Glial Regulation of α7-Type Nicotinic Acetylcholine Receptor Expression in Cultured Rat Cortical Neurons

Abstract: Primary embryonic cortical cultures were used as an in vitro model to evaluate the influence of glia on developmental expression of α7-type nicotinic acetylcholine receptors in rat brain. In cells cultured in serum-containing medium without mitotic inhibitors, specific ¹²⁵I-α-bungarotoxin binding to α7-type nicotinic receptors was maximal 4–8 days after plating. Treatment with 5'-fluorodeoxyuridine (80 µ M ) from 1 to 3 days in vitro significantly reduced glial proliferation and concomitantly increased ¹²⁵I-α-bungarotoxin binding, whereas plating onto a glial bed layer decreased binding. There was no significant binding to pure glial cultures. Treatment-induced changes in neuronal binding resulted from alterations in receptor density, with no change in affinity. 5'-Fluorodeoxyuridine treatment also increased cellular expression of α7 receptor mRNA but had no effect on N -[³H]methylscopolamine binding to muscarinic receptors. Glial conditioned medium decreased ¹²⁵I-α-bungarotoxin binding in both control and 5'-fluorodeoxyuridine-treated cultures, suggesting the release of a soluble factor that inhibits α7-type nicotinic receptor expression. An additional mechanism of glial regulation may involve removal of glutamate from the surrounding medium, as added glutamate (200 µ M ) increased ¹²⁵I-α-bungarotoxin binding in astrocyte-poor cultures but not in those that were astrocyte enriched. These results suggest that glia may serve a physiological role in regulating α7-type nicotinic receptors in developing brain.     

Glutamate Uptake Stimulates Na+,K+-ATPase Activity in Astrocytes via Activation of a Distinct Subunit Highly Sensitive to Ouabain

Abstract: The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na⁺-dependent transporter. Evidence had been provided that Na⁺,K⁺-ATPase might be involved in this process. We have now measured the activity of Na⁺,K⁺-ATPase in cultured astrocytes, using ouabain-sensitive ⁸⁶Rb uptake as an index. l -Glutamate increases glial Na⁺,K⁺-ATPase activity in a concentration-dependent manner with an EC₅₀ = 67 µ M . Both l - and d -aspartate, but not d -glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na⁺,K⁺-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain ( K _0.5 = 113 µ M ), compatible with the presence of an α₁ isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain ( K _0.5 = 20 n M ), thus revealing a high-affinity site akin to the α₂ isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na⁺,K⁺-ATPase that can be mobilized in response to increased neuronal activity.     

Benzodiazepine Treatment Causes Uncoupling of Recombinant GABAA Receptors Expressed in Stably Transfected Cells

Abstract: GABA_A and benzodiazepine receptors are allosterically coupled, and occupation of either receptor site increases the affinity of the other. Chronic exposure of primary neuronal cultures to benzodiazepine agonists reduces these allosteric interactions. Neurons express multiple GABA_A receptor subunits, and it has been suggested that uncoupling is due to changes in the subunit composition of the receptor. To determine if uncoupling could be observed with expression of defined subunits, mouse Ltk⁻ cells stably transfected with GABA_A receptors (bovine α1, β1, and γ2L subunits) were treated with flunitrazepam (Flu) or clonazepam. The increase in [³H]Flu binding affinity caused by GABA (GABA shift or coupling) was significantly reduced in cells treated chronically with the benzodiazepines, whereas the K _D and B _max of [³H]Flu binding were unaffected. The uncoupling caused by clonazepam treatment occurred rapidly with a t _1/2 of ∼30 min. The EC₅₀ for clonazepam treatment was ∼0.3 µ M , and cotreatment with the benzodiazepine antagonist Ro 15-1788 (5.6 µ M ) prevented the effect of clonazepam. The uncoupling observed in this system was not accompanied by receptor internalization, is unlikely to be due to changes in receptor subunit composition, and probably represents posttranslational changes. The rapid regulation of allosteric coupling by benzodiazepine treatment of the stably transfected cells should provide insights to the mechanisms of coupling between GABA_A and benzodiazepine receptors as well as benzodiazepine tolerance.     

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.     

NMDA Receptor-Mediated Neurotoxicity: A Paradoxical Requirement for Extracellular Mg2+ in Na+/Ca2+-Free Solutions in Rat Cortical Neurons In Vitro

Abstract: Accumulation of intracellular Ca²⁺ is known to be critically important for the expression of NMDA receptor-mediated glutamate neurotoxicity. We have observed, however, that glutamate can also increase the neuronal intracellular Mg²⁺ concentration on activation of NMDA receptors. Here, we used conditions that elevate intracellular Mg²⁺ content independently of Ca²⁺ to investigate the potential role of Mg²⁺ in excitotoxicity in rat cortical neurons in vitro. In Ca²⁺-free solutions in which the Na⁺ was replaced by N -methyl- d -glucamine or Tris (but not choline), which also contained 9 m M Mg²⁺, exposure to 100 µ M glutamate or 200 µ M NMDA for 20 min produced delayed neuronal cell death. Neurotoxicity was correlated to the extracellular Mg²⁺ concentration and could be blocked by addition of NMDA receptor antagonists during, but not immediately following, agonist exposure. Finally, we observed that rat cortical neurons grown under different serum conditions develop an altered sensitivity to Mg²⁺-dependent NMDA receptor-mediated toxicity. Thus, the increase in intracellular Mg²⁺ concentration following NMDA receptor stimulation may be an underestimated component critical for the expression of certain forms of excitotoxic injury.     

γ-Aminobutyric AcidA Receptor Function Is Desensitised in Rat Cultured Cerebellar Granule Cells Following Chronic Flunitrazepam Treatment

Abstract: This study examined γ-aminobutyric acid_A (GABA_A) receptor function in cultured rat cerebellar granule cells by using microphysiometry following chronic flunitrazepam exposure, and correlated the findings with the α1 and β2/3 subunit protein expression and [³H]muscimol binding after the same treatment paradigm. Flunitrazepam treatment reduced ( p < 0.05) the maximal GABA-stimulated increase in extracellular acidification rate ( E _max) (16.5 ± 1.2% and 11.3 ± 1.0%, 2-day control and treated cells, respectively; 17.4 ± 1.0% and 9.9 ± 0.7%, 7-day control and treated cells, respectively; best-fit E _max± SEM, n = 7), without affecting the GABA concentration required to elicit 50% of maximal response (EC₅₀) (1.2 ± 1.7 and 2.3 ± 1.8 µ M , 2-day control and treated cells, respectively; 1.7 ± 1.5 and 1.5 ± 1.5 µ M , 7-day control and treated cells, respectively; best-fit EC₅₀± SEM, n = 7). Flunitrazepam exposure also abolished the flunitrazepam potentiation of the GABA response, caused a transient reduction of the GABA_A receptor α1 and β2/3 subunit proteins over the initial 2 days, but did not alter [³H]muscimol binding compared with vehicle-treated cells. The results suggest that changes in GABA_A receptor subunit protein expression, rather than loss of [³H]muscimol binding sites, underlie the chronic flunitrazepam-mediated desensitisation of GABA_A receptor function.     

The Metabotropic Glutamate Receptor mGluR4, but Not mGluR1α, Is Palmitoylated when Expressed in BHK Cells

Abstract: Several G protein-coupled receptors have been shown to be palmitoylated, and for some of these receptors the covalent attachment of palmitate has been implicated in the regulation of receptor-G protein coupling. The metabotropic glutamate receptor (mGluR) family forms a distinct group of G protein-coupled receptors, and the possibility that these may also be palmitoylated has been examined. Clonal baby hamster kidney (BHK) cells permanently transfected with the mGluR4 and mGluR1α subtypes were labelled with [³H]palmitic acid. The cells were lysed, the receptors were immuno-precipitated with specific antipeptide antibodies, and the immunoprecipitates were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. The palmitoylated, endogenously expressed G protein α-subunit α_q could be immunoprecipitated from [³H]palmitate-labelled BHK cells expressing mGluR1α using a specific antipeptide antibody, but in the same cell lysates no detectable [³H]palmitate-labelled mGluR1α was found. This suggests that this mGluR subtype, associated with stimulation of phospholipase C, is not palmitoylated. In contrast, mGluR4, which is coupled to inhibition of adenylyl cyclase, was found to be labelled with [³H]palmitic acid, and the palmitate was quantitatively removed by treatment with 1 M hydroxylamine, suggesting attachment of the palmitate through a thioester bond. Stimulation with maximal doses of the neurotransmitter glutamate for 1, 5, or 10 min appeared to have no effect on the level of receptor palmitoylation.     

Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

Influence of Cannabinoids on Electrically Evoked Dopamine Release and Cyclic AMP Generation in the Rat Striatum

Abstract: Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 {[1α,2β( R )5α]-(−)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol}, and the specific antagonist SR 141716 [ N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [³H]dopamine-prelabelled striatal slices were also investigated. CP 55940 (1 µ M ) and anandamide (10 µ M ) caused significant reductions in forskolin-stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 µ M ). CP 55940 (1 µ M ) had no effect on either KCl- or neurotransmitter-stimulated ³H-inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [³H]dopamine-prelabelled striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.     

Enteric Glia Exhibit P2U Receptors that Increase Cytosolic Calcium by a Phospholipase C-Dependent Mechanism

Abstract: Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 m M ), glutamate (100 µ M ), norepinephrine (10 µ M ), and substance P (1 µ M ) did not increase the intracellular calcium concentration ([Ca²⁺]_i) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 µ M ), bradykinin (11%; 10 µ M ), and histamine (31%; 100 µ M ), whereas 100% of glia responded to ATP (100 µ M ). ATP-evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca²⁺]_i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca²⁺]_i evoked, ATP = UTP > ADP > β,γ-methyleneadenosine 5'-triphosphate ≫ 2-methylthioadenosine 5'-triphosphate = α,β-methyleneadenosine 5'-triphosphate = AMP = adenosine, suggesting a glial P_2U receptor. Depletion of d - myo -inositol 1,4,5-trisphosphate-sensitive calcium stores by thapsigargin (10 µ M ) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U-73122 (10 µ M ), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12-myristate 13-acetate (100 n M ), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca²⁺]_i. Our data suggest that glial responses to ATP are mediated by a P_2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores.     

Endothelin Evokes Efflux of Glutamate in Cultures of Rat Astrocytes

Abstract: Excessive release of glutamate, from glial cells as well as neurons, is thought to be a major cause of neuronal death in ischemia. To investigate glutamate release from glial cells, we measured glutamate efflux from cultures of rat astrocytes preloaded with l -[³H]-glutamate. Glutamate efflux was induced by either 60 m M KCl or Na⁺-free medium, suggesting that the efflux is due to the reversed operation of a Na⁺- and K⁺-coupled glutamate uptake machinery. While investigating various neuropeptides and neurotransmitters, we found that endothelin (ET) specifically induced efflux of glutamate. Northern blot analysis and binding study showed that the ET type B receptor (ET_B-R) subtype was expressed two to three times more densely than the ET type A receptor (ET_A-R) in astrocytes. The ET_B-R antagonist IRL 2500 partially inhibited efflux of glutamate induced by 1 n M ET-1 in a concentration-dependent manner, causing a maximal inhibition of 60% at 1 µ M . However, the ET_A-R antagonist BQ-123 did not cause significant inhibition even at 10 µ M . Combination of both antagonists completely inhibited the ET-1-induced efflux. These results indicate that both receptor subtypes are involved in efflux of glutamate with a major contribution from the ET_B-R. Our findings suggest that ET, which is known to be released in ischemia, may exacerbate neurodegeneration by stimulating efflux of glutamate.     

[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.     

The δ-Opioid Receptor in SK-N-BE Human Neuroblastoma Cell Line Undergoes Heterologous Desensitization

Abstract: The human neuroblastoma cell line SK-N-BE expresses δ-opioid receptors negatively coupled to adenylyl cyclase. Prolonged treatment (2 h) of the cells with 100 n M etorphine leads to an almost complete desensitization (8.2 ± 5.9 vs. 45.8 ± 8.7% for the control). Other receptors negatively coupled to adenylyl cyclase, namely, D2-dopaminergic, α₂-adrenergic, and m2/m4-muscarinic, were identified by screening of these cells, and it was shown that prolonged treatment (2 h) with 1 µ M 2-bromo-α-ergocryptine or 1 µ M arterenol resulted in a marked desensitization of D2-dopaminergic and α₂-adrenergic receptors, respectively. Cross-desensitization experiments revealed that pretreatment with etorphine desensitized with the same efficiency the δ-opioid receptor and the D2-dopaminergic receptor, and pretreatment with 2-bromo-α-ergocryptine also desensitized both receptors. In contrast, pretreatment with etorphine desensitized only partly the α₂-adrenergic receptor response, whereas pretreatment with 1 µ M arterenol partly desensitized the δ-opioid receptor response. It is concluded that the δ-opioid receptor-mediated inhibitory response of adenylyl cyclase undergoes heterologous desensitization, and it is suggested that δ-opioid and D2-dopaminergic receptors are coupled to adenylyl cyclase via a G_i2 protein, whereas α₂-adrenergic receptor could be coupled to the enzyme via two G proteins, G_i2 and another member of the G_i/G_o family.     

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.     

Ca2+ and Reactive Oxygen Species in Staurosporine-Induced Neuronal Apoptosis

Abstract: Staurosporine (0.03–0.5 µ M ) induced a dose-dependent, apoptotic degeneration in cultured rat hippocampal neurons that was sensitive to 24-h pretreatments with the protein synthesis inhibitor cycloheximide (1 µ M ) or the cell cycle inhibitor mimosine (100 µ M ). To investigate the role of Ca²⁺ and reactive oxygen species in staurosporine-induced neuronal apoptosis, we overexpressed calbindin D_28K, a Ca²⁺ binding protein, and Cu/Zn superoxide dismutase, an antioxidative enzyme, in the hippocampal neurons using adenovirus-mediated gene transfer. Infection of the cultures with the recombinant adenoviruses (100 multiplicity of infection) resulted in a stable expression of the respective proteins assessed 48 h later. Overexpression of both calbindin D_28K and Cu/Zn superoxide dismutase significantly reduced staurosporine neurotoxicity compared with control cultures infected with a β-galactosidase overexpressing adenovirus. Staurosporine-induced neuronal apoptosis was also significantly reduced when the culture medium was supplemented with 10 or 30 m M K⁺, suggesting that Ca²⁺ influx via voltage-sensitive Ca²⁺ channels reduces this apoptotic cell death. In contrast, neither the glutamate receptor agonist NMDA (1–10 µ M ) nor the NMDA receptor antagonist dizocilpine (MK-801; 1 µ M ) was able to reduce staurosporine neurotoxicity. Cultures treated with the antioxidants U-74500A (1–10 µ M ) and N -acetylcysteine (100 µ M ) also demonstrated reduced staurosporine neurotoxicity. These results suggest a fundamental role for both Ca²⁺ and reactive oxygen species in staurosporine-induced neuronal apoptosis.     

Glutamate Activates Phospholipase D in Hippocampal Slices of Newborn and Adult Rats

Abstract: Phospholipase D (PLD) is activated by many neuro-transmitters in a novel signal transduction pathway. In the present work, PLD activity was studied comparatively in hippocampal slices of newborn and adult rats. Basal PLD activity in adult rats was almost three times higher than in newborn rats. In newborn rats, L-glutamate and 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid (1 S ,3 R -ACPD) time- and concentrationdependently enhanced the formation of [³H]phosphatidylpropanol ([³H]PP) and of [³H]phosphatidic acid in the presence of 2% propanol. N -MethylD-aspartate and kainate (both 1 m M ) caused small, but significant increases (∼50%). whereas α-amino-3-hydroxy-5-methylisoxazole-4-propionate (100 μ M ) was ineffective. Maximally effective concentrations of glutamate (1 m M ) and of 1 S ,3 R -ACPD (300 μ M ) increased the PLD activity to almost 300% of basal activity; the EC₅₀ values were 199 and 47 μ M , respectively. Glutamate receptor antagonists, such as DL-2-amino-3-phosphonopropionic acid (AP3). DL-2-aminc-5-phosphonovalenic acid, and kynurenate (all 1 m M ) did not inhibit the glutamate-evoked increase of PP formation. In slices of adult rats, the response to 1 S ,3 R -ACPD was significant, but small, whereas glutamate was effective only in the presence of the glutamate uptake inhibitor L-aspartate-β-hydroxarnate. It is concluded that glutamate activates PLD in rat hippocampus through an AP3-resistant metabotropic receptor. This effect is subject to ontogenetic development, with one important factor being glutamate uptake.     

NMDA Receptor Subtype Selectivity: Eliprodil, Polyamine Spider Toxins, Dextromethorphan, and Desipramine Selectively Block NMDA-Evoked Striatal Acetylcholine but Not Spermidine Release

Abstract: NMDA receptor stimulation concomitantly increases the release of [¹⁴C]acetylcholine and [³H]spermidine from rat striatal slices in vitro. The NMDA-induced release of both acetylcholine and spermidine was blocked with equal potency by the NMDA channel blocker phencyclidine (0.1–10 µ M ). However, certain other channel blockers, including dextromethorphan (1–100 µ M ), which antagonized NMDA-evoked acetylcholine release without affecting NMDA-evoked spermidine release, and dextrorphan (1–100 µ M ) and memantine (1–100 µ M ), which block NMDA-evoked acetylcholine release more potently than NMDA-evoked spermidine release, showed greater selectivity of action. As previously shown for ifenprodil, eliprodil (SL82.0715; 1–100 µ M ) blocked NMDA-evoked acetylcholine but not spermidine release. This selectivity is also observed for other agents interacting with the polyamine site(s) on the NMDA receptor, including arcaine (1–1,000 µ M ), philanthotoxin₃₄₃, and argiotoxin₆₃₆ (10 µ M ) and was also noted for desipramine (1–100 µ M ). The NMDA-induced release of acetylcholine and spermidine is likely to be mediated by different native NMDA receptor subtypes, and several NMDA antagonists may be candidates for a selective action at a particular NMDA receptor subtype.     

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.     

Guanine Nucleotide Regulatory Proteins, Gq and Gi1/2, Mediate Platelet-Activating Factor-Stimulated Phosphoinositide Metabolism in Immortalized Hippocampal Cells

Abstract: Platelet-activating factor (PAF) may be a neuromodulator involved in neural cell differentiation, cerebral inflammation, and ischemia. The PAF receptor is a member of the G protein-coupled receptor superfamily. In the present study, we sought to define the specific G protein(s) that mediate PAF-stimulated phosphoinositide (PI) metabolism in an immortalized hippocampal cell line, HN33.11. PAF increased the production of ³H-labeled inositol phosphates (IPs) with EC₅₀ values of 1.2–1.5 n M . The effect of PAF on ³H-IPs formation was completely blocked by the PAF antagonist BN 50739 at a concentration of 300 n M . Pertussis toxin pretreatment attenuated PAF-stimulated ³H-IPs production by 20–30% ( p < 0.05). Consistent with a role for G_i1/2 in this response, antiserum against G_αi1/2 blocked the response to a similar degree. Pretreatment of permeabilized cells with G_αq/11 antiserum attenuated the response by 70% ( p < 0.05), suggesting a role for G_q/11 in mediating the PAF response in this cell line. Stimulation with PAF increased [α-³²P]-GTP binding to both G_αq and G_αi1/2 proteins. Moreover, specific [³H]PAF binding sites coprecipitated with G_αq and G_αi1/2 proteins. The results suggest that PAF-stimulated PI metabolism in HN33.11 cells is mediated by both G_q and G_i1/2 proteins.     

Dopamine Receptor Stimulation Decreases Cytosolic γ Protein Kinase C Immunoreactivity in Rat Hippocampal Slices: Evidence for Increased Ca2+-Dependent Proteolysis

Abstract: The effect of dopamine (DA) receptor stimulation on the distribution of γ protein kinase C (γPKC) in hippocampal slices was assessed. Nanomolar concentrations of DA decreased cytosolic γPKC (56%) without altering membrane γPKC levels, resulting in decreased total γPKC immunoreactivity. The maximal decrease in cytosolic γPKC occurred at 20 min of incubation and was significantly blocked by the D₁ DA antagonist SCH 23390 (10⁻⁶ M ) but not by the D₂ antagonist sulpiride (10⁻⁵ M ). The D₁ agonists SKF 38393 and A 77636 mimicked the effect of DA with similar responses produced at 10 µ M and 1 n M , respectively. The D₂ agonist quinpirole had no effect on γPKC immunoreactivity, thus indicating that this dopaminergic response is mediated through a D₁-like receptor. DA had no effect on α, δ, or ζPKC isozyme immunoreactivity in the same hippocampal preparations. The DA-induced decrease in cytosolic γPKC immunoreactivity was blocked by the Ca²⁺-dependent protease inhibitor N -acetyl-Leu-Leu-norleucinal (100 µ M ) and by the inorganic Ca²⁺ channel blocker Co²⁺. The data suggest that DA stimulates a D₁-like DA receptor, which increases the influx of Ca²⁺ and activates the Ca²⁺-dependent proteolysis of γPKC.