Deramciclane inhibits N-methyl-D-aspartate receptor function

Effects of the novel anxiolytic drug deramciclane on excitatory amino acid release and transmembrane Ca²⁺ ion flux processes were compared in rat cerebrocortical homogenates containing resealed plasmalemma fragments and nerve endings. Deramciclane (10 μM) significantly inhibited [³H]D-aspartate release and transmembrane Ca²⁺ flux to N-methyl-D-aspartate in the absence of Mg²⁺. By contrast, inhibition of [³H]D-aspartate release and transmembrane Ca²⁺ flux evoked by 0.1 mM (S)-α-amino-3-hydroxy-5-methyl-4-isoxazole propionate in the presence of Mg²⁺ and 10 μM cyclothiazide by 10 μM deramciclane was not significant. In the presence of N-methyl-D-aspartate receptor antagonists, deramciclane (10 μM) did not inhibit [³H]D-aspartate release to N-methyl-D-aspartate. These results suggest an involvement of the inhibition of a presynaptic N-methyl-D-aspartate receptor in the anxiolytic properties of deramciclane.     

N-methyl-D-aspartate autoreceptors respond to low and high agonist concentrations by facilitating, respectively, exocytosis and carrier-mediated release of glutamate in rat hippocampus

Presynaptic NMDA autoreceptors regulating glutamate release have rarely been investigated. High-micromolar N-methyl-D-aspartate (NMDA) was reported to elicit glutamate release from hippocampal synaptosomes in a Ca(2+)-independent manner by reversal of excitatory amino acid transporters. The aim of this work was to characterize excitatory amino acid release evoked by low-micromolar NMDA from glutamatergic axon terminals. Purified rat hippocampal synaptosomes were prelabelled with [(3)H]D-aspartate ([(3)H]D-ASP) and exposed in superfusion to varying concentrations of NMDA in the presence of 1 microM glycine. The release of [(3)H]D-ASP and also that of endogenous glutamate provoked by 10 microM NMDA were external Ca(2+) dependent and sensitive to the NMDA channel blocker MK-801 but insensitive to the glutamate transporter inhibitor DL-TBOA, which, on the contrary, prevented the Ca(2+)-independent release evoked by 100 microM NMDA. The NMDA (10 microM) response was blocked by 1 nM Zn(2+) and 1 microM ifenprodil, compatible with the involvement of a NR1/NR2A/NR2B assembly, although the presence of two separate receptor populations, i.e., NR1/NR2A and NR1/NR2B, cannot be excluded. This response was strongly antagonized by submicromolar (0.01-1 microM) concentrations of kynurenic acid and was mimicked by quinolinic acid (1-100 microM) plus 1 microM glycine. Finally, the HIV-1 protein gp120 potently mimicked the NMDA co-agonists glycine and D-serine, being significantly effective at 30 pM. In conclusion, glutamatergic nerve terminals possess NMDA autoreceptors mediating different types of release when activated by different agonist concentrations: low-micromolar glutamate would potentiate glutamate exocytosis, whereas higher glutamate concentrations would also provoke carrier-mediated release.     

K(+)-Evoked [(3)H]D-aspartate release in rat spinal cord synaptosomes: modulation by neuropeptide Y and calcium channel antagonists

This study was conducted to investigate mechanisms regulating the release of [(3)H]D-aspartate (or endogenous glutamate) in the rat spinal cord. Presynaptic modulation of glutamate release was studied in superfused synaptosomes depolarized with 20 mM KCl. Calcium-channel antagonists, omega-conotoxin GVIA (omega-CgTx GVIA; N-type), nifedipine (L-type), and omega-conotoxin MVIIC (omega-CmTx MVIIC; P/Q type), were used to characterize the voltage-operated Ca(2+) channels (VOCCs) involved in this release. Nifedipine had no significant effect on the K(+)-evoked release of [(3)H]D-aspartate, but the omega-conotoxins GVIA and MVIIC produced dose-dependent inhibitory effects that were additive. The most substantial reduction (54.30% +/- 4.40%) was seen with omega-CgTx GVIA, indicating that N-type channels play a major role in the release of glutamate in this tissue. We investigated the effects of neuropeptide Y (NPY), NPY(13-36), and [Leu(31)][Pro(34)]NPY on Ca(2+)-dependent, K(+)-evoked [(3)H]D-aspartate release. NPY and NPY(13-36) equipotently inhibited the release of glutamate in a concentration-dependent manner. The half-maximal response was observed at about 12 nM; maximal inhibition of 44.22% +/- 4.60% was achieved with 0.3 microM. The selective GABA(B) agonist (-)baclofen inhibited K(+)-evoked [(3)H]D-aspartate release from superfused spinal cord synaptosomes by 50.00% +/- 4.80% at 10 microM. When NPY(13-36) and (-)baclofen were used together at maximal doses, their release-inhibiting effects were not additive. In addition, neither of the agonists was able to enhance the inhibition produced by pretreating the synaptosomes with the selective inhibitor of N-type VOCCs omega-CgTx GVIA. These results are consistent with the hypothesis that presynaptic Y(2)-like and GABA(B) receptors regulate glutamate release by blocking Ca(2+) currents through N-type VOCCs. Characterization of the receptors that can inhibit the release of glutamate may provide useful information for treatment of conditions characterized by excessive glutamatergic transmission in the spinal cord.     

Dopamine D(1) receptor facilitation of depolarization-induced release of gamma-amino-butyric acid in rat striatum is mediated by the cAMP/PKA pathway and involves P/Q-type calcium channels

Transmission in the "direct" pathway through the basal ganglia, which has an important role in the control of motor movement, is markedly facilitated by the concurrent activation of dopamine D(1) receptors. Consistent with this, Ca(2+)-dependent, depolarization-induced release of [(3)H]-GABA from striatal slices from rats pretreated with reserpine was greatly increased in the presence of 1 microM SKF 38393, a dopamine D(1)-like receptor agonist. The effect of SKF 38393 was mimicked by 1 mM 8-bromo-cyclic AMP (Br-cAMP) and inhibited by the protein kinase A (PKA) inhibitor H-89, mean inhibition 92% +/- 4% with 10 microM H-89 (n = 3). The effects of SKF 38393 and Br-cAMP were not additive. The stimulatory effects of SKF 38393 and Br-cAMP were practically abolished in the presence of the histamine H(3) receptor agonist immepip (1 microM). The depolarization-induced release of [(3)H]-GABA in the presence of SKF 38393 was not significantly inhibited by 5 microM nimodipine, an L-type Ca(2+) channel blocker, or by 0.3 microM omega-conotoxin MVIIA, a selective blocker of N-type channels. However, preincubation of the slices with 0.95 microM omega-agatoxin TK, a P/Q-type channel blocker, followed by washing before changing to a depolarizing medium containing SKF 38393, resulted in a marked inhibition of the stimulated release of [(3)H]-GABA, mean 68% +/- 4% (n = 3). These observations provide evidence that dopamine D(1) agonist facilitation of the depolarization-induced release of GABA from striatal terminals is mediated by the cAMP/PKA pathway and involves mainly P/Q-type Ca(2+) channels.     

Role of intracellular Ca(2+) stores shaping normal activity in brain.

The role of intracellular Ca(2+) stores in the control of brain activity was investigated in microdialysis experiments by monitoring changes in the extracellular concentration of amino acids (AA) in the hippocampus of the rat after intracerebroventricular (icv) administration of the intracellular Ca(2+) release blocker, dantrolene in vivo, as well as in D-aspartate release and transmembrane Ca(2+) flux measurements in dantrolene-treated (50 microM) hippocampal homogenates containing resealed plasmalemma fragments and nerve endings in vitro. Microdialysis data demonstrate that icv injection of 0.6 mM dantrolene significantly decreases ( approximately 20%) the background (Glu) in the hippocampus. Both the (Glu; approximately 300%) and the inhibitory effect of dantrolene thereupon ( approximately 50%) was significantly increased when 0.5 mM of the Glu uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid, was dialysed into the hippocampus. NMDA and (S)-AMPA induced [(3)H]-D-aspartate release in hippocampal homogenates. Preincubation of these homogenates with 50 microM dantrolene was found to reduce the response to NMDA, but not to (S)-AMPA, in a NMDA-dependent manner. Increased rates of transmembrane influx and efflux of Ca(2+) in hippocampal homogenates with half-times of 4 ms and 200 ms, respectively, can be observed by the addition of 100 microM NMDA as recorded using a stopped-flow UV/fluorescence spectrometer in combination with the Ca(2+) indicator dye, bisfura-2. Both the Ca(2+) influx and efflux rates of the NMDA response were reduced (25-fold and >5-fold, respectively) in homogenates preloaded with 50 microM dantrolene. These results suggest a role for NMDA-inducible intracellular Ca(2+) stores in the control of normal brain activity in vivo.     

Differential effect of quinpirole and 7-OH-DPAT on the spontaneous [(3)H]-dopamine efflux from rat striatal synaptosomes

The effect of quinpirole and 7-OH-DAPT, two D(2)-like agonists, were examined using superfused rat striatal synaptosomes to study the autoregulation of spontaneous [(3)H]-dopamine ([(3)H]-DA) release. Basal [(3)H]-DA efflux was Ca(2+)-dependent by approximately 45% and was inhibited by cadmium 10 microM by 24%. Quinpirole (1 nM to 3 microM) inhibited spontaneous [(3)H]-DA efflux in a concentration-dependent manner (pEC(50) = 7.56 +/- 0.07 and E(max) = 26 +/- 0.09%) and this effect was competitively antagonized by haloperidol (0.3-1 nM) (apparent pA(2) = 9.61 +/- 0.08). In addition, activation of the D(2) DA autoreceptor by quinpirole only modulates the calcium-dependent component of [(3)H]-DA efflux. Low concentrations of a putative-selective D(3) DA agonist, (+/-)-7-OH-DPAT (0.03-0.1 microM), inhibited spontaneous [(3)H]-DA release by 13% (P < 0.05), but higher drug concentrations (> or =1 microM) increased basal [(3)H]-DA efflux in a concentration-dependent, nonsaturable, but reversible manner. Haloperidol (1-10 nM) reversed the (+/-)-7-OH-DPAT-induced inhibition, but not the increase in [(3)H]-DA outflow. The effect of (+/-)-7-OH-DPAT was mimicked by (+)-7-OH-DPAT. However, another putative D(3) DA agonist, PD 128,907 (1 nM to 3 microM), decreased spontaneous tritium efflux (maximal inhibition of 19 +/- 3.06% at 3 microM, P < 0.01). The effect of 7-OH-DPAT 10 microM was independent of the presence of extracellular Ca(2+), since its effect on basal [(3)H]-DA outflow was not significantly modified in a 200 nM free-Ca(2+) medium. In addition, the 7-OH-DPAT-induced enhancement of basal [(3)H]-DA efflux does not involve depolarization of nerve terminals or the reversal of the DA uptake system, as tetrodotoxin (1 microM) and nomifensine (1microM) did not modify the effect of 7-OH-DPAT 10 microM. The present data indicate that activation of D(2) DA autoreceptor subtype by quinpirole inhibits Ca(2+)-dependent spontaneous [(3)H]-DA efflux. 7-OH-DPAT activates the D(2) DA autoreceptor at low concentrations, whereas its action in releasing [(3)H]-DA effect is not receptor-mediated and could involve other mechanisms other than either conventional vesicular exocytosis or the DA uptake system.     

Modulation of Mg(2+)-dependent [3H]TCP binding by L-glutamate, glycine, and guanine nucleotides in rat cerebral cortex.

Biochemical and electrophysiological studies have demonstrated that phencyclidine (PCP) recognition site exists in the ion channel of the N-methyl-D-aspartate (NMDA) receptor ion channel complex. Using an extensively washed rat cortical membrane preparation, the effects of Mg2+ and guanylylimidodiphosphate (GppNHp) were examined on the binding of [3H]-N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP). Low concentrations of Mg2+ (EC50 = 11 microM) stimulated [3H]TCP binding under the basal condition and high concentrations of Mg2+ (IC50 = 1 mM) inhibited it. In the presence of 10 microM L-glutamate and 10 microM glycine, their EC50 values for Mg2+ enhancement of [3H]TCP binding were markedly reduced (to 1.9 microM or 8.4 microM), respectively. By contrast, the IC50 values for Mg2+ inhibition of [3H]TCP binding were reduced in the presence of L-glutamate, but not glycine. Furthermore, a stimulatory effect of Mg2+ on [3H]TCP binding was additional to the [3H]TCP binding stimulated by a maximally effective concentration of L-glutamate (10 microM) or glycine (10 microM). In the kinetic study, 300 microM Mg2+ produced an increase in the rates of both association and dissociation of [3H]TCP. Similar results were obtained with L-glutamate (10 microM) and glycine (10 microM); 10 mM Mg2+ also caused an acceleration of the association rate but strongly decreased [3H]TCP binding at equilibrium. Compared with [3H]TCP binding under the basal condition, K+ (10 mM) alone decreased the maximal binding without producing any change in the association rate; 10 mM K+ also significantly decreased Mg(2+)-stimulated [3H]TCP binding but caused no change in the acceleration of the association rate caused by Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in NPY-mediated modulation of hippocampal [3H]D-aspartate outflow in the kindling model of epilepsy

The anticonvulsant effect of NPY may depend on Y(2) and/or Y(5) receptor-mediated inhibition of glutamate release in critical areas, such as the hippocampus. However, Y(2) and Y(5) receptor levels have been reported to increase and decrease, respectively, in the epileptic hippocampus, implicating that the profile of NPY effects may change accordingly. The aim of this study was to evaluate the differential effects of NPY on glutamate release in the normal and in the epileptic hippocampus. Thus, we pharmacologically characterized the effects of NPY on the release of [(3)H]D-aspartate, a valid marker of endogenous glutamate, from synaptosomes prepared from the whole hippocampus and from the three hippocampal subregions (dentate gyrus and CA1 and CA3 subfields) of control and kindled rats, killed 1 week after the last stimulus-evoked seizure. In the whole hippocampus, NPY does not significantly affect stimulus-evoked [(3)H]D-aspartate overflow. In synaptosomes prepared from control rats, NPY significantly inhibited 15 mM K(+)-evoked [(3)H]D-aspartate overflow only in the CA1 subfield (approx. -30%). Both Y(2) and Y(5) receptor antagonists (respectively, 1 microM BIIE0246 and 1 microM CGP71683A) prevented this effect, suggesting the involvement of both receptor types. In contrast, in synaptosomes prepared from kindled rats NPY significantly inhibited 15 mM K(+)-evoked [(3)H]D-aspartate overflow in the CA1 subfield and in the dentate gyrus (approx. -30%). Only the Y(2) (not the Y(5)) antagonist prevented these effects. These data indicate a critical role for the Y(2) receptor in the inhibitory control of glutamate release in the kindled hippocampus and, thus, suggest that the anticonvulsant effect of NPY in the epileptic brain is most likely Y(2), but not Y(5), receptor-mediated.     

Depolarization-induced release of [(3)H]D-aspartate from GABAergic neurons caused by reversal of glutamate transporters.

Cultured neocortical neurons, which predominantly consist of GABAergic neurons exhibit a pronounced stimulus-coupled GABA release. Since the cultures may contain a small population of glutamatergic neurons and the GABAergic neurons have a high content of glutamate it was of interest to examine if glutamate in addition to gamma-aminobutyric acid (GABA) could be released from these cultures. The neurons were preloaded with [(3)H]D-aspartate and subsequently its release was followed during depolarization induced by a high potassium concentration or the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor agonists, AMPA and kainate. Depolarization of the neurons with 55 mM potassium increased the release of [(3)H]D-aspartate by more than 10-fold. When the non-specific calcium-channel blockers cobalt or lanthanum were included in the stimulation buffer with potassium, the release of [(3)H]D-aspartate was decreased by about 40%. These results indicated that some of the released [(3)H]D-aspartate might originate from a vesicular pool. When AMPA was applied to the neurons, the release of [(3)H]D-aspartate was increased 2-fold and could not be prevented or decreased by addition of cobalt. Since AMPA has a rapid desensitizing effect on AMPA receptors, it was examined whether AMPA under non-desensitizing conditions was able to induce an increased release of [(3)H]D-aspartate as compared to the conditions of applying AMPA alone. The desensitization of AMPA receptors was blocked by 6-chloro-3,4-dihydro-3-(2-norbornen-5-yl)-2H-1,2, 4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide). Under the non-desensitizing conditions, the AMPA-induced release of [(3)H]D-aspartate was highly enhanced showing about a 10-fold increase over basal release. Addition of cobalt or lanthanum did not decrease the amount of [(3)H]D-aspartate released, indicating that the release originated from a cytoplasmic pool. Kainate, which induces an almost non-desensitizing effect on AMPA receptors, showed similar results as observed for AMPA under non-desensitizing conditions. The NMDA receptor antagonist (5R,10 S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) had only minor effects on the [(3)H]D-aspartate release induced by AMPA and kainate. Thus, the depolarization-induced release of [(3)H]D-aspartate from cultured GABAergic neurons appears to be caused mainly by reversal of the glutamate transporters.     

On the role of calcium ions in the presynaptic alpha-receptor mediated inhibition of [3H]noradrenaline release from rat brain cortex synaptosomes

Rat brain cortex synaptosomes, previously labeled by incubation with [3H]noradrenaline ([3H]NA) were continuously superfused with Krebs-Ringer media. Release of [3H]NA was induced by superfusion with medium containing either 15 mM K+, 20 microM veratrine or 1 microM of the calcium-ionophore A 23187 and was strongly dependent on the concentration of Ca2+ in the medium. Noradrenaline (1 microM, in the presence of the uptake inhibitor desipramine) inhibited K+-induced [3H]NA release by activation of presynaptic alpha-receptors. When the Ca2+-concentration in the medium was reduced, or the Mg2+-concentration increased, [3H]NA release appeared to be more susceptible to alpha-receptor mediated inhibition. Noradrenaline (1 microM) inhibited [3H]NA release induced by 15 mM K+, in the presence of 0.075 Ca2+ and 10 mM Mg2+, by 86%. Veratrine-induced release was also inhibited by alpha-receptor activation. However, [3H]NA release induced by the calcium-ionophore was not affected by alpha-receptor agonists. These results strongly support the view that alpha-receptor activation results in a decrease of the availability of Ca2+ for stimulus-secretion coupling processes. Presumably this is effected by an inhibition of voltage-sensitive calcium channels in the neuronal membrane associated with neurotransmitter release.     

Exposure to N-methyl-D-aspartate increases release of arachidonic acid in primary cultures of rat hippocampal neurons and not in astrocytes

The release of [3H]arachidonic acid (ARA) was investigated from prelabelled primary cultures of hippocampal neurons and astroglial cells. The activation of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors resulted in a dose-dependent stimulation of [3H]ARA release. The half maximal effect was obtained at about 15 microM NMDA, whereas the maximum concentration (50 microM NMDA) produced about a 2-fold increase in 7-day-old cultures. This elevation in [3H]ARA release was blocked in a dose-related manner by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV), and by Mg2+ which blocks NMDA receptor-linked Ca2+ ion channels. The removal of external Ca2+ inhibited NMDA-induced release, whereas treatment with calcimycin (A 23187, a Ca2+ ionophore) greatly increased the [3H]ARA release. The inhibitors of phospholipase A2, nordihydroguaiaretic acid and mepacrine, decreased the NMDA-dependent [3H]ARA release in a dose-related manner, maximum inhibition reaching to about 90% at high doses. Entry of Ca2+ brought about by opening the voltage-sensitive channels by high K+ had no effect on the release of [3H]ARA, indicating that NMDA gated channels are situated in a part of the neuron where Ca2+ entry through this route is more efficiently coupled to the activation of phospholipase A2. Treatment with NMDA had no significant effect on [3H]ARA release in hippocampal astroglial cells as opposed to neurons. This was not due to inability of astrocytes to release ARA, for ATP still evoked [3H]ARA release, and this was markedly inhibited by mepacrine. It is suggested that ARA act as both intracellular and intercellular messengers in the functioning of NMDA receptors in synaptic transmission and plasticity in the hippocampus.     

The bradykinin receptor--a putative receptor-operated channel in PC12 cells: studies of neurotransmitter release and inositol phosphate accumulation.

Bradykinin (BK) induced [3H]norepinephrine [( 3H]NE) release and phosphatidylinositol turnover were investigated in PC12 cells. Induction of [3H]NE release by BK is mediated by activation of BK-B2-receptors, as determined using type specific BK receptor antagonists. BK induces [3H]NE release with a half maximal effective concentration of 30 +/- 0.5 nM, and reaches maximal net fractional release of 9.0 +/- 1% with 200 nM BK. The BK-induced release is Ca2+ dependent, reaching maximal release at 1.0 mM Ca2+, is pertussis toxin insensitive (1 microgram/ml), slightly increased by a dibutyryl cAMP (1 mM) and not affected by inhibitors of the cyclooxygenase or lipoxygenase pathways. Voltage-sensitive Ca2+ channel blockers, verapamil (10 microM), nifedipine (10 microM), and omega-conotoxin (CgTx 10 nM), do not block the BK-induced release. However, a considerable inhibitory effect was obtained by divalent cations Co2+ (ED50 = 0.2 mM) and Ni2+ (ED50(2)+ = 1 mM). These results indicate the involvement of a Ca2+ channel in the BK-mediated release which is different from the L- or N-type voltage sensitive calcium channels. Whereas [Ca2+]ex is essential for the BK-induction of catecholamine release, the rise in level of InsP's induced by BK in the presence or in the absence of [Ca2+]ex is similar up to concentration of 1 microM. This indicates that the rise in InsP's induced by BK is not sufficient to cause neurotransmitter release. Moreover, subsequent addition of Ca2+ to BK-stimulated cells in Ca(2+)-free medium yields no release. Hence, no activity triggered by BK alone could be further stimulated by Ca2+ for induction of release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabotropic glutamate receptors modulate [(3)H]acetylcholine release from cultured amacrine-like neurons.

Retinal amacrine cells express metabotropic glutamate receptors (mGluRs), but their physiological role is unknown. We investigated the effect of mGluR on [(3)H]acetylcholine release ([(3)H]ACh) from cultured chick amacrine-like neurons. Activation of group III mGluR with the agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) inhibited [(3)H]ACh release evoked by 25 mM KCl in a dose-dependent manner, and this effect was sensitive to pertussis toxin. In contrast, activation of group I or II mGluR with (S)-3, 5-dihydroxyphenylglycine (DHPG) and (2S,2'R,3'R)-2-(2', 3'-dicarboxycyclopropyl)glycine (DCG-IV), respectively, did not affect significantly [(3)H]ACh release. The effect of L-AP4 on [(3)H]ACh release was sensitive to nitrendipine, suggesting that it is, at least in part, due to inhibition of L-type Ca(2+) channels. Activation of group III mGluR also partly inhibited omega-conotoxin GVIA-sensitive Ca(2+) channels, coupled to [(3)H]ACh release. The L-AP4 did not affect the cAMP levels measured in amacrine-like neurons depolarized with 25 mM KCl or stimulated with forskolin, indicating that the effect of group III mGluR on [(3)H]ACh release is not due to inhibition of adenylyl cyclase activity. Inhibition of protein kinase A with KT-5720 was without effect on [(3)H]ACh release evoked by 25 mM KCl, further indicating that the effect of group III mGluR on [(3)H]ACh release cannot be attributed to the inhibition of the kinase. The effect of L-AP4 on [(3)H]ACh release was reversed by DHPG or by DCG-IV, and activation of group II mGluR also partially inhibited cAMP production stimulated by forskolin. Taken together, our results show that the effect of group III mGluR on [(3)H]ACh release may be due to a direct inhibition of L- and N-type Ca(2+) channels and is modulated by group I and group II mGluR.     

Manganese inhibits NMDA receptor channel function: implications to psychiatric and cognitive effects

Humans exposed to excess levels of manganese (Mn(2+)) express psychiatric problems and deficits in attention and learning and memory. However, there is a paucity of knowledge on molecular mechanisms by which Mn(2+) produces such effects. We now report that Mn(2+) is a potent inhibitor of [(3)H]-MK-801 binding to the NMDA receptor channel in rat neuronal membrane preparations. The inhibition of [(3)H]-MK-801 to the NMDA receptor channel by Mn(2+) was activity-dependent since Mn(2+) was a more potent inhibitor in the presence of the NMDA receptor co-agonists glutamate and glycine (K(i)=35.9+/-3.1 microM) than in their absence (K(i)=157.1+/-6.5 microM). We also show that Mn(2+) is a NMDA receptor channel blocker since its inhibition of [(3)H]-MK-801 binding to the NMDA receptor channel is competitive in nature. That is, Mn(2+) significantly increased the affinity constant (K(d)) with no significant effect on the maximal number of [(3)H]-MK-801 binding sites (B(max)). Under stimulating conditions, Mn(2+) was equipotent in inhibiting [(3)H]-MK-801 binding to NMDA receptors expressed in neuronal membrane preparations from different brain regions. However, under basal, non-stimulated conditions, Mn(2+) was more potent in inhibiting NMDA receptors in the cerebellum than other brain regions. We have previously shown that chronic Mn(2+) exposure in non-human primates increases Cu(2+), but not zinc or iron concentrations in the basal ganglia [Guilarte TR, Chen M-K, McGlothan JL, Verina T, Wong DF, Zhou Y, Alexander M, Rohde CA, Syversen T, Decamp E, Koser AJ, Fritz S, Gonczi H, Anderson DW, Schneider JS. Nigrostriatal dopamine system dysfunction and subtle motor deficits in manganese-exposed non-human primates. Exp Neurol 2006a;202:381-90]. Therefore, we also tested the inhibitory effects of Cu(2+) on [(3)H]-MK-801 binding to the NMDA receptor channel. The data shows that Cu(2+) in the presence of glutamate and glycine is a more potent inhibitor of the NMDA receptor than Mn(2+). Our findings suggest that the inhibitory effect of Mn(2+) and/or Cu(2+) on the NMDA receptor may produce a deficit in glutamatergic transmission in the brain of individuals exposed to excess levels of Mn(2+) and produce neurological dysfunction.     

Characterization of Ca-Channels Responsible for K-Evoked [H]Noradrenaline Release from Rat Brain Cortex Synaptosomes and Their Response to Amyotrophic Lateral Sclerosis IgGs

The contribution of the different Ca(2+)-channel subtypes to the K(+)-evoked [(3)H]noradrenaline release from rat cerebral cortex synaptosomes has been investigated. In the same experimental model, it was also verified whether the calcium-mediated neurotransmitter release is influenced by IgGs purified from sera of seven patients affected by sporadic amyotrophic lateral sclerosis. Synaptosome treatment with 3.0 microM nifedipine or 2.0 microM calciseptine, which block L-type channels, slightly decreased [(3)H]noradrenaline release, the reduction being 7 and 13% of the control values, respectively. The blockade of N-type Ca(2+)-channels with omega-conotoxin-GVIA (0.001-1.0 microM) induced a concentration-dependent reduction of the neurotransmitter release, with maximum effect of 34%. omega-Agatoxin-IVA failed to significantly affect the studied release, which was instead markedly reduced by omega-conotoxin-MVIIC. After the blockade of N-type channels with maximal concentrations of omega-conotoxin-GVIA, 3.0 microM omega-conotoxin-MVIIC reduced the release by 58%. Synaptosome treatment with amyotrophic lateral sclerosis IgGs enhanced the K(+)-evoked [(3)H]noradrenaline release, which was mostly mediated by P/Q- and N-type Ca(2+)-channels. The increase induced by pathologic IgGs (0.2 mg/ml) ranged from 11 to 62% for the different patients, and it was concentration-dependent. The basal release was instead unaffected by IgG treatment. The results of the present study suggest that the K(+)-evoked [(3)H]noradrenaline release from brain cortex synaptosomes is mainly mediated by activation of P/Q- and N-type Ca(2+)-channels. Autoantibodies present in the sera of patients affected by sporadic amyotrophic lateral sclerosis may interact with these channels by producing an increased calcium influx, with consequent enhancement of the neurotransmitter release. Preliminary results of the present study have been published in abstract form (Martire et al., 1997, Pharmacol. Res. 35:9).     

Activation of P2X(7) receptors induced [(3)H]GABA release from the RBA-2 type-2 astrocyte cell line through a Cl(-)/HCO(3)(-)-dependent mechanism.

ATP is an important signaling molecule in the nervous system and it's signaling is mediated through the metabotropic P2Y and ionotropic P2X receptors. ATP is known to stimulate Ca(2+) influx and phospholipase D (PLD) activity in the type-2 astrocyte cell line, RBA-2; in this study, we show that the release of preloaded [(3)H]GABA from RBA-2 cells is mediated through the P2X(7) receptors. ATP and the ATP analogue 3'-O-(4-benoylbenoyl)-adenosine-5'-triphosphate (BzATP) both stimulated [(3)H]GABA release in a concentration dependent manner, while the nonselective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), the P2X(7)-sensitive antagonist oxidized ATP (oATP), and high extracellular Mg(2+) all inhibited the ATP-stimulated [(3)H]GABA release. The ATP-stimulated [(3)H]GABA release was not affected neither by removing extracellular Na(+) nor by changes in the intracellular or extracellular Ca(2+) concentration. The GABA transporter inhibitors nipecotic acid and beta-alanine also had no effect. The ATP-stimulated [(3)H]GABA release was blocked, however, when media Cl(-) was replaced with gluconate and when extracellular HCO(3)(-) was removed. The Cl(-) channel/exchanger blockers 4,4'-diisothiocyanatostilbene-2',2'-disulfonic acid (DIDS) and 4-acetamido-4'- isothiocyanatostilbene-2',2'-disulfonic acids (SITS), but not diphenylamine-2-carboxylic acid (DPC) and furosemide, blocked the ATP-stimulated [(3)H]GABA release. The anionic selectivity of the process was F(-) > Cl(-) > Br(-) which is the same as that reported for volume-sensitive Cl(-) conductance. Treating cells with phorbol-12-myristate 13-acetate (PMA), forskolin, dibutyryl-cAMP, PD98059, neomycin, and D609 all inhibited the ATP-stimulated [(3)H]GABA release. We concluded that in RBA-2 cells, ATP stimulates [(3)H]GABA release through the P2X(7) receptors via a Cl(-)/HCO(3)(-)-dependent mechanism that is regulated by PKC, PKA, MEK/ERK, and PLD.     

Modulatory effects of aluminum, calcium, lithium, magnesium, and zinc ions on [3H]MK-801 binding in human cerebral cortex

The independent and combined effects of Ca2+, Mg2+, Zn2+, Al3+ and Li+ on [3H]MK-801 binding in human cerebral cortical membranes were studied to further characterize the modulatory effects of metal ions on the N-methyl-D-aspartate (NMDA) receptor-ionophore. Glycine, in the presence of glutamate, significantly intensified the Mg2+ inhibition of [3H]MK-801 binding whereas it masked the Ca2+ enhancement and slightly diminished the Zn2+ inhibition. Both Ca2+ and Mg2+ reduced the Zn2+ inhibitory potency. Aluminum demonstrated a potent, relatively glycine-insensitive inhibition of [3H]MK-801 binding as an amorphous Al(OH)3 polymer rather than as the free ion. Cationic modulation of the NMDA receptor-ionophore appears to be regulated at multiple sites which have significant allosteric interactions.     

Activation of gamma-aminobutyric acid GAT-1 transporters on glutamatergic terminals of mouse spinal cord mediates glutamate release through anion channels and by transporter reversal

The effects of gamma-aminobutyric acid (GABA) on the release of glutamate from mouse spinal cord nerve endings have been studied using superfused synaptosomes. GABA elicited a concentration-dependent release of [3H]D-aspartate ([3H]D-ASP; EC50= 3.76 microM). Neither muscimol nor (-)baclofen mimicked GABA, excluding receptor involvement. The GABA-evoked release was strictly Na+ dependent and was prevented by the GABA transporter inhibitor SKF89976A, suggesting involvement of GAT-1 transporters located on glutamatergic nerve terminals. GABA also potentiated the spontaneous release of endogenous glutamate; an effect sensitive to SKF89976A and low-Na+-containing medium. Confocal microscopy shows that the GABA transporter GAT-1 is coexpressed with the vesicular glutamate transporter vGLUT-1 and with the plasma membrane glutamate transporter EAAT2 in a substantial portion of synaptosomal particles. The GABA effect was external Ca2+ independent and was not decreased when cytosolic Ca2+ ions were chelated by BAPTA. The glutamate transporter blocker DL-TBOA or dihydrokainate inhibited in part (approximately 35%) the GABA (10 microM)-evoked [3H]D-ASP release; this release was strongly reduced by the anion channel blockers niflumic acid and NPPB. GABA, up to 30 microM, was unable to augment significantly the basal release of [3H]glycine from spinal cord synaptosomes, indicating selectivity for glutamatergic transmission. It is concluded that GABA GAT-1 transporters and glutamate transporters coexist on the same spinal cord glutamatergic terminals. Activation of these GABA transporters elicits release of glutamate partially by reversal of glutamate transporters present on glutamatergic terminals and largely through anion channels.     

Subtypes of excitatory amino acid receptors involved in the stimulation of [3H]dopamine release from cell cultures of rat ventral mesencephalon

N-methyl-D-aspartic acid (NMDA), quisqualic acid (QUIS), and kainic acid (KAIN), respective agonists for three excitatory amino acid (EAA) receptor subtypes, stimulated [3H]dopamine ([3H]DA) release from dissociated cell cultures of fetal rat ventral mesencephalon. Release evoked by all three agonists was Ca2(+)-dependent and inhibited by broad-spectrum antagonists (D,L-cis-2,3-piperidine dicarboxylic acid [PDA] and kynurenic acid [KYN]). However, both of these antagonists were more potent against KAIN than against QUIS and only KAIN-evoked release was blocked by gamma-D-glutamyl-aminomethyl sulfonic acid (GAMS, IC50 700 microM). NMDA-stimulated [3H]DA release was selectively inhibited by competitive (3-[2-carboxypiperazine-4-yl]propyl-1-phosphonic acid [CPP] and D,L-2-amino-5-phosphonovaleric acid [APV]) and non-competitive (phencyclidine and MK-801) NMDA receptor antagonists. In 1.2 mM Mg2+, NMDA-stimulated [3H]DA release was Na(+)-dependent and inhibited by tetrodotoxin (TTX, 2 microM) or by the local anaesthetic, lidocaine (200 microM). However, in 0 Mg 2+, NMDA-evoked release was not inhibited by TTX or lidocaine. Thus, TTX-sensitivity of the NMDA response in 1.2 mM Mg2+ apparently occurs because Na(+)-action potentials are required to alleviate a Mg2+ blockade. Neither QUIS- nor KAIN-evoked release was affected by Mg2+ or TTX. When extracellular NaCl was replaced by sucrose or Na2SO4, the QUIS response was increased. KAIN-evoked release was unaffected by the sucrose substitution and was attenuated in the Na2SO4-containing buffer. It is concluded that NMDA and QUIS/KAIN release [3H]DA via separate receptor subtypes.