Stimulatory Effects of Protein Kinase C and Calmodulin Kinase II on N-Methyl-d-Aspartate Receptor/Channels in the Postsynaptic Density of Rat Brain

Abstract: To clarify the regulatory mechanism of the N -methyl- d -aspartate (NMDA) receptor/channel by several protein kinases, we examined the effects of purified type II of protein kinase C (PKC-II), endogenous Ca²⁺/calmodulin-dependent protein kinase II (CaMK-II), and purified cyclic AMP-dependent protein kinase on NMDA receptor/ channel activity in the postsynaptic density (PSD) of rat brain. Purified PKC-II and endogenous CaMK-II catalyzed the phosphorylation of 80–200-kDa proteins in the PSD and l -glutamate-(or NMDA)-induced increase of (+)-5-[³H]methyl-10, 11-dihydro-5 H -dibenzo[a, d]cyclohepten-5, 10-imine maleate ([³H]MK-801; open channel blocker for NMDA receptor/channel) binding activity was significantly enhanced. However, the pretreatment of PKC-II-and CaMK-II-catalyzed phosphorylation did not change the binding activity of l -[³H]glutamate, cis -4-[³H](phospho-nomethyl)piperidine-2-carboxylate ([³H]CGS-19755; competitive NMDA receptor antagonist), [³H]glycine, α-[³H]-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, or [³H]-kainate in the PSD. Pretreatment with PKC-II-and CaMK-II-catalyzed phosphorylation enhanced l -glutamate-induced increase of [³H]MK-801 binding additionally, although purified cyclic AMP-dependent protein kinase did not change l -glutamate-induced [³H]MK-801 binding. From these results, it is suggested that PKC-II and/or CaMK-II appears to induce the phosphorylation of the channel domain of the NMDA receptor/channel in the PSD and then cause an enhancement of Ca²⁺ influx through the channel.     

Identification of Strychnine Binding Sites in the Rat Retina

Abstract: [³H]Strychnine specifically binds to membrane fractions isolated from rat retinae. The binding is saturable, with an apparent dissociation constant, K _D, of 14.3 × 10⁻⁹ M and 205 fmol bound/mg protein. Specific binding is time-dependent and proportional to protein concentration. Glycine and taurine are equally potent inhibitors of [³H]strychnine binding ( K _i= 4 × 10⁻⁵ M); no other amino acids endogenously present in the retina inhibited [³H]strychnine binding.     

Comparison of Solubilised Kainate and α-Amino-3-Hydroxy-5-Methylisoxazolepropionate Binding Sites in Chick Cerebellum

Abstract: [³H]Kainate bound to chick cerebellar membranes with a K _D of 0.6 μ M and with an exceptionally high B max of 165 pmol/mg of protein. In octylglucoside-solubilised extracts, the affinity of [³H]kainate was reduced ( K _D= 2.7 μ M ), but the B _max was relatively unchanged (130 pmol/mg of protein). The rank potency of competitive ligands was domoate > kainate > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > glutamate. Binding sites for α-[³H]amino-3-hydroxy-5-methylisoxazolepropionate ([³H]AMPA) were much less abundant, with K _D and B _max values in membranes of 86 n M and I pmol/mg of protein, respectively. The affinity of [³H]AMPA binding was also reduced on solubilisation ( K _D= 465 n M ), but there was an increase in the B _max (1.7 pmol/mg of protein). Quisqualate and CNQX were the most effective displacers of [³H]AMPA binding, but kainate was also a relatively potent inhibitor. However, in contrast to the displacement profile for [³H]kainate, domoate was markedly less potent than kainate at displacing [³H]AMPA. These results suggest that [³H]AMPA binds to a small subset of the kainate sites that, unlike the majority of the [³H]kainate binding protein, which has been reported to be located in the Bergmann glia, may represent neuronal unitary non- N -methyl-D-aspartate receptors.     

[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.     

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.     

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.     

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.     

Binding Characteristics and Interactions of Depressant Drugs with [35S]t-Butylbicyclophosphorothionate, a Ligand that Binds to the Picrotoxinin Site

Abstract: [³⁵S]r-Butylbicyclophosphorothionate (TBPT), a cage convulsant with picrotoxinin-like activity, binds to rat brain membranes to a single site with an apparent K_D of 25.1 ± 5.6 n M and a B_max of 1.40 ± 0.22 pmol/mg protein. TBPT binding to rat brain membranes was inhibited by a variety of convulsant, depressant, anxiolytic, and anticonvulsant drugs that had previously been shown to inhibit [³H]a-dihydropicrotoxinin binding. Depressant drugs such as pentobarbital and the nonbarbiturate (+)etomidate inhibited TBPT binding in an uncompetitive manner. Thus, pentobarbital and (+)etomidate decreased both the affinity and the number of binding sites of TBPT to whole brain membranes. The IC₅₀ values of (+)etomidate (9 μ M ) and pentobarbital (90 μ M ) are similar to the EC₅₀ values at which they enhance both [³H]-γ-aminobutyric acid and [³H]diazepam binding in cerebral cortex membranes. RO5–4864, which has recently been shown to be a convulsant, also inhibited TBPT binding (IC₅₀= 10 μ M ). These results suggest that TBPT binds to the picrotoxinin site and further supports the notion that the picrotoxinin site is an important modulatory site at the benzodiazepine-GABA receptor-ionophore complex.     

[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.     

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.     

Allosteric Modulation of [3H]CGP 39653 Binding by Glycine in Rat Brain

Abstract: D,L-(E)-2-Amino-4-propyl-5-phosphono-3-pen-tenoic acid (CGP 39653). a new, high-affinity, selective NMDA receptor antagonist, interacts with rat cortical membranes in a saturable way and apparently to a single binding site, with a K_D of 10.7 nM and a receptor density of 2.6 pmol/mg of protein. Displacement analysis of [³H]CGP 39653 binding shows a pharmacological profile similar to that reported for another NMDA antagonist, 3-[(±)-2-carboxypiperazin-4-yI]propyl-1-phosphonic acid (CPP). Glycine, however, is able to discriminate between the two ligands; in fact, it does not affect [³H]CPP binding but inhibits [³H]CGP 39653 binding in a biphasic way. D-Serine, another agonist at the strychnine-insensitive glycine binding site of the NMDA receptor complex, inhibits [³H]CGP 39653 binding in the same way as glycine, with a potency that correlates with its binding affinity at the glycine site. In addition, 7-chlorokynurenic acid, an antagonist at the glycine site, is able to reverse the displacement of [³H]CGP 39653 by glycine in a dose-dependent manner. Furthermore, the dissociation rate constant of [³H]CGP 39653 is enhanced in the presence of glycine, whereas the presence of NMDA receptor ligands does not modify the rate of dissociation of [³H]CGP 39653 from the receptor. These results indicate that part of the binding of the NMDA antagonist CGP 39653 can be potently modified by glycine through an allosteric mechanism, and suggest the existence of two antagonist preferring NMDA receptor subtypes that are differentially modulated through the glycine binding site.     

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.     

High Level Expression of the NMDAR1 Glutamate Receptor Subunit in Electroporated COS Cells

Abstract: The rat N -methyl- d -aspartate (NMDA) glutamate receptor subunit NR1-1a was transiently expressed in COS cells using the technique of electroporation, which was fivefold more efficient than the calcium phosphate precipitation method of transfection. The glycine site antagonist 5,7-[³H]dichlorokynurenic acid labeled a single high-affinity site ( K _D = 29.6 ± 6 n M ; B _max = 19.4 ± 1.6 pmol/mg of protein) in membranes derived from COS cells electroporated with NR1-1a. In contrast to previous reports using transiently transfected human embryonic kidney 293 cells, binding of the noncompetitive antagonist (+)-5-[³H]methyl-10,11-dihydro-5 H -dibenzo[ a,d ]-cyclohepten-5,10-imine ([³H]MK-801) was not detected in NR1-1a-transfected COS cells. Although immunofluorescent labeling of electroporated COS cells demonstrated that the NR1-1a protein appears to be associated with the cell membrane, neither NMDA nor glutamate effected an increase in intracellular calcium concentration in fura-2-loaded cells, suggesting that homomeric NR1-1a receptors do not act as functional ligand-gated ion channels. Therefore, COS cells appear to differ from Xenopus oocytes with respect to the transient expression of functional homomeric NR1 receptors. Although expression of NR1-1a is sufficient to reconstitute a glycine binding site with wild-type affinity for antagonists in COS cells, recombinant homomeric NR1-1a receptors do not display properties that are characteristic of native NMDA receptors, such as permeability to Ca²⁺ and channel occupancy by MK-801, when expressed in this mammalian cell line.     

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.     

Characterization of [3H]Met-Enkephalin-Arg6-Phe7 Binding to Opioid Receptors in Frog Brain Membrane Preparations

Abstract: A tritiated heptapeptide, [³H]Tyr-Gly-Gly-Phe-Met-Arg-Phe ([³H]Met-enkephalin-Arg⁶-Phe⁷), with high specific radioactivity has been synthesized in order to characterize its opioid binding activity to frog brain membrane fractions. The apparent K _D value of the radioligand calculated from homologous displacement experiments was 3.4 n M , and the maximal number of specific binding sites was 630 fmol/mg of protein. The K _D determined from equilibrium saturation binding studies was found to be 3.6 n M . However, the Hill coefficient was far below unity ( n _H = 0.43), which suggests the presence of a second, lower affinity binding site. The presence of this binding component is strengthened by the displacement experiments performed with levorphanol and some other ligands. It is assumed that the lower affinity site has no opiate character. The rank order of potency of the applied ligands in competing reversibly with [³H]Met-enkephalin-Arg⁶-Phe⁷ binding reflects a κ₂- and/or δ-subtype specificity of the heptapeptide. Binding to a κ₁ and/or μ site of opioid receptors is excluded, but the existence of a novel endogenous opiate receptor subtype for Met-enkephalin-Arg⁶-Phe⁷ in frogs cannot be ruled out. The [³H]Met-enkephalin-Arg⁶-Phe⁷ binding was inhibited by both sodium ions and GppNHp, which suggests the opioid agonist character of the heptapeptide.     

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.     

NMDA-mediated modulation of dopamine release is modified in rat prefrontal cortex and nucleus accumbens after chronic nicotine treatment

In this study, we investigate the effects of chronic administration of (−)nicotine on the function of the NMDA-mediated modulation of [³H]dopamine (DA) release in rat prefrontal cortex (PFC) and nucleus accumbens (NAc). In the PFC synaptosomes NMDA in a concentration-dependent manner evoked [³H]DA release in rats chronically treated with vehicle (14 days) with an EC₅₀ of 13.1 ± 2.0 μM. The NMDA-evoked overflow of the [³H]DA in PFC nerve endings of rats treated with (−)nicotine was significantly lower (−43%) than in vehicle treated rats. The EC₅₀ was 9.0 ± 1.4 μM. Exposure of NAc synaptosomes of rats treated with vehicle to NMDA produced an increase in [³H]DA overflow with an EC₅₀ of 14.5 ± 5.5 μM. This effect was significantly enhanced in chronically treated animals. The EC₅₀ was 10.5 ± 0.5 μM. The K⁺-evoked release of [³H]DA was not modified by the (−)nicotine administration. Both the changes of the NMDA-evoked [³H]DA overflow in the NAc and PFC disappeared after 14 days withdrawal. The results show that chronic (−)nicotine differentially affects the NMDA-mediated [³H]DA release in the PFC and NAc of the rat.     

Comparison of [3H]WIN 35,428 Binding, a Marker for Dopamine Transporter, in Embryonic Mesencephalic Neuronal Cultures with Striatal Membranes of Adult Rats

Abstract: In contrast to striatal membranes of adult rats, where high- ( K _D1= 34 n M ) and low- ( K _D2= 48,400 n M ) affinity binding sites for [³H]WIN 35,428 are present, in primary cultures of ventral mesencephalon neurons (CVMNs) only low-affinity binding sites were found ( K _D= 336,000 n M ). The binding of [³H]WIN 35,428 in CVMNs prepared from rat embryos was reversible, saturable, and located in cytosol. Although dopamine (DA) uptake blockers inhibited [³H]DA uptake at nanomolar concentrations in CVMNs, the displacement of [³H]WIN 35,428 binding in CVMNs by DA uptake inhibitors required 100-8,000 times higher concentrations than were needed to displace [³H]WIN 35,428 binding in striatal membranes. Piperazine derivatives, e.g., GBR-12909, GBR-12935, and rimcazole, inhibited [³H]WIN 35,428 binding in CVMNs more effectively than did cocaine, WIN 35,428, mazindol, nomifensine, or benztropin. A positive correlation ( r = 0.779; p < 0.001) was found between drug affinities for the striatal membrane sites labeled by [³H]WIN 35,428 and their abilities to inhibit DA uptake in CVMNs, whereas no correlation existed between the IC₅₀ values of drugs that inhibited [³H]WIN 35,428 binding and [³H]DA uptake in CVMNs. The cytosolic [³H]WIN 35,428 binding sites may be a piperazine acceptor and may not be involved in the regulation of the DA transporter.     

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.