Protective effect of H2O2 against subsequent H2O2-induced cytotoxicity involves activation of the PI3K-Akt signaling pathway.

Preconditioning of sublethal ischemia implies a cytoprotective mechanism against subsequent ischemia­induced cell death; however, the precise mechanism by which preconditioning protects against ischemic injury is not known. In the present study, we clarified whether pretreatment with a sublethal concentration of H2O2 could counter subsequent H2O2-induced cytotoxicity and also investigated the mechanisms of the cytoprotective effect of a sublethal concentration of H2O2. Using the MTT reduction assay and Calcein-AM staining assay, we showed that pretreatment with H2O2 (10 µM, 24 hr) of COS7 cells partially protected cells against subsequent H2O2 (6 mM, 1 hr) - induced cytotoxicity. The phosphorylation of Akt/PKB, a downstream target of phosphatydylinositol-3 kinase (PI3K), at Ser473 was augmented by H2O2 (10 µM) administration. This augmentation peaked at 10 minutes after H2O2 (10 µM) treatment and fell to the basal level at 24 hr. A blocker of PI3K, LY294002, significantly attenuated H2O2 (10 µM, 24 hr) - induced cytoprotection. In addition, pretreatment with LY294002 reduced H2O2 (10 µM, 10 min)-induced phosphorylation of Akt at Ser473. These findings suggest that a sublethal concentration of H2O2 exerts a cytoprotective effect against subsequent H2O2-induced cell death and that this cytoprotective effect of H2O2 is mediated by activation of the PI3K-Akt signaling pathway.

Patch sensor detection of glutamate release evoked by a single electrical shock.

We tried to detect minimal stimulation-induced glutamate overflow from the surface of a hippocampal slice using an outside-out patch electrode excised from pyramidal cell membranes. The amplitude of the stimulation-induced patch current was dependent on the distance between the slice surface and the tip of patch sensor. The current-voltage relations of the stimulation-induced patch current were similar to those of the current evoked puff by application of L-glutamate to the patch. This indicates that the stimulation-induced patch current was produced by glutamate released from presynaptic terminals, and thus this technique may be useful in the study of transmitter release evoked by minimal electrical stimulation in brain slices.

Thrombin induces striatal neurotoxicity depending on mitogen-activated protein kinase pathways in vivo.

Intracerebral hemorrhage represents stroke characterized by formation and expansion of hematoma within brain parenchyma. Blood-derived factors released from hematoma are considered to be involved in poor prognosis of this disorder. We previously reported that thrombin, a blood-derived serine protease, induced cytotoxicity in the cerebral cortex and the striatum in organotypic slice cultures, which depended on mitogen-activated protein kinase (MAPK) pathways. Here we investigated the mechanisms of thrombin cytotoxicity in the striatum in vivo. Thrombin microinjected into the striatum of adult rats induced neuronal death and microglial activation around the injection site. Neuronal loss without any sign of nuclear fragmentation was observed as early as 4 h after thrombin injection, which was followed by gradual neuronal death exhibiting nuclear fragmentation. Thrombin-induced damage assessed at 72 h after injection was partially but significantly reduced by concomitant administration of inhibitors of MAPK pathways. Activation of extracellular signal-regulated kinase (ERK) and p38 MAPK in response to thrombin was verified by Western blot analysis. Moreover, phosphorylated ERK and p38 MAPK were localized prominently in reactive microglia, and inhibition of microglial activation by minocycline attenuated thrombin-induced damage, suggesting that reactive microglia were responsible for thrombin-induced neuronal death. Thus, MAPK pathways and microglial activation may serve as therapeutic targets of pathogenic conditions associated with hemorrhagic stroke.

Differential expression of small heat shock proteins in reactive astrocytes after focal ischemia: possible role of beta-adrenergic receptor.

Small heat shock proteins (sHSPs), a family of HSPs, are known to accumulate in the CNS, mainly in astrocytes, in several pathological conditions such as Alexander's disease, Alzheimer's disease, and Creutzfeldt-Jakob disease. sHSPs may act not only as molecular chaperones, protecting against various stress stimuli, but may also play a physiological role in regulating cell differentiation and proliferation. In the present study, we have demonstrated that transient focal ischemia in rats dramatically induced HSP27 but not alpha B-crystallin (alphaBC), both of which are members of sHSPs, in reactive astrocytes. In contrast, in vitro chemical ischemic stress induced both HSP27 and alphaBC in cultured glial cells to the same extent. Dibutyryl cAMP (dBcAMP) and isoproterenol, a beta-adrenergic receptor (betaAR) agonist, enhanced HSP27 expression but suppressed alphaBC, and changed the shape of the cells to a stellate form. dBcAMP and isoproterenol inhibited cell proliferation under normal conditions. An increase in betaAR-like immunoreactivity was also observed in reactive astrocytes in vivo. These results, together with recent findings that betaAR plays an important role in glial scar formation in vivo, raise the possibility that betaAR activation modulates sHSP expression after focal ischemia and is involved in the transformation of astrocytes to their reactive form.

Positive regulation of capacitative Ca2+ entry by intracellular Ca2+ in Xenopus oocytes expressing rat TRP4.

We have investigated the role of intracellular Ca2+ in the opening of capacitative Ca2+ entry (CCE) channels formed with rat TRP4 (rTRP4) using Xenopus oocytes. In rTRP4-expressing oocytes pretreated with thapsigargin, perfusion with A23187, a Ca2+ ionophore, significantly potentiated the delayed phase of the CCE-mediated Cl- current response evoked by extracellular perfusion with Ca2+, without affecting the transient phase of CCE response. In control oocytes, the potentiation of delayed CCE response by A23187 was not significant. Using cut-open recording in combination with artificial intracellular perfusion of oocytes, CCE-mediated Cl- response was recorded at controlled cytosolic Ca2+ concentrations. Intracellular perfusion with a Ca2+ free solution containing 10 mM EGTA abolished most of the CCE responses of both non-injected and rTRP4-expressing oocytes. The native CCE response was not fully recovered by subsequent increases in the intracellular Ca2+ concentration up to 300 nM. However, CCE response of the rTRP4-expressing oocytes was restored at an internal Ca2+ concentration of 110 nM. Blockade of endogenous Cl- channels with anion channel blocker isolated Ca2+ current flowing through CCE channels and clarified the difference in the sensitivity to an internal Ca2+ concentration. These findings indicate that recombinant CCE channels formed with rTRP4 are positively regulated by cytosolic Ca2+ at higher sensitivity compared to oocyte-endogenous CCE channels.

Localization of fractalkine and CX3CR1 mRNAs in rat brain: does fractalkine play a role in signaling from neuron to microglia?

Localization of the mRNAs for fractalkine, a CX3C chemokine, and for its receptor CX3CR1 was investigated in the rat brain. In situ hybridization study revealed that fractalkine mRNA was dominantly expressed in neuronal cells particularly in the olfactory bulb, cerebral cortex, hippocampus, caudate putamen and nucleus accumbens. In vitro study using enriched neuronal or glial culture supported the dominant expression of fractalkine mRNA in neurons. On the other hand, CX3CR1 mRNA was dominantly expressed in glial cells throughout the whole brain. The in vitro study suggested the cells expressing CX3CR1 mRNA are microglia, not astrocytes or neurons. Fractalkine appears to function as a signal molecule from neuron to microglia.

Inhibitory effects of thyrotropin-releasing hormone on neuronal activity in the nucleus accumbens.

The effects of thyrotropin-releasing hormone (TRH) were examined on neuronal activity in the nucleus accumbens, receiving an input from the parafascicular nucleus of the thalamus or the hippocampus, in chloral hydrate-anesthetized rats, using a microiontophoretic technique. The spikes produced by stimulation of the parafascicular nucleus were predominantly and dose-dependently inhibited during iontophoretic application of TRH. When the effects of TRH and dopamine were tested on the same neurons of the nucleus accumbens, inhibition of the generation of spikes by both drugs was observed in most neurons. In contrast, spikes elicited by stimulation of the hippocampus in most neurons of the nucleus accumbens were not affected by TRH or dopamine. Both TRH- and dopamine-induced inhibition of the spikes induced by stimulation of the parafascicular nucleus was antagonized by simultaneous application of haloperidol. In animals treated with reserpine, inhibition of the generation of spikes upon stimulation of the parafascicular nucleus did not occur in any neurons in the nucleus accumbens during application of TRH, whereas the dopamine-induced inhibition was still observed. These results suggest that inhibitory effects of TRH on the neurons of the nucleus accumbens receiving an input from the parafascicular nucleus are mediated by dopamine released from the dopaminergic nerve terminals located in the nucleus accumbens.

Inhibition of hippocampal CA1 neurons by 5-hydroxytryptamine, derived from the dorsal raphe nucleus and the 5-hydroxytryptamine1A agonist SM-3997.

Electrophysiological studies, using chloral hydrate-anesthetized rats, were undertaken to determine whether hippocampal pyramidal neurons, receiving input from the medial septal nucleus, were affected by 5-hydroxytryptamine (5-HT) derived from the dorsal raphe nucleus. The pyramidal neurons in the CA1 region of the hippocampus were classified into short- and long-latency neurons, based on their response to stimulation of the medial septal nucleus. Microiontophoretically applied atropine inhibited the generation of spikes upon stimulation of the medial septal nucleus in short-latency neurons, but had no effect on long-latency neurons. In the short-latency neurons, the stimulation-induced spikes of the medial septal nucleus were inhibited by conditioning stimuli applied to the dorsal raphe nucleus and iontophoretic application of 5-HT and the 5-HT1A agonists, SM-3997 (3 a alpha,4 beta,7 beta,7a alpha-hexahydro-2-(4-(4-(2-pyrimidinyl)-1- piperazinyl)-butyl)-4,7-methano-1H-isoindole-1,3(2H)-dione dihydrogen citrate) and 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin). The conditioning effect of the dorsal raphe nucleus was antagonized by methysergide. However, in the long-latency neurons, the spikes elicited by stimulation of the medial septal nucleus were not affected by the conditioning stimulation of the dorsal raphe nucleus, or iontophoretically applied 5-HT. These results indicate that 5-HT, originating in the dorsal raphe nucleus inhibited hippocampal pyramidal neurons receiving cholinergic input from the medial septal nucleus, but not those receiving non-cholinergic input from the medial septal nucleus. The drug SM-3997 inhibited the activity of hippocampal pyramidal neurons, that receive excitatory cholinergic input from the medial septal nucleus by acting on 5-HT1A receptors.

Protective effects of 1 alpha,25-(OH)(2)D(3) against the neurotoxicity of glutamate and reactive oxygen species in mesencephalic culture.

This study was undertaken to determine whether 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)(2)D(3)], an active metabolite of vitamin D, protects dopaminergic neurons against the neurotoxic effects of glutamate and dopaminergic toxins using rat mesecephalic culture. Brief glutamate exposure elicited cytotoxicity in both dopaminergic and non-dopaminergic neurons. Pretreatment, but not co-administration, of 1 alpha,25-(OH)(2)D(3) protected both types of neurons against the cytotoxicity of glutamate in a concentration- and time-dependent manner. The neuroprotective effect of 1 alpha,25-(OH)(2)D(3) was inhibited by the protein synthesis inhibitor, cycloheximide. To investigate the mechanisms of these neuroprotective effects, we examined the effects of 1 alpha,25-(OH)(2)D(3) on neurotoxicity induced by calcium ionophore and reactive oxygen species (ROS). Pretreatment with 1 alpha,25-(OH)(2)D(3) protected both types of neurons against the cytotoxicity induced by A23187 in a concentration-dependent manner. Furthermore, 24-h pretreatment with 1 alpha,25-(OH)(2)D(3) concentration-dependently protected both types of neurons from ROS-induced cytotoxicity. A 24-h incubation with 1 alpha,25-(OH)(2)D(3) inhibited the increase in intracellular ROS level following H(2)O(2) exposure. A 24-h exposure to 1-methyl-4-phenylpyridium ion (MPP(+)) or 6-hydroxydopamine (6-OHDA) exerted selective neurotoxicity on dopaminergic neurons, and these neurotoxic effects were ameliorated by 1 alpha,25-(OH)(2)D(3). These results suggest that 1 alpha,25-(OH)(2)D(3) provides protection of dopaminergic neurons against cytotoxicity induced by glutamate and dopaminergic toxins by facilitating cellular functions that reduce oxidative stress.

Protective effects of methylcobalamin, a vitamin B12 analog, against glutamate-induced neurotoxicity in retinal cell culture.

PURPOSE: To examine the effects of methylcobalamin on glutamate-induced neurotoxicity in the cultured retinal neurons. METHODS: Primary cultures obtained from the fetal rat retina (gestation days 16 to 19) were used for the experiment. The neurotoxicity was assessed quantitatively using the trypan blue exclusion method. RESULTS: Glutamate neurotoxicity was prevented by chronic exposure to methylcobalamin and S-adenosylmethionine (SAM), which is formed in the metabolic pathway of methylcobalamin. Chronic exposure to methylcobalamin and SAM also inhibited the neurotoxicity induced by sodium nitroprusside that release nitric oxide. By contrast, acute exposure to methylcobalamin did not protect retinal neurons against glutamate neurotoxicity. CONCLUSIONS: Chronic administration of methylcobalamin protects cultured retinal neurons against N-methyl-D-aspartate-receptor-mediated glutamate neurotoxicity, probably by altering the membrane properties through SAM-mediated methylation.

Protective action of zinc against glutamate neurotoxicity in cultured retinal neurons.

PURPOSE: To examine the effects of Zn2+ on glutamate-induced neurotoxicity in cultured retinal neurons. METHODS: Primary cultures obtained from fetal rat retinas (16 to 19 days gestation) were used. The neurotoxic effects of excitatory amino acids were quantitatively assessed using the trypan blue exclusion method. RESULTS: A brief exposure of retinal cultures to glutamate or N-methyl-D-aspartate (NMDA) induced delayed cell death. Zn2+ at concentrations of 3 to 30 microM ameliorated glutamate- and NMDA-induced neurotoxicity in a dose-dependent manner. By contrast, neurotoxicity induced by a 1-hour exposure to kainate was not affected by Zn2+. CONCLUSIONS: These findings demonstrate that Zn2+ protects retinal neurons from NMDA receptor-mediated glutamate neurotoxicity.

Protective effects of FK506 against glutamate-induced neurotoxicity in retinal cell culture.

PURPOSE: To examine the effects of FK506 on glutamate neurotoxicity in cultured retinal neurons. METHODS: Experiments were performed with primary retinal cultures obtained from 17- to 19-day-old rat fetuses. To assess the effects of FK506 and other drugs on glutamate neurotoxicity, cultures were treated with a drug beginning 10 minutes before application of glutamate and continuing during the subsequent 10 minutes of glutamate exposure. The treated cells were then incubated for 1 hour in a drug-free and glutamate-free medium. After a 1-hour incubation, cell viability was quantitatively measured by the trypan blue exclusion method. RESULTS: Brief exposure to glutamate markedly decreased cell viability. FK506 protected against glutamate neurotoxicity in a dose-dependent manner. Rapamycin is a competitive inhibitor of FK506 that binds FK506 binding protein. Simultaneous application of rapamycin and FK506 negated the protective effects of FK506. Cyclosporin A, which binds and inhibits calcineurin, mimicked the protective effects of FK506. Treatment with FK506 did not affect the intracellular maximum Ca2+ concentration induced by glutamate application. Although FK506 exhibited protective action against Ca2+ ionophore-induced neurotoxicity, it had no effect on nitric oxide-induced neurotoxicity. Treatment with FK506 reduced the activity of nitric oxide synthase (NOS). CONCLUSION: FK506 protected against glutamate neurotoxicity by inhibiting NOS activity in cultured retinal neurons.

Protective action of dopamine against glutamate neurotoxicity in the retina.

PURPOSE: The electrophysiologic study using patch-clamp techniques demonstrated that NMDA-induced currents had properties similar to those recorded in the brain. METHODS: Primary cultures obtained from the fetal rat retina (gestation days 16 to 19) were used for the experiment. Immunocytochemical and electrophysiologic studies were done to identify the cultured cells. The neurotoxic effects of glutamate or N-methyl-D-aspartate (NMDA) on the retinal cultures were quantitatively assessed using the trypan blue exclusion method. RESULTS: The immunocytochemical study revealed that the major component of the rat retinal cultures was neurons including amacrine cells. The electrophysiologic study using patch-clamp techniques demonstrated that exposure to NMDA-induced currents with properties characteristic of those recorded in the brain. Brief exposure of these neurons to glutamate or NMDA induced delayed cell death. Glutamate neurotoxicity was prevented by the application of dopamine and forskolin. The protective action of dopamine was antagonized by a D1 receptor antagonist (SCH 23390) but not by D2 receptor antagonists (domperidone and sulpiride). A D1 receptor agonist (SKF 38393) protected glutamate-induced neurotoxicity in a concentration-dependent manner, whereas a D2 receptor agonist (quinpirole) did not affect it. CONCLUSIONS: These findings demonstrate that dopamine protects retinal neuronal cells against NMDA receptor-mediated glutamate neurotoxicity via D1 receptors.

Protective effect of bradykinin against glutamate neurotoxicity in cultured rat retinal neurons.

PURPOSE: To identify the localization and expression of bradykinin (BK)-B2 receptors in rat retina and examine the effects of BK on glutamate-induced neurotoxicity using cultured rat retinal neurons. METHODS: An immunohistochemical study using a specific antibody against BK-B2 receptor was performed with rat retina. Primary cultures were obtained from the retina of fetal rats (gestation day 17-19). Expression of BK-B2 receptor mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA obtained from cultured retinal neurons. Cultured cells were exposed to glutamate (1 mM) for 10 minutes and followed by incubation in glutamate-free medium for 1 hour. The effects of BK were assessed by simultaneous application of BK with glutamate. The neurotoxic effects on retinal cultures were quantitatively assessed by the trypan blue exclusion method. RESULTS: Immunohistochemical study demonstrated that BK-B2 receptors were expressed in the ganglion cell, inner nuclear layers, and outer nuclear layers. Furthermore, BK-B2 receptor mRNA expression was observed in cultured retinal neurons. Cell viability was markedly reduced by 10-minute exposure to 1 mM glutamate followed by a 1-hour incubation in glutamate-free medium. Simultaneous application of BK at concentrations of 0.001 to 1 microM with glutamate demonstrated dose-dependent protection against glutamate neurotoxicity. The protective action of BK (1 microM) was inhibited by simultaneous application of BK-B2 receptor antagonist, Hoe140 (1 microM). Furthermore, 1 microM BK had protective effects on neurotoxicity induced by 1 microM ionomycin, a calcium ionophore, and sodium nitroprusside (SNP, 500 microM), a nitric oxide (NO)-generating agent. However, BK did not inhibit neurotoxicity induced by 3-morpholinosydnonimine (SIN-1, 10 microM), an NO and oxygen radical donor. CONCLUSIONS: These results suggest that BK-B2 receptors were distributed in rat retinas and cultured retinal neurons and that BK had a protective action against glutamate neurotoxicity through BK-B2 receptors in cultured retinal neurons. It is suggested that BK-induced protection against glutamate neurotoxicity took place downstream to NO generation and upstream to oxygen radical generation.

Inhibition of Ca2+ channel current by mu- and kappa-opioid receptors coexpressed in Xenopus oocytes: desensitization dependence on Ca2+ channel alpha 1 subunits.

1. Desensitization of mu- and kappa-opioid receptor-mediated inhibition of voltage-dependent Ca2+ channels was studied in a Xenopus oocyte translation system. 2. In the oocytes coexpressing kappa-opioid receptors with N- or Q-type Ca2+ channel alpha 1 and beta subunits, the kappa-agonist, U50488H, inhibited both neuronal Ca2+ channel current responses in a pertussis toxin-sensitive manner and the inhibition was reduced by prolonged agonist exposure. 3. More than 10 min was required to halve the inhibition of Q-type channels by the kappa-agonist. However, the half-life for the inhibition of N-type channels was only 6 +/- 1 min. In addition, in the oocytes coexpressing mu-opioid receptors with N-type or Q-type channels, the uncoupling rate of the mu-receptor-mediated inhibition of N-channels was also faster than that of Q-type channels. 4. In the oocytes coexpressing both mu- and kappa-receptors with N-type channels, stimulation of either receptor resulted in a cross-desensitization of the subsequent response to the other agonist. Treatment of oocytes with either H-8 (100 microM), staurosporine (400 nM), okadaic acid (200 nM), phorbol myristate acetate (5 nM) or forskolin (50 microM) plus phosphodiesterase inhibitor did not affect either the desensitization or the agonist-evoked inhibition of Ca2+ channels. 5. These results suggest that the rate of rapid desensitization is dependent on the alpha 1 subtype of the neuronal Ca2+ channel, and that a common phosphorylation-independent mechanism underlies the heterologous desensitization between opioid receptor subtypes.

Mobilization of intracellular Ca2+ and stimulation of cyclic AMP production by kappa opioid receptors expressed in Xenopus oocytes.

The intracellular metabotropic pathway, following kappa opioid receptor activation, was investigated in the Xenopus oocyte translation system. When oocytes were injected with cRNA for kappa opioid receptor cDNA, U50488H rarely evoked phospholipase C-mediated, oscillatory Cl- current responses. However, after the oocytes were incubated with staurosporine, both the occurrence and the amplitude of U50488H-evoked responses were increased. The U50488H-evoked response was antagonized by naloxone and inhibited by pretreatment of the oocytes with pertussis toxin. When oocytes were coinjected with RNAs encoding kappa opioid receptor and cystic fibrosis transmembrane conductance regulator (CFTR), treatment of the oocytes with forskolin and 3-isobutyl-1-methylxanthine (IBMX) evoked a smooth-shaped Cl- current flowing through the CFTR channels. The forskolin/IBMX-evoked response was never inhibited but was greatly potentiated in the presence of U50488H, indicating stimulation of adenylyl cyclase by U50488H. This U50488H-induced potentiation of CFTR channel opening was antagonized by naloxone and inhibited by pretreatment with pertussis toxin. These results suggest that kappa opioid receptors mobilize intracellular Ca2+ and stimulate cyclic AMP production by coupling positively to both phospholipase C and adenylyl cyclase via pertussis toxin-sensitive GTP-binding proteins in the oocytes.

Suppressive effect of zinc ion on iNOS expression induced by interferon-gamma or tumor necrosis factor-alpha in murine keratinocytes.

Zinc, an essential metal, is a critical component of zinc binding proteins such as zinc fingers, zinc enzymes and metallothioneins. Recently, evidence for its anti-inflammatory property in skin has been accumulating, as shown in the treatment of acne, alopecia and zinc deficiency. In cutaneous inflammations, a large amount of nitric oxide (NO) is produced through induction of inducible nitric oxide synthase (iNOS) under the influence of proinflammatory cytokines, resulting in tissue damages in skin, as clarified in other organs. Therefore, we asked if the effect of zinc on NO production and/or on iNOS expression in keratinocytes may explain the anti-inflammatory property of zinc in skin. Accordingly, we sought to determine in this study whether zinc ion may have effect on IFN-gamma or TNF-alpha induced NO production and iNOS expression in cultured murine keratinocytes. Ten microM of zinc ion remarkably suppressed cytokine-induced NO production in keratinocytes. Furthermore, zinc ion also suppressed cytokine-induced iNOS expression in the protein level as well as in the messenger RNA level. These results suggest the possibility that the suppressive effect of zinc ion on cytokine-induced NO production in keratinocytes may be in part implicated in the anti-inflammatory property of zinc in some of skin disorders.

Nicotine-induced protection against glutamate cytotoxicity. Nicotinic cholinergic receptor-mediated inhibition of nitric oxide formation.

Cortical neurodegeneration in Alzheimer's disease (AD) is suggested to be attributable not only to beta-protein but also to glutamate. Although degeneration of cholinergic projection to the cerebral cortex is recognized to be one of the most prominent pathological changes in AD, there is only limited information concerning the cholinergic interaction with the cortical neurodegeneration. This study was performed to examine the protective effect of nicotine against glutamate-induced cytotoxicity using rat cultured cortical neurons. The cell viability was significantly reduced when cultures were briefly exposed to glutamate or N-methyl-D-aspartate (NMDA). The simultaneous addition of nicotine did not reduce glutamate cytotoxicity. In contrast, the simultaneous application of NMDA receptor antagonists such as MK-801 reduced glutamate cytotoxicity. Incubating the cultures with nicotine (10 microM) for 0.5-24 h prior to glutamate exposure reduced its cytotoxicity. Neuroprotection by nicotine was dependent on both the concentration and incubation period. In contrast to nicotine, muscarine (10 microM) weakly potentiated glutamate cytotoxicity. The neuroprotective effect of nicotine against glutamate cytotoxicity was antagonized by hexamethonium but not by artopine. Nicotine prevented NMDA cytotoxicity but did not affect cytotoxicity induced by either kainate or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA). Cell viability was significantly reduced by a brief exposure of cultures to ionomycin, a calcium ionophore. Ionomycin-induced cytotoxicity was abolished by removing Ca2+ from incubating medium. S-nitrosocysteine (SNOC), which spontaneously releases nitric oxide (NO), also induced delayed cell death. Nicotine prevented ionomycin-induced cytotoxicity without affecting SNOC-induced cytotoxicity. These results suggest that nicotinic cholinergic receptor stimulation induces neuroprotection against glutamate cytotoxicity by its inhibitory action on NO-formation. Therefore, we propose that acetylcholine, acting through nicotinic cholinergic receptors, can function as a putative neuroprotective factor against neurodegeneration caused by the excessive release of glutamate and/or NMDA receptor activation.

Ca2+ channel inhibition by kappa opioid receptors expressed in Xenopus oocytes.

Functional coupling between kappa opioid receptors and voltage-dependent Ca2+ channels was studied in the Xenopus oocyte translation system, in which specific RNAs encoding rat kappa opioid receptor, rabbit BI-2 alpha 1 subunit, and human beta subunit were co-injected. Perfusion of the oocytes with U50488H inhibited depolarization-evoked Ba2+ current (IBa) in a reversible manner, showing maximal inhibition of 25% at 1 microM (IC50 = 31 nM). The inhibitory effect of U50488H was desensitized by pre-exposure of the oocytes to U50488H and abolished by the kappa opioid antagonist nor-binaltorphimine and by overnight pretreatment with pertussis toxin. Agents affecting the activity of protein kinase A or C did not affect the U50488H-induced inhibition of IBa. These findings suggest that kappa opioid receptors inhibit the activity of neuronal Ca2+ channels via GTP-binding proteins, without the participation of protein kinase A or C.

Protection of cultured spinal motor neurons by estradiol.

Estrogens have been reported to exert neuroprotection in the brain, but there have been no reports of such neuroprotection in spinal motor neurons, the neurons selectively involved in amyotrophic lateral sclerosis (ALS). In this study, we demonstrated that 17beta-estradiol and its biologically inactive stereoisomer, 17alpha-estradiol, prevented glutamate- and nitric oxide (NO)-induced selective motor neuronal death observed in primary cultures of the rat spinal cord. The dose of estradiols required for motor neuron protection was greatly reduced by co-administration with glutathione. The results of this study shows that estradiol protects spinal motor neurons from excitotoxic insults in vitro, and may have application as a treatment for ALS.