Effects of Non-NMDA Receptor Modulators on [3H]Dopamine Release from Rat Mesencephalic Cells in Primary Culture

Abstract: The effects of AMPA and kainate on [³H]dopamine release from fetal (embryonic day 15) rat mesencephalic neurons in primary culture were enhanced markedly in a dose-dependent fashion by cyclothiazide, a recently described inhibitor of AMPA receptor desensitization. The EC₅₀ value for cyclothiazide was 2.2 ± 0.8 µ M . The release of [³H]dopamine induced by both AMPA (or kainic acid) and the combination of AMPA (or kainic acid) with cyclothiazide was antagonized by specific antagonists like 6-cyano-7-nitroquinoxaline-2,3-dione or the noncompetitive benzodiazepine GYKI 52466. Unlike cyclothiazide, the lectin concanavalin A did not stimulate [³H]dopamine release. These results established the involvement of AMPA-preferring receptors on [³H]dopamine release from rat mesencephalic neurons in primary culture and provided further evidence for the existence of regulatory allosteric sites on AMPA receptor subunits.     

Platelet-Activating Factor: Diminished Acetylcholine Release from Rat Brain Slices Is Mediated by a Gi Protein

Abstract: The effect of platelet-activating factor (PAF) on neurotransmitter release from rat brain slices prelabeled with [³H]acetylcholine ([³H]ACh), [³H]norepinephrine ([³H]NE), or [³H]serotonin ([³H]5-HT) was studied. PAF inhibited K⁺ depolarization-induced [³H]ACh release in slices of brain cortex and hippocampus by up to 59% at 10 n M but did not inhibit [³H]ACh release in striatal slices. PAF did not affect 5-HT or NE release from cortical brain slices. The inhibition of K⁺-evoked [³H]ACh release induced by PAF was prevented by pretreating tissues with several structurally different PAF receptor antagonists. The effect of PAF was reversible and was not affected by pretreating brain slices with tetrodotoxin. PAF-induced inhibition of [³H]ACh release was blocked 90 ± 3 and 86 ± 2% by pertussis toxin and by anti-Gαi_1/2 antiserum incorporated into cortical synaptosomes, respectively. The results suggest that PAF inhibits depolarization-induced ACh release in brain slices via a Gαi_1/2 protein-mediated action and that PAF may serve as a neuromodulator of brain cholinergic system.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

Allosteric Interactions Among Agonists and Antagonists at 5-Hydroxytryptamine3 Receptors

Abstract: Cooperation in the action of agonists suggests that there are multiple binding sites on 5-hydroxytryptamine₃ (5-HT₃) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5-HT₃ receptors. The affinities of competitive 5-HT₃ receptor antagonists were similar regardless of whether the receptors were labeled with [³H]RS-42358, [³H]granisetron, or 1-( m -[³H]chlorophenyl)biguanide ([³H]mCPG). By contrast, the affinities of the agonists 5-HT, mCPG, and phenylbiguanide were approximately 10-fold higher when the receptors were labeled with [³H]mCPG. The dissociation of [³H]mCPG, [³H]RS-42358, and [³H]RS-25259, but not [³H]granisetron, from both cloned and native 5-HT₃ receptors was markedly slower in the presence of 5-HT or 2-methyl-5-HT than in the presence of antagonists such as RS-42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5-HT₃ receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor.     

Characterization of the Binding of [3H]NS 257, a Novel Competitive AMPA Receptor Antagonist, to Rat Brain Membranes and Brain Sections

Abstract: The binding of [³H]NS 257 {1,2,3,6,7,8-hexahydro-3-(hydroxyimino)- N,N -[³H]dimethyl-7-methyl-2-oxobenzo[2,1- b :3,4- c '] dipyrrole-5-sulfonamide} to rat cortical membranes was characterized in the absence and presence of thiocyanate. Specific [³H]NS 257 binding was saturable and reversible, and the stimulating effect of thiocyanate on binding was optimal at 100 m M . In the presence of thiocyanate [³H]NS 257 bound to a single population of binding sites with an affinity of 225 ± 8 n M and a binding site density of 0.61 ± 0.04 pmol/mg of original tissue. Thiocyanate increased the affinity of the binding site labeled by [³H]NS 257 for both α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and l -glutamate by a factor of 20 and 5, respectively. However, the affinity of the agonist domoate and the antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo( f )-quinoxaline (NBQX) was decreased in the presence of thiocyanate. Apparently, the affinities of antagonists as well as agonists for the AMPA receptor can be either increased or decreased by thiocyanate. The rank order of potency of the putative agonists quisqualate > AMPA > l -glutamate > domoate > kainate and of the antagonists NBQX > CNQX is consistent with the labeling of AMPA receptors. Autoradiographic studies showed that the distribution of [³H]NS 257 binding sites in rat brain was similar to that of [³H]AMPA binding sites. NS 257 is the first AMPA antagonist to be described showing an increased affinity for the AMPA receptor in the presence of thiocyanate.     

Pharmacology and Regional Distribution of the Binding of 6-[3H]Nitro-7-Sulphamoylbenzo[f]-Quinoxaline-2,3-Dione to Rat Brain

Abstract: 6-Nitro-7-sulphamoylbenzo[ f ]quinoxaline-2,3-dione (NBQX) is a competitive antagonist selective for α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. Here we report the pharmacological characteristics and anatomical distribution of [³H]NBQX binding to rat brain. The association rate of [³H]NBQX to rat cerebrocortical membranes was rapid, with peak binding occurring within 10 min at 0°C. The off-rate was also rapid, with near-complete dissociation of the radioligand within 5 min of addition of 1 m M unlabelled l -glutamate. [³H]NBQX bound to a single class of sites with K _D and B _max values of 47 n M and 2.6 pmol mg⁻¹ of protein, respectively. The rank order of inhibition of [³H]NBQX binding by AMPA receptor ligands was NBQX ≫ 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) ≥ ( S )-5-fluorowillardiine ≥ AMPA ≫ l -glutamate. The chaotrope KSCN had no effect on the IC₅₀ value of unlabelled NBQX displacement of [³H]NBQX binding. The kainate receptor-selective ligands NS102 and kainate were only very weak displacers. It is interesting that NBQX and CNQX displaced significantly more [³H]NBQX than any of the agonists tested. Autoradiographic analysis of the binding of [³H]NBQX to coronal sections showed a distribution compatible with that of [³H]AMPA binding. These data indicate that [³H]NBQX provides a useful novel tool to characterise the antagonist binding properties of AMPA receptors.     

Irreversible Inhibition of High-Affinity [3H]Kainate Binding by a Novel Photoactivatable Analogue: (2'S,3'S,4'R)-2'-Carboxy-4'-(2-Diazo-1-Oxo-3,3,3-Trifluoropropyl)-3'-Pyrrolidinyl Acetate

Abstract: A photolabile trifluoromethyldiazoketone derivative of kainate (KA), (2' S ,3' S ,4' R )-2'-carboxy-4'-(2-diazo-1-oxo-3,3,3-trifluoropropyl)-3'-pyrrolidinyl acetate (DZKA), was synthesized and evaluated as an irreversible inhibitor of the high-affinity KA site on rat forebrain synaptic plasma membranes (SPMs). In the absence of UV irradiation, DZKA preferentially blocked [³H]KA binding with an IC₅₀ of 0.63 µ M , a concentration that produced little or no inhibition at AMPA or NMDA sites. At 100 µ M , however, DZKA inhibited [³H]AMPA and l -[³H]glutamate binding by ∼50%. When examined electrophysiologically in HEK293 cells expressing human KA (GluR6) or AMPA (GluR1) subtypes, DZKA acted preferentially at KA receptors as a weak agonist. DZKA also exhibited little or no excitotoxic activity in mixed rat cortical cultures. Irreversible inhibition was assessed by pretreating SPMs with DZKA (50 µ M ) in the presence of UV irradiation, removing unbound DZKA, and then assaying the reisolated SPMs for radioligand binding. This protocol produced a selective and irreversible loss of ∼50% of the [³H]KA sites. The binding was recoverable in SPMs pretreated with DZKA or UV alone. Coincubation with l -glutamate prevented the loss in [³H]KA binding, suggesting that the inactivation occurred at or near the ligand binding site. These results are consistent with the action of DZKA as a photoaffinity ligand for the KA site and identify the analogue as a valuable probe for future investigations of receptor structure and function.     

Modulation of Histamine Release and Synthesis in the Brain Mediated by α2-Adrenoceptors

Abstract: The adrenergic regulation of histamine release was studied in rat brain slices labeled with L-[³H]histidine. Noradrenaline in increasing concentrations progressively inhibited K⁺-evoked [³H]histamine release from cortical slices, whereas phenylephrine and isoprenaline were ineffective. Yohimbine, a preferential α2-adrenoceptor antagonist, reversed the noradrenaline effect in an apparently competitive manner and with a mean K i value of 30 n M . Phentolamine reversed the noradrenaline effect with a similar potency, whereas propranolol was ineffective. The imidazolines clo-nidine and oxymetazoline acted as partial agonists, oxymeta-zoline even behaving as an apparent antagonist. In vivo clo-nidine also inhibited [³H]histamine formation in cerebral cortex, an effect reversed by the administration of yohimbine. However, yohimbine failed to increase significantly [³H]histamine release in vitro and [³H]histamine formation in vivo, suggesting that adrenergic receptors are not activated by endogenous noradrenaline released under basal conditions. It is concluded that adrenergic α₂-adrenoceptors presumably located on histaminergic axons control release and synthesis of histamine in the brain.     

Exposure to an enriched environment selectively increases the functional response of the pre-synaptic NMDA receptors which modulate noradrenaline release in mouse hippocampus

We evaluated the impact of environmental training on the functions of pre-synaptic glutamatergic NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and nicotinic receptors expressed by hippocampal noradrenergic nerve terminals. Synaptosomes isolated from the hippocampi of mice housed in enriched (EE) or standard (SE) environment were labeled with [³H]noradrenaline ([³H]NA) and tritium release was monitored during exposure in superfusion to NMDA, AMPA, epibatidine or high K⁺. NMDA -evoked [³H]NA release from EE hippocampal synaptosomes was significantly higher than that from SE synaptosomes, while the [³H]NA overflow elicited by 100 μM AMPA, 1 μM epibatidine or (9, 15, 25 mM) KCl was unchanged. In EE mice, the apparent affinity of NMDA or glycine was unmodified, while the efficacy was significantly augmented. Sensitivity to non-selective or subtype-selective NMDA receptor antagonists (MK-801, ifenprodil and Zn²⁺ ions) was not modified in EE. Finally, the analysis of NMDA receptor subunit mRNA expression in noradrenergic cell bodies of the locus coeruleus showed that NR1, NR2A, NR2B and NR2D subunits were unchanged, while NR2C decreased significantly in EE mice as compared to SE mice. Functional up-regulation of the pre-synaptic NMDA receptors modulating NA release might contribute to the improved learning and memory found in animals exposed to an EE.     

Binding Characteristics of a Potent AMPA Receptor Antagonist [3H]Ro 48-8587 in Rat Brain

Abstract: A new AMPA receptor antagonist, Ro 48-8587, was characterized pharmacologically in vitro. It is highly potent and selective for AMPA receptors as shown by its effects on [³H]AMPA, [³H]kainate, and [³H]MK-801 binding to rat brain membranes and on AMPA- or NMDA-induced depolarization in rat cortical wedges. [³H]Ro 48-8587 bound with a high affinity ( K _D = 3 n M ) to a single population of binding sites with a B _max of 1 pmol/mg of protein in rat whole brain membranes. [³H]Ro 48-8587 binding to rat whole brain membranes was inhibited by several compounds with the following rank order of potency: Ro 48-8587 > 6-nitro-7-sulphamoylbenzo[ f ]quinoxaline-2,3-dione (NBQX) > YM 90K > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > quisqualate > AMPA > glutamate > kainate > NMDA. The distribution and abundance of specific binding sites (∼95% of total) in sections of rat CNS, revealed by quantitative receptor radioautography and image analysis, indicated a very discrete localization. Highest binding values were observed in cortical layers (binding in layers 1 and 2 > binding in layers 3–6), hippocampal formation, striatum, dorsal septum, reticular thalamic nucleus, cerebellar molecular layer, and spinal cord dorsal horn. At 1 n M , the values for specific binding were highest in the cortical layers 1 and 2 and lowest in the brainstem (∼2.6 and 0.4 pmol/mg of protein, respectively). Ro 48-8587 is a potent and selective AMPA receptor antagonist with improved binding characteristics (higher affinity, selectivity, and specific binding) compared with those previously reported.     

Desensitization of Nicotine-Stimulated [3H]Dopamine Release from Mouse Striatal Synaptosomes

Potential desensitization of brain nicotinic receptors was studied using a [³H]dopamine release assay. Nicotine-stimulated [³H]dopamine release from mouse striatal synaptosomes was concentration-dependent with an EC₅₀ of 0.33 ± 0.13 μ M and a Hill coefficient of 1.44 ± 0.18. Desensitization by activating concentrations of nicotine had a similar EC₅₀ and a half-time of 35 s. Concentrations of nicotine that evoked little release also induced a concentration-dependent desensitization (EC₅₀=6.9 plusmn; 3.6 n M , t_1/2= 1.6-2.0 min, n _H=1.02 ± 0.01). Both types of desensitization produced a maximum 75% decrease in [³H]dopamine release. Recovery from desensitization after exposure to low or activating concentrations of nicotine was time-dependent with half-times of 6.1 min and 12.4 min, respectively. Constants determined for binding of [³H]nicotine to striatal membrane at 22°C included a K _Dof 3.7 ± 0.5 n M , B_max of 67.5 ± 2.2 fmol/mg, and Hill coefficient of 1.07 ± 0.06. Association of nicotine with membrane binding sites was biphasic with half-times of 9 s and 1.8 min. The fast rate process contributed 37% of the total reaction. Dissociation was a uniphasic process with a half-time of 1.6 min. Comparison of constants determined by the release and binding assays indicated that the [³H]-nicotine binding site could be the presynaptic receptor involved in [³H]dopamine release in mouse striatal synaptosomes.     

Dopamine Agonist-Mediated Inhibition of Acetylcholine Release in Rat Striatum Is Modified by Thyroid Hormone Status

Abstract: K⁺-evoked acetyl[³H]choline ([³H]ACh) release was inhibited in a concentration-dependent manner by apomorphine and the D₂ agonist quinpirole in striatal slices prepared from euthyroid and hypothyroid rats. However, there was a significant increase in the maximum inhibition observed with both agonists in the hypothyroid compared with the euthyroid group, which paralleled the increased D₂ agonist sensitivity reported for stereotyped behavior. The D₂ antagonist raclopride decreased, and the D, antagonist SCH 23390 increased, the inhibition of [³H]ACh release by apomorphine, confirming an inhibitory role for D₂ receptors and an opposing role for D₁ receptors. Because there is no difference in D₁ or D₂ receptor concentration between the euthyroid and hypothyroid groups, it is suggested that thyroid hormone modulation of D₂ receptor sensitivity affects a receptor-mediated event. Following intrastriatal injection of pertussis toxin (PTX), apomorphine no longer inhibited [³H]ACh release. In fact, increased [³H]- ACh release was observed, an effect reduced by SCH 23390, providing evidence that D₁ receptors enhance [³H]- ACh release, and confirming that a PTX-sensitive G protein mediates the D₂ response. As it has been reported that thyroid hormones modulate G protein expression, this mechanism may underlie their effect on dopamine agonist- mediated inhibition of ACh.     

Nicotine-Stimulated Release of [3H]Norepinephrine from Fetal Rat Locus Coeruleus Cells in Culture

Abstract: Acute nicotine administration stimulated [³H]norepinephrine ([³H]NE) release from cultured fetal locus coeruleus (LC) cells. The effect was concentration dependent, with an EC₅₀ of 0.9 µ M , and was abolished by removal of calcium from, or addition of tetrodotoxin (500 n M ) to, the assay buffer. Other nicotinic receptor agonists stimulated [³H]NE release, with the rank order of potency being (±)-epibatidine > (−)-nicotine > 1,1-dimethyl-4-phenylpiperazinium (DMPP). Whereas (−)-nicotine and (±)-epibatidine exhibited equal maximal responses, DMPP was a partial agonist and (−)-cytisine had no agonist activity. Nicotine-stimulated release of [³H]NE was blocked by nicotinic receptor antagonists, with an order of potency of mecamylamine > lobeline > cytisine > methyllycaconitine > dihydro-β-erythroidine. The pharmacological profile of this nicotinic receptor is largely consistent with that described previously for an α4β2 subunit combination, although discrepancies in the efficacies of agonists were observed. No additivity in NMDA- and nicotine-stimulated [³H]NE release was observed, suggesting a common signal transduction mechanism. However, the pharmacological characteristics of MK-801 blockade of nicotine-induced responses were not consistent with those of an NMDA receptor. We therefore conclude that nicotine directly releases [³H]NE from LC cells and does not act indirectly via activation of glutamate release.     

Aluminum, Iron, and Zinc Ions Promote Aggregation of Physiological Concentrations of β-Amyloid Peptide

Abstract: Mechanisms of non-NMDA receptor-mediated excitotoxicity were studied in embryonic rat hippocampal cultures using kainic acid (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) as agonists. Under basal culture conditions, overnight treatment with AMPA resulted in negligible excitotoxicity as assessed by phase-contrast microscopy and measurement of lactate dehydrogenase (LDH) release. In contrast, similar treatment with KA resulted in marked excitotoxic morphologic changes and release of LDH. Cotreatment of cultures with AMPA but not NMDA effectively blocked KA toxicity, suggesting that AMPA-induced rapid desensitization of the AMPA/KA receptor could account for the lack of prominent direct toxicity as well as AMPA's ability to block KA toxicity. To test this hypothesis, cultures were briefly pretreated with 10 μ M cyclothiazide, a drug reported to block desensitization of the AMPA/KA receptor, and then exposed overnight to cyclothiazide plus AMPA and/or KA. Cyclothiazide-treated cultures were now vulnerable to AMPA as well as KA; moreover, AMPA was unable to block KA toxicity completely, suggesting that cyclothiazide impaired AMPA/KA receptor desensitization. These and related studies suggest that a regulatory site may exist on the AMPA/KA receptor that modulates non-NMDA receptor-mediated excitotoxicity.     

AMPA Receptor Development in Rat Telencephalon: [3H]AMPA Binding and Western Blot Studies

Abstract: Telencephalic membranes from rats of different embryonic (E16, E19) and postnatal (P2, P7, P14, adult) ages were assessed for α-[³H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([³H]AMPA) binding and for immunoreactivity levels of AMPA receptor subunits (GluR1, GluR2/3, and GluR4). In addition, the synaptic markers synaptophysin and NCAM₁₄₀ (a neural cell adhesion molecule isoform) were examined by immunoblot. The density of [³H]AMPA binding sites increased steadily with advancing age. This increase was due mainly to the development of the large low-affinity component ( K _D = 400 n M ) that dominates the [³H]AMPA binding profile of adult rat brain membranes. As resolved by two-site regression analysis, the high-affinity component ( K _D = 15 n M ) of the [³H]AMPA binding increased by approximately twofold from E16 to adult, whereas the low-affinity component increased by 25-fold. Staining for GluR1 and GluR2/3 increased steadily with increasing age at all time points examined; synaptophysin and NCAM₁₄₀ exhibited similar ontogenic immunostaining profiles. GluR4 immunoreactivity was first evident at P14 and increased by adulthood. These results indicate that AMPA receptor density increases steadily during development and that this increase is coincident with the ontogenic expression of other synaptic components. Furthermore, there is a shift toward a preponderance of low-affinity [³H]AMPA binding, which occurs during the period when AMPA receptors are being sorted into postsynaptic regions, suggesting that some element of the postsynaptic membrane environment modulates AMPA receptor properties.     

Increased Activation of L-Type Voltage-Dependent Calcium Channels Is Associated with Glycine Enhancement of N-Methyl-d-Aspartate-Stimulated Dopamine Release in Global Cerebral Ischemia/Reperfusion

Abstract: We investigated the relationships among N -methyl- d -aspartate, glycine, L-type voltage-dependent calcium channels, and [³H]dopamine release in a canine model of global cerebral ischemia/reperfusion. The binding of [³H]PN200-110 ([³H]isradipine) to L-type voltage-dependent calcium channels, that open as a consequence of N -methyl- d -aspartate-induced changes in membrane potential, was approximately doubled in striatal membranes prepared from ischemic animals relative to controls, and remained significantly elevated at 30 min and 2 h of reperfusion. These changes coincided temporally with changes in the ability of the voltage-sensitive calcium channel blocker nitrendipine to inhibit glycine enhancement of N -methyl- d -aspartate-stimulated [³H]dopamine release in striatal slices prepared from the same animals. Compared with nonischemic controls, N -methyl- d -aspartate-stimulated [³H]dopamine release was increased in ischemic animals and remained increased throughout reperfusion up to at least 24 h. Glycine enhanced N -methyl- d -aspartate-stimulated release in all treatment groups. The enhancement of N -methyl- d -aspartate-stimulated dopamine release by glycine was reduced by the inclusion of nitrendipine in striatal slices from ischemic and 30-min reperfused animals. These data suggest that glycine may facilitate opening of the voltage-dependent calcium channels activated by N -methyl- d -aspartate and that this facilitation is blocked by the antagonist nitrendipine.     

Interactions Between N-Acetylaspartylglutamate and AMPA, Kainate, and NMDA Binding Sites

Abstract: The structure of N -acetylaspartylglutamate (NAAG) suggests this neuronal dipeptide as a candidate for interaction with discrete subclasses of ionotropic and metabotropic acidic amino acid receptors. A substantial difficulty in the assay of these interactions is posed by membrane-bound peptidase activity that converts the dipeptide to glutamate and N -acetylaspartate, molecules that will interfere with receptor assays. We have developed two sets of unique receptor assay conditions and applied one standard assay to measure the interactions, under equilibrium binding conditions, of [³H]kainate, [³H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([³H]AMPA), and [³H]CGS-19755 with the three classes (kainate, quisqualate, and N -methyl- d -aspartate) of ionotropic glutamate receptors, while inhibiting peptidase activity against NAAG. Under these conditions, NAAG exhibits apparent inhibition constants (IC₅₀) of 500, 790, and 8.8 µ M in the kainate, AMPA, and CGS-19755 receptor binding assays, respectively. Glutamate was substantially more effective and less specific in these competition assays, with inhibition constants of 0.36, 1.1, and 0.37 µ M . These data support the hypothesis that, relative to glutamate, NAAG functions as a specific, low potency agonist at N -methyl- d -aspartate subclass of ionotropic acidic amino acid receptors, but the peptide is not likely to activate directly the kainate or quisqualate subclasses of excitatory ionotropic receptors under physiologic conditions.     

Stimulation of [3H]GABA and β-[3H]Alanine Release from Rat Brain Slices by cis-4-Aminocrotonic Acid

Abstract: cis -4-Aminocrotonic acid (CACA; 100 µ M ), an analogue of GABA in a folded conformation, stimulated the passive release of [³H]GABA from slices of rat cerebellum, cerebral cortex, retina, and spinal cord and of β-[³H]alanine from slices of cerebellum and spinal cord without influencing potassium-evoked release. In contrast, CACA (100 µ M ) did not stimulate the passive release of [³H]taurine from slices of cerebellum and spinal cord or of d -[³H]aspartate from slices of cerebellum and did not influence potassium-evoked release of [³H]taurine from the cerebellum and spinal cord and d -[³H]aspartate from the cerebellum. These results suggest that the effects of CACA on GABA and β-alanine release are due to CACA acting as a substrate for a β-alanine-sensitive GABA transport system, consistent with CACA inhibiting the uptake of β-[³H]alanine into slices of rat cerebellum and cerebral cortex. The observed K _i for CACA against β-[³H]alanine uptake in the cerebellum was 750 ± 60 µ M . CACA appears to be 10-fold weaker as a substrate for the transporter system than as an agonist for the GABA_c receptor. The effects of CACA on GABA and β-alanine release provide indirect evidence for a GABA transporter in cerebellum, cerebral cortex, retina, and spinal cord that transports GABA, β-alanine, CACA, and nipecotic acid that has a similar pharmacological profile to that of the GABA transporter, GAT-3, cloned from rat CNS. The structural similarities of GABA, β-alanine, CACA, and nipecotic acid are demonstrated by computer-aided molecular modeling, providing information on the possible conformations of these substances being transported by a common carrier protein.     

Role of Intercellular and Intracellular Communication by Nitric Oxide in Coupling of Muscarinic Receptors to Activation of Guanylate Cyclase in Neuronal Cells

Abstract: Muscarinic receptor-mediated cyclic GMP formation and release of nitric oxide (NO) (or a precursor thereof) were compared in mouse neuroblastoma N1E-115 cells. [³H]Cyclic GMP was assayed in cells prelabeled with [³H]guanine. Release of NO upon the addition of muscarinic agonists to unlabeled neuroblastoma cells (NO donor cells) was quantitated indirectly by its ability to increase the [³H]cyclic GMP level in labeled cells whose muscarinic receptors were inactivated by irreversible alkylation (NO detector cells). Carbachol increased NO release in a concentration-dependent manner, with half-maximal stimulation at 173 μ M (compared to 96 μ M for direct activation of cyclic GMP formation). The maximal effect of carbachol in stimulating release of NO when measured indirectly was lower than that in elevating [³H]cyclic GMP directly in donor cells. Hemoglobin was more effective in blocking the actions of released NO than in attenuating direct stimulation of [³H]cyclic GMP synthesis. There was a good correlation between the ability of a series of muscarinic agonists to release NO or to activate [³H]cyclic GMP formation directly, and the potency of pirenzepine in inhibiting the two responses. Furthermore, there was a similar magnitude of desensitization of both responses by prolonged receptor activation or stimulation of protein kinase C. NO release was also regulated in relation to the cellular growth phase. A model is proposed in which a fraction of NO generated upon receptor activation does not diffuse extracellularly and stimulates cyclic GMP synthesis within the same cell where it is formed (locally acting NO). The remainder of NO that is extruded extracellularly might travel to neighboring cells (neurotransmitter NO) or might be taken back into the cells of origin (homing NO).     

Regional Distribution of α-[3H]Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Binding Sites in Rat Brain: Effect of Chemical Modification of SH– Groups in Tissue Sections

Abstract: Previous studies have shown that chemical modifications of sulfhydryl (SH–) groups with mercurial compounds in rat brain membrane preparations increase the binding of α -[³H]-amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([³H]AMPA), a ligand for the quisqualate/AMPA type of glutamate receptors. In the present study we investigated the regional distribution of SH– group modification by quantitative analysis of autoradiographic images of [³H]AMPA binding in tissue sections. We also compared the effect of SH– group modification to that of the chaotropic ion thiocyanate (SCN⁻) which has been generally utilized to study [³H]AMPA binding sites. Low levels of binding sites were observed in the absence of potassium thiocyanate (KSCN), with binding predominantly found in telencephalic structures. The presence of KSCN induced a relatively uniform and large (four- to fivefold) increase in binding throughout the different brain structures. Pretreatment of the tissue sections with the SH– group reagent p -chloromercuriphenylsulfonic acid produced a 0.5- to 1.5-fold increase in [³H]AMPA binding. The enhanced binding displayed a regional variation with the largest increase in binding observed in the outer layer of the parietal cortex whereas the lowest increase occurred in the striatum. These results indicate that SH– group modification of tissue sections produces an increase in [³H]AMPA binding similar to that observed in detergent-treated membrane preparations. Moreover they reveal that [³H]AMPA binding sites in different brain regions vary in their susceptibility to modification by SH– reagents, suggesting the existence in brain of a heterogeneous distribution of quisqualate/AMPA receptor subtypes.