Human, Bovine, and Rabbit Retinal Glutamate-Induced [3H]D-Aspartate Release: Role in Excitotoxicity

The pharmacological basis of glutamate-induced [³H]D-aspartate release was investigated in isolated human, bovine and rabbit retinas. Isolated mammalian retinas were preloaded with [³H]D-aspartate and then prepared for studies of neurotransmitter release using the superfusion method. Release of [³H]D-aspartate was elicited by K⁺ (50 mM) or by L-glutamate. In bovine retinas, L-glutamate, but not D-glutamate induced an overflow of [³H]D-aspartate that was partially inhibited by low external calcium, -conotoxin (10 nM) or nitrendipine (1 M). Metabotropic glutamate receptor (GLUR) agonists also evoked [³H]D-aspartate release in both bovine and human retinas whereas polyamines only enhanced the excitatory effects of L-glutamate on [³H]D-aspartate release. Antagonists of GLURs and the polyamine site inhibited L-glutamate evoked [³H]D-aspartate overflow with the following rank order of potency: MCPG >ifenprodil > AP-5 > arcaine> MK-801. In conclusion, L-glutamate-induces a stereoselective, calcium-dependent release of [³H]D-aspartate from isolated mammalian retinas that can be mimicked by GLUR agonists (and blocked by both receptor and polyamine site antagonists).     

Temporal Variation in the Interaction Between Calcium Channel Blockers and Morphine-Induced Analgesia

Circadian variations in the interaction between calcium channel blockers and morphine-induced analgesia were determined by the mouse hot-plate test. Calcium channel blockers diltiazem, verapamil, or nicardipine alone did not display any significant analgesic effect, but all of them potentiated morphine-induced analgesia when injected 30 min prior to morphine at most of the injection times. In terms of percent absolute potentiation, they produced more potentiation during the light period than darkness. Their potentiating effects decreased abruptly during darkness, and around the midtime of the dark period no significant potentiation of morphine-induced analgesia was observed. It is concluded that these fluctuations in the magnitude of interaction between calcium channel blockers and morphine must be taken into consideration particularly in studies dealing with the role of calcium in analgesia.     

Protective Effect of L-type Calcium Channel Blockers Against Haloperidol-induced Orofacial Dyskinesia: A Behavioural, Biochemical and Neurochemical Study

Haloperidol is a classical neuroleptic drug that is still in use and can lead to abnormal motor activity such as tardive dyskinesia (TD) following repeated administration. TD has no effective therapy yet. There is involvement of calcium in triggering the oxidative damage and excitotoxicity, both of which play central role in haloperidol-induced orofacial dyskinesia and associated alterations. The present study was carried out to investigate the protective effect of calcium channel blockers [verapamil (10 and 20 mg/kg), diltiazem (10 and 20 mg/kg), nifedipine (10 and 20 mg/kg) and nimodipine (10 and 20 mg/kg)] against haloperidol induced orofacial dyskinesia and associated behavioural, biochemical and neurochemical alterations in rats. Chronic administration of haloperidol (1 mg/kg i.p., 21 days) resulted in a significant increase in orofacial dyskinetic movements and significant decrease in % retention, coupled with the marked increase in lipid peroxidation and superoxide anion generation where as significant decrease in non protein thiols and endogenous antioxidant enzyme (SOD and catalase) levels in rat brain striatum homogenates. All these deleterious effects of haloperidol were significantly attenuated by co-administration of different calcium channel blockers. Neurochemically, chronic administration of haloperidol resulted in significant decrease in levels of catecholamines (dopamine, serotonin) and their metabolites (HVA and HIAA) but increased turnover of dopamine and serotonin. Co-administration of most effective doses of verapamil, diltiazem, nifedipine and nimodipine significantly attenuated these neurochemical changes. Results of the present study indicate that haloperidol-induced calcium ion influx is involved in the pathogenesis of tardive dyskinesia and calcium channel blockers should be tested in clinical trials with nifedipine as the most promising one.     

Evaluation of Drugs Acting at Glutamate Transporters in Organotypic Hippocampal Cultures: New Evidence on Substrates and Blockers in Excitotoxicity

Removal of L-glutamate (Glu) from the synapse is critical to maintain normal transmission and to prevent excitotoxicity, and is performed exclusively by excitatory amino acid transporters (EAATs). We investigated the effects of substrates and blockers of EAATs on extracellular Glu and cellular viability in organotypic cultures of rat hippocampus. Seven-day treatment with a range of drugs (L-trans-pyrrolidine-2,4-dicarboxylate, (2S,4R)-4-methyl-glutamate, (±)-threo-3-methylglutamate and DL-threo--benzyloxyaspartate), in the presence of 300 M added Glu, resulted in increased extracellular Glu and a significant correlation between Glu concentration and cellular injury (as indicated by lactate dehydrogenase release). In contrast, (2S,3S,4R)-2-(carboxycyclopropyl)glycine (L-CCG-III) exerted a novel neuroprotection against this toxicity, and elevations in extracellular Glu were not toxic in the presence of this compound. Similar results were obtained following two-week treatment of cultures without added Glu. Whilst blockade of GLT-1 alone was relatively ineffective in producing excitotoxic injury, heteroexchange of Glu by EAAT substrates may exacerbate excitotoxicity.     

Excitatory Amino Acid-Induced Toxicity in Chick Retina: Amino Acid Release, Histology, and Effects of Chloride Channel Blockers

Acute excitotoxicity in embryonic chick retina and the ability of Cl- channel blockers to prevent toxicity were evaluated by measurement of endogenous amino acid release and histology. Treatment of retina with kainate, quisqualate, or N-methyl-D-aspartate resulted in a large dose-dependent release of gamma-aminobutyric acid and taurine, moderate release of glutamine and alanine, and no measurable release of glutamate or aspartate. Concentrations inducing maximal gamma-aminobutyric acid release were 50 microM quisquaalate, 100 microM kainate, and 100 microM N-methyl-D-aspartate. Treatment with 1 mM glutamate resulted in significant gamma-aminobutyric acid release, as well as an elevation in medium aspartate levels. Typical excitotoxic retinal lesions were produced by the agonists and, at the lower concentrations tested, revealed a regional sensitivity. There was a positive correlation between the amount of gamma-aminobutyric acid release and the extent of tissue swelling, suggesting that release may be secondary to toxic cellular events. Omission of Cl- completely blocked cytotoxic effects due to kainate or glutamate. Likewise, addition of the Cl-/bicarbonate anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonate at 600 microM protected retina from cytotoxic damage from all excitotoxic analogs and restored amino acid levels to baseline values. Furosemide, which blocks Na+/K+/2Cl- cotransport, was only minimally effective in reducing amino acid release induced by the agonists. Consistent with the latter, histological examination showed the continued presence of the lesion but with general reduction of cellular edema. These results indicate that although influx of Cl- is a central component of the acute excitotoxic phenomenon, mechanisms other than passive Cl- flux may be involved.     

基因多态性对钙离子通道阻滞剂类抗高血压药物药动学及疗效的影响

钙离子通道阻滞剂是常用的一种高血压治疗药物,其中又以二氢吡啶类钙离子通道阻滞剂为主要代表,常见的有硝苯地平、氨氯地平、非洛地平等。然而,不同的患者个体对此类抗高血压药物可产生不同的降压效果,这种差异是环境和遗传共同作用的结果。基因多态性决定了药物代谢酶、转运体以及作用受体的差异,是导致药物疗效差异的重要原因之一。综述药物代谢酶、转运体和作用受体的基因多态性对钙离子通道阻滞剂类抗高血压药物的药动学和疗效的影响。     

Dopamine Receptor 2 Regulates L-Type Voltage-Gated Calcium Channel in Primary Cultured Mouse Midbrain Neural Network

1. Synchronous oscillation of intracellular Ca²⁺ in the central nervous system is essential for neural development. We previously reported that endogenous dopamine was involved with synchronous Ca²⁺ oscillation of primary cultured midbrain neurons, and that regulation of dopamine in synchronous oscillation was distinctly different through dopamine receptor 1 (D1R) and 2 (D2R): the action of dopamine through D1R or D2R was facilitative or suppressive, respectively, to the Ca²⁺ influx of synchronous oscillation.2. In the present study, we confirmed that the suppressive effects of D2R were mediated by the regulation of the L-type voltage-gated Ca²⁺ channel, not by the regulation of NMDA receptor on the Ca²⁺ influx in the midbrain neural network showing synchronous oscillation.3. This evidence promotes better understanding of the regulation of neural activity by endogenous dopamine in networked neurons.     

Nicotinamide Pathway-Dependent Sirt1 Activation Restores Calcium Homeostasis to Achieve Neuroprotection in Spinocerebellar Ataxia Type 7

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Effect of Organic and Inorganic Calcium Channel Blockers on γ-Amino-n-Butyiic Acid Release Induced by Monensin and Veratrine in the Absence of External Calcium

The effects of two organic Ca2+ antagonists (verapamil and nitrendipine) and of two inorganic Ca2+ channel blockers (Co2+ and ruthenium red) on the Na+-dependent release of gamma-amino-n-butyric acid (GABA) triggered by veratrine and monensin in the absence of external Ca2+ were studied in mouse brain synaptosomes. Ca2+-independent release of GABA stimulated by the Na+ channel activator veratrine was inhibited with micromolar concentrations of verapamil and nitrendipine. In contrast, GABA release induced by the Na+ ionophore monensin was insensitive to the organic Ca2+ antagonists. Verapamil also failed to modify A23187-stimulated release of GABA in the presence of Ca2+ but inhibited high K+-induced release of the transmitter. Co2+ partially diminished veratrine-induced release but did not change monensin-induced release. Releasing responses to monensin and veratrine were insensitive to ruthenium red, which inhibited the Ca2+-dependent component of GABA release evoked by high K+ depolarization. These data demonstrate that the mechanism of inducing GABA release is different for veratrine and monensin, as evidenced by their differing sensitivities to inhibition by Ca2+ channel antagonists and organic Ca2+ blockers. It is concluded that voltage-sensitive Ca2+ channels of the presynaptic membrane are not involved in the inhibitory action of Ca2+ antagonists on the Na+-dependent, Ca2+-independent mechanism of GABA release.     

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.     

The Peripheral-Type Benzodiazepine Receptor Ligands [3H]Ro 5-4864 and [3H]PK 11195 Bind to the Retina of Rabbit, but Not of Turtle

Abstract: The binding of [³H]flunitrazepam, [³H]RO 5-4864, and [³H]PK 11195 to membrane preparations of the retina was studied in the turtle and rabbit. Only a single population of [³H]flunitrazepam binding sites was detected in the turtle, whereas two populations appeared to be present in the rabbit. No specific binding for [³H]RO 5-4864 and [³H]PK 11195 could be detected in the turtle. In rabbit, both ligands bound with high affinity, revealing a significant population of binding sites (K_D values of 24 ± 2.3 and 2.2 ± 0.8 nM, and B_max values of 440 ± 35 and 1,482 ± 110 fmol/mg of protein, respectively). The binding was temperature - and protein-dependent. Displacement studies showed a similar rank order of potency of various unlabeled ligands against both [³H]RO 5-4864 and [³H]PK 11195 (PK 11195 > Ro 5-4864 > flunitrazepam > flumazenil). These results suggest that peripheral-type benzodiazepine receptors are present in the retina of the rabbit, but not of the turtle.     

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.     

Neurite Outgrowth Stimulated by L1 Requires Calcium Influx into Neurons but is Not Associated with Changes in Steady State Levels of Calcium in Growth Cones

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N- type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.     

Neurite Outgrowth Stimulated by L1 Requires Calcium Influx into Neurons but is Not Associated with Changes in Steady State Levels of Calcium in Growth Cones

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N- type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.     

Opioid Receptor and Calcium Channel Regulation of Adenylyl Cyclase,Modulated By GM1, in NG108-15 Cells: Competitive Interactions

GM1 ganglioside was previously shown to function as a specific regulator of excitatory opioid activity in dorsal root ganglion neurons and F11 hybrid cells, as seen in its facilitation of opioid-induced activation of adenylyl cyclase and its ability to dramatically reduce the threshold opioid concentration required to prolong the action potential duration. The elevated levels of GM1 resulting from chronic opioid exposure of F11 cells were postulated to cause the ensuing opioid excitatory supersensitivity. We now show that GM1 promotes opioid (DADLE)-induced activation of adenylyl cyclase in NG108-15 cells which possess the -type of receptor. In keeping with previous studies of other systems, this can be envisioned as conformational interaction of GM1 with the receptor that results in uncoupling of the receptor from G_i and facilitated coupling to G_s. This would also account for the observation that DADLE-induced attenuation of forskolin-stimulated adenylyl cyclase was reversed by GM1, provided the cells were not pretreated with pertussis toxin. When the cells were so pretreated, GM1 evoked an unexpected attenuation of forskolin-stimulated adenylyl cyclase attributed to GM1-promoted influx of calcium which was postulated to inhibit a calcium-sensitive form of adenylyl cyclase. This is concordant with several studies showing GM1 to be a potent modulator of calcium flux. Pertussis toxin in these experiments exerted dual effects, one being to promote interaction of the -opioid receptor with G_s through inactivation of G_i, and the other to enhance the GM1-promoted influx of calcium by inactivation of G_o; the latter is postulated to function as constitutive inhibitor of the relevant calcium channel. NG108-15 cells thus provide an interesting example of competitive interaction between two GM1-regulated systems involving enhancement of both opioid receptor excitatory activity and calcium influx.     

Effects of Local Administration of L-, N-, and P/Q-Type Calcium Channel Blockers on Spontaneous Dopamine Release in the Striatum and the Substantia Nigra: A Microdialysis Study in Rat

Abstract: The pivotal role for voltage-sensitive calcium channels in initiating synaptic transmitter release is undisputed, but it is only partly known to what extent the different subtypes contribute in vivo. Their importance for the dendritic release of dopamine has not been investigated in vivo previously. To evaluate comprehensively the relative importance of different voltage-sensitive calcium channel subtypes for striatal dopamine release, and to further investigate the mechanism of dendritic dopamine release in the reticulate part of substantia nigra, dopamine was measured by in vivo microdialysis in the striatum or substantia nigra of awake rats. The calcium channel blockers nimodipine, ω-conotoxin-GVIA, ω-agatoxin-IVA, and neomycin were administered locally through the dialysis probes and compared with calcium-free perfusion. Results indicate that dopamine release in the striatum is mainly dependent on N- and P/Q-type channels, but the dendritic dopamine release in the substantia nigra is mediated mainly by some other calcium-dependent mechanism, for example, calcium mobilization through T-, O-, or R-type calcium channels. A portion of the dendritic release is calcium independent but can be inhibited partially by neomycin, which might suggest a role for inositol 4,5-bisphosphate breakdown products.     

Inhibition of Ischemia-Induced Dopamine Release by ω-Conotoxin, a Calcium Channel Blocker, in the Striatum of Spontaneously Hypertensive Rats: In Vivo Brain Dialysis Study

The effect of omega-conotoxin GVIA (CgTX), an N-and L-type voltage-sensitive calcium channel (VSCC) blocker, on the release of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum before and during transient cerebral ischemia in spontaneously hypertensive rats was studied using an in vivo brain dialysis technique. Continuous perfusion of CgTX in the striatum was started 20 min before ischemia and concentrations of dopamine and DOPAC in the dialysate were measured using HPLC with an electro-chemical detector. Before ischemia, both 10 and 100 microM CgTX significantly lowered the concentration of dopamine, to 49% of the basal values. DOPAC concentrations also decreased significantly, by 28 and 17%, respectively. Forebrain ischemia, produced by bilateral carotid artery occlusion, reduced striatal blood flow to less than 6% of the resting value in each group. During 20 min of ischemia, the vehicle group showed a marked increase in dopamine (175 times the basal concentration). In the 10 or 100 microM CgTX perfusion group, in contrast, dopamine release was significantly attenuated, to 38 or 29% of the vehicle group, respectively. DOPAC concentrations decreased during ischemia to 58% of the basal value in the vehicle group and 49% in both CgTX groups. These results indicate that the massive release of striatal dopamine during ischemia depends largely on the influx of extracellular calcium via CgTX-sensitive VSCCs.