal receptors are similar and that NMDA and KA act at distinct excitatory amino acid receptor subtypes. 3. [3H]-noradrenaline release induced by both NMDA and KA was similarly inhibited (approximately 30%) by omega-conotoxin GVIA. In contrast, PN 200-110 had no significant effect, although there was a tendency towards inhibition. 4. The results suggest that although NMDA- and KA-receptors are pharmacologically distinct, the N-type, but not the L-type, VSCC plays a small but significant role in neurotransmitter release induced by both NMDA and KA. It remains to be determined whether the N-type VSCC are involved in the physiological and/or pathological manifestations of excitatory amino acid receptor stimulation.

Interaction of phencyclidine with voltage-dependent potassium channels in cultured rat hippocampal neurons: comparison with block of the NMDA receptor-ionophore complex

Whole-cell voltage-clamp recording techniques were used to investigate the blockade of voltage-dependent K+ channels by phencyclidine (PCP) in cultured rat hippocampal neurons. All recordings were carried out in the presence of tetrodotoxin (1-2 microM) to eliminate Na+ currents. Step depolarization from a holding potential of -40 mV activated a slowly rising, minimally inactivating K+ current (IK). PCP (0.5-1000 microM) caused a reduction in the maximum conductance of IK [IC50(+30 mV), 22 microM] without altering its voltage dependency. The PCP block of IK diminished at depolarized potentials. Analysis according to the scheme of Woodhull (1973) suggested that block occurs via binding to an acceptor site (presumably within the channel pore) that senses 40-50% of the transmembrane electrostatic field. PCP had no effect on the kinetic properties of IK and the block failed to show use dependency, suggesting that PCP may bind to the IK channel via a hydrophobic mechanism not requiring open channels. For comparison, we also investigated the effect of PCP on the transient K+ current, IA, activated by step depolarization following a 200 msec prepulse to -90 mV (20 mM tetraethylammonium was present in the bathing solution to reduce IK). In contrast to the potent blocking action of PCP on IK, the drug only affected IA at high concentrations [IC50(+30 mV), 224 microM]. At concentrations causing substantial block (300-500 microM), PCP produced an acceleration in the IA inactivation rate, and, for brief (5-6 msec) depolarizing steps, the suppression of IA was use dependent. These observations suggest that PCP block of IA requires open channels. PCP reduced inward current responses induced by the excitatory amino acid agonist N-methyl-D-aspartate (NMDA) at substantially lower concentrations than those required for its effects on K+ channels [IC50(-60 mV), 0.45 microM]. The PCP-like dioxadrol stereoisomer dexoxadrol (10 microM) blocked NMDA-evoked inward current responses, while its behaviorally inactive enantiomer levoxadrol did not. Dexoxadrol and levoxadrol also blocked IK in a stereoselective fashion (IC50's, 73 and 260 microM, respectively), whereas the sigma ligands (+)- and (-)-SKF 10,047 and (+)-3-[3-hydroxyphenyl]-N-(1-propyl)piperidine [(+)-3-PPP] had little effect on the current (IC50's, greater than 300-500 microM). We conclude that PCP causes a selective, voltage-dependent block of IK in hippocampal neurons via a PCP- and not a sigma-type acceptor site.(ABSTRACT TRUNCATED AT 400 WORDS)

NMDA Antagonists Differentiate Epileptogenesis from Seizure Expression in an in Vitro Model

In an electrographic model of seizures in the hippocampal slice, both of the N-methyl-D-aspartate (NMDA) antagonists 2-amino-5-phosphonovaleric acid and 5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate (MK-801) prevented the progressive development of seizures but did not block previously induced seizures. Thus, a process dependent on the NMDA receptor-ionophore complex establishes a long-lasting, seizure-prone state; thereafter the seizures depend on non-NMDA receptor-ionophore mechanisms. This suggests that there is an important distinction between epileptogenesis and seizure expression and between antiepileptogenic and anticonvulsant pharmacological agents.

Formula omitted] promotes the survival of cerebellar granule cells: Pharmacological characterization

The survival of cerebellar granule cells in culture is promoted by chronic exposure to N- methyl- d-aspartate (NMDA). The effect is due to the stimulation of ‘conventional’ NMDA receptor-ionophore complex: it is concentration dependent, voltage dependent and blocked by the selective antagonists d-2-amino-5-phosphonovalerate, d-2-amino-7-phosphonoheptanoate, dextromethorphan and (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-iminemaleate (MK 801). The most potent antagonist tested was MK-801. In contrast, non-selective antagonists, including kynurenate, were much less effective. Further, the trophic effect of NMDA is not reproduced by ibotenate or quinolinate at the concentration range tested. It could also be shown that glutamate released into the culture medium is responsible for limited cell survival in the absence of NMDA.

Effects of nicotinic agonists and antagonists on N‐methyl‐D‐aspartate‐induced 3H‐norepinephrine release and 3H‐(1‐[1‐(2‐thienyl)cyclohexyl]‐piperidine) binding in rat hippocampus

The nicotinic agonists dimethylphenylpiperazinium iodide (DMPP) and carbachol (CARB) as well as (-)nicotine [-)NIC) were tested alone and in combination with N-methyl-D-aspartate (NMDA) for their abilities to enhance the efflux of 3H-norepinephrine (NE) from slices of rat hippocampus. CARB and (-)NIC produced small, transient increases in NE efflux, while DMPP produced larger, longlasting increases. Inasmuch as the nicotinic antagonists mecamylamine (MECA) and hexamethonium (C6) did not consistently inhibit the increases in NE efflux produced by these agonists, the role of a nicotinic receptor in mediating these responses is uncertain. CARB and DMPP enhanced the ability of NMDA to stimulate NE release, while (-)NIC did not. MECA, but not C6, was found to selectively antagonize NMDA-stimulated NE release that did not appear to involve a nicotinic receptor. Binding studies indicated that MECA and the related nicotinic antagonist pempidine produced an inhibition of NMDA-stimulated NE release by an action at the PCP receptor that is known to be linked to the NMDA receptor-ionophore complex. These data suggest that the actions of these ganglionic blocking agents on excitatory responses in the hippocampus involve inhibition of excitatory amino acid as well as nicotinic receptors.