[3H]SCH 58261, a Selective Adenosine A2A Receptor Antagonist, Is a Useful Ligand in Autoradiographic Studies

Abstract: We have characterized the new potent and selective nonxanthine adenosine A_2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A_2A receptors ([³H]CGS 21680) or A₁ receptors ( N ⁶-[³H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A_2A versus A₁ receptors ( K _i values of 1.2 n M versus 0.8 µ M ). Moreover, receptor autoradiography showed that [³H]SCH 58261, in concentrations below 2 n M , binds only to the dopamine-rich regions of the rat brain, with a K _D value of 1.4 (0.8–1.8) n M . The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 n M , the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [³H]SCH 58261 with the following estimated K _i values (n M ): 2-hex-1-ynyl-5'- N -ethylcarboxamidoadenosine, 3.9 (1.8–8.4); CGS 21680, 130 (42–405); N ⁶-cyclohexyladenosine, 9,985 (3,169–31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 µ M ) or Mg²⁺ (10 m M ). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [³H]CGS 21680.     

[3H]LY303870, a Novel Nonpeptide Radioligand for the NK-1 Receptor

Abstract: We synthesized a potent and selective antagonist radioligand for the neurokinin (NK)-1 receptor and characterized its binding to guinea pig striatal membranes. ( R ) - N - [2 - [Acetyl[³H₃][(2 - methoxyphenyl) - methyl]amino] - 1 - (1 H - indol - 3 - ylmethyl)ethyl][1,4' - bipiperidine]-1'-acetamide ([³H]LY303870) binds to a single class of sites with an equilibrium K _D of 0.22 n M and a B _max of 723 fmol/mg of protein. Unlabeled LY303870 potently inhibited the binding with an IC₅₀ of 0.56 n M , whereas the less active ( S )-enantiomer (LY306155) was substantially less potent. The nonpeptide NK-1 antagonists (±)-CP96,345 and (±)-RP 67580 had IC₅₀ values of 0.74 and 49 n M , respectively. Substance P (SP) was also a potent inhibitor with with an IC₅₀ of 3.1 n M . The inhibition by SP could be separated into two components: a high-affinity component with a K _i of 0.53 n M and a lower-affinity component with a K _i of 155 n M . Addition of 100 µ M guanylyl 5'-imidodiphosphate [Gpp(NH)p] in the incubation increased the relative amount of the low-affinity agonist state of the receptor. Consistent with the antagonist properties of LY303870, the dissociation rate of [³H]LY303870 was not changed by the presence of 100 µ M Gpp(NH)p. The distribution of [³H]LY303870 binding sites in the guinea pig brain closely matched the distribution of NK-1 receptors labeled by [³H]SP. Therefore, [³H]LY303870 is a potent and selective antagonist radioligand for NK-1 receptors in guinea pig brain. In addition, regulation of NK-1 agonist affinity by guanine nucleotides is similar to that seen for monoaminergic receptors.     

[3H]Dizocilpine Association Kinetics Distinguish Stimulatory and Inhibitory Polyamine Sites of N-Methyl-d-Aspartate Receptors

Abstract: Spermine and other polyamines both stimulate and inhibit N -methyl- d -aspartate receptor function, probably by interacting with two separate sites. To characterize these two actions, the effect of spermine on the binding kinetics of the channel blocker [³H]dizocilpine was studied in the presence of glutamate and glycine. Low concentrations (10 µ M ) of spermine increased the association and dissociation rates without modifying equilibrium binding, indicating that spermine increases the accessibility of [³H]dizocilpine to the channel by interacting with a high-affinity, stimulatory site. At higher concentrations (1 m M ), spermine markedly decreased equilibrium [³H]-dizocilpine binding by decreasing both affinity and B _max, indicating that spermine allosterically inhibits binding by interacting with a second, low-affinity site. The presumed polyamine antagonists arcaine, diethylenetriamine, and 1,10-diaminodecane completely inhibited equilibrium [³H]dizocilpine binding, probably by interacting with the inhibitory polyamine site or other sites, but not with the stimulatory polyamine site. Low concentrations (10 µ M ) of ifenprodil completely reversed the increase in association rate produced by spermine, whereas higher concentrations (IC₅₀ = 123 µ M ) inhibited equilibrium binding, indicating that ifenprodil is both a potent antagonist of the stimulatory site and a low-affinity ligand of the inhibitory site. The polyamine agonists spermine, spermidine, and neomycin interacted with the inhibitory site, but produced only partial inhibition of equilibrium [³H]dizocilpine binding.     

Modulation of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid (AMPA) Binding Sites by Nitric Oxide

Abstract: Nitric oxide release is reported to be involved in physiological processes associated with altered sensitivity of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) class of glutamate receptor. A series of compounds liberating nitric oxide were therefore tested for their ability to modulate in vitro the characteristics of [³H]AMPA binding to sections of rat brain. Pretreatment of forebrain or cerebellar sections with sodium nitroprusside (1 m M ), S -nitroso- N -acetylpenicillamine (SNAP, 200 µ M ), glyceryl trinitrate (1 µ M ), or isosorbide dinitrate (0.5 m M ) all increased the binding of 3 n M [³H]AMPA by 15–30%. These actions were reproduced by 8-bromo-cyclic GMP (200 µ M ) in the cerebellum but not in the forebrain. In a similar manner, the effect of SNAP was attenuated by an inhibitor of cyclic GMP-dependent protein kinase in the cerebellum but not in the forebrain. The elevated [³H]AMPA binding observed after pretreatment with SNAP was caused by an increase in binding affinity, but the capacity of the sites was unchanged. Autoradiographic analysis showed that forebrain binding was enhanced in the cerebral cortex and hippocampus but not in the striatum. Nitric oxide therefore appears to be able to increase the affinity of AMPA binding sites via two distinct mechanisms in different brain areas. This action may contribute to synaptic plasticity associated with nitric oxide release.     

Characterization of 5,7-Dichlorokynurenate-Insensitive d-[3H]Serine Binding to Synaptosomal Fraction Isolated from Rat Brain Tissues

Abstract: To explore target sites for endogenous d -serine that are different from the glycine site of the N -methyl- d -aspartate (NMDA) type glutamate receptor, we have studied the binding of d -[³H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 µ M ) of the glycine site antagonist 5,7-dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 m M unlabeled d -serine. Association, dissociation, and saturation experiments indicated that d -[³H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a K _D of 614 n M and a B _max of 2.07 pmol/mg of protein. d -Serine, l -serine, and glycine produced a total inhibition of the specific DCK-insensitive d -[³H]serine binding to the cerebellum with similar K _i values. Strychnine and 7-chlorokynurenate failed to inhibit the binding at 10 µ M . The profiles of displacement of the DCK-insensitive d -[³H]serine binding by various amino acids and glutamate and glycine receptor-related compounds differ from those of any other defined recognition sites. DCK-insensitive d -[³H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK-insensitive d -[³H]serine binding site could be a novel candidate for a target for endogenous d -serine in mammalian brains.     

Differential Regulation by Guanine Nucleotides of Opiate Agonist and Antagonist Receptor Interactions

Abstract: Guanine nucleotides differentiate binding of tritium-labeled agonists and antagonists to rat brain membranes. In the absence of sodium, GTP (50 μM) decreased binding of [³H]-labeled agonists by 20–60% and [³H]-labeled antagonists by 0–20%. In the presence of 100 mM-NaCl, GTP had no effect on antagonist binding, but decreased agonist binding by 60–95%. GMP was less potent than either GTP or GDP in decreasing agonist binding. GTP (50 μM) reduced high-affinity [³H]dihydromorphine sites by 52% and low-affinity sites by 55%. Without sodium, GTP reduced high-affinity [³H]-naloxone sites by 36%; in the presence of 100 mM-NaCl, GTP had no effect on either high- or low-affinity [³H]naloxone sites. GTP increased the association rate of [³H]dihydromorphine twofold and the dissociation rate by fourfold, while having no effect on association or dissociation rates of the antagonist [³H]diprenorphine. The affinities of uniabeled antagonists in inhibiting [³H]-diprenorphine binding were not affected by GTP or sodium, but the affinities of agonists were reduced 40- 120-fold, with met- and leu-enkephalin affinities reduced by the greatest degree. GTP and sodium lowered [³H]dihydromorphine binding in an additive fashion, while divalent cations, especially manganese, reversed the effects of GTP on [³H]-labeled agonist binding by stimulating membrane-bound phosphatases that hydrolyze GTP to GMP and guanosine. These results suggest that by affecting binding of agonists, but not antagonists, GTP may regulate opiate receptor interactions with their physiological effectors.     

Characterization of Ligand Binding to the Cannabinoid Receptor of Rat Brain Membranes Using a Novel Method: Application to Anandamide

Abstract: Ligand binding to the cannabinoid receptor of brain membranes has been characterized using [³H]CP 55,940 and the Multiscreen Filtration System. Binding of [³H]CP 55,940 is saturable and reaches equilibrium by 45 min at room temperature. At a concentration of 10 µg of membrane protein/well, the K _D for [³H]CP 55,940 is 461 p M and the B _max is 860 fmol/mg of protein. The apparent K _D of [³H]CP 55,940 is dependent upon tissue protein concentration, increasing to 2,450 p M at 100 µg of membrane protein. Binding of [³H]CP 55,940 is dependent upon the concentration of bovine serum albumin in the buffer; the highest ratio of specific to nonspecific binding occurs between 0.5 and 1.0 mg/ml. The K _i of anandamide, a putative endogenous ligand of the cannabinoid receptor, is 1.3 µ M in buffer alone and 143 n M in the presence of 0.15 m M phenylmethylsulfonyl fluoride. When [¹⁴C]anandamide is incubated with rat forebrain membranes at room temperature, it is degraded to arachidonic acid; the hydrolysis is inhibited by 0.15 m M phenylmethylsulfonyl fluoride. These results support the hypothesis that anandamide is a high-affinity ligand of the cannabinoid receptor and that it is rapidly degraded by membrane fractions.     

Aminergic Receptors in Drosophila melanogaster: Properties of [3H]Dihydroergocryptine Binding Sites

Abstract: [³H]Dihydroergocryptine ([³H]DHE) binds to a particulate preparation from Drosophila melanogaster heads at a level of 2.4 ± 0.4 pmol/mg protein, with an apparent dissociation constant of 2.0 ± 0.5 n M . The binding sites are inactivated by heat, pronase treatment, detergents, and sulfhydryl and disulfide reagents. [³H]DHE binding is inhibited by low concentrations of serotonergic and α-adrenergic ligands. The specificity of the binding sites, as revealed by displacement studies, differs from that of [³H]DHE binding sites in various vertebrate tissues. The [³H]DHE binding sites may correspond to serotonergic receptors, and possibly, to additional classes of receptors for putative neurotransmitters in Drosophila .     

Up-Regulation of the Neuronal Form of Nitric Oxide Synthase in Response to Prolonged Muscarinic M1 Receptor Stimulation

Abstract: It is generally believed that the neuronal form of nitric oxide synthase (nNOS) is constitutively expressed and that regulation of this enzyme's activity is mediated solely by changes in cytosolic calcium concentration. Serendipitously, however, we observed that pretreatment of Chinese hamster ovary (CHO) cells, which coexpress muscarinic M₁ receptors and nNOS, with 3.3 µ M or 1 m M carbachol (CCh) for 48 h resulted in marked enhancement of maximal muscarinic receptor-stimulated nNOS activity as determined by l -[³H]citrulline and cyclic [³H]GMP production. This was accompanied by a decrease in the potency of CCh. Muscarinic receptor density was reduced in the agonist-pretreated cells, as determined by specific [ N-methyl -³H]scopolamine methyl chloride binding, whereas competition binding studies revealed no changes in agonist affinity. Both receptor-stimulated inositol phosphate formation and elevation of intracellular calcium concentrations were found to be desensitized in agonist-pretreated cells in a manner dependent on CCh pretreatment concentration. It is interesting that ionomycin-stimulated nNOS activity was greater in CCh-pretreated cells. Also, western analysis revealed increased nNOS immunoreactivity in pretreated cells. A similar increase in nNOS immunoreactivity following agonist treatment was demonstrated in N1E-115 neuroblastoma cells, which endogenously express nNOS and muscarinic M₁ receptors. Thus, the enhancement of maximal receptor-stimulated nNOS activity following agonist pretreatment can be attributed to up-regulation of nNOS. It is interesting that this augmentation of the response takes place in spite of receptor down-regulation and desensitization of multiple steps involved in nNOS activation.     

M1 Muscarinic Acetylcholine Receptor in Cultured Rat Neostriatum Regulates Phosphoinositide Hydrolysis

Abstract: : Muscarinic acetylcholine receptor expression and function in cultured rat neostriatal neurons were examined. All experiments were performed on intact neurons grown in vitro for 12-14 days. The muscarinic antagonist N-[³H]methylscopolamine ([³H]NMS) binds to a single site in cultures with a K_D of 89 pM and a B_max of 187 fmol/mg of protein, or 32,000 sites/neuron. Competition studies using [³H]NMS were performed to determine what receptor sur > types were present. Nonlinear analysis of competition curves was best described with a single binding site for atropine, pirenzepine, and AF-DX 116 {11-[[2-[(diethylamino)-methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one}, with K_i values of 0.6, 62, and 758 nM, respectively. These results indicate that the muscarinic receptors present in neostriatal cultures are of the M₁subtype, having high affinity for pirenzepine and low affinity for AF-DX 116. In contrast with antagonists, carbachol displaced [³H]NMS from two sites with K_i values of 6.5 and 147 μM, with the higher-affinity form predominant (83% of sites). The M₁ receptor subtype was linked to phosphoinositide turnover. Carbachol stimulated the formation of phosphoinositides with an EC₅₀ of 37 μM and was antagonized by atropine. At equimolar doses, pirenzepine was more potent than AF-DX 116 at antagonizing the response.     

Rapid Communication: cis-Methyldioxolane Specifically Recognizes the m2 Muscarinic Receptor

Abstract: cis -Methyldioxolane (CD) is a muscarinic receptor agonist. [³H] CD has been used to label a subpopulation of muscarinic receptors described as exhibiting high agonist affinity. Pharmacological evidence suggests that the population of receptors labeled by [³H] CD consists of m2 and/or m4 subtypes; however, no studies have directly addressed the subtype selectivity of [³H] CD. The present study characterizes binding of this ligand to individual human receptor subtypes expressed in transfected Chinese hamster ovary cells. Results indicate that [³H] CD binds with high affinity only to Hm2 receptors but not to all Hm2 receptors. Twenty-eight percent of Hm2 receptors bound [³H] CD with a K _D of 3.5 ± 0.5 nM. Binding was eliminated in the presence of guanosine 5'- O -(3-thiotriphosphate), indicating that the Hm2 receptors labeled by [³H] CD are those that are associated with GDP-bound G protein. Binding of [³H] CD by only a subpopulation of Hm2 receptors is in agreement with data generated from studies of [³H] CD binding in mammalian brain. Because muscarinic receptors have been implicated to play a role in the pathogenesis of both Alzheimer's and Parkinson's disease, as well as the neurotoxicity of organophosphorus compounds, knowledge of the binding specificity of the muscarinic agonist [³H] CD should aid research in these areas.     

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.     

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.     

Protection by Alcuronium of Muscarinic Receptors Against Chemical Inactivation and Location of the Allosteric Binding Site for Alcuronium

Abstract: We have found earlier that the neuromuscular blocker alcuronium binds to cardiac muscarinic receptors simultaneously with their specific antagonist [³H]methyl- N -scopolamine ([³H]NMS) and allosterically increases their affinity to this ligand. Nothing is known about the allosteric site with which alcuronium interacts. To gain an insight, we have now investigated how the binding of [³H]NMS is affected by agents known to modify specific residues in proteins and how their effects are altered by alcuronium. Reagents that covalently modify the tyrosyl residues ( p -nitrobenzenesulfonyl fluoride and 4-chloro-7-nitrobenzofurazan) and the carboxyl groups of aspartate and glutamate [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N' -dicyclohexylcarbodiimide, and N -ethyl-5-phenylisoxazolium-3'-sulfonate] blocked the binding of [³H]NMS to receptors in rat heart atria. Their action was probably due to the modification of tyrosyl and aspartyl residues directly in the muscarinic binding sites because it was antagonized by atropine and carbamoylcholine. Alcuronium and gallamine, another allosteric ligand, also protected the [³H]NMS binding sites against the inactivation by tyrosine- and carboxyl-directed chemical modifiers just as well as by benzilylcholine mustard, known to attach covalently to the muscarinic binding sites. Protection by alcuronium has also been observed on cerebrocortical muscarinic receptors. The effect of alcuronium indicates that the drug interferes with the access of chemical modifiers to the muscarinic sites. In view of the unspecific nature of most of the modifiers used (with regard to muscarinic mechanisms), the protection by alcuronium appears to be best explained on the assumption that the drug binds in close vicinity of the "classical" muscarinic site and sterically blocks the access to this site.     

Inhibition of [3H]MK-801 Binding by Ferrous (II) but Not Ferric (III) Ions in a Manner Different from That by Sodium Nitroprusside (II) in Rat Brain Synaptic Membranes

Abstract: The addition of sodium nitroprusside (SNP) significantly inhibited binding of (+)-5-[³H]methyl-10,11-dihydro-5 H -dibenzo[ a,d ]cyclohepten-5,10-imine ([³H]MK-801) to an ion channel associated with the N -methyl- d -aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of >1 µ M in rat brain synaptic membranes not extensively washed. However, neither S -nitroso- N -acetylpenicillamine nor S -nitroso- l -glutathione inhibited binding even at 100 µ M . Of the two compounds structurally related to SNP (II), similarly potent inhibition was induced by potassium ferrocyanide (II) but not by potassium ferricyanide (III). In addition, ferrous chloride (II) induced much more potent inhibition of binding than ferric chloride (III), at a similar concentration range. In contrast, iron chelators prevented the inhibition by ferrous chloride (II) without markedly affecting that by SNP (II) and potassium ferrocyanide (II). Pretreatment with ferrous chloride (II) also led to potent inhibition of [³H]MK-801 binding in a manner insensitive to subsequent addition of the iron chelators. Pretreatment with Triton X-100 resulted in significant potentiation of the ability of ferrous chloride (II) to inhibit [³H]MK-801 binding irrespective of the addition of agonists, moreover, although binding of other radioligands to the non-NMDA receptors was unaltered after pretreatment first with Triton X-100 and then with ferrous chloride (II). These results suggest that ferrous ions (II) may interfere selectively with opening processes of the NMDA channel through mechanisms entirely different from those underlying the inhibition by both SNP (II) and potassium ferrocyanide (II) in rat brain.     

Serotonin Stimulation of 5-HT4 Receptors Indirectly Enhances In Vivo Dopamine Release in the Rat Striatum

Abstract: Serotonin (5-HT) applied at 1, 3, and 10 µ M into the striatum of halothane-anesthetized rats by in vivo microdialysis enhanced dopamine (DA) outflow up to 173, 283, and 584% of baseline values, respectively. The 5-HT effect was partially reduced by 1 or 10 µ M GR 125,487, a 5-HT₄ antagonist, and by 100 µ M DAU 6285, a 5-HT_3/4 antagonist, whereas the 5-HT_1/2/6 antagonist methiothepin (50 µ M ) was ineffective. In the presence of tetrodotoxin the effect of 1 µ M 5-HT was not affected by 5-HT₄ antagonists. In addition, tetrodotoxin abolished the increase in DA release induced by the 5-HT₄ agonist ( S )-zacopride (100 µ M ). In striatal synaptosomes, 1 and 10 µ M 5-HT increased the outflow of newly synthesized [³H]DA up to 163 and 635% of control values, respectively. The 5-HT₄ agonists BIMU 8 and ( S )-zacopride (1 and 10 µ M ) failed to modify [³H]DA outflow, whereas 5-methoxytryptamine (5-MeOT) at 10 µ M increased it (62%). In prelabeled [³H]DA synaptosomes, 1 µ M 5-HT, but not ( S )-zacopride (1 and 10 µ M ), increased [³H]DA outflow. DAU 6285 (10 µ M ) failed to modify the enhancement of newly synthesized [³H]DA outflow induced by 5-MeOT or 5-HT (1 µ M ), whereas the effect of 5-HT was reduced to the same extent by the DA reuptake inhibitor nomifensine (1 µ M ) alone or in the presence of DAU 6285. These results show that striatal 5-HT₄ receptors are involved in the 5-HT-induced enhancement of striatal DA release in vivo and that they are not located on striatal DA terminals.     

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).     

Accumulation of N-Arachidonoylethanolamine (Anandamide) into Cerebellar Granule Cells Occurs via Facilitated Diffusion

Abstract: N -Arachidonoylethanolamine (anandamide, AEA) is a putative endogenous ligand of the cannabinoid receptor. Intact cerebellar granule neurons in primary culture rapidly accumulate AEA. [³H]AEA accumulation by cerebellar granule cells is dependent on incubation time ( t _1/2 of 2.6 ± 0.8 min at 37°C) and temperature. The accumulation of AEA is saturable and has an apparent K _m of 41 ± 15 µ M and a V _max of 0.61 ± 0.04 nmol/min/10⁶ cells. [³H]AEA accumulation by cerebellar granule cells is significantly reduced by 200 µ M phloretin (57.4 ± 4% of control) in a noncompetitive manner. [³H]AEA accumulation is not inhibited by either ouabain or removal of extracellular sodium. [³H]AEA accumulation is fairly selective for AEA among other naturally occurring N -acylethanolamines; only N -oleoylethanolamine significantly inhibited [³H]AEA accumulation at a concentration of 10 µ M . The ethanolamides of palmitic acid and linolenic acid were inactive at 10 µ M . N -Arachidonoylbenzylamine and N -arachidonoylpropylamine, but not arachidonic acid, 15-hydroxy-AEA, or 12-hydroxy-AEA, compete for AEA accumulation. When cells are preloaded with [³H]AEA, temperature-dependent efflux occurs with a half-life of 1.9 ± 1.0 min. Phloretin does not inhibit [³H]AEA efflux from cells. These results suggest that AEA is accumulated by cerebellar granule cells by a protein-mediated transport process that has the characteristics of facilitated diffusion.     

Ethanol-Induced Inhibition of [3H]Thienylcyclohexylpiperidine (TCP) Binding to NMDA Receptors in Brain Synaptic Membranes and to a Purified Protein Complex

Abstract: N -Methyl- d -asparate receptors (NMDARs) are a major target of ethanol effects in the nervous system. Haloperidol-insensitive, but dizocilpine (MK-801)-sensitive, binding of N -[1-(2-[³H]thienyl)cyclohexyl]piperidine ([³H]TCP) to synaptic membranes has the characteristics of ligand interaction with the ion channel of NMDARs. In the present studies, ethanol produced a concentration-dependent decrease in the maximal activation of [³H]TCP binding to synaptic membranes by NMDA and Gly, but a moderate change in the activation by l -Glu when l -Glu was present at concentrations < 100 µ M . However, ethanol (100 m M ) inhibited completely the activation of [³H]TCP binding produced by high concentrations of l -Glu (200–400 µ M ). It also inhibited strongly the activation of [³H]TCP binding by spermidine or spermidine plus Gly. In a purified complex of proteins that has l -Glu-, Gly-, and [³H]TCP-binding sites, ethanol (100 m M ) decreased significantly the maximal activation of [³H]TCP binding produced by either l -Glu or Gly. Activation constants ( K _act) for l -Glu and Gly acting on the purified complex were 12 and 28 µ M, respectively. Ethanol had no significant effect on the K _act of l -Glu but caused an increase in the K _act of Gly. These studies have identified at least one protein complex in neuronal membranes whose response to both l -Glu and Gly is inhibited by ethanol. These findings may explain some of the effects of acute and chronic ethanol treatment on the function and expression of the subunits of this complex in brain neurons.     

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.