Dissociation of [3H]L-glutamate uptake from L-glutamate-induced [3H]D-aspartate release by 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic acid and 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid, two conformationally constrained aspartate and glutamate analogs

We characterized the interaction of two conformationally constrained aspartate and glutamate analogs, 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic acid (HIP-A) and 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B), with excitatory amino acid transporters (EAATs) in rat brain cortex synaptosomes. HIP-A and HIP-B were potent and noncompetitive inhibitors of [(3)H]L-glutamate uptake, with IC(50) values (17-18 microM) very similar to that of the potent EAAT inhibitor dl-threo-beta-benzyloxyaspartic acid (TBOA). The two compounds had little effect in inducing [(3)H]D-aspartate release from superfused synaptosomes but they potently inhibited l-glutamate-induced [(3)H]D-aspartate release, thus behaving as EAAT blockers, not substrates, in a manner similar to those of TBOA and dihydrokainate (DHK). HIP-A and HIP-B, but not TBOA and DHK, unexpectedly inhibited L-glutamate-induced [(3)H]D-aspartate release with IC(50) values (1.2-1.6 microM) 10 times lower than those required to inhibit [(3)H]L-glutamate uptake. There is therefore a concentration window (1-3 microM) in which the two compounds significantly inhibited l-glutamate-induced release with very little effect on L-glutamate uptake. This selective inhibitory effect required quite long preincubation (>5 min) of synaptosomes with the drugs. At these low concentrations, however, HIP-A and HIP-B had no effect on the EAAT-mediated [(3)H]d-aspartate release induced by altering the ion gradients, indicating that they specifically affect some L-glutamate-triggered process(es)--different from L-glutamate translocation itself--responsible for the induction of reverse transport. These data are inconsistent with the classic model of facilitated exchange-diffusion and provide the first evidence that EAAT-mediated substrate uptake and substrate-induced EAAT-mediated reverse transport are independent. Compounds such as HIP-A and HIP-B could be useful to further clarify the mechanisms underlying these operating modes of transporters.     

Differentiation of substrate and nonsubstrate inhibitors of the high-affinity, sodium-dependent glutamate transporters

Within the mammalian central nervous system, the efficient removal of L-glutamate from the extracellular space by excitatory amino acid transporters (EAATs) has been postulated to contribute to signal termination, the recycling of transmitter, and the maintenance of L-glutamate at concentrations below those that are excitotoxic. The development of potent and selective inhibitors of the EAATs has contributed greatly to the understanding of the functional roles of these transporters. In the present study, we use a library of conformationally constrained glutamate analogs to address two key issues: the differentiation of substrates from nontransportable inhibitors and the comparison of the pharmacological profile of synaptosomal uptake with those of the individual EAAT clones. We demonstrate that the process of transporter-mediated heteroexchange can be exploited in synaptosomes to rapidly distinguish transportable from nontransportable inhibitors. Using this approach, we demonstrate that 2,4-methanopyrrolidine-2,4-dicarboxylate, cis-1-aminocyclobutane-1,3-dicarboxylate, and L-trans-2, 4-pyrrolidine dicarboxylate act as substrates for the rat forebrain synaptosomal glutamate uptake system. In contrast, L-anti-endo-3, 4-methanopyrrolidine-3,4-dicarboxylate, L-trans-2,3-pyrrolidine dicarboxylate, and dihydrokainate proved to be competitive inhibitors of D-[(3)H]aspartate uptake that exhibited little or no activity as substrates. When these same compounds were characterized for substrate activity by recording currents in voltage-clamped Xenopus laevis oocytes expressing the human transporter clones EAAT1, EAAT2, or EAAT3, it was found that the pharmacological profile of the synaptosomal system exhibited the greatest similarity with the EAAT2 subtype, a transporter believed to be expressed primarily on glial cells.     

Activation of alpha 6 GABAA receptors on depolarized cerebellar parallel fibers elicits glutamate release through anion channels.

Rat cerebellar synaptosomes labeled with [3H]D-aspartate ([3H]D-ASP) were exposed in superfusion to muscimol. The GABA(A) receptor agonist did not affect [3H]D-ASP basal release or the overflow provoked by 15mM K(+); muscimol potentiated the 35mM K(+)-evoked overflow of [3H]D-ASP or endogenous glutamate. Membrane potential measured by Rhodamine 6G fluorescence was -65mV under resting conditions and -32mV in the presence of 35mM K(+). The membrane potential was not significantly affected by muscimol. The muscimol effect on the K(+)(35mM)-evoked [3H]D-ASP overflow was not inhibited by omitting external Ca(2+) or by entrapping BAPTA to chelate cytosolic Ca(2+). Muscimol lost its ability to release glutamate following superfusion with D-aspartate to deplete cytosolic glutamate by heteroexchange suggesting that GABA(A) receptor activation elicits release of cytosolic glutamate. The non-transportable glutamate carrier blockers dihydrokainate or DL-TBOA did not reduce the muscimol potentiation. This was abolished by the anion channel blockers niflumic acid and NPPB. To conclude, when cerebellar parallel fiber terminals are sufficiently depolarized, activation of alpha6 GABA(A) receptors on these terminals mediates glutamate release in addition to that evoked by depolarization. This extra-release does not occur by exocytosis or transporter reversal but involves the opening of anion channels present on parallel fiber terminals.     

Characterization of glutamate efflux from preoptic area synaptosomes.

Treatment of ovariectomized rats in vivo with ovarian steroids has been found to influence the efflux of glutamate and gamma-aminobutyric acid from preoptic area synaptosomes incubated in vitro. Since these studies indicated a possible role of the glutamate carrier in steroid-modulated release of amino acids, the present studies examined the characteristics of efflux of glutamate and of the carrier system for glutamate in synaptosomes of the preoptic area derived from ovariectomized hormone-treated rats. The efflux of [3H]glutamate from preoptic area synaptosomes, was induced by glutamate and by the glutamate carrier agonist, D-aspartate; the putative glutamate carrier antagonist dihydrokainate failed to block this efflux. Dihydrokainate inhibited the uptake of glutamate but it was less effective than D-aspartate. The excitatory amino acid receptor agonists, N-methyl-D-aspartate and kainate were without effect while quisqualate modestly stimulated the efflux of [3H]glutamate. Efflux of [3H]glutamate, induced by glutamate itself or by D-aspartate was not blocked by the excitatory amino acid receptor antagonists, D-2-amino-5-phosphonovaleric acid, 6,7-dinitroquinoxaline-2,3-dione or kynurenate. Glutamate-induced efflux of [3H]glutamate did not require external Ca2+. Glutamate altered neither the basal nor the potassium-induced increases in the intrasynaptosomal concentration of Ca2+ as measured by the fura-2 method. Glutamate-induced efflux of [3H]glutamate was blocked by the putative chloride channel antagonist, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. It is concluded that the glutamate-induced efflux of [3H]glutamate in synaptosomes of the preoptic area is a carrier-mediated process that does not require activation of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamate efflux from human cerebrocortical slices during ischemia: vesicular-like mode of glutamate release and sensitivity to A(2A) adenosine receptor blockade

Glutamate extracellular accumulation is an early event in brain ischemia triggering excitotoxic neuron damage. We have investigated how to control the glutamate efflux from human cerebrocortical slices superfused in conditions simulating an acute ischemic insult (oxygen and glucose deprivation). The efflux of previously accumulated [3H]D-aspartate or endogenous glutamate increased starting 18 min after exposure to ischemia and returned almost to basal values in 6 min reperfusion with standard medium. Superfusion with Ca2+-free, EGTA (0.5 mM)-containing medium or with medium containing tetrodotoxin (TTX; 0.5 microM) inhibited the ischemia (24 min)-evoked [3H]D-aspartate efflux by about 50% and 65%, respectively. The ischemia (24 or 36 min)-evoked efflux of [3H]D-aspartate or endogenous glutamate was reduced at least 40% by the adenosine A(2A) receptor antagonist SCH 58261 (1 microM); the compound was effective when added up to 15 min after exposure to ischemia. No effect of SCH 58261 on the ischemia-evoked [3H]D-aspartate was found in Ca2+-free, EGTA-containing medium. To conclude, a significant component of the ischemia-evoked glutamate efflux in human cerebrocortical slices seems to occur by a vesicular-like mechanism. Endogenously released adenosine is likely to activate A(2A) receptors that enhance vesicular-like glutamate release during ischemia; A(2A) receptor antagonists would deserve consideration for their neuroprotective potential.     

Low concentrations of potassium inhibit the Na-dependent [3H]glutamate binding to rat hippocampal membranes

Potassium at low concentrations was found to inhibit the Na-dependent [3H]L-glutamate binding to rat hippocampal synaptic membranes. This inhibition was probably due to a competition between potassium and sodium at the ionic locus of the recognition site for glutamate of the uptake process. In addition, potassium inhibited the high-affinity [3H]L-glutamate uptake in hippocampal synaptosomes. These results provide an additional mechanism for the spreading of depression which accompanies seizure activity in the hippocampus.     

L-glutamate stimulates the efflux of newly taken up glutamine from astroglia but not from synaptosomes of the rat

Bulk isolated rat astrocytes loaded with [14C] glutamine (Gln) were superfused on glass fiber filters with a standard Krebs-Henseleit medium until the basal efflux of the radioactivity became constant. The samples were then pulse-treated with: L-glutamate (Glu), gamma-aminobutyric acid (GABA), L-aspartate (Asp), cold Gln, or catecholamines, each at 0.5 mM concentration. Of the neurotransmitters tested, only Glu stimulated the efflux of radio-labelled Gln. The effect of Glu was sodium-dependent and was not mimicked by the Glu-receptor agonists: N-methyl-D-aspartate (NMDA), quisqualate (Quis) or kainate (KA), indicating a transport-mediated mechanism. Glu did not stimulate the efflux of newly taken up glutamine from either crude or purified brain synaptosomes, which is in keeping with the existing evidence that astrocytes function as a glutamine donor to other compartments of the central nervous system.     

Characterization of the interactions of N-acetyl-aspartyl-glutamate with [3H]L-glutamate receptors

The specific binding of [³H]L-glutamate and its displacement by N-acetyl-aspartyl glutamate, a peptide endogenous to brain, has been examined in nine regions of the central nervous system. N-acetyl-aspartyl-glutamate caused only a partial displacement of [³H]L-glutamate specific binding with an uneven regional distribution of maximal inhibition, ranging from 61% in the thalamus to 40% in the cerebral cortex and the hippocampus. The maximal displacement of specifically bound [³H]L-glutamate by N-acetyl-aspartyl-glutamate was not significantly affected by calcium added to chloride containing buffer; however, in the absence of chloride or calcium, no significant displacement of [³H]L-glutamate by N-acetyl-aspartyl-glutamate was observed. N-Acetyl-aspartyl-glutamate displayed the highest affinity for the chloride-dependent sites labeled by [³H]L-glutamate among all peptide analogues examined. These results suggest that N-acetyl-aspartyl-glutamate may play a role as an endogenous excitatory peptide in the mammalian central nervous system and raise the question whether endogenous brain peptides enriched in acidic amino acids may serve as excitatory transmitters.     

Presynaptic alpha7 and non-alpha7 nicotinic acetylcholine receptors modulate [3H]d-aspartate release from rat frontal cortex in vitro

The presynaptic nicotinic modulation of glutamatergic transmission in the CNS has been associated with activation of the alpha7 subtype of nicotinic acetylcholine receptor (nAChR) in sub-cortical regions, whereas in the frontal cortex, non-alpha7 nAChRs have been implicated. The aim of this investigation was to directly characterise nAChR-evoked release of excitatory amino acids from rat frontal cortex, by monitoring the release of [3H]D-aspartate from superfused synaptosomes or minces. Co-administration of a nAChR agonist with a depolarising stimulus enhanced [3H]D-aspartate release above the effect of depolarising agent alone. This enhancement was blocked by the nicotinic antagonist mecamylamine. Other experiments revealed that in the absence of a depolarising stimulus, the nAChR agonists nicotine, epibatidine and anatoxin-a could evoke the release of [3H]D-aspartate in a Ca2+- and concentration-dependant manner. Differential sensitivity to the alpha7- and beta2*-selective nAChR antagonists alpha-bungarotoxin (alpha-Bgt) and dihydro-beta-erythroidine (DHbetaE) implicated two nAChR subtypes (alpha7 and beta2*), and this was supported by using the subtype-selective agonists choline (10 mM; alpha7 selective, blocked by alpha-Bgt but not by DHbetaE) and 5-Iodo-A-85380 (10 nM; beta2*-selective, blocked by DHbetaE but not by alpha-Bgt). Immunocytochemistry showed that alpha-Bgt labelling was associated with structures immunopositive for vesicular glutamate transporters, in both frontal cortex sections and synaptosome preparations, supporting the presence of alpha7 nAChR on glutamatergic terminals in rat frontal cortex.     

Glycine taken up through GLYT1 and GLYT2 heterotransporters into glutamatergic axon terminals of mouse spinal cord elicits release of glutamate by homotransporter reversal and through anion channels

Glycine concentration-dependently elicited [3H]D-aspartate ([3H]D-ASP) release from superfused mouse spinal cord synaptosomes. Glycine effect was insensitive to strychnine or 5,7-dichlorokynurenic acid, but was prevented by the glycine transporter blocker glycyldodecylamide. Glycine also evoked release of endogenous glutamate, which was sensitive to glycyldodecylamide and abolished in low-Na+ medium. Experiments with purified synaptosomes and gliasomes show that the glycine-evoked [3H]D-ASP release largely originates from glutamatergic nerve terminals. The glycine-evoked [3H]D-ASP release was halved by NFPS, a selective blocker of GLYT1 transporters, or by Org 25543, a selective GLYT2 blocker, and almost abolished by a mixture of the two, suggesting that activation of GLYT1 and GLYT2 present on glutamatergic terminals triggers the release of [3H]D-ASP. Accordingly, confocal microscopy experiments show localization of GLYT1 and GLYT2 in purified synaptosomes immuno-stained for the vesicular glutamate transporter vGLUT1. The glycine effect was independent of extra- and intraterminal Ca2+ ions. It was partly inhibited by the glutamate transporter blocker DL-TBOA and largely prevented by the anion channel blockers niflumic acid and NPPB. To conclude, transporters for glycine (GLYT1 or/and GLYT2) and for glutamate coexist on the same spinal cord glutamatergic terminals. Activation of glycine heterotransporters elicits glutamate release partly by homotransporter reversal and largely through anion channels.     

Dopamine and apomorphine do not modulate the uptake of [3H]D-aspartate in the rat striatum in-vitro

Sodium-dependent [3H]D-aspartate uptake was measured in rat striatal homogenates. The uptake was inhibited by both L- and D-glutamate, with IC50 values of 5.6 and 224 microM, respectively. Dopamine (10(-7)-10(-4) M), apomorphine (10(-7) M), sulpiride (10(-6) M) or a combination of dopamine and sulpiride were found not to affect the observed uptake of [3H]D-aspartate. Thus, the in-vitro dopaminergic modulation of high affinity glutamate uptake reported in the literature is not found when [3H]D-aspartate is used instead of [3H]L-glutamate.     

A pharmacological review of competitive inhibitors and substrates of high-affinity, sodium-dependent glutamate transport in the central nervous system

The acidic amino acid L-glutamate acts as both a primary excitatory neurotransmitter and a potential neurotoxin within the mammalian central nervous system. Functionally juxtaposed between these neurophysiological and pathological actions are an assorted group of integral membrane transporter proteins that rapidly and efficiently sequester glutamate into cellular and subcellular compartments. While multiple systems exist that are capable of mediating the uptake of L-glutamate, the high-affinity, sodium-dependent transporters have emerged as the most prominent players in the CNS with respect to terminating the excitatory signal, recycling the transmitter, and regulating extracellular levels of glutamate below those which could induce excitotoxic pathology. The focus of the present review is on the pharmacological specificity of these sodium-dependent transporters and, more specifically, on the competitive inhibitors that have been used to delineate the chemical requirements for binding and translocation. Analogues of glutamate that are conformationally constrained as a consequence of either the addition of substituents to the carbon backbone of glutamate or aspartate (e.g., beta-hydroxyaspartate or methylglutamate derivatives) or the incorporation of ring systems (e.g., (carboxycyclopropyl)glycines, aminocyclobutane dicarboxylates, or pyrrolidine dicarboxylates), have been especially valuable in these efforts. In this review, a particular emphasis is placed on the identification of analogues that exhibit preferential activity among the recently cloned transporter subtypes and on the differentiation of substrates from non-transportable inhibitors.     

Somatostatin inhibits glutamate release from mouse cerebrocortical nerve endings through presynaptic sst2 receptors linked to the adenylyl cyclase-protein kinase A pathway

The effects of somatostatin (SRIF, somatotropin release inhibiting factor) on the release of glutamate have been investigated using superfused mouse cerebrocortical synaptosomes. SRIF-14 inhibited the K+ (12 mM)-evoked overflow of preaccumulated [3H]D-aspartate as well as that of endogenous glutamate. Cyanamid 154806, a selective sst2 receptor antagonist, but not BIM-23056, an antagonist at sst5 receptors, prevented the SRIF-14 effect. Octreotide and L779976, selective agonists at sst2 receptors, mimicked SRIF-14, whereas L797591, L796778, L803087 and L362855, selective agonists at sst1, sst3, sst4 and sst5 receptor subtypes, were inactive. Activation of sst2 receptors seems to involve inhibition of the adenylyl cyclase-protein kinase A pathway present in glutamatergic terminals since the adenylyl cyclase inhibitor MDL-12,330A and the protein kinase A inhibitor H89 prevented the K+-evoked [3H]D-aspartate overflow. Consistent with the involvement of adenylyl cyclase, depolarization with 12 mM K+ increased synaptosomal cyclic AMP (cAMP) content, while forskolin, an adenylyl cyclase activator, potentiated basal [3H]D-aspartate release in an octreotide-, MDL-12,330A- and H89-sensitive manner. To conclude, glutamatergic cerebrocortical nerve endings possess release-inhibiting sst2 receptors which represent potential targets for new drugs able to mitigate the effects of excessive glutamate transmission.     

Excessive and precocious glutamate release in a mouse model of amyotrophic lateral sclerosis

The release of [3H]D-aspartate ([3H]D-ASP) or [3H]GABA evoked by glycine and that of [3H]D-ASP or [3H]glycine evoked by GABA from spinal cord synaptosomes were studied in SOD1-G93A(+) mice, a transgenic model of amyotrophic lateral sclerosis, SOD1(+) mice and SOD1(-)/G93A(-) animals. Mutant mice were killed at advanced phase of pathology or during the presymptomatic period. In SOD1(-)/G93A(-) or SOD1(+) mice glycine evoked [(3)H]d-ASP and [(3)H]GABA release, while GABA caused [3H]D-ASP, but not [3H]glycine, release. The glycine-evoked release of [3H]D-ASP, but not that of [3H]GABA, and the GABA-evoked [3H]D-ASP release, but not that of [3H]glycine, were more pronounced in SOD1-G93A(+) than in SOD1(+) mice. Furthermore, these potentiations were already present in asymptomatic 30- to 40-day-old mice. Basal [3H]D-ASP release was also higher in SOD1-G93A(+) than SOD1(+) or SOD1(-)/G93A(-) mice. The release of endogenous glutamate and GABA was also enhanced in asymptomatic animals; the glycine-evoked release of endogenous glutamate, but not of endogenous GABA, was higher in SOD1-G93A(+) than in SOD1(+) animals. The effects of glycine and GABA were insensitive to receptor blockers, but sensitive to transporter inhibitors, indicating coexistence of glutamate and glycine transporters and of glutamate and GABA transporters on glutamate-releasing terminals. The glutamate release machinery seems excessively functional in SOD1-G93A(+) animals.     

Autoregulation of acetylcholine release in isolated hippocampal nerve endings

The existence of presynaptic autoreceptors modulating acetylcholine release from central cholinergic nerve endings was investigated by using rat hippocampal synaptosomes in a superfusion system. The presence of exogenous acetylcholine, carbachol or oxotremorine in the superfusion fluid produced a dose-dependent inhibition of the release of [³H]acetylcholine elicited by 15 mM KCl in synaptosomes prelabeled with tritiated choline. The inhibition was counteracted by atropine. Another well known muscarinic agonist, bethanechol, had no effect on [³H]acetylcholine release. Our results indicate that central cholinergic nerve terminals possess autoreceptors of the muscarinic type for the control of acetylcholine release. Moreover, differences seem to exist between pre- and postsynaptic muscarinic receptors in the central nervous system.     

The mechanism by which monoamine oxidase inhibitors give rise to a non-calcium-dependent component in the depolarization-induced release of 5-HT from rat brain synaptosomes.

1. The effects of the monoamine oxidase inhibitors pargyline and nialamide on the Ca2+-dependency of [3H]-5-hydroxytryptamine release from superfused rat brain synaptosomes has been studied in order to evaluate the discrepancies that have occasionally been observed in studying transmitter release by in vivo and in vitro techniques. 2. The application of K+ pulses of low concentration (12.5-20 mM) caused an essentially Ca2+-dependent release of [3H]-5-HT. However, at K+ concentrations above 30 mM, a small non-Ca2+-dependent component appeared. 3. At high concentrations of K+ (30-55 mM), nialamide (18 microM) or pargyline (7 microM) increased the amount of [3H]-5-HT released which could be accounted for by an increase in the non-Ca2+-dependent component of release. 4. The elevation of the non-Ca2+-dependent component of release caused by the monoamine oxidase inhibitors was totally abolished by the inhibitors of the plasma membrane 5-HT carrier, chlomipramine (500 nM), citalopram (50 nM) and fluoxetine (1 microM). 5. The results suggest that the non-Ca2+-dependent component of release seen with high depolarizing concentrations of K+, particularly in the presence of monoamine oxidase inhibitors, is caused by the efflux of [3H]-5-HT through the plasma membrane carrier which seems to be activated during depolarization. 6. The significance of these findings to the physiological in vivo situation, and to the use of in vitro preparations in the study of transmitter release is discussed.     

Evidence for the presence of glutamatergic receptors in adult Schistosoma mansoni

Several studies have suggested that L-glutamate is a putative neurotransmitter in helminths. The present study investigated the presence of non-N-methyl-D-aspartate (NMDA) ionotropic receptors for glutamate in four subcellular fractions from adult male Schistosoma mansoni. Low-affinity (K(d)=221+/-80 nM) binding sites for [3H]kainic acid (KA) were detected in the heterogeneous (P(1)) fraction, which contains pieces of unbroken worm tissues, tegument, nuclei, and some vesicles. This binding was inhibited by classical glutamatergic ligands in the following order of potency: KA>L-glutamate>alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)>quisqualate congruent with 6,7-dinitroquinoline-2,3-dione (DNQX). However, neither NMDA, a selective agonist for NMDA receptors, nor DL-threo-beta-hydroxyaspartate (THA) and 1-trans-pyrollidine-2-dicarboxylic acid (PDC), inhibitors of high-affinity glutamate transporters, modified [3H]KA binding to the P(1) fraction. In addition, no specific binding for 10nM [3H]AMPA was detected in any subcellular fraction from S. mansoni. These results suggested the presence of KA receptors in adult male worms. This is supported by the evidence that direct application of 10 microM KA to whole worms produced a corkscrew-like coiling of their bodies, modifying the motility of the worms. The KA-induced response, measured as a decrease of the body area, was time-dependent and reversible. PDC was ineffective at blocking the KA effects, indicating that KA does not depend on high-affinity glutamate transporters to reach its site of action. On the other hand, DNQX, the non-NMDA antagonist, was able to partially inhibit KA-induced responses. As a whole, the present data support the presence of a glutamatergic signaling pathway in this parasite.     

Release of 5-hydroxytryptamine from serotonergic nerve endings by alpha-methyl-meta-tyramine

Preparations of synaptosomes (P₂) from the telencephalon of the pigeon and rat were used to study the effects of α-methyl-m-tyramine (α-MMTA) and metaraminol on the efflux of radioactive serotonin (5-HT). The preparations were first incubated in the presence of 2.5 × 10^?8 M [³H]-5-HT to selectively label serotonergic nerve endings. Both α-MMTA and metaraminol in combination at a concentration of 0.15, 0.030 and 0.006 mM each significantly increased the efflux of [³H]-5-HT over control values in preparations from both the pigeon and rat. These two compounds together at the two concentrations also appeared to decrease the efflux of [³H]-5-hydroxyindoleacetic acid. At a concentration of 0.012 mM, α-MMTA significantly increased the efflux of [³H]-5-HT whereas metaraminol when tested alone at this concentration did not appear to have an effect on the release of [³H]-5-HT. The results are discussed in terms of the behavioral effects produced by α-methyl-meta-tyrosine in pigeons working on a multiple FR 50 FI 10 schedule of reinforcement.     

Effects of methylmercury on neurotransmitter release from rat brain synaptosomes

Although the effects of methylmercury (MeHg) at the neuromuscular junction have been well characterized, similar studies employing CNS preparations and transmitters have been limited. We found that MeHg (0.5-5.0 microM) produced a concentration-dependent increase in the spontaneous release of [3H]dopamine. gamma-[3H]aminobutyric acid, and [3H]acetylcholine from synaptosomes isolated from rat brain striatum, cortex, and hippocampus, respectively. At these same concentrations MeHg did not attenuate calcium-dependent depolarization-evoked 3H-transmitter release. MeHg did not appear to induce calcium influx into the nerve terminal since the increase in release persists in the absence of extrasynaptosomal calcium. The increase in spontaneous transmitter release induced by MeHg persisted in the presence of low extrasynaptosomal sodium, suggesting that MeHg's effects on release are not mediated by either Na+, K+-ATPase inhibition or selective increases in membrane sodium permeability. MeHg produced only a very small increase in 45Ca efflux from synaptosomes preloaded with 45Ca, whereas these same MeHg concentrations produced large increases in 45Ca efflux from preloaded isolated mitochondria. MeHg did increase the efflux of [3H]deoxyglucose phosphate from synaptosomes. An increase in the efflux of [3H]deoxyglucose phosphate is believed to reflect an increase in neuronal membrane permeability. The quantitative and temporal aspects of the MeHg-induced [3H]-deoxyglucose phosphate efflux were similar to those observed for MeHg-induced neurotransmitter release. These data suggest that the increase in spontaneous transmitter release induced by MeHg is mainly the result of transmitter leakage that occurs subsequent to MeHg-induced increases in synaptosomal membrane permeability. However, these results cannot exclude possible effects of MeHg on intrasynaptosomal calcium homeostasis.     

Effects of inorganic mercury on [3H]dopamine release and calcium homeostasis in rat striatal synaptosomes

Inorganic mercury (Hg2+) in vitro increases spontaneous transmitter release from nerve terminals. The mechanisms of action are not well understood but may involve alterations in intraterminal Ca2+ dynamics. In this study we describe actions of Hg2+ in vitro on isolated mammalian CNS striatal nerve terminals (synaptosomes). Cobalt (2 mM) completely blocked the effect of 2 microM Hg2+ on spontaneous [3H]dopamine release. Cadmium (100 microM) was equipotent to Co2+ in blocking depolarization-dependent [3H]dopamine release, but did not alter the 2 microM Hg2(+)-induced spontaneous [3H]dopamine release. Depolarization-dependent [3H]dopamine release was not altered by 5 microM Hg2+. It appears that the site of action of Hg2+ on spontaneous [3H]dopamine release is not the Ca2+ channel. The effects of Hg2+ on intraterminal ionized Ca2+ [( Ca2+]i) were evaluated using the Ca2(+)-specific fluorescent probe, fura-2. Hg2+ (1-8 microM) had no effect on [Ca2+]i in 1.2 mM Ca2(+)-containing buffers. In nominal Ca2+ media, 4 and 8 microM Hg2+ significantly decreased [Ca2+]i. Following exposure to 4 and 8 microM Hg2+ the quenching of extrasynaptosomal fura-2 by Mn2+ was increased, suggesting that Hg2+ facilitated the leakage of fura-2. This apparent leakage was probably due to a nonspecific increase in membrane permeability since 2 microM Hg2+ produced a Co2(+)-insensitive increase in [3H]deoxyglucose phosphate efflux. Hg2+ did not increase the leakage of either lactate dehydrogenase or soluble protein from synaptosomes. Hg2+ produced a concentration-dependent (1-8 microM) increase in 45Ca2+ efflux from superfused synaptosomes which was insensitive to blockade either by 2 mM Co2+ or by 100 microM Cd2+. These data suggest that the transmitter releasing action of Hg2+ involves interactions with sites that also interact with Co2+ but not with Cd2+. Furthermore, Hg2+ may have direct transmitter releasing actions (i.e., Ca2(+)-mimetic properties), as well as nonspecific actions on plasma membrane permeability which may not necessarily be linked to [3H]dopamine release.