Extracellular protons differentially potentiate the responses of native AMPA receptor subtypes regulating neurotransmitter release

1. The effects of pH changes on the basal and evoked release of [(3)H]noradrenaline ([(3)H]NA) and [(3)H]5-hydrohytryptamine ([(3)H]5-HT) from hippocampal synaptosomes and of [(3)H]dopamine ([(3)H]DA) and [(3)H]acetylcholine ([(3)H]ACh) from striatal and cortical synaptosomes were investigated in rat brain. 2. Changing pH between 6.4 and 8.0 did not affect the spontaneous release of the four [(3)H]neurotransmitters; alkalinization to pH 8.8 significantly enhanced release. Acidification to pH 6.4 augmented the AMPA-evoked overflows of [(3)H]NA, [(3)H]5-HT and [(3)H]DA, but not that of [(3)H]ACh. In contrast, lowering pH to 6.4 decreased the K(+)-evoked overflows of [(3)H]NA, [(3)H]5-HT, [(3)H]DA and [(3)H]ACh. 3. AMPA released transmitters in a Ca(2+)-dependent, exocytotic manner since its effects, at pH 7.4 or 6.4, were abolished by omitting external Ca(2+) or by depleting vesicular transmitter stores with bafilomycin A1. AMPA did not evoke carrier-mediated release because the uptake blockers nisoxetine, 6-nitroquipazine, GBR12909 and hemicholinium-3 could not inhibit the AMPA-induced release of [(3)H]NA, [(3)H]5-HT, [(3)H]DA and [(3)H]ACh. 4. Extraterminal acidification to pH 6.4 prevented the potentiating effect of cyclothiazide on the AMPA-evoked release of [(3)H]NA, [(3)H]DA and [(3)H]5-HT, whereas the proton-insensitive AMPA-evoked release of [(3)H]ACh, previously found to be cyclothiazide-insensitive at pH 7.4 was cyclothiazide-resistant also at pH 6.4. 5. To conclude, the cyclothiazide-sensitive AMPA receptors mediating release of NA, 5-HT and DA, but not the cyclothiazide-insensitive AMPA receptors mediating the release of ACh, become more responsive when external pH is lowered to pathophysiologically relevant values. The results with cyclothiazide suggest that H(+) ions may prevent desensitization of some AMPA receptor subtypes.     

Differential desensitization of ionotropic non-NMDA receptors having distinct neuronal location and function

The release of tritium from rat hippocampal synaptosomes prelabeled with [³H]noradrenaline ([³H]NA) or [³H]5-hydroxytryptamine ([³H]5-HT) and from rat neocortex synaptosomes prelabeled with [³H]choline and the release of endogenous GABA and glutamate from rat neocortex synaptosomes were monitored during superfusion with media containing varying concentrations of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. Concentration-dependent release potentiations were elicited by both excitatory amino acids (EAAs) in all the transmitter systems investigated. The releases evoked by 100 μM AMPA were, in all cases, almost totally dependent on external Ca²⁺ and sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX), indicating involvement of non-NMDA receptors. When cyclothiazide, a drug able to prevent desensitization of AMPA-preferring receptors, was added to the superfusion medium (at 1 or 10 μM) concomitantly with 100 μM AMPA or kainate, the EAA-evoked release of [³H]NA was significantly enhanced. Concanavalin A, a lectin thought to prevent desensitization of kainate-preferring receptors, had no effect (up to 10 μM) on the release of [³H]NA evoked by AMPA or kainate. The effect of cyclothiazide was lost if, after an 8-min pretreatment, the drug was removed just before the AMPA stimulus. When added concomitantly with the EAAs, cyclothiazide potentiated the release of [³H]5-HT elicited by AMPA and, less so, that evoked by kainate. Concanavalin A was ineffective. Neither cyclothiazide (1 or 10 μM) nor concanavalin A (3 or 10 μM) could affect the release of [³H]ACh or endogenous GABA provoked by 100 μM AMPA or kainate, suggesting that the receptors involved do not desensitize. Exposure of neocortex synaptosomes to AMPA or kainate concomitantly with cyclothiazide caused endogenous glutamate release that did not differ from that evoked by the EAAs alone. In contrast, the effects of AMPA and kainate were potentiated by concanavalin A. The activity of the lectin (3 μM) persisted when it was applied for 8 min and then removed before the AMPA or kainate (100 μM) pulse. When hippocampal synaptosomes prelabeled with [³H]NA were subjected to three subsequent AMPA (100 μM) stimuli (S₁, S₂ and S₃), the release of [³H]NA decreased dramatically from S₁ to S₃ (S₃/S₁ = 0.14 ± 0.04); a significant ‘protection’ of the AMPA effect was offered by 1 μM cyclothiazide (S₃/S₁ = 0.36 ± 0.06). This value did not differ from the S₃/S₁ ratio (0.38 ± 0.04) obtained in parallel experiments with 12 mM K⁺. The release evoked by high-K⁺ was insensitive to cyclothiazide. Finally, the effect of AMPA on the release of [³H]ACh did not respond to cyclothiazide also during three subsequent stimuli with 100 μM AMPA. To conclude: a) ionotropic non-NMDA receptors mediating enhancement of NA, 5-HT, ACh, GABA and glutamate release exist on the corresponding nerve terminals; b) the receptors present on noradrenergic and serotonergic neurons are AMPA-preferring receptors, whereas the glutamate autoreceptors resemble most the kainate-preferring subtype; the receptors mediating ACh and GABA release can not be subclassified at present; c) desensitization may not be a property of all non-NMDA ionotropic receptors. The receptors here characterized represent five models of native non-NMDA receptors suitable for pharmacological and molecular studies. Received: 28 January 1997 / Accepted: 14 April 1997     

Modulation of [3H]serotonin release by dihydropyridines in spinal cord synaptosomes.

The release of [3H]monoamines from preloaded synaptosomes from spinal cord is K(+)-dependent and can be modulated by L-type Ca2+ channel agonists such as the 1,4-dihydropyridine (1,4-DHP), Bay K 8644. Whereas the basal release of [3H]monoamines was not altered by Bay K 8644, K(+)-stimulated release of [3H]norepinephrine was enhanced 35% and [3H]serotonin 50%. Modulation of release by Bay K 8644 was dependent on the K+ concentration in the medium, being present only at submaximal depolarization with 15 mM K+. Enhanced release in the presence of Bay K 8644 was concentration-dependent and Ca2(+)-dependent. Ca2(+)-independent release induced by fenfluramine was not enhanced by Bay K 8644. Both nimodipine and nitrendipine, 1,4-DHP antagonists, produced a concentration-dependent block of the Bay K 8644-induced monoamine release and had no independent effect on basal or K(+)-stimulated release. omega-Conotoxin GVIA (omega-CgTx) produced a concentration dependent decrease of K(+)-stimulated serotonin release, which antagonized the stimulatory effect of low concentrations of Bay K 8644. However, omega-CgTx did not alter the enhancement of K(+)-stimulated release at higher concentrations of Bay K 8644. The data from the present work establish the conditions for modulation of K(+)-evoked monoamine release in spinal cord by 1,4-DHP agonists and suggest a role for the L-type voltage dependent Ca2+ channel in this process.     

Aniracetam, 1-BCP and cyclothiazide differentially modulate the function of NMDA and AMPA receptors mediating enhancement of noradrenaline release in rat hippocampal slices

Aniracetam, 1-(1,3-benzodioxol-5-yl-carbo-nyl)piperidine (1-BCP) and cyclothiazide, three compounds considered to enhance cognition through modulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, were evaluated in the ‘kynurenate test’, a biochemical assay in which some nootropics have been shown to prevent the antagonism by kynurenic acid of the N-methyl-d-aspartate (NMDA)-evoked [³H]noradrenaline ([³H]NA) release from rat hippocampal slices. Aniracetam attenuated the kynurenate (100 μM) antagonism of the [³H]NA release elicited by 100 μM NMDA with high potency (EC₅₀≤0.1 μM). Cyclothiazide and 1-BCP were about 10 and 100 times less potent than aniracetam, respectively. The effect of aniracetam persisted in the presence of the AMPA receptor antagonist 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) added at 5 μM, a concentration that did not affect NMDA receptors; in contrast, NBQX reduced the effect of 1-BCP and abolished that of cyclothiazide. The AMPA-evoked release of [³H]NA from hippocampal slices or synaptosomes was enhanced by cyclothiazide, less potently by 1-BCP and weakly by aniracetam. High concentrations of kynurenate (1 mM) antagonized the AMPA-evoked [³H]NA release in slices; this antagonism was attenuated by 1 μM cyclo-thiazide and reversed to an enhancement of AMPA-evoked [³H]NA release by 10 μM of the drug, but was insensitive to 1-BCP or aniracetam. It is concluded that aniracetam exerts a dual effect on glutamatergic transmission: modulation of NMDA receptor function at nanomolar concentrations, and modulation of AMPA receptors at high micromolar concentrations. As to cyclothiazide and 1-BCP, our data concur with the idea that both compounds largely act through AMPA receptors, although an NMDA component may be involved in the effect of 1-BCP. Received: 14 September 1998 / Accepted: 19 January 1999     

Effects of barbiturates and ethanol on muscimol-induced release of [3H]-D-aspartate from rodent cerebellum

The K+-stimulated release of [3H]-D-aspartate and [14C]-GABA from synaptosomal (P2) fractions prepared from rat cerebellum was studied. Muscimol enhanced the release of [3H]-D-aspartate by 60-75% and the release of [14C]-GABA by 20-35%. Muscimol also enhanced the release of [3H]-D-aspartate from P2 fractions prepared from swine and mouse cerebellum. Pentobarbital, an anesthetic barbiturate, had no effect on basal or K+-stimulated release of [3H]-D-aspartate or [14C]-GABA but potentiated the enhancement of [3H]-D-aspartate release by muscimol. The EC50 was approx. 50 microM. The S(-)-isomer of pentobarbital was more potent than the R(+)-isomer in potentiating the action of muscimol, in agreement with the anesthetic potencies of the isomers. Phenobarbital, an anticonvulsant barbiturate, enhanced release of [3H]-D-aspartate and [14C]-GABA in the absence of muscimol. In contrast, the convulsant barbiturate 5-ethyl-5-(2'-cyclohexylidene-ethyl)barbituric acid (CHEB) caused a significant increase in basal release of [3H]-D-aspartate and [14C]-GABA in the absence of muscimol. Diazepam and ethanol had no effect on the release of [3H]-D-aspartate and did not potentiate the action of muscimol. These experiments provide biochemical evidence for an enhancement of the action of GABA by anesthetic barbiturates. This effect appears to be mediated through a benzodiazepine-insensitive presynaptic GABA receptor.     

Hippocampal AMPA autoreceptors positively coupled to NMDA autoreceptors traffic in a constitutive manner and undergo adaptative changes following enriched environment training

α-Amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) autoreceptors exist on glutamate hippocampal terminals. Aimed at investigating whether these autoreceptors traffic constitutively, (S)AMPA-evoked [³H]D-ASP release from synaptosomes enriched with peptides that impede the interaction of GluA2 subunits with cytosolic proteins involved in receptor movements [namely Glutamate Receptor-Interacting Protein (GRIP), Protein Interacting with C kinase 1 (PICK1), N-ethyl-maleimide-Sensitive Fusion protein NSF proteins] was monitored. (S)AMPA alone had no effect on the spontaneous release of [³H]D-ASP from control synaptosomes, but became efficacious in the presence of cyclothiazide or when preventing GluA2/GRIP/PICK1, but not GluA2/NSF, interaction. Hippocampal glutamatergic terminals also possess NMDA autoreceptors. 10 μM NMDA/1 μM glycine-induced [³H]D-ASP release was concentration-dependently increased by (S)AMPA. Cyclothiazide potentiated the 10 μM NMDA/1 μM glycine/50 μM (S)AMPA-induced [³H]D-ASP overflow, while NBQX halved and MK-801 abolished it, suggesting NMDA-AMPA autoreceptor cross-talk. Western Blot analysis of sub-synaptic fractions confirmed presynaptic GluN2B-GluA2/3 co-localization. Impeding GluA2/GRIP/PICK1 interaction facilitated the NMDA/glycine/(S)AMPA-induced release of [³H]D-ASP, while competing for GluA2/NSF interaction reduced it, indicating that NMDA receptor favours AMPA receptor insertion in synaptosomal plasmamembranes. Finally, rearing mice in enriched environment unveiled the (S)AMPA-induced release of [³H]D-ASP, but leaved unmodified that caused by NMDA/glycine. The NBQX-sensitive, 50 μM (S)AMPA-evoked release of [³H]D-ASP was insensitive to cyclothiazide and to peptide interfering with GluA2/GRIP/PICK1 interaction but was addictive to that caused by NMDA/glycine. Presynaptic GluA2/3 immunoreactivity in EE hippocampal terminals was increased, while GluN2B was unchanged. We conclude that hippocampal AMPA autoreceptors positively coupled to NMDA autoreceptors traffic in a constitutive manner and undergo functional up-regulation in EE animals.     

(S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide: (S18986-1) a positive modulator of AMPA receptors enhances (S)-AMPA-mediated [3H]noradrenaline release from rat hippocampal and frontal cortex slices

The present study describes the effect of (S)-2,3-dihydro-[3, 4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S18986-1), a positive allosteric modulator of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors with cognitive-enhancing effects, on (S)-AMPA-induced [3H]noradrenaline release in rat hippocampal and frontal cortex slices. (S)-AMPA significantly increased [3H]noradrenaline release in rat hippocampus and frontal cortex slices, whereas S18986-1 (3-1000 microM) alone, was inactive. However, S18986-1 between 30 and 1000 microM potently enhanced (+200%) (S)-AMPA-mediated [3H]noradrenaline release in both hippocampal and frontal cortex slices. The capacity of S18986-1 to potentiate [3H]noradrenaline release was specific for AMPA receptors as S18986-1 failed to potentiate either kainate and N-methyl-D-aspartate (NMDA)-mediated release of [3H]noradrenaline in rat hippocampal slices. Moreover, 1, 2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) and 1-(4-aminophenyl)-3-methylcarbamoyl-4-methyl-3, 4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-53655) but not (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine ((+)-MK-801), inhibited (S)-AMPA and S18986-induced stimulation of (S)-AMPA-mediated [3H]noradrenaline release. In addition, S18986-1-induced stimulation of (S)-AMPA-evoked [3H]noradrenaline release was markedly attenuated in the presence of tetrodotoxin (1 microM) and in Ca(2+)-free buffer. S18986-1 enhanced (S)-AMPA-mediated [3H]noradrenaline release to a greater extent than its corresponding (R)-enantiomer S19024-1 and racemic mixture S17951-1. However, positive allosteric modulators of AMPA receptors such as aniracetam failed to potentiate AMPA-mediated noradrenaline release in hippocampal slices, whereas cyclothiazide potently enhanced (S)-AMPA-mediated [3H]noradrenaline release. These results suggest that the capacity of S18986-1 to enhance AMPA receptor-mediated release of noradrenaline in rat hippocampus and frontal cortex, could contribute to the cognition enhancing mechanisms of S18986-1.     

Protein kinase C modulates the release of [3H]5-hydroxytryptamine in the spinal cord of the rat: the role of L-type voltage-dependent calcium channels.

The present studies examined the relationship between protein kinase C (PKC) and L-type voltage-dependent calcium channels in modulating the release of neurotransmitter from K(+)-depolarized rat spinal cord synaptosomes. Activators of PKC, such as phorbol 12-myristate 13-acetate (PMA), mezerein and oleoyl acetylglycerol produced a concentration-dependent potentiation of K(+)-induced release of [3H]5-hydroxytryptamine ([3H]5-HT). Enhanced release was dependent on the concentration of both Ca2+ and K+ in the superfusion medium. Calcium-independent release of [3H]5-HT or release induced by the Ca2+ ionophore were unaffected by PKC activators. Calcium-dependent release of [3H]5-HT, evoked by K+, was enhanced under similar conditions by the L-type Ca2+ channel agonists Bay K 8644 and (+)-SDZ 202-791. Nimodipine, an L-type Ca2+ channel antagonist, while having no independent effect on K(+)-induced release of [3H]5-HT, abolished the potentiative effects of Bay K 8644 and PMA. Similarly, the PKC inhibitors, polymyxin B and staurosporine, blocked effects of both PMA and Bay K 8644 on K(+)-stimulated release of [3H]5-HT. Neither PMA nor Bay K 8644 altered the uptake of [3H]5-HT. These results suggest that PKC-dependent mechanisms utilize calcium influx, via the L-type calcium channel, to modulate release of neurotransmitter and indicate a possible functional link between PKC and L-type voltage-dependent calcium channels in the spinal cord.     

Inhibition of neuronal Ca(2+) influx by gabapentin and subsequent reduction of neurotransmitter release from rat neocortical slices

Cytosolic calcium ion concentrations ([Ca(2+)](i)) were measured in rat neocortical synaptosomes using fura-2, and depolarization of synaptosomal membranes was induced by K(+) (30 mM). The release of the endogenous excitatory amino acids glutamate and aspartate was evoked by K(+) (50 mM) and determined by HPLC. The release of [(3)H]-noradrenaline from rat neocortical synaptosomes or slices was evoked by K(+) (15 and 25 mM) and measured by liquid scintillation counting. Gabapentin produced a concentration-dependent inhibition of the K(+)-induced [Ca(2+)](i) increase in synaptosomes (IC(50)=14 microM; maximal inhibition by 36%). The inhibitory effect of gabapentin was abolished in the presence of the P/Q-type Ca(2+) channel blocker omega-agatoxin IVA, but not by the N-type Ca(2+) channel antagonist omega-conotoxin GVIA. Gabapentin (100 microM) decreased the K(+)-evoked release of endogenous aspartate and glutamate in neocortical slices by 16 and 18%, respectively. Gabapentin reduced the K(+)-evoked [(3)H]-noradrenaline release in neocortical slices (IC(50)=48 microM; maximal inhibition of 46%) but not from synaptosomes. In the presence of the AMPA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2, 3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide (NBQX), gabapentin did not reduce [(3)H]-noradrenaline release. Gabapentin did, however, cause inhibition in the presence of the NMDA receptor antagonist DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 37849). Gabapentin is concluded to reduce the depolarization-induced [Ca(2+)](i) increase in excitatory amino acid nerve terminals by inhibiting P/Q-type Ca(2+) channels; this decreased Ca(2+) influx subsequently attenuates K(+)-evoked excitatory amino acid release. The latter effect leads to a reduced activation of AMPA receptors which contribute to K(+)-evoked noradrenaline release from noradrenergic varicosities, resulting in an indirect inhibition of noradrenaline release.     

Phencyclidine and dizocilpine modulate dopamine release from rat nucleus accumbens via sigma receptors

Phencyclidine (PCP) binds to many sites in brain, including PCP receptors located within the N-methyl-D-aspartate (NMDA) receptor-operated cation channel and sigma (sigma) receptors. In this study, we compare mechanisms by which PCP, dizocilpine (MK-801), the prototypical sigma receptor agonist (+)-pentazocine, and the proposed endogenous sigma receptor ligand neuropeptide Y regulate potassium (K(+))-stimulated [3H]dopamine release from slices of rat nucleus accumbens. (+)-Pentazocine inhibits K(+)-stimulated [3H]dopamine release, and neuropeptide Y enhances it. Both effects are blocked by sigma(1) and neuropeptide Y receptor antagonists, suggesting possible inverse agonism at a subpopulation of sigma/neuropeptide Y receptors. In contrast, PCP and MK-801 both enhance K(+)-stimulated [3H]dopamine release via sigma(1) and sigma(2) receptor subtypes, as demonstrated by antagonist sensitivity. Regulation of release by both (+)-pentazocine and neuropeptide Y persists in the presence of tetrodotoxin suggests that the sigma/neuropeptide Y receptors mediating the modulation are located presynaptically on dopaminergic nerve terminals, but tetrodotoxin eliminates regulation by PCP and MK-801, suggesting that receptors mediating their effects are located upstream from dopaminergic nerve terminals.     

Trafficking of presynaptic AMPA receptors mediating neurotransmitter release: neuronal selectivity and relationships with sensitivity to cyclothiazide

Postsynaptic glutamate AMPA receptors (AMPARs) can recycle between plasma membrane and intracellular pools. In contrast, trafficking of presynaptic AMPARs has not been investigated. AMPAR surface expression involves interactions between the GluR2 carboxy tail and various proteins including glutamate receptor-interacting protein (GRIP), AMPA receptor-binding protein (ABP), protein interacting with C kinase 1 (PICK1), N-ethyl-maleimide-sensitive fusion protein (NSF). Here, peptides known to selectively block the above interactions were entrapped into synaptosomes to study the effects on the AMPA-evoked release of [3H]noradrenaline ([3H]NA) and [3H]acetylcholine ([3H]ACh) from rat hippocampal and cortical synaptosomes, respectively. Internalization of pep2-SVKI to prevent GluR2-GRIP/ABP/PICK1 interactions potentiated the AMPA-evoked release of [3H]NA but left unmodified that of [3H]ACh. Similar potentiation was caused by pep2-AVKI, the blocker of GluR2-PICK1 interaction. Conversely, a decrease in the AMPA-evoked release of [3H]NA, but not of [3H]ACh, was caused by pep2m, a selective blocker of the GluR2-NSF interaction. In the presence of pep2-SVKI the presynaptic AMPARs on noradrenergic terminals lost sensitivity to cyclothiazide. AMPARs releasing [3H]ACh, but not those releasing [3H]NA, were sensitive to spermine, suggesting that they are GluR2-lacking AMPARs. To conclude: (i) release-regulating presynaptic AMPARs constitutively cycle in isolated nerve terminals; (ii) the process exhibits neuronal selectivity; (iii) AMPAR trafficking and desensitization may be interrelated.     

5-HT3 receptors are not involved in the modulation of the K(+)-evoked release of [3H]5-HT from spinal cord synaptosomes of rat.

The ability of 5-HT3 receptor agonists to modulate the resting efflux or K(+)-evoked release of [3H]5-HT from superfused synaptosomes from the spinal cord of the rat was investigated. Phenylbiguanide did not alter the resting efflux of [3H]5-HIAA or [3H]5-HT or modify the K(+)-evoked release of [3H]5-HT. 2-Methyl-5-HT (10 microM) caused an increase in resting efflux of [3H]5-HIAA, an effect that was blocked by the inhibitor of the uptake of 5-HT fluoxetine. No effect on K(+)-evoked release of tritium was observed. Bufotenine (100-1000 nM) increased the resting efflux of [3H]5-HT and [3H]5-HIAA. These effects were not antagonized by the 5-HT3 antagonist ICS 205-930 but were antagonized by fluoxetine. The drug ICS 205-930 (1 microM) did not alter resting efflux or block the ability of serotonin (30 and 100 nM) to decrease K(+)-evoked release of tritium. Quipazine, a potent antagonist of peripheral 5-HT3 receptors (subnanomolar concentrations), was also unable to alter resting or K(+)-evoked release of [3H]5-HT. It did, however, attenuate the inhibitory effect 5-HT on K(+)-evoked release. The concentrations required were in the micromolar range, consistent with the ability of the drug to antagonize the 5-HT1B autoreceptor. These results support the idea that 5-HT3 receptors do not act as nerve terminal autoreceptors in the spinal cord of the rat.     

Studies on the mechanism of [3H]-noradrenaline release from SH-SY5Y cells: the role of Ca2+ and cyclic AMP.

1. The roles of both Ca2+ and adenosine 3':5'-cyclic monophosphate (cyclic AMP) in carbachol and K(+)-stimulated [3H]-noradrenaline release from SH-SY5Y human neuroblastoma cells were examined. 2. Both carbachol and K+ caused a time- and dose-related stimulation of [3H]-noradrenaline release. The release event in perfused cells was monophasic. Half-maximum stimulation measured in statically incubated (3 min) cells was 38 +/- 4 microM and 63 +/- 4 mM respectively. K+ (100 mM, added)-evoked release was greater than that produced by carbachol (1 mM). 3. Both carbachol and K+ caused a time- and dose (measured at 3 min)-related stimulation of cyclic AMP formation with half-maximum stimulation occurring at 5 +/- 1 microM and 49 +/- 2 mM respectively. In contrast to its effects on release, carbachol produced a greater stimulation of cyclic AMP formation than K+. 4. K(+)-stimulated [3H]-noradrenaline release was entirely dependent on Ca2+ entry as 2.5 mM Ni2+ abolished release. However, carbachol-evoked (1 mM) release appeared to be unaffected by Ni2+ pretreatment. 5. These data suggest that in SH-SY5Y cells, elevated cyclic AMP levels are not directly involved in [3H]-noradrenaline release. In addition, carbachol-stimulated release is largely independent of extracellular Ca2+ possibly implying a role for intracellular stored Ca2+ in the release process.     

Cholecystokinin (CCK) increases GABA release in the rat anterior nucleus accumbens via CCKB receptors located on glutamatergic interneurons

The effects of cholecystokinin sulfate octapeptide (CCK-8S) on [3H]gamma-aminobutyric acid (GABA) release have been studied in the anterior side of the rat nucleus accumbens on tissue punches exposed in superfusion to 30 mM KCl. CCK-8S in a concentration dependent manner (10-3000 nM) increased K+-evoked [3H]GABA release (EC50=192 nM). The increase caused by 1 microM CCK-8S ranged from 37% to 42%. CR 2945, (beta-[2-[[2-(8-azaspiro[4.5]dec-8-ylcarbonyl)-4,6-dimethylp henyl]-amino]-2-oxoethyl]-(R)-1-naphthalenepropanoic acid), a potent and selective nonpeptidergic CCK(B) antagonist, concentration-dependently blocked CCK-8S effect (IC50=2.16 nM). CCK-8S-induced increase in [3H]GABA overflow was completely blocked by 1 microM tetrodotoxin. Both the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) and the N-methyl-D-aspartic acid (NMDA) receptor antagonist dizocilpine (MK-801) antagonized the CCK-8S effect. By contrast, (+)-bicuculline, a GABA(A) receptor antagonist, was completely ineffective. Phaclofen, a selective GABA(B) antagonist, increased K+-evoked [3H]GABA release but did not affect the facilitative effect of CCK-8S. Moreover, tetrodotoxin failed to block AMPA-evoked [3H]GABA release but completely prevented the effect of NMDA (Mg2+ free conditions). The data presented suggest that CCK(B) receptors modulating [3H]GABA release from anterior accumbal punches may not be present on GABAergic terminals but could be located on glutamatergic interneurons.     

Novel receptor site involved in enhancement of stimulus-induced acetylcholine, dopamine, and serotonin release.

The cognitive enhancer DuP 996 [3,3-bis(4-pyrindinylmethyl)-1-phenylindolin-2-one] and its structural analogs enhance the K(+)-stimulated release of acetylcholine, dopamine, and serotonin in brain slices, without effect on basal release. A novel receptor site labeled by [3H]DuP 996 has been identified. The [3H]DuP 996 binding site has a Kd of 19 nM and a Bmax of 102 fmol/mg of protein. Binding to this site is specific, saturable, reversible, and time, pH, and temperature dependent. Specific binding is decreased by treatment with trypsin and not affected by phospholipase C. Specific binding is inhibited by Ca2+ and increased by Mn2+ but not affected by Na+, K+, or Mg2+. The [3H]DuP 996 binding sites are heterogeneously distributed in brain, with striatum and hypothalamus having highest density and cerebellum lowest. The [3H]DuP 996 binding site does not belong to any known class of receptor site, because [3H]DuP 996 binding could not be displaced by a broad variety of standard pharmacological agents and neuropeptides. Physiological significance of this binding site is suggested by the excellent correlation between the binding affinity for this site and the potency to enhance K(+)-stimulated release of acetylcholine for a series of DuP 996 analogs. Ligands for this receptor site may have therapeutic potential for the treatment of cognitive deficits and neurodegenerative diseases.     

A single residue contributes sensitivity to allosteric modulation of AMPA receptors by LY395153

Previous studies have shown that a single point mutation (S(750)Q) in the splice variant region of rat Glu(1) subunits can eliminate positive allosteric modulation by cyclothiazide. The present study investigated the effects of mutating the equivalent residue (S(776)Q) in the human Glu(4) subunit on the activity and binding of a novel AMPA receptor potentiator, LY395153 (N-2-(4-benzamidophenylpropyl-2-propanesulfonamide)). The mutation markedly attenuated, but did not eliminate, potentiation by LY395153 and cyclothiazide. In addition, binding of [3H]LY395153 was significantly reduced by this mutation. These effects occurred in the absence of any change in the response to glutamate or the binding of a competitive AMPA receptor antagonist, [3H]Ro 48-8587 ([2,4,5-3H]9-imidazol-1-yl-8-nitro-2,3,5,6-tetrahydro[1,2,4]-triazolo[1,5-c]quinazoline-2,5-dione triethylammonium salt). Collectively, these results demonstrate that structurally diverse classes of potentiators are sensitive to mutations of this single Ser residue, suggesting that binding to this residue may be necessary for positive allosteric modulation of AMPA receptors.     

Influence of antipsychotics and serotonin antagonists on presynaptic receptors modulating the release of serotonin in synaptosomes of the nucleus accumbens of rats

In the nucleus accumbens of rats the release of [3H]serotonin (5-HT) from superfused synaptosomes stimulated by 30 mM K+ was investigated. In the presence of 40 microM of the uptake inhibitor cocaine the release of [3H]5-HT was inhibited by 5-HT in a concentration-dependent manner (IC50 = 0.45 microM). The maximum inhibitory effect of 5-HT was 54% of controls. The inhibition of K+-stimulated release of [3H]5-HT induced by 5-HT was antagonized completely by methiothepine and clozapine, respectively, whereas methysergide had only a weak antagonizing effect in a concentration of 20 microM or less, haloperidol was ineffective. Furthermore, the synaptosomal K+-stimulated release of [3H]5-HT was also inhibited by dopamine (DA) in a concentration-dependent manner (IC50 = 0.1 microM). This inhibitory effect was antagonized by antipsychotic drugs, the rank order of antagonistic potencies was sulpiride greater than haloperidol greater than clozapine; methiothepine was ineffective. The experimental system (the K+-stimulated synaptosomal release of [3H]5-HT seems to be a suitable model for differentiating dopaminergic and/or serotonergic components of antipsychotics or other drugs on presynaptic receptors.     

Inhibition of neuronal Ca(2+) influx by gabapentin and pregabalin in the human neocortex.

Gabapentin and pregabalin (S-(+)-3-isobutylgaba) produced concentration-dependent inhibitions of the K(+)-induced [Ca(2+)](i) increase in fura-2-loaded human neocortical synaptosomes (IC(50)=17 microM for both compounds; respective maximal inhibitions of 37 and 35%). The weaker enantiomer of pregabalin, R-(-)-3-isobutylgaba, was inactive. These findings were consistent with the potency of these drugs to inhibit [(3)H]-gabapentin binding to human neocortical membranes. The inhibitory effect of gabapentin on the K(+)-induced [Ca(2+)](i) increase was prevented by the P/Q-type voltage-gated Ca(2+) channel blocker omega-agatoxin IVA. The alpha 2 delta-1, alpha 2 delta-2, and alpha 2 delta-3 subunits of voltage-gated Ca(2+) channels, presumed sites of gabapentin and pregabalin action, were detected with immunoblots of human neocortical synaptosomes. The K(+)-evoked release of [(3)H]-noradrenaline from human neocortical slices was inhibited by gabapentin (maximal inhibition of 31%); this effect was prevented by the AMPA receptor antagonist NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide). Gabapentin and pregabalin may bind to the Ca(2+) channel alpha 2 delta subunit to selectively attenuate depolarization-induced Ca(2+) influx of presynaptic P/Q-type Ca(2+) channels; this results in decreased glutamate/aspartate release from excitatory amino acid nerve terminals leading to a reduced activation of AMPA heteroreceptors on noradrenergic nerve terminals.     

Effects of glutamate transport substrates and glutamate receptor ligands on the activity of Na-/K(+)-ATPase in brain tissue in vitro

1. It has been suggested that Na+/K(+)-ATPase and Na(+)-dependent glutamate transport (GluT) are tightly linked in brain tissue. In the present study, we have investigated Na+/K(+)-ATPase activity using Rb+ uptake by 'minislices' (prisms) of the cerebral cortex. This preparation preserves the morphology of neurons, synapses and astrocytes and is known to possess potent GluT that has been well characterized. Uptake of Rb+ was determined by estimating Rb+ in aqueous extracts of the minislices, using atomic absorption spectroscopy. 2. We determined the potencies of several known substrates/inhibitors of GluT, such as L-trans-pyrrolidine-2,4-dicarboxylate (LtPDC), DL-threo-3-benzyloxyaspartic acid, (2S,3S,4R)-2-(carboxycyclopropyl)-glycine (L-CCG III) and L-anti,endo-3,4-methanopyrrolidine dicarboxylic acid, as inhibitors of [3H]-L-glutamate uptake by cortical prisms. In addition, we established the susceptibility of GluT, measured as [3H]-L-glutamate uptake in brain cortical prisms, to the inhibition of Na+/K(+)-ATPase by ouabain. Then, we tested the hypothesis that the Na+/K(+)-ATPase (measured as Rb+ uptake) can respond to changes in the activity of GluT produced by using GluT substrates as GluT-specific pharmacological tools. 3. The Na+/K(+)-ATPase inhibitor ouabain completely blocked Rb+ uptake (IC50 = 17 micromol/L), but it also potently inhibited a fraction of GluT (approximately 50% of [3H]-L-glutamate uptake was eliminated; IC50 < 1 micromol/L). 4. None of the most commonly used GluT substrates and inhibitors, such as L-aspartate, D-aspartate, L-CCG III and LtPDC (all at 500 micromol/L), produced any significant changes in Rb+ uptake. 5. The N-methyl-D-aspartate (NMDA) receptor agonists (R,S)-(tetrazol-5-yl)-glycine and NMDA decreased Rb+ uptake in a manner compatible with their known neurotoxic actions. 6. None of the agonists or antagonists for any of the other major classes of glutamate receptors caused significant changes in Rb+ uptake. 7. We conclude that, even if a subpopulation of glutamate transporters in the rat cerebral cortex may be intimately linked to a fraction of Na+/K(+)-ATPase, it is not possible, under the present experimental conditions, to detect regulation of Na+/K(+)-ATPase by GluT.