Ethanol inhibits N-methyl-D-aspartate-stimulated [3H]norepinephrine release from rat cortical slices

The effects of ethanol on N-methyl-D-aspartate (NMDA)-stimulated [3H]norepinephrine (NE) release from rat cortical slices was studied. NMDA-stimulated [3H]NE release was inhibited by tetrodotoxin, Mg++ and 2-amino-5-phosphonopentanoic acid, indicating that NMDA receptors in the cortex have characteristics similar to those observed using electrophysiological studies. Ethanol (60-200 mM) decreased the release of [3H]NE evoked by 100 microM NMDA in a concentration-dependent manner (32-52% inhibition), but it did not significantly alter the basal release. The inhibitory effect of 100 mM ethanol was due to a reduction in the maximal response with no significant change in the EC50 for NMDA. Pretreatment of the slices with 100 mM ethanol up to 6 min did not alter the magnitude of inhibition. The inhibition of NMDA-stimulated [3H]NE release due to ethanol was reversible after a 13-min recovery period. The presence of ethanol did not significantly affect the IC50 for Mg++ inhibition of NMDA-stimulated [3H]NE release (23 +/- 3 microM). Glycine (10-300 microM) potentiated the release of [3H]NE stimulated by 250 microM NMDA, and 60 mM ethanol did not alter this effect of glycine. Ethanol (100 mM) inhibited the release of [3H]NE evoked by 18.9 mM KCl in the presence or absence of 2-amino-5-phosphonopentanoic acid, but had no effect on release induced by 49.1 mM KCl. Tetrodotoxin (0.3 mM) significantly decreased the release of [3H] NE evoked by 23.2 mM KCl, and 60 to 200 mM ethanol did not alter this release. These results suggest that NMDA receptors in rat cortical slices are located on nerve cell bodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-methyl-D-aspartic acid (NMDA) and non-NMDA receptors regulating hippocampal norepinephrine release. II. Evidence for functional cooperation and for coexistence on the same axon terminal.

The possible interactions between activation of N-methyl-D-aspartic acid (NMDA) receptors and non-NMDA receptors regulating the release of [3H]norepinephrine [( 3H]NE) have been investigated in superfused synaptosomes from rat hippocampus. NMDA--at a concentration (100 microM) which, in a medium containing 1.2 mM Mg++ ions, did not evoke [3H]NE release--acquired releasing activity in the presence of equimolar concentrations of quisqualic acid (QA), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. The [3H] NE release evoked by NMDA plus QA in the presence of Mg++ ions was Ca(++)-dependent, partly tetrodotoxin-sensitive, inhibited by clonidine but insensitive to desipramine. The NMDA receptor antagonists D-2-amino-5-phosphonopentanoic acid (D-AP5) and (+)-5-methyl-10,11-dihydro-5-H-dibenzo[a,d]cycloepten-5,10-imine (MK-801) antagonized the NMDA-induced [3H]NE release in Mg(++)-free medium; the IC50 values amounted, respectively, to 81.4 microM and to 1.11 microM. When NMDA was tested in the presence of QA and Mg++ ions, the affinity of D-AP5 was enormously increased (IC50 = 40 nM; i.e., more than 6 orders of magnitude); the affinity of MK-801 was found to be augmented by 350-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

CPP, a selective N-methyl-D-aspartate (NMDA)-type receptor antagonist: characterization in vitro and in vivo

3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) was synthesized as a rigid analog of 2-amino-7-phosphonoheptanoate, a previously known antagonist at the N-methyl-D-aspartate (NMDA) preferring, or NMDA-type, of excitatory amino acid receptor. CPP was found to be a potent, selective and competitive antagonist of NMDA-type receptors. CPP antagonized with an IC50 of 8 muM [3H]ACh release which was evoked from rat striatal brain slices by NMDA (50 muM). In contrast, the release of [3H]ACh evoked by elevated KCI was not inhibited by CPP even at a concentration of 100 muM. The antagonism by CPP of NMDA-evoked [3H]ACh release was competitive, with a pA2 of 5.66 for CPP, compared with a pA2 value of 5.22 for 2-amino-7-phosphonoheptanoate. CPP affected neither the uptake of L-[3H]glutamate nor the inhibition by aconitine of L-[3H]glutamate uptake, suggesting a lack of membrane-stabilizing or local anesthetic effects, and also suggesting that CPP itself may not be taken up through the L-glutamate membrane transporter. Moreover, [3H] CPP was not accumulated by synaptosomes (P2 fraction) which avidly accumulate L-[3H]glutamate, supporting the concept that this NMDA-type receptor antagonist acts at an NMDA-type receptor on the external surface of the plasma membrane. CPP (10 muM) failed to interact with any of 21 other putative neurotransmitter receptors including alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding (quisqualate-type receptor) and [3H]kainate binding (kainate-type receptor). Audiogenic convulsions in DBA/2 mice were blocked by CPP (ED50 = 1.5 mg/kg i.p.) as were NMDA-induced seizures in CF-1 mice (ED50 = 1.9 mg/kg i.p.). In both strains, CPP impaired the traction reflex at higher doses (ED50 = 6.8 mg/kg and 6.1 mg/kg and 6.1 mg/kg i.p. for DBA/2 and CF-1, respectively). The traction reflex impairment by CPP may be due to muscle relaxant effects of the compound, an explanation supported by the finding that CPP reduced muscle tone as assessed by electromyogram measurement in animals whose muscle tone had been increased by opiate administration. Finally, cerebellar cyclic GMP levels, known to be sensitive to neurotransmission via NMDA-type receptors, were decreased by CPP (ED50 = 4.7 mg/kg i.p.) in mice. In conclusion, based upon the competitive antagonism by CPP of NMDA-evoked [3H] ACh release in vitro and the antagonism of NMDA-induced convulsions in vivo, the data presented are consistent with competitive antagonism of NMDA-type receptors.     

Heterogeneity of the N-methyl-D-aspartate receptor ionophore complex in rat brain, as revealed by ligand binding techniques.

The polyamine spermidine markedly potentiated the binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) to open ion channels associated with an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, in the presence of a maximally effective concentration of L-glutamic acid (Glu) and glycine (Gly), in Triton-treated preparations of synaptic membranes from the cerebral cortex as well as hippocampus of rat brain, without significantly affecting that in cerebellar synaptic membranes. Among several ligands for the binding sites of [3H]MK-801, the binding was most potently displaced by (+)-MK-801, followed by N-[1-(2-thienyl)cyclohexyl]piperidine, (-)-MK-801, phencyclidine, cyclazocine, ketamine, ketocyclazocine, N-allylnormetazocine and pentazocine, in a rank order of decreasing potency, in hippocampal synaptic membranes. However, the abilities of these ligands to displace the binding were much lower in cerebellar membranes than in hippocampal membranes. Competitive NMDA antagonists induced a much more potent inhibition of [3H] MK-801 binding in the hippocampus than in the cerebellum, whereas competitive Gly antagonists elicited a similarly potent inhibition of the binding in both structures. Moreover, NMDA antagonists caused a greater than 10 times more potent displacement of [3H]Glu binding to the NMDA recognition site in hippocampal membranes than in cerebellar membranes, whereas NMDA agonists similarly displaced [3H]Glu binding in both central regions. Gly agonists elicited an equivalent displacement of strychnine-insensitive [3H]Gly binding in both membrane preparations, whereas Gly antagonists more potently displaced [3H]Gly binding in the hippocampus than in the cerebellum. These results suggest possible heterogeneity of the respective domains within the NMDA receptor ionophore complex, in terms of differential sensitivity to isosteric and allosteric ligands.     

Regulation of [3H]dopamine release from guinea pig striatum by NMDA receptor/channel activators and inhibitors.

Excitatory amino acids, that interact with the N-methyl-D-aspartate (NMDA) receptor stimulate release of [3H]dopamine [3H]DA) from the striatum of the guinea pig and rat in a concentration-dependent manner. DA release was measured in the presence of domperidone and nomifensine to avoid complications associated with autoreceptor alteration of and reuptake of released DA. This release is inhibited by magnesium. Therefore, all experiments were performed in the absence of this ion. The competitive NMDA antagonists D-(-)2-amino-5-phosphonopentanoic acid and 3-[(+-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid and the noncompetitive antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine and phencyclidine also inhibit NMDA-stimulated release. Glycine enhances NMDA-stimulated release and can release [3H]DA in the absence of added NMDA. Release stimulated by glycine alone is not affected by 3-[(+-)-2-carboxypiperazine-4-yl]-propyl-1-phosphonic acid. Conversely, if the glycine antagonist 3-amino-1-hydroxy-2-pyrrolidone or 6-cyano-7-nitroquinoxaline-2,3-dione is included, NMDA elicits less release of [3H]DA. This inhibition can be overcome by increasing the concentration of glycine. The kappa-selective opioid agonist trans-(+-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzene-acetamide is also capable of inhibiting the NMDA-stimulated release of [3H]DA from guinea pig and rat striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological profile of NPC 12626, a novel, competitive N-methyl-D-aspartate receptor antagonist

The novel compound 2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid (NPC 12626) was evaluated for activity in a variety of tests associated with receptors for excitatory amino acids. NPC 12626 failed to inhibit the specific binding of RS-[3H] amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid or [3H] kainic acid to brain membranes in vitro but displaced both agonist and antagonist binding to N-methyl-D-aspartic acid (NMDA) receptors. Like cis-(+/-)-3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid, NPC 12626 competitively blocked NMDA-induced enhancement of [3H]-1-thienylcyclohexyl)piperidine binding. In the voltage-clamped frog oocyte expression system, NPC 12626 was a competitive inhibitor of NMDA-evoked inward current with a pA2 of 6.24. After both i.c.v. or i.p. administration, NPC 12626 was a potent anticonvulsant in the pentylenetetrazol, maximal electroshock and NMDA seizure models. Furthermore, low doses (25 mg/kg) of NPC 12626 given i.v. were effective in preventing damage to the CA1 region of hippocampus in the gerbil model of global ischemia. Unlike the noncompetitive NMDA antagonist, phencyclidine, but like cis-(+/-)-3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid and pentobarbital, NPC 12626 only partially substituted for phencyclidine in a drug discrimination study. The results of the current study indicate that NPC 12626 is a novel, systemically active and competitive NMDA receptor antagonist.     

Characterization of L-glutamate action on the release of endogenous dopamine from the rat caudate-putamen

In the present study the effect of L-glutamic acid (L-Glu), N-methyl-D-aspartic acid (NMDA), kainic acid (KA) and quisqualic acid (QUIS) on the release of endogenous dopamine (DA) from slices of the rat caudate-putamen was investigated. DA was measured by high-performance liquid chromatography coupled to an electrochemical detector. L-Glu, NMDA, KA and QUIS, in the absence of Mg++, produced a dose-related, Ca++-dependent increase in DA release. The order of agonist efficacy was L-Glu greater than NMDA greater than KA = QUIS. D-2-amino-7-phosphonoheptanoic acid (0.5 mM), but not L-2-amino-7-phosphonoheptanoic acid, antagonized the action of L-Glu and NMDA, but did not modify the effect of KA or QUIS. Tetrodotoxin (0.1 microM) partially inhibited the stimulatory effect of KA and QUIS, but not that of L-Glu or NMDA. Mg++ (1.2 mM) abolished the excitatory effect of NMDA, significantly reduced the action of L-Glu, but did not influence the action of KA or QUIS. The inhibitory action of Mg++ on the L-Glu-induced DA release was reversed when L-Glu was coupled to high concentrations of K+. N-allylnormetazocine (SKF-10,047), a benzmorphan agent, produced a stereospecific inhibition of L-Glu-induced DA release. This inhibition was also produced by 1-[1-(2-thienyl)cyclohexyl]piperidine, a phencyclidine receptor ligand, but not by 1,3-di-O-tolylguanidine, a sigma receptor-selective ligand. The results of this study show that L-Glu increases DA release predominantly by activation of the NMDA receptor located presynaptically on dopaminergic afferents.(ABSTRACT TRUNCATED AT 250 WORDS)     

An antagonist/partial agonist at the polyamine recognition site of the N-methyl-D-aspartate receptor that alters the properties of the glutamate recognition site.

The effects of N-(3-aminopropyl)-1,10-diaminodecane (APDA10) on the N-methyl-D-aspartate (NMDA) receptor/ion channel complex were investigated. In the presence of 100 microM glutamate and 100 microM glycine, APDA10 had biphasic effects on the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten5,10-imin e (MK-801) to NMDA receptors on well washed synaptic plasma membranes. The maximal stimulation of binding by APDA10 was less than that seen with spermine. In the presence of glutamate and glycine, APDA10 attenuated the stimulatory effect of spermine and the inhibitory effect of 1,10-diaminodecane. In the nominal absence of glutamate and glycine, APDA10 had no effect on the binding of [3H]MK-801, but antagonized the stimulatory effect of spermine on the binding of [3H] MK-801. These data suggest that APDA10 acts as a mixed antagonist/partial agonist at the polyamine recognition site, and that the partial agonist properties of APDA10 are dependent on the activation state of the receptor complex. An increase in the potency of the glutamate site antagonists D-2-amino-5-phosphonovaleric acid and 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid for inhibiting the binding of [3H]MK-801 was seen in the presence of APDA10. APDA10 also increased the affinity of binding of [3H]3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid to the NMDA receptor complex but had no effect on the binding of [3H]glycine. These data suggest that the polyamine APDA10 may alter the properties of the glutamate recognition site on the NMDA receptor complex.     

Pentobarbital-like discriminative stimulus effects of N-methyl-D-aspartate antagonists

The discriminative stimulus effects of competitive and noncompetitive N-methyl-D-aspartate (NMDA) antagonists were compared in rats trained to discriminate sodium pentobarbital (5.0 mg/kg i.p.) from saline under a two-lever fixed ratio 32 schedule of food reinforcement. The competitive NMDA antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) substituted for pentobarbital at doses that did not disrupt rates of responding. The proposed competitive NMDA antagonist NPC 12626 [2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid] also substituted for pentobarbital. The benzodiazepine antagonist Ro15-1788 did not antagonize the pentobarbital-like discriminative stimulus effects of CPP. The noncompetitive NMDA antagonists phencyclidine and MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate] produced a maximum average of only 42 and 38%, respectively, pentobarbital-lever responding at doses that also substantially reduced response rates. These results suggest that the competitive NMDA antagonists CPP and NPC 12626 share discriminative stimulus properties with pentobarbital. However, the pentobarbital-like discriminative stimulus effects of CPP are probably not mediated through interaction with benzodiazepine receptors sensitive to Ro15-1788. In addition, because phencyclidine and MK-801 did not fully substitute for pentobarbital, these results provide further evidence for differences in the discriminative stimulus properties of competitive and noncompetitive NMDA antagonists.     

3H]CGS 21680, a selective A2 adenosine receptor agonist directly labels A2 receptors in rat brain

Characterization of the adenosine A2 receptor has been limited due to the lack of available ligands which have high affinity and selectivity for this adenosine receptor subtype. In the present study, the binding of a highly A2-selective agonist radioligand, [3H]CGS 21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosine) is described. [3H]CGS 21680 specific binding to rat striatal membranes was saturable, reversible and dependent upon protein concentration. Saturation studies revealed that [3H]CGS 21680 bound with high affinity (Kd = 15.5 nM) and limited capacity (apparent Bmax = 375 fmol/mg of protein) to a single class of recognition sites. Estimates of ligand affinity (16 nM) determined from association and dissociation kinetic experiments were in close agreement with the results from the saturation studies. [3H]CGS 21680 binding was greatest in striatal membranes with negligible specific binding obtained in rat cortical membranes. Adenosine agonists ligands competed for the binding of 5 nM [3H]CGS 21680 to striatal membranes with the following order of activity; CGS 21680 = 5'-N-ethylcarboxamidoadenosine greater than 2-phenylaminoadenosine (CV-1808) = 5'-N-methylcarboxamidoadenosine = 2-chloroadenosine greater than R-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than N6cyclopentyltheophylline greater than S-phenylisopropyladenosine. The nonxanthine adenosine antagonist, CGS 15943A, was the most active compound in inhibiting the binding of [3H]CGS 21680. Other adenosine antagonists inhibited binding in the following order; xanthine amine congener = (1,3-dipropyl-8-(2-amino-4-chloro)phenylxanthine greater than 1,3-dipropyl-8-cyclopentylxanthine greater than 1,3-diethyl-8-phenylxanthine greater than 8-phenyltheophylline greater than 8-cyclopentyltheophylline = xanthine carboxylic acid congener greater than 8-parasulfophenyltheophylline greater than theophylline greater than caffeine. The pharmacological profile of both adenosine agonist and antagonist compounds to compete for the binding of [3H]CGS 21680 was consistent with a selective interaction at the high affinity adenosine A2 receptor. A high positive correlation (r = 0.98, P less than .01) was observed between the pharmacological profile of adenosine ligands to inhibit the binding of [3H]CGS 21680 and the selective binding of [3H]NECA (+50 nM CPA) to high affinity A2 receptors. However, some differences between these assays were found for compounds which have moderate affinity and nonselective actions at both the A1 and A2 adenosine receptor subtypes. Unlike data obtained with nonselective adenosine ligands, the present results indicate that [3H]CGS 21680 directly labels the high affinity A2 receptor in rat brain without the need to block binding activity at the A1 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ifenprodil and SL 82.0715 as cerebral anti-ischemic agents. II. Evidence for N-methyl-D-aspartate receptor antagonist properties

The effects of the anti-ischemic agents ifenprodil and its derivative SL 82.0715 ((+/-)-alpha-(4-chlorophenyl)-4-[(4-fluorophenyl) methyl]-1-piperidineethanol] have been analyzed in a number of models indicative of N-methyl-D-aspartate (NMDA) antagonistic potential in vitro and in vivo. Ifenprodil and SL 82.0715 potently and noncompetitively antagonize the stimulatory effects of NMDA on cyclic GMP production in immature rat cerebellar slices (IC50 values, 0.4 and 10 microM, respectively), as well as the NMDA-evoked [3H]acetylcholine release in adult rat striatal slices (IC50 values, 1.6 and 6.6 microM, respectively). Ifenprodil is 10 times more potent than (+/-)3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) but less active than the reference noncompetitive NMDA channel blockers [MK 801, ((+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine ], phencyclidine and 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP)] in these models. Ifenprodil and SL 82.0715 partially displace (maximal displacement 40-50% at 10 microM) the NMDA receptor ligand [3H]CPP from its binding site to rat brain membranes (IC50 values, 0.1 and 0.3 microM, respectively) in a noncompetitive manner; in the micromolar range the two agents also partially displace the NMDA channel ligand [3H]TCP from its binding site to rat brain membranes, and noncompetitively antagonize the L-glutamate-induced increase in [3H]TCP binding. Ifenprodil (0.01-1 microM) partially antagonizes the depolarizing effects of NMDA on the immature rat hemisected spinal cord in vitro. In mouse cultured spinal cord neurons, ifenprodil dose-dependently antagonizes the depolarizing effects of micropressure applied NMDA. Inhibition of the effects of NMDA in this model by ifenprodil and SL 82.0715 is noncompetitive. In vivo and after systemic i.p. administration, ifenprodil and SL 82.0715 antagonize the stimulatory effects of intrastriatally dialyzed NMDA on striatal dopamine release in rats (ID50 values, 0.9 and 0.3 mg/kg, respectively), and block the harmaline-evoked increase in cerebellar cyclic GMP production in mice (ID50 values, 3 and 4 mg/kg, respectively). These results indicate that ifenprodil is a noncompetitive NMDA antagonist which has a mechanism of action distinct from either the reference competitive NMDA receptor antagonists (CPP and 2-amino-5-phosphonovalerate) or the noncompetitive NMDA channel blockers (phencyclidine, TCP and MK 801). The potent NMDA antagonistic effects of the ifenprodil class of compounds are likely to be related to the demonstrated anti-ischemic potential of these compounds.     

3H]zacopride binding to 5-hydroxytryptamine3 sites on partially purified rabbit enteric neuronal membranes

5-Hydroxytryptamine3 (5-HT3) receptors are present in both central and peripheral neuronal tissues but radioligand binding studies have thus far been limited to crude membranes from brain and vagus nerve. The present studies describe the isolation and characterization from the rabbit small bowel of neuronal membranes enriched in binding sites for the potent 5-HT3 ligand, [3H]zacopride. The number of specific [3H]zacopride binding sites per milligram of protein was increased 6-fold in a 10,000 to 100,000 x g membrane fraction as compared to the homogenate. [3H]Zacopride bound to these membranes with high specificity (greater than 90%), exhibited high affinity for a homogeneous population of binding sites (Kd = 0.3 nM) and its binding was inhibited competitively by other 5-HT3 compounds with the following rank order of potency: ICS 205-930 greater than GR 38032F greater than or equal to quipazine greater than BRL 24924 approximately MDL 72222 much greater than metoclopramide greater than 2-CH3-5-HT3. On a discontinuous sucrose gradient, specific [3H]zacopride binding was increased an additional 3.5-fold and copurified with three plasma membrane markers. Fractionation on a continuous sucrose gradient demonstrated that specific [3H]zacopride binding was associated with the enteric neuronal plasma membranes. Comparative studies in rabbit vagus nerve also demonstrated a large number (maximum binding = 148 fmol/mg of protein) of high affinity [3H]zacopride binding sites (Kd = 0.4 nM), in membranes that exhibited a density and binding characteristics similar to those from enteric neurons. Thus, membranes enriched in 5-HT3 binding sites can be isolated from both enteric and vagus neurons and [3H]zacopride is a potent ligand useful for characterization of these sites.     

Characterization of [3H]quinpirole binding to D2-like dopamine receptors in rat brain.

The putative D2 dopamine receptor agonist quinpirole (LY 171,555) is the most widely used D2 agonist in in vivo and in vitro studies of D2 receptor-mediated effects. In addition, quinpirole may have even higher affinity for the recently described D3 dopamine receptor. The present study describes the in vitro binding properties of newly developed [3H]quinpirole in rat brain. [3H]Quinpirole binding was characterized in striatal membrane homogenate preparations using a filtration assay. Nonspecific binding was defined by 1 microM (+)-butaclamol. Specific [3H]quinpirole binding was saturable, and dependent on temperature, membrane concentration, sodium concentration and guanine nucleotides. Saturation analysis revealed high affinity binding characteristics (KD = 2.3 +/- 0.3 nM) which were confirmed by association-dissociation kinetics. The pharmacological profile of [3H]quinpirole binding in striatum was: (-)-N-n-propylnorapomorphine (+/-)-2-amino-6,7-dihydroxyl-1,2,3,4-tetrahydronaphthalene greater than or equal to quinpirole greater than apomorphine greater than bromocriptine greater than dopamine greater than SKF 38393 much greater than 5-hydroxytryptamine for putative dopamine agonists; spiperone greater than (+)-butaclamol greater than haloperidol greater than (-)-sulpiride greater than clozapine greater than SCH 23390 much greater than cinanserin for antagonists. [3H]Quinpirole binding exhibited stereoselectivity: (-)-sulpiride greater than (+)-sulpiride and (+)-butaclamol greater than (-)-butaclamol. This pharmacological profile is similar, though-not identical, to that observed for [3H] spiperone-labeled D2 receptors. The regional distribution of [3H]quinpirole binding sites roughly paralleled the distribution of [3H]spiperone binding sites, with greatest densities present in the striatum, nucleus accumbens and olfactory tubercles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation between the excitatory and "excitotoxic" effects of quinolinic acid analogs on the striatal cholinergic interneuron

Analogs of quinolinic acid were tested for excitatory properties in evoking neurotransmitter release from striatal cholinergic interneurons and for their ability to lesion these same neurons in vivo (excitotoxin activity). The ability of these analogs to inhibit the specific binding of several ligands thought to label excitatory amino acid receptors was also investigated. Dipicolinic acid (2,6-pyridine dicarboxylic acid) was found to be as potent and as efficacious as quinolinic acid (2,3-pyridine dicarboxylic acid) at N-methyl-D-aspartate (NMDA)-type receptors mediating [3H]acetylcholine release from striatal slices. However, unlike quinolinate, the structure of NMDA is not superimposable upon that of dipicolinic acid. Moreover, unlike quinolinic acid, dipicolinic acid injected intrastriatally did not produce detectable excitotoxic lesions. Most unexpectedly, phthalic acid (1,2-benzene dicarboxylic acid), which lacks a nitrogen, also evoked [3H]acetylcholine release from striatal slices, apparently by acting at NMDA-type receptors. Phthalic acid was equipotent to quinolinic acid and dipicolinic acid but possessed less intrinsic activity than these compounds in evoking [3H]acetylcholine release. Despite its lack of a nitrogen and low intrinsic activity, intrastriatal injection of phthalic acid produced axon-sparing lesions of intrinsic cell bodies, like quinolinic acid as assessed by neurochemical and histologic methods. Quinolinic acid, dipicolinic acid and phthalic acid were moderately potent inhibitors (Kl = approximately equal to 100 microM) of the specific binding of 2-[3H]amino-7-phosphonoheptanoic acid a compound thought to be a competitive antagonist of NMDA-type receptors. In contrast, these three compounds failed to inhibit the chloride-dependent or chloride-independent binding of L-[3H]glutamate or [3H]kainic acid. The present results suggest a major dissociation between the structure-activity relationships for "excitotoxicity" vis-à-vis excitation as reflected in the [3H]acetylcholine release model, for compounds acting at NMDA-type receptors.     

Native N-methyl-D-aspartate receptors containing NR2A and NR2B subunits have pharmacologically distinct competitive antagonist binding sites

The pharmacological properties of native N-methyl-D-aspartate (NMDA) receptors were determined in rat brain sections with quantitative autoradiography of [(3)H](E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP39653) binding. With five competitive antagonists as displacers, two subpopulations of binding sites were observed in the horizontal plane of section examined. These two populations corresponded anatomically to NR2A and NR2B subunits. Quantitative analysis of NR2A-like and NR2B-like binding sites was enabled by examining the cerebellar granule cell layer, which expresses NR2A and NR2C subunits, and the medial striatum, which predominately expresses NR2B subunits. The antagonists (R)-(E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid and (R)-2-amino-5-phosphonopentanoate (D-AP5) displayed similar affinities at cerebellar NMDA receptors and medial striatal NMDA receptors. In contrast, the NMDA receptor antagonists (+/-)-6-(1H-Tetrazol-5-ylmethyl)decahydroisoquinoline- 3-carboxylic acid, (S)-alpha-amino-5-(phosphonomethyl)[1,1'-biphenyl]-3-propanoic acid, and (+/-)-cis-4-(4-phenylbenzoyl) piperazine-2,3-dicarboxylic acid displayed varied, higher affinities at medial striatal NMDA receptors than at cerebellar NMDA receptors. For the five antagonists, there was a strong correlation (r = 0.9) between the cerebellar K(i)/medial striatum K(i) ratio and the NR2A K(i)/NR2B K(i) ratio for recombinant receptors. Thus, [(3)H]CGP39653 labels two pharmacologically distinct populations of NMDA receptors that have pharmacological and anatomical properties consistent with NR2A and NR2B subunits. Because native NR2A- and NR2B-containing receptors are pharmacologically distinct, it should be possible to develop NR2A- and NR2B-selective glutamate site antagonists.     

Glycine-evoked neurotransmitter release from rat hippocampal brain slices: evidence for the involvement of glutaminergic transmission

Glycine caused a concentration-dependent evoked release of [3H]norepinephrine from rat hippocampal brain slices. Other amino acids evoked [3H]norepinephrine release with a rank order of potency: L-serine greater than or equal to glycine greater than beta-alanine greater than D-serine. Strychnine inhibited [3H]norepinephrine release evoked by both glycine and L-serine, but was less effective in inhibiting the release evoked by N-methyl-D-aspartate (NMDA) and kainic acid. Inhibitors of the NMDA receptor/ionophore complex, MK-801, CPP and Mg++, as well as the strychnine-insensitive glycine receptor antagonist, HA-966, caused an incomplete inhibition (maximum approximately 60%) of glycine-evoked [3H]norepinephrine release. The potencies with which MK-801, CPP and Mg++ inhibited glycine- and NMDA-evoked [3H]norepinephrine release were very similar. The combination of MK-801 plus kynurenic acid, a nonselective glutamate receptor antagonist, caused no greater inhibition of glycine-evoked release than MK-801, alone. omega-Conotoxin GVIA, an inhibitor of neuronal L- and N-type voltage-sensitive calcium channels, inhibited glycine-evoked [3H]norepinephrine release by approximately 50%, whereas the L-channel inhibitor PN 200-110 had no significant effect. The combination of MK-801 plus omega-conotoxin GVIA caused only a slightly greater inhibition (P greater than .05) of glycine-evoked release than MK-801 alone. Tetrodotoxin inhibited glycine-evoked release of [3H]norepinephrine by approximately 75%. The inhibitory effects of tetrodotoxin and omega-conotoxin GVIA suggest that voltage-sensitive sodium channels and N-type voltage-sensitive calcium channels are important mediators of glycine-evoked release of [3H]norepinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of 3H-leukotriene D4 binding to guinea pig lung receptors by the novel leukotriene antagonist ICI 198,615

The specific binding of [3H]5(S)hydroxy-6(R)-S-cysteinylglycyl -7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid ([3H]LTD4) to receptors on guinea pig lung parenchymal membranes and its inhibition by ICI 198,615, a representative example of a new class of leukotriene antagonists, was characterized by a receptor-ligand binding assay. [3H]LTD4 bound specifically and rapidly (Kon = 0.29 +/- 0.6 nM-1.min-1) reaching equilibrium within 15 min. The rate of binding was greatly inhibited in the presence of ICI 198,615. Excess LTD4 or ICI 198,615 slowly (t1/2 = 20 min) dissociated about 70% of the receptor-bound [3H]LTD4, whereas in combination with GTP analogs, both induced a rapid (t1/2 less than 5 min) and full dissociation. Equilibrium saturation analysis of [3H]LTD4 binding demonstrated a saturable (Bmax = 1014 +/- 174 fmol/mg) and high affinity (Kd = 0.43 +/- 0.09 nM) binding site. A high degree of stereoselectivity was demonstrated with inhibition of binding by the stereoisomers of LTD4: S,R much greater than R,R greater than R,S much greater than S,S. The rank order for inhibition of binding by peptide leukotriene was: LTD4 greater than 5(S)-hydroxy-6(R)-S-cysteinyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid much greater than 5(S)hydroxy-6(R)-S-glutathionyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (potency ratios were: 1:4:590). In competition assays, ICI 198,615 competitively inhibited binding of [3H]LTD4 (Ki = 0.27 +/- 0.16 nM) and was 2300-fold and 3100-fold more potent than LY171883 or FPL55712. These data, together with results obtained previously in functional receptor assays, illustrate that this new class of leukotriene antagonists are the most potent and selective competitive antagonists of LTD4 receptors yet described.     

Effects of cocaine and related drugs in nonhuman primates. I. [3H]cocaine binding sites in caudate-putamen

Specific binding sites for [3H]cocaine were identified in caudate-putamen membranes prepared from nonhuman primate brains (Macaca fascicularis and Saimiri sciureus). Saturation of the sites was determined in competition studies using a fixed concentration of [3H]cocaine (2.7 nM) and increasing concentrations of unlabeled cocaine (1 pM-100 microM). Computer resolution of the shallow displacement curve (nH, 0.58) revealed that a two-component binding model [Kd1, 19.2 nM, maximum binding1 (Bmax1), 28.3 pmol/g of tissue; Kd2, 1120 nM, Bmax2, 431 pmol/g of tissue] was statistically preferred over a one-component model (K.50, 283 nM, Bmax, 471 pmol/g of tissue). Binding of [3H]cocaine was NaCl-dependent, with specific binding reduced by 72% when NaCl (100 mM) was omitted from the incubation medium. [3H]Cocaine was displaced stereoselectively by the enantiomers of cocaine and by the diastereoisomers of cocaine and its phenyltropane analog. Cocaine congeners displaced specifically bound [3H]cocaine with IC50 values ranging from 17 nM to over 100 microM in the following rank order of potency: WIN 35,428 greater than WIN 35,065-2 greater than (-)-cocaine greater than WIN 35,981 greater than (-)-norcocaine greater than WIN 35,140 greater than (+)-cocaine, (+)-pseudococaine greater than 3 alpha-tropanyl-1H-indole-carboxylic acid ester greater than 1 alpha H-3 alpha-5 alpha H-tropan-3-yl-3,5-dichlorobenzoate greater than benzoylecgonine, benzoylnorecgonine and (-)-pseudococaine. Several monoamine uptake inhibitors structurally unrelated to cocaine also displaced [3H]cocaine with IC50 values ranging from 1.6 nM to 50 microM. The rank order of potency was: ( +/- )-trans-3-(3',4'-dichlorophenyl)-N-methyl-1-indanamine greater than mazindol greater than nomifensine greater than methylphenidate 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]- 4-(3-phenylpropyl)piperazine, N-[1-(2- benzo(b)thiophenyl)cyclohexyl]piperidine greater than (-)-cocaine greater than 1-amino-4-phenylbicyclo-[2,2,2]-octane greater than bupropion, nisoxetine greater than desipramine, talsupram greater than citalopram. Other drugs, including the dopamine releasing agent (+)-amphetamine and the dopamine receptor agonists (-)-apomorphine, (+)-4-propyl-9-hydroxy-naphthoxazine, quinpirole and SKF 38393 were weak displacers of [3H]cocaine. Monoamine neurotransmitters also were relatively weak, but dopamine was considerably more potent than either norepinephrine or serotonin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Release of endogenous norepinephrine from rat hypothalamus by stimulation of N-methyl-D-aspartic acid receptors.

Release of endogenous dopamine and norepinephrine (NE) from rat hypothalamic slices superfused with Mg(++)-free medium in the presence of nomifensine and tyrosine was measured by high-performance liquid chromatography coupled to an electrochemical detector. Superfusion with L-glutamic acid or N-methyl-D-aspartic acid elicited a concentration-dependent release of NE but not of dopamine. The release of NE was transient, returning toward basal values despite the continued presence of the amino acid. Superfusion with 20 mM K+ caused a release of NE that declined at a slower rate. Mg++, DL-2-amino-5-phosphonopentanoic acid and MK-801 (D-5-methyl-10,11,dihydro-5H-dibenzo[a,d] cyclohepten-5-10-imine maleate), but not 6-cyano-7-nitroquinoxaline-2,3-dione, inhibited the L-glutamic acid-evoked release of NE. The release of NE by L-glutamic acid was virtually abolished by tetrodotoxin and by elimination of Ca++ from and inclusion of 2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid in the superfusion medium. Repeated L-glutamic acid applications displayed a decreased response, whereas repeated exposure to 20 mM K+ did not. Exposure to L-glutamic acid in the absence of Ca++ (plus 2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) or in the presence of DL-2-amino-5-phosphonopentanoic acid did not reduce the effects seen on subsequent exposure to L-glutamic acid. Exposure to L-glutamic acid in the absence of Mg++ reduced the effect of a subsequent exposure to L-glutamic acid. These observations provide evidence for an indirect modulation of rat hypothalamic endogenous NE by the N-methyl-D-aspartate receptor.     

Pharmacological properties of the N-methyl-D-aspartate receptor system coupled to the evoked release of gamma-[3H] aminobutyric acid from striatal neurons in primary culture

The actions of a series of endogenous excitatory amino acid (EAA) agonists and synthetic antagonists at the N-methyl-D-aspartate (NMDA) receptor system coupled to the evoked release of gamma-[3H]aminobutyric acid (GABA) from purified populations of striatal neurons in primary culture were examined. EAA agonists displayed the following rank order of potency in evoking [3H]GABA release: glutamate greater than homocysteate greater than aspartate, NMDA greater than cysteine sulfinate. Glutamate, homocysteate and cysteine sulfinate were equieffective, whereas at saturating concentrations, aspartate and NMDA reached 75 and 65%, respectively, of the maximum efficacy of the former three agonists. The release of [3H]GABA evoked by 100 microM NMDA was attenuated in a dose-dependent manner by the following antagonists (IC50, micromolar): MK-801 (0.067), phencyclidine (0.151), CGS-19755 (3.31), 2-aminophosphonovalerate (18.8), kynurenate (100) and gamma-D-glutamylglycine (100). The antagonist properties of MK-801 and phencyclidine were not competitive with NMDA, whereas NMDA dose-response curves performed in the absence and presence of increasing concentrations of CGS-19755 resulted in parallel rightward shifts (pA2 = 5.95). CGS-19755 produced similar rightward shifts of the homocysteate dose-response curve (pA2 = 5.89). At glutamate concentrations less than 100 microM, CGS-19755 and 2-aminophosphonovalerate were potent antagonists of glutamate-evoked release; however, at glutamate concentrations greater than 100 microM these agents were ineffective blockers. The blockade of NMDA-evoked release of [3H]GABA by kynurenate was not competitive in nature.(ABSTRACT TRUNCATED AT 250 WORDS)