thiourea and 2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline

Cerebellar Purkinje neurons have intracellular regulatory systems including Ca2+-binding proteins, intracellular Ca2+ stores, Ca2+-ATPase and Na+-Ca2+ exchanger (NCX) that keep intracellular Ca2+ concentration ([Ca2+]i) in physiological range. Among these, NCX interacts with AMPA receptors, activation of which induces cerebellar synaptic plasticity. And the activation of metabotropic glutamate receptor 1 (mGluR1) is also involved in the induction of cerebellar long-term depression. The interaction of NCX with mGluR1 is not known yet. Thus, in this study, the functional relationship between NCX and mGluR1 in modulating the [Ca2+]i in rat Purkinje neurons was investigated. The interaction between NCX and mGluR1 in Purkinje neurons was studied by measuring intracellular Ca2+ transients induced by an agonist of group I mGluRs, 3,5-dihydroxyphenylglycine (DHPG). The DHPG-induced Ca2+ transient was significantly reduced by treatments of NCX inhibitors, bepridil and KB-R7943. When cells were pretreated with antisense oligodeoxynucleotides of NCX, the DHPG-induced Ca2+ transient was also inhibited. These results suggest that NCX modulates the activity of mGluR1 in cerebellar Purkinje neurons. Therefore, NCX appears to play an important role in the physiological function of cerebellar Purkinje neurons such as synaptic plasticity.

Topics: Animals; Animals, Newborn; Bepridil; Calcium; Calcium Channel Blockers; Cells, Cultured; Cerebellum; Drug Interactions; Enzyme Activation; Excitatory Amino Acid Antagonists; Methoxyhydroxyphenylglycol; Oligodeoxyribonucleotides, Antisense; Purkinje Cells; Quinoxalines; Rats; Receptors, Metabotropic Glutamate; Sodium-Calcium Exchanger; Thiourea

Variations in intracellular calcium activity ([Ca2+]i) play crucial roles in information processing in Purkinje neurons such as synaptic plasticity. Although Na+-Ca2+ exchanger (NCX) has been shown to participate in the regulation of homeostasis and secretion in neuronal cells, the physiological role of NCX in Purkinje neurons, such as a role in cerebellar synaptic plasticity, is not well understood. NCX in acutely dissociated rat Purkinje neurons was identified by double staining with anti-calbindin D-28k antibody and anti-NCX antibody. The physiological activity of NCX was examined by measuring transient intracellular Ca2+ changes resulting from the Ca2+ influx via reverse mode of NCX (with 0 mM Na+/2.5 mM Ca2+ solutions) and the efflux via the forward mode of NCX (with 140 mM Na+/0 mM Ca2+ solutions). This transient increase in Ca2+ concentration was not elicited in the cells pretreated with NCX antisense oligodeoxynucleotides. And the Ca2+ influx resulting from the reverse mode of NCX was significantly reduced by 2-[2-[4-(4-nitrobenyloxy) phenyl] ethyl] isothiourea methanesulfonate, while the Ca2+ efflux via forward mode was inhibited by bepridil. The physiological role of NCX in synaptic function was studied by measuring Ca2+ transients induced by alpha-amino-3-hydroxy-5-methyl-4-isoxazolone-propionate (AMPA) receptor activation. This AMPA-evoked response was decreased with the inhibition of NCX forward mode and also, to less degree, with the inhibition of reverse mode. In antisense oligodeoxynucleotides pretreated cells, the AMPA-evoked response was also reduced, as was the case in NCX-inhibitor treated cells. The inhibition of NCX activity had depressant effects on Ca2+ transients induced by AMPA receptor activation. These results suggest that NCX plays a physiological role in modulating the activity of cerebellar Purkinje neurons, such as synaptic plasticity, via interaction with AMPA receptors in Purkinje neurons.

Topics: Agatoxins; Animals; Animals, Newborn; Bepridil; Calbindins; Calcium; Calcium Channel Blockers; Cells, Cultured; Cerebellum; Diagnostic Imaging; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fluorescent Antibody Technique; Glutamic Acid; Isoxazoles; Microscopy, Confocal; Oligonucleotides, Antisense; Polyamines; Propionates; Purkinje Cells; Quinoxalines; Rats; S100 Calcium Binding Protein G; Sodium; Sodium Channel Blockers; Sodium-Calcium Exchanger; Thiourea

Intracellular ATP supply and ion homeostasis determine neuronal survival and degeneration after ischemic stroke. The present study provides a systematic investigation in organotypic hippocampal slice cultures of the influence of experimental ischemia, induced by oxygen-glucose-deprivation (OGD). The pathways controlling intracellular Na(+) and Ca(2+) concentration ([Na(+)](i) and [Ca(2+)](i)) and their inhibition were correlated with delayed cell death or protection. OGD induced a marked decrease in the ATP level and a transient elevation of [Ca(2+)](i) and [Na(+)](i) in cell soma of pyramidal neurons. ATP level, [Na(+)](i) and [Ca(2+)](i) rapidly recovered after reintroduction of oxygen and glucose. Pharmacological analysis showed that the OGD-induced [Ca(2+)](i) elevation in neuronal cell soma resulted from activation of both N-methyl-d-aspartate (NMDA)-glutamate receptors and Na(+)/Ca(2+) exchangers, while the abnormal [Na(+)](i) elevation during OGD was due to Na(+) influx through voltage-dependent Na(+) channels. In hippocampal slices, cellular degeneration occurring 24 h after OGD, selectively affected the pyramidal cell population through apoptotic and non-apoptotic cell death. OGD-induced cell loss was mediated by activation of ionotropic glutamate receptors, voltage-dependent Na(+) channels, and both plasma membrane and mitochondrial Na(+)/Ca(2+) exchangers. Thus, we show that neuroprotection induced by blockade of NMDA receptors and plasma membrane Na(+)/Ca(2+) exchangers is mediated by reduction of Ca(2+) entry into neuronal soma, whereas neuroprotection induced by blockade of AMPA/kainate receptors and mitochondrial Na(+)/Ca(2+) exchangers might result from reduced Na(+) entry at dendrites level.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Boron Compounds; Calcium; Calcium Channel Blockers; Cell Death; Clonazepam; Dantrolene; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Fura-2; Glucose; Hippocampus; Hypoxia; In Situ Nick-End Labeling; Indoles; Intracellular Space; Ion Exchange; Lidocaine; Mibefradil; Nimodipine; Organ Culture Techniques; Quinoxalines; Rats; Rats, Wistar; Sodium; Sodium Channel Blockers; Thiazepines; Thiourea; Time Factors

Ca(2+) influx via reversed K(+)-dependent (NCKX) and/or K(+)-independent (NCX) plasmalemmal Na(+)/Ca(2+) exchangers may play a role in neuronal death following global brain ischemia to which CA1 neurons are particularly vulnerable. Therefore, this work tested whether the rates of Ca(2+) influx via reversed NCKX or NCX in cultured rat CA1 neurons differ from those in forebrain neurons (FNs) or cerebellar granule cells (CGCs). The NCKX-mediated Ca(2+) influx was several times more rapid in CA1 neurons than in FNs or CGCs and was not affected by Na(+)/Ca(2+) exchange inhibitors, KB-R7943 or bepridil. NCKX reversal inhibitors are not yet available. Their development would greatly facilitate further testing the role of NCKX in ischemic death of CA1 neurons.

Topics: Animals; Calcium; Cells, Cultured; Cesium; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Female; Gluconates; Gramicidin; Hippocampus; Ion Transport; Lithium; Male; Meglumine; Neurons; Potassium; Pregnancy; Quinoxalines; Rats; Sodium; Sodium-Calcium Exchanger; Thiourea

A prominent feature of cerebral ischemia is the excessive intracellular accumulation of both Na(+) and Ca(2+), which results in subsequent cell death. A large number of studies have focused on pathways involved in the increase of the intracellular Ca(2+) concentration [Ca(2+)](i), whereas the elevation of intracellular Na(+) has received less attention. In the present study we investigated the effects of inhibitors of different Na(+) channels and of the Na(+)/Ca(2+) exchanger, which couples the Na(+) to the Ca(2+) gradient, on ischemic damage in organotypic hippocampal slice cultures. The synaptically evoked population spike in the CA1 region was taken as a functional measure of neuronal integrity. Neuronal cell death was assessed by propidium iodide staining. The Na(+) channel blocker tetrodotoxin, and the NMDA receptor blocker MK 801, but not the AMPA/kainate receptor blocker NBQX prevented ischemic cell death. The novel Na(+)/Ca(2+) exchange inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), which preferentially acts on the reverse mode of the exchanger, leading to Ca(2+) accumulation, also reduced neuronal damage. At higher concentrations, KB-R7943 also inhibits Ca(2+) extrusion by the forward mode of the exchanger and exaggerates neuronal cell death. Neuroprotection by KB-R7943 may be due to reducing the [Ca(2+)](i) increase caused by the exchanger.

Topics: Animals; Brain Ischemia; Cell Death; Culture Techniques; Dizocilpine Maleate; Electrophysiology; Hippocampus; Homeostasis; Hypoglycemia; Hypoxia; Neurons; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Sodium; Sodium Channel Blockers; Sodium Channels; Sodium-Calcium Exchanger; Tetrodotoxin; Thiourea