A comparison of N-methyl-D-aspartate-evoked release of adenosine and [3H]norepinephrine from rat cortical slices

Tetrodotoxin reduced N-methyl-D-aspartate (NMDA)-evoked release of adenosine by 35% but virtually abolished [3H]norepinephrine release. Although [3H]norepinephrine release from rat cortical slices evoked by 500 microM NMDA was abolished by 1.2 mM Mg++, which produces a voltage-sensitive, uncompetitive block of NMDA-channels, adenosine release was increased in the presence of Mg++. Partial depolarization with 12 mM K+ relieved the Mg++ block of 500 microM NMDA-evoked [3H]norepinephrine release but did not affect adenosine release, indicating that a Mg++ requirement for the adenosine release process per se cannot account for this discrepancy. NMDA was 33 times more potent in releasing adenosine than [3H]norepinephrine. At submaximal concentrations of NMDA (10 and 20 microM), adenosine release was augmented in Mg+(+)-free medium. Although a high concentration of the uncompetitive NMDA antagonist MK-801 [(+)-5-methyl-10,11,dihydro-5H-dibenzo[a,d]cyclohepten-5-10-imine maleate] (3 microM) blocked NMDA-evoked release of [3H]norepinephrine and adenosine, a lower concentration (300 nM) decreased NMDA-evoked [3H]norepinephrine release by 66% without affecting adenosine release. These findings suggest that maximal adenosine release occurs when relatively few NMDA receptors are activated, raising the possibility that spare receptors exist for NMDA-evoked adenosine release. Rather than acting as a protectant against excessive NMDA excitation, released adenosine might provide an inhibitory threshold which must be overcome for NMDA-mediated neurotransmission to proceed.     

Activity of putative cognition enhancers in kynurenate test performed with human neocortex slices.

Some cognition enhancers were previously shown to potently prevent antagonism of the N-methyl-D-aspartate (NMDA)-evoked release of norepinephrine (NE) brought about in slices of rat hippocampus by kynurenic acid, an endogenous NMDA receptor blocker. We have examined the impact of putative nootropic agents in the kynurenate test performed with slices of human cerebral cortex from patients undergoing neurosurgery. In slices of human neocortex, local application of NMDA evoked release of [3H]NE; the effect of NMDA was antagonized by several NMDA receptor antagonists, including kynurenic acid. The antagonism of the NMDA-evoked [3H]NE release produced by 300 microM kynurenate was potently (EC50 <10 microM) prevented by most of the nootropics tested, including aniracetam, oxiracetam, D-cycloserine, and the glutamate analog CR 2249 (but not its enantiomer CR 2361). Nicotine or tacrine (up to 10 microM) did not show any effect in the kynurenate test. Nicotine (30-100 microM) itself increased the release of [3H]NE; interestingly, the nicotine-evoked overflow was blocked not only by the nicotin receptor antagonist mecamylamine but also by NMDA receptor antagonists, suggesting an indirect mechanism mediated by glutamate/aspartate release. To conclude, the similarities between the data obtained here with human neocortex slices and those previously obtained in the rat indicate that the kynurenate test performed with rat brain slices may represent a useful biochemical assay to study cognition-enhancing drugs.     

A further evaluation of the effects of K+ depolarization on glutamate-evoked [3H]dopamine release from striatal slices.

Exogenous glutamate will evoke dopamine (DA) release from striatal slices in vitro. To further characterize glutamate-evoked DA release from striatal slices, experiments were designed to: 1) determine if sufficient endogenous glutamate can be released in vitro to presynaptically mediate [3H]DA release in the absence of Mg++ and 2) reevaluate how K+ depolarization affects glutamate-evoked [3H]DA release. Removal of Mg++ to potentiate N-methyl-D-aspartate (NMDA) receptor-mediated DA release increased 15 mM K(+)-evoked [3H]DA release to about 200% of control. The potentiation of this release was probably not mediated by NMDA receptors because it was not blocked by the glutamate receptor antagonists MK-801, 6,7-dinitroquinoxalinedione (DNQX) or kynurenate. Furthermore, the removal of Mg++ increased DA release substantially (200%) in the presence of 5 microM sulpiride and 10 microM nomifensine, indicating that DA reuptake and DA D2 autoreceptors are not primarily responsible for increased DA release. In the absence of Mg++, depolarization produced by 20 mM or greater [K+] inhibited DA released by exogenous glutamate, whereas a much higher [K+] was necessary to evoke endogenous glutamate release. In the presence of 1.5 mM Mg++, a reduction of the "Mg++ blockade" of NMDA receptors by 15 mM K+ depolarization during glutamate-evoked DA release was evaluated with and without the DA reuptake inhibitor nomifensine and the DA D2 antagonist sulpiride. DA released by K+ depolarization (Mg++ present) was markedly increased by 1 mM glutamate, but this effect was only partially reversed by kynurenate or high concentrations of either MK-801 (25 microM) or DNQX (100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Inhibition by 5,6-dihydroxy-2-dimethylaminotetralin (M7) of noradrenergic neurotransmission in the rabbit hypothalamus: role of alpha-2 adrenoceptors and of dopamine receptors

Rabbit hypothalamic slices were prelabeled with [3H]norepinephrine and transmitter release elicited by electrical stimulation. In the presence of 10 microM cocaine and in a low Ca++ medium (0.65 mM), exposure for 8 min to exogenous dopamine (0.01-1 microM) inhibited, in a concentration-dependent manner, the electrically evoked release of [3H]norepinephrine. This inhibitory effect of dopamine on [3H]norepinephrine release was antagonized by the dopamine receptor antagonist S-sulpiride (1 microM), but remained unchanged in the presence of the alpha-2 adrenoceptor antagonists idazoxan (1 microM) or yohimbine (0.1 microM). These results indicate that, in a low Ca++ medium, exposure to dopamine decreased [3H]norepinephrine overflow in rabbit hypothalamic slices through the exclusive activation of presynaptic inhibitory dopamine receptors. M7 (5,6-dihydroxy-2-dimethylaminotetralin) is a potent agonist at central presynaptic dopamine autoreceptors and at peripheral alpha-2 adrenoceptors. Exposure to M7 in a normal Ca++ medium, inhibited in a concentration-dependent manner the electrically evoked release of [3H]norepinephrine without affecting the spontaneous outflow of radioactivity. The slope of the concentration-effect curve for these inhibitory effects of M7 was rather flat and the maximal inhibition obtained was 80%. The selective D2 receptor antagonist S-sulpiride (1 microM) failed to produce a significant shift to the right in the concentration-effect curve for the inhibitory effects of M7 on [3H]norepinephrine release. The preferential alpha-2 adrenoceptor antagonist yohimbine (0.1 microM) significantly antagonized the inhibition of [3H]norepinephrine release elicited by 0.01 microM M7, but not for higher concentrations of this aminotetraline.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

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)     

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)     

Sigma1 receptor agonist-mediated regulation of N-methyl-D-aspartate-stimulated [3H]dopamine release is dependent upon protein kinase C

We have previously shown that sigma1 receptor agonists inhibit N-methyl-D-aspartate (NMDA)-stimulated [3H]dopamine from slices of rat striatum in a concentration-related manner and that the inhibition is reversed by sigma1 receptor-selective and nonsubtype-selective sigma receptor antagonists. Based on previous evidence from our laboratory as well as other laboratories, we hypothesized that sigma1 receptors might use a protein kinase C (PKC) signaling pathway to modulate stimulated dopamine release. We tested several inhibitors of PKC isozymes, as well as a phospholipase C inhibitor for their effects on sigma1 receptor agonist-mediated regulation of [3H]dopamine release. Although none of the inhibitors tested affected the ability of NMDA to stimulate [3H]dopamine release, they all abolished regulation by the sigma1 receptor agonist (+)-pentazocine in a concentration-related manner. We also found that prior exposure to 1 microM phorbol 2-myristate 13-acetate for 30 min abolished regulation by (+)-pentazocine. We concluded that an intact PKC system was required for sigma1 agonist-mediated regulation of NMDA-stimulated [3H]dopamine release from rat striatal slices. Based on the pharmacological profile of the PKC inhibitors tested, as well as reports in the literature on PKC     

Phencyclidine selectively inhibits N-methyl-D-aspartate-induced hippocampal [3H]norepinephrine release

We have reported previously that phencyclidine (PCP) antagonizes N-methyl-D-aspartate (NMDA)-induced release of dopamine and acetylcholine from slices of rat striatum and nucleus accumbens. In the present experiments, we examined the effect of PCP on NMDA and kainic acid (KA)-induced release of [3H]norepinephrine (NE) from superfused rat hippocampal slices. NMDA and KA stimulated the efflux of NE with EC50 values of 192 and 245 microM, respectively. The presence of 1.2 mM MgCl2 in the buffer abolished NMDA-induced release but had little effect on KA-induced release. PCP inhibited the release of [3H]NE induced by 100 microM NMDA with an IC50 of 46 nM, but had no effect on the release of NE stimulated by 300 microM KA. 2-Aminophosphonovalerate antagonized NMDA-induced release, producing a parallel shift to the right in the concentration-response curve. However, PCP shifted the concentration-response curve to the right in a nonparallel fashion. Drugs with PCP-like properties, such as dexoxadrol and cyclazocine, inhibited NMDA-induced release, whereas related drugs such as levoxadrol, ethylketocyclazocine and morphine, which are not PCP-like, had no effect. These data suggest that PCP is a potent, selective, noncompetitive inhibitor of amino acid-induced [3H]NE release and that this action of PCP is mediated through the PCP/sigma receptor.     

Interaction between serotonin uptake inhibitors and alpha-2 adrenergic heteroreceptors in the rat hypothalamus

The effectiveness of presynaptic receptor agonists to inhibit the electrically evoked release of [3H]monoamines from brain slices is attenuated in the presence of blockade of neuronal uptake for the serotonin (5-HT) and the norepinephrine (NE) systems. There is controversy, however, as to the existence of a functional link between the presynaptic receptors and the neuronal uptake carriers. An alternative hypothesis involves competition for the presynaptic receptor sites between the exogenous agonist and the released neurotransmitter. In order to examine the proposed functional interaction, we studied the alpha-2 adrenoceptor-mediated inhibition of the electrically evoked release of [3H]-5-HT from slices of the rat hypothalamus, a model in which endogenous NE does not activate the alpha-2 heteroreceptors located on 5-HT terminals. The inhibitors of 5-HT uptake, citalopram (0.01-1 microM) and paroxetine (1 microM), which by themselves did not modify [3H]-5-HT release, antagonized the inhibition of [3H]-5-HT overflow produced by UK 14.304, an alpha-2 adrenoceptor agonist. The inhibition of the electrically evoked release of [3H]-5-HT by exogenous NE (0.1-1 microM) was also attenuated in the presence of citalopram. In contrast, citalopram did not modify the electrically evoked release of [3H]-NE or the inhibition of [3H]-NE release mediated by UK 14.304. When the 5-HT autoreceptor was blocked by cyanopindolol, the inhibitory effect of UK 14.304 on [3H]-5-HT release was unaltered in the presence of citalopram.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spermidine reverses arcaine's inhibition of N-methyl-D-aspartate-induced hippocampal [3H]norepinephrine release

The inhibition of N-methyl-D-aspartate (NMDA)-induced [3H]norepinephrine ([3HNE) release by a putrescine analog was studied. We report that arcaine, diguanidinobutane, a putative competitive polyamine antagonist, completely and noncompetitively antagonized NMDA-induced [3H]NE release from rat hippocampal minces with an IC50 value of 102 microM. Arcaine did not alter kainate- or potassium-induced [3H]NE release suggesting a specific effect on NMDA-mediated responses. Spermidine did not alter NMDA-induced [3H]NE release, nor did it reverse the effect of arcaine when introduced in a normal physiologic superfusion buffer. However, spermidine reversed the effect of arcaine when superfusing with buffer that contained 5% (v/v) of the organic solvent dimethylsulfoxide. This finding suggests that the polyamine site may be located at the intracellular surface of the cell membrane. Our results provide the first evidence for polyamine modulation of the NMDA receptor ionophore complex in a functional physiologic system.     

Guanine nucleotides are competitive inhibitors of N-methyl-D-aspartate at its receptor site both in vitro and in vivo

Guanine nucleotides were shown to alter N-methyl-d-aspartate (NMDA) receptor-effector coupling by competitive antagonism at the glutamate binding site, rather than via interaction with an intracellularly located GTP-binding protein. Thus, in contrast to known G-protein linked receptors, micromolar concentrations of guanine nucleotides and their analogs decreased both agonist [( 3H]glutamate) and antagonist [( 3H]-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid binding to the NMDA receptor complex. The most potent compound, the GDP analog guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), was studied in detail. GDP beta S exhibited almost 200-fold selectivity for the glutamate recognition site vs. the strychnine-insensitive glycine binding site. IC50 values were 2.7 +/- 1.4 and 484 +/- 97 microM, respectively. GDP beta S also inhibited N-[1-(2-thienyl)cyclohexyl-3H]piperidine binding (IC50 was 28.0 +/- 3.7 microM) in an NMDA-reversible fashion. [3H]-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid saturation binding studies revealed an increase in Kd from 263 +/- 49 (control) to 552 +/- 134 nM (8 microM GDP beta S) without any change in maximum binding (4.94 +/- 0.34 and 5.19 +/- 0.58 pmol/mg of protein, respectively). GDP beta S was also a competitive inhibitor of the following NMDA-stimulated responses: elevation of cyclic GMP in neonatal rat cerebellar slices, release of preloaded [3H]norepinephrine from superfused rat hippocampal slices and elevation of cytosolic calcium concentration in fura-2-loaded cultured rat forebrain neurons. IC50 values were 78.4, 53.4 and 1.6 microM, respectively. Finally, GDP beta S resembled known NMDA receptor antagonists in its ability to block NMDA receptor-induced seizures after i.c.v. administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ifenprodil and SL 82.0715 as cerebral antiischemic agents. III. Evidence for antagonistic effects at the polyamine modulatory site within the N-methyl-D-aspartate receptor complex

Ifenprodil and SL 82.0715 are noncompetitive N-methyl-D-aspartate (NMDA) antagonists whose inhibitory actions are not explained by antagonistic effects at any of the three commonly recognized sites within the NMDA receptor complex (recognition, channel and modulatory glycine sites). We presently show that ifenprodil and SL 82.0715 antagonize the effects of NMDA via a selective action at the recently described polyamine modulatory site. Spermine and spermidine (0.5-100 microM) increase the binding of [3H]1-[1-(2-thienyl)cyclohexyl] piperidine to washed rat forebrain membranes in the presence of glutamate (10 microM). This effect is antagonized by ifenprodil and SL 82.0715 (0.1-10 microM) at concentrations which do not displace [3H]1-(2-thienyl)cyclohexyl] piperidine in the absence of added polyamine. Spermine and spermidine (up to 100 microM) do not significantly alter the binding of [3H]glycine but increase the binding of the NMDA recognition site ligand [3H](+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid. Ifenprodil and SL 82.0715 (0.1-10 microM) antagonize this effect; ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-10-imine maleate) or 7-chlorokynurenate (100 microM) are ineffective. In immature rat cerebellar slices, spermine and spermidine (10-1000 microM) potentiate the maximal effects of NMDA (80-160 microM) on cyclic GMP production. Spermine (100-1000 microM) reverses the antagonistic effects of ifenprodil (0.15-50 microM) but not of ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-10-imine acid or kynurenate on the NMDA receptor-mediated increase in cyclic GMP levels. Ifenprodil (0.01-1 microM) potently but only partially antagonizes the depolarizing effects of NMDA (10 microM) on the immature rat spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)     

Met-enkephalin release from slices of the rat striatum and globus pallidus: stimulation by excitatory amino acids.

The present study evaluated the effects of high K+ and four excitatory amino acids (EAAs) on the release of met-enkephalin-like immunoreactivity (ME-i.r.) from slices of the rat striatum and globus pallidus. High K+ (15-50 mM) increased the release of ME-i.r. in a concentration-dependent manner in both regions, the release response in the globus pallidus being consistently greater than in the striatum. This release was highly Ca(++)-dependent and was significantly enhanced in the absence of external Mg++. D-2-Amino-7-phosphonoheptanoic acid (0.5 mM), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, did not alter this enhanced action of K+, suggesting that the activation of NMDA receptors by an endogenous agonist did not contribute to the enhancement. Exposure of pallidal or striatal slices to four EAA receptor agonists, NMDA, L-glutamate, kainate (KA) and quisqualate, increased the release of ME-i.r. above the base line, an effect that was Ca(++)-dependent. Both L-glutamate and NMDA, at concentrations of 1 and 5 mM, produced a graded increase in the ME-i.r. release, but a higher concentration (10 mM) produced a lower release. In both regions the NMDA (5 mM)-evoked release was effectively inhibited by Mg++ (1.2 mM), 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) (5 microM), a competitive NMDA receptor antagonist and thienylcyclohexylpiperidine (10 microM), a noncompetitive NMDA receptor antagonist. Tetrodotoxin (0.3 microM), a Na+ channel blocker, did not affect the NMDA-evoked release of ME-i.r. in the striatum, but decreased it by 52% in the globus pallidus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-2 adrenoceptors modulate [3H]dopamine release from rabbit retina

In the rabbit retina, preloaded in vitro with [3H]dopamine, calcium-dependent release of radioactivity was elicited by a 1-min period of field stimulation at 3 Hz (20 mA, 2 msec). In the presence of the catecholamine uptake inhibitor nomifensine (30 microM), unlabeled catecholamines (0.01-3 microM), namely, dopamine, norepinephrine and epinephrine, inhibited in a concentration-dependent manner the field stimulation-evoked release of [3H]dopamine from the retina. The concentrations of dopamine, norepinephrine or epinephrine which inhibited by 50% the release of [3H]dopamine (IC50) were 0.30, 0.25 and 0.25 microM, respectively. In the presence of 30 microM nomifensine, S-sulpiride (1 microM) significantly increased the calcium-dependent release of [3H]dopamine, suggesting that this dopamine antagonist blocks a receptor tonically activated by endogenous dopamine in the rabbit retina. In contrast, the alpha receptor antagonist phentolamine (1 microM) alone did not affect the release of [3H]dopamine from the retina. The inhibitory effect of norepinephrine and epinephrine on [3H]dopamine overflow was not modified by S-sulpiride which, on the contrary, selectively antagonized the effect of exogenous dopamine. Phentolamine (1 microM) competitively antagonized the inhibitory effect of norepinephrine and epinephrine on [3H]dopamine release, suggesting that these catecholamines activate alpha adrenoceptors in retina. In the absence of nomifensine, the selective alpha-2 agonist clonidine (IC50 = 0.056 microM) inhibited the stimulation-evoked release of [3H]dopamine from retina, whereas the alpha agonist methoxamine was without effect. The inhibitory effect of clonidine was antagonized by yohimbine (1 microM), but not prazosin, suggesting that the release modulating alpha receptors of the retina are of the alpha-2 subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effects of the antiparkinsonian drugs memantine and amantadine on N-methyl-D-aspartate-evoked acetylcholine release in the rabbit caudate nucleus in vitro.

Slices of the rabbit caudate nucleus were incubated with [3H]choline or [3H]dopamine and then superfused continuously with Mg(++)-free medium. Stimulation with N-methyl-D-aspartate (NMDA), alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA), L-glutamate and kainic acid (in that rank order of potencies) caused a concentration-dependent increase in [3H]ACh efflux, which was abolished in the presence of Mg++. This kind of release was Ca(++)-dependent and tetrodotoxin-sensitive. In contrast, NMDA was hardly effective in stimulating [3H]ACh release from hippocampal or cortical slices, as well as [3H]dopamine release from slices of rabbit caudate nucleus. Hence, the presence of cell bodies of stimulated neurons seems to be a prerequisite for the induction of release via NMDA receptors. Dizocilpine [(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate] at nanomolar concentrations, as well as memantine and amantadine at low micromolar concentrations, inhibited the L-glutamate- and NMDA-evoked [3H]ACh release in a concentration-dependent, noncompetitive and use-dependent manner. Also (+/-)-2-amino-5-phosphopentanoic acid at micromolar concentrations depressed the L-glutamate- and NMDA-induced release, acting, however, in a competitive manner. It is concluded that, by antagonizing NMDA receptor-mediated ACh release, memantine and amantadine may act as functional "anticholinergics" when administered clinically to treat Parkinson's disease.     

Changes in sensitivity of release modulating dopamine autoreceptors after chronic treatment with haloperidol

The release of recently taken up [3H]dopamine ([3H]DA) elicited by electrical stimulation (3 Hz, 2 min, 16 mA) from slices of the rabbit caudate nucleus is inhibited by apomorphine (0.01-0.1 microM) in a concentration-dependent manner. This action is mediated through the activation of presynaptic inhibitory DA autoreceptors. The inhibition of [3H]DA release by apomorphine (0.1 microM) was antagonized 2 hr, but not 24 hr after the single administration of haloperidol (1 mg/kg s.c.). After 2 days of withdrawal after 28 days of chronic treatment with haloperidol (1 mg/kg s.c.) once daily, apomorphine (0.01-0.1 microM) was more effective in inhibiting [3H]DA release elicited by electrical stimulation when compared with rabbits injected chronically with either the vehicle for haloperidol or with saline. In superfused slices of the rabbit caudate nucleus, exposure to S-sulpiride (0.1 and 1 microM) increased in a concentration-dependent manner the release of [3H] DA elicited by electrical stimulation. After 28 days of chronic treatment with haloperidol, the facilitation of [3H]DA release by S-sulpiride was significantly reduced when compared with the controls. The inhibition of central noradrenergic transmission by DA receptor agonists was studied in hypothalamic slices prelabeled with [3H]norepinephrine ([3H-NE]). Apomorphine (0.01-1 microM) inhibited the electrically evoked (5 Hz, 2 min, 26 mA) release of [3H]NE from hypothalamic slices of untreated rabbits. The sensitivity to the inhibitory effect of apomorphine on [3H]NE overflow remained unaffected after 2 days of withdrawal following 28 days of chronic treatment with haloperidol. In summary, our results indicate that chronic haloperidol administration induces changes in sensitivity of the DA autoreceptors regulating dopaminergic neurotransmission but does not affect the sensitivity of DA receptors modulating NE release in the central nervous system. These results suggest that the DA autoreceptors that regulate dopaminergic neurotransmission may play a physiological role in the modulation of transmitter release and consequently are susceptible to the development of changes in sensitivity after chronic receptor blockade. The possible implication of changes in sensitivity of the DA autoreceptor during the treatment of schizophrenia with neuroleptics is discussed.     

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.