Angiotensin receptor type 1 antagonists protect against neuronal injury induced by oxygen-glucose depletion.

BACKGROUND AND PURPOSE: Several clinical trials and in vivo animal experiments have suggested that blockade of angiotensin receptor type 1 (AT(1)) improves ischaemic outcomes. However, the mechanism(s) underlying these effects has not been elucidated. Here, we have investigated the protective effects of pretreatment with AT(1) receptor antagonists, losartan or telmisartan, against ischaemic insult to neurons in vitro. EXPERIMENTAL APPROACH: Primary rat neuron-astrocyte co-cultures and astrocyte-defined medium (ADM)-cultured pure astrocyte cultures were prepared. Ischaemic injury was modelled by oxygen-glucose depletion (OGD) and lactate dehydrogenase release after OGD was measured with or without AT(1) receptor antagonists or agonists (L162313), AT(2) receptor antagonist (PD123319) or agonist (CGP-42112A) pretreatment, for 48 h. Activity of glutamate transporter 1 (GLT-1) was evaluated by [(3)H]-glutamate uptake assays, after AT(1) receptor agonists or antagonists. Immunoblot and real-time PCR were used for analysis of protein and mRNA levels of GLT-1. KEY RESULTS: AT(1) receptor agonists augmented OGD-induced cellular damage, which was attenuated by AT(1) receptor antagonists. AT(1) receptor antagonists also suppressed OGD-induced extracellular glutamate release, reactive oxygen species production and nitric oxide generation. GLT-1 expression and glutamate uptake activity were significantly enhanced by AT(1) receptor antagonists and impaired by AT(1) receptor agonists. AT(1) receptor stimulation suppressed both ADM-induced GLT-1 protein expression and mRNA levels. AT(1)b receptor knock-down with siRNA enhanced GLT-1 expression. In postnatal (P1-P21) rat brains, protein levels of GLT-1 and AT(1) receptors were inversely correlated. CONCLUSIONS AND IMPLICATIONS: Suppression of AT(1) receptor stimulation induced GLT-1 up-regulation, which ameliorated effects of ischaemic injury.

Neuroprotection by donepezil against glutamate excitotoxicity involves stimulation of alpha7 nicotinic receptors and internalization of NMDA receptors.

BACKGROUND AND PURPOSE: Glutamate excitotoxicity may be involved in ischaemic injury to the CNS and some neurodegenerative diseases, such as Alzheimer's disease. Donepezil, an acetylcholinesterase (AChE) inhibitor, exerts neuroprotective effects. Here we demonstrated a novel mechanism underlying the neuroprotection induced by donepezil. EXPERIMENTAL APPROACH: Cell damage in primary rat neuron cultures was quantified by lactate dehydrogenase release. Morphological changes associated with neuroprotective effects of nicotine and AChE inhibitors were assessed by immunostaining. Cell surface levels of the glutamate receptor sub-units, NR1 and NR2A, were analyzed using biotinylation. Immunoblot was used to measure protein levels of cleaved caspase-3, total NR1, total NR2A and phosphorylated NR1. Immunoprecipitation was used to measure association of NR1 with the post-synaptic protein, PSD-95. Intracellular Ca(2+) concentrations were measured with fura 2-acetoxymethylester. Caspase 3-like activity was measured using enzyme substrate, 7-amino-4-methylcoumarin (AMC)-DEVD. KEY RESULTS: Levels of NR1, a core subunit of the NMDA receptor, on the cell surface were significantly reduced by donepezil. In addition, glutamate-mediated Ca(2+) entry was significantly attenuated by donepezil. Methyllycaconitine, an inhibitor of alpha7 nicotinic acetylcholine receptors (nAChR), inhibited the donepezil-induced attenuation of glutamate-mediated Ca(2+) entry. LY294002, a phosphatidyl inositol 3-kinase (PI3K) inhibitor, had no effect on attenuation of glutamate-mediated Ca(2+) entry induced by donepezil. CONCLUSIONS AND IMPLICATIONS: Decreased glutamate toxicity through down-regulation of NMDA receptors, following stimulation of alpha7 nAChRs, could be another mechanism underlying neuroprotection by donepezil, in addition to up-regulating the PI3K-Akt cascade or defensive system.

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.

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.

Effects of selegiline on antioxidant systems in the nigrostriatum in rat.

Selegiline, a therapeutic agent of Parkinson's disease, is known to have neuroprotective properties that may involve its regulatory effects on antioxidant enzymes. We evaluated effects of selegiline on activities of catalase (CAT), Cu,Zn-superoxide dismutase (SOD1) and Mn-SOD (SOD2) in the striatum, cortex and hippocampus of 8- and 25-week-old rats, and on SOD activities and glutathione levels in mesencephalic slice cultures. Selegiline (2 mg/kg) significantly increased CAT and SOD2 activities in the striatum, but not in the cortex and hippocampus, of 25-week-old rats. In contrast, selegiline failed to increase CAT and SOD activities in three brain regions of 8-week-old rats, whereas L: -dopa significantly increased SOD1 activity in the striatum. In slice cultures, selegiline increased SOD1 and SOD2 activities with a maximal effective concentration of 10(-8) and 10(-10) M, respectively. Moreover, selegiline significantly increased glutathione level. These results suggest that selegiline can decrease oxidative stress in nigrostriatum by augmenting various antioxidant systems, each of which responds optimally to different concentrations of selegiline.

Implication of prostaglandin E(2) in TNF-alpha-induced release of m-calpain from HCS-2/8 chondrocytes. Inhibition of m-calpain release by NSAIDs.

OBJECTIVE: Calpains are known as Ca(2+)-dependent intracellular neutral cysteine proteases. However, m-calpain is detected in synovial fluid of arthritic joints and is shown to possess the proteoglycanase activity in vitro. The mechanism of m-calpain release into the extracellular spaces during arthritis has not yet been well characterized. In the present study, we have analyzed m-calpain release from cultured chondrocytes stimulated by a proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on m-calpain release were also examined. METHODS: Human chondrocytic HCS-2/8 cells were stimulated by TNF-alpha in the presence or absence of an NSAID. m-Calpain in the cells and culture medium was quantified by Western blot analysis using an anti-m-calpain antibody. Western blots were subjected to densitometric analysis and band intensities were determined. RESULTS: TNF-alpha (10 ng/ml) stimulated m-calpain release with transient increase in cellular m-calpain in HCS-2/8 cells. NSAIDs examined (aspirin, loxoprofen-SRS, diclofenac sodium, indomethacin and NS398) inhibited m-calpain release and production of prostaglandin E(2) (PGE(2)) induced by 10 ng/ml TNF-alpha. Exogenously added PGE(2) accelerated the release of m-calpain in response to a lower concentration of TNF-alpha (1 ng/ml). AH6809, an EP1/2 antagonist, but not SC19220 (an EP1 antagonist), effectively inhibited TNF-alpha-induced m-calpain release. In contrast, butaprost, an EP2 agonist, accelerated release of m-calpain by 1 ng/ml TNF-alpha. CONCLUSIONS: These results suggest that TNF-alpha stimulates upregulation and release of m-calpain in chondrocytic HCS-2/8 cells, and that stimulation of EP2-PGE(2) receptor by produced PGE(2) is deeply involved in this process.

Activation of inositol 1,4,5-trisphosphate receptor is essential for the opening of mouse TRP5 channels.

We studied the opening mechanism of Ca(2+)-permeable channels formed with mouse transient receptor potential type 5 (mTRP5) using Xenopus oocytes. After stimulation of coexpressed muscarinic M(1) receptors with acetylcholine (ACh) in a Ca(2+)-free solution, switching to 2 mM Ca(2+)-containing solution evoked a large Cl(-) current, which reflects the opening of endogenous Ca(2+)-dependent Cl(-) channels following Ca(2+) entry through the expressed channels. The ACh-evoked response was not affected by a depletion of Ca(2+) store with thapsigargin but was inhibited by preinjection of antisense oligodeoxynucleotides (ODNs) to G(q), G(11), or both. The mTRP5 channel response was also induced by a direct activation of G proteins with injection of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S). The ACh- and GTP gamma S-evoked responses were inhibited by either pretreatment with a phospholipase C inhibitor, U73122, or an inositol-1,4,5-trisphosphate (IP(3)) receptor inhibitor, xestospongin C (XeC). An activation of IP(3) receptors with injection of adenophostin A (AdA) evoked the mTRP5 channel response in a dose-dependent manner. The AdA-evoked response was not suppressed by preinjection of antisense ODNs to G(q/11) or U73122 but was suppressed by either preinjection of XeC or a peptide mimicking the IP(3) binding domain of Xenopus IP(3) receptor. These findings suggest that the activation of IP(3) receptor is essential for the opening of mTRP5 channels, and that neither G proteins, phosphoinositide metabolism, nor depletion of the Ca(2+) store directly modifies the IP(3) receptor-linked opening of mTRP5 channels.

(-)-1-(Benzofuran-2-yl)-2-propylaminopentane enhances locomotor activity in rats due to its ability to induce dopamine release.

"Catecholaminergic and serotoninergic activity enhancer" effects are newly found mechanisms of action of a class of compound that enhance impulse propagation-mediated release of catecholamines and serotonin in the brain. In the present study, (-)-1-(benzofuran-2-yl)-2-propylaminopentane hydrochloride [(-)-BPAP HCl], a compound with selective and potent "catecholaminergic and serotoninergic activity enhancer" effects, was tested for its efficacy to potentiate locomotor activity in normal rats and to attenuate hypolocomotion in reserpine-treated rats. (-)-BPAP HCl potentiated locomotor activity in non-habituated rats during a 2-h observation period dose-dependently (0.3-10 mg/kg). (-)-BPAP HCl (1-3 mg/kg) was also effective to reverse reserpine-induced hypolocomotion. The effects of (-)-BPAP HCl in normal and reserpine-treated rats were attenuated by the dopamine D1 receptor antagonist, R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390), suggesting that the effects of (-)-BPAP HCl were mediated by activation of the dopaminergic system. In addition, the administration of (-)-BPAP HCl increased ipsilateral turning in unilaterally 6-hydroxydopamine-lesioned rats, implying presynaptic activation of nigrostriatal dopaminergic terminals by (-)-BPAP HCl. Furthermore, although antiparkinsonian agents, such as apomorphine and amantadine, failed to improve reserpine-induced ptosis, (-)-BPAP HCl significantly improved ptosis. These findings suggested that a "catecholaminergic and serotoninergic activity enhancer" compound, (-)-BPAP, stimulates motor function in rats and improves motor deficits in animal models of Parkinson's disease due to its ability to induce dopamine release.

Nongenomic antiapoptotic signal transduction by estrogen in cultured cortical neurons.

Estrogen replacement therapy in menopausal women has been suggested to be beneficial in preventing the progression of cognitive impairment in Alzheimer disease. We demonstrated previously that the phosphatidylinositol 3-kinase (PI3-K)/Akt signal transduction pathway plays a pivotal role on the neuroprotection provided by 17beta-estradiol against acute glutamate toxicity. In the present study, we investigated the mechanism of neuroprotection against apoptosis because acute glutamate toxicity predominantly induced necrosis. 17beta-estradiol provided neuroprotection against apoptosis induced by staurosporine. This neuroprotection was inhibited by pretreatment with a PI3-K inhibitor, LY294002. An estrogen receptor specific antagonist, ICI182780, also suppressed the neuroprotection provided by 17beta-estradiol. Western blotting analysis demonstrated that treatment with 17beta-estradiol induced the phosphorylation of Akt within 5 min, which was suppressed by pretreatment with LY294002 and ICI182780. Furthermore, 17beta-estradiol induced phosphorylation of the cAMP response element binding protein (CREB) at Ser(133) within 15 min and then upregulated Bcl-2 in a PI3-K/Akt-dependent manner. Because CREB is known to be a transcription factor for Bcl-2, these results suggest that 17beta-estradiol exerts its antiapoptotic effects by CREB phosphorylation and Bcl-2 upregulation via nongenomic activation of the PI3-K/Akt pathway in cultured cortical neurons.

Binding of G alpha(o) N terminus is responsible for the voltage-resistant inhibition of alpha(1A) (P/Q-type, Ca(v)2.1) Ca(2+) channels.

G-protein-mediated inhibition of presynaptic voltage-dependent Ca(2+) channels is comprised of voltage-dependent and -resistant components. The former is caused by a direct interaction of Ca(2+) channel alpha(1) subunits with G beta gamma, whereas the latter has not been characterized well. Here, we show that the N terminus of G alpha(o) is critical for the interaction with the C terminus of the alpha(1A) channel subunit, and that the binding induces the voltage-resistant inhibition. An alpha(1A) C-terminal peptide, an antiserum raised against G alpha(o) N terminus, and a G alpha(o) N-terminal peptide all attenuated the voltage-resistant inhibition of alpha(1A) currents. Furthermore, the N terminus of G alpha(o) bound to the C terminus of alpha(1A) in vitro, which was prevented either by the alpha(1A) channel C-terminal or G alpha(o) N-terminal peptide. Although the C-terminal domain of the alpha(1B) channel showed similar ability in the binding with G alpha(o) N terminus, the above mentioned treatments were ineffective in the alpha(1B) channel current. These findings demonstrate that the voltage-resistant inhibition of the P/Q-type, alpha(1A) channel is caused by the interaction between the C-terminal domain of Ca(2+) channel alpha(1A) subunit and the N-terminal region of G alpha(o).

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.

Alpha-synuclein protein is not scavenged in neuronal loss induced by kainic acid or focal ischemia.

Alpha-synuclein, a presynaptic protein, is markedly included in Lewy bodies (LB) in Parkinson's and LB diseases. In this study, neuronal loss and the activation of glial cells such as microglia and astrocytes were induced by neurodegenerative insults such as the injection of kainic acid and occlusion of the middle cerebral artery. In contrast, immunoreactivity for alpha-synuclein did not change even at 7 days after these insults. These results suggest that alpha-synuclein protein may be so scarcely scavenged by glial cells that it readily condenses in neurodegenerative regions.

alpha 7 nicotinic receptor transduces signals to phosphatidylinositol 3-kinase to block A beta-amyloid-induced neurotoxicity.

Multiple lines of evidence, from molecular and cellular to epidemiological, have implicated nicotinic transmission in the pathogenesis of Alzheimer's disease (AD). Here we show the signal transduction mechanism involved in nicotinic receptor-mediated protection against beta-amyloid-enhanced glutamate neurotoxicity. Nicotine-induced protection was suppressed by an alpha7 nicotinic receptor antagonist (alpha-bungarotoxin), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002 and wortmannin), and a Src inhibitor (PP2). Levels of phosphorylated Akt, an effector of PI3K, and Bcl-2 were increased by nicotine. The alpha7 nicotinic receptor was physically associated with the PI3K p85 subunit and Fyn. These findings indicate that the alpha7 nicotinic receptor transduces signals to PI3K in a cascade, which ultimately contributes to a neuroprotective effect. This might form the basis of a new treatment for AD.

Superoxide dismutase activity in organotypic midbrain-striatum co-cultures is associated with resistance of dopaminergic neurons to excitotoxicity.

We have previously demonstrated that dopaminergic neurons in midbrain-striatum slice co-cultures are more resistant to NMDA cytotoxicity than the same neuronal population in single midbrain slice cultures. Here, we show that dopaminergic neurons in midbrain-striatum co-cultures also exhibit resistance to the cytotoxicity of nitric oxide donors, 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC-18) and 3-morpholinosydnonimine (SIN-1). The cytotoxicity of NMDA (30 microM) in single cultures was significantly attenuated by the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (100 microM), whereas the toxicity in co-cultures was not. The levels of tyrosine residue nitration of tyrosine hydroxylase, a hallmark of the occurence of peroxynitrite anion in dopaminergic neurons, were lower in co-cultures than those in single cultures. Single cultures and co-cultures did not show appreciable differences in the number or distribution of NOS-containing neurons as assessed by NADPH diaphorase histochemistry. On the other hand, midbrain slices cultured with striatal slices showed higher levels of superoxide dismutase (SOD) activity as well as increased protein levels of Cu,Zn-SOD, than midbrain slices cultured alone. These results suggested that the generation of NO is involved in NMDA cytotoxicity on dopaminergic neurons, and that increased activity of SOD in co-cultures renders dopaminergic neurons resistant to NMDA cytotoxicity by preventing the formation of peroxynitrite.

N-methyl-D-aspartate receptor-mediated mitochondrial Ca(2+) overload in acute excitotoxic motor neuron death: a mechanism distinct from chronic neurotoxicity after Ca(2+) influx.

Mitochondrial uptake of Ca(2+) has recently been found to play an important role in glutamate-induced neurotoxicity (GNT) as well as in the activation of Ca(2+)-dependent molecules, such as calmodulin and neuronal nitric oxide synthase (nNOS), in the cytoplasm. Prolonged exposure to glutamate injures motor neurons predominantly through the activation of Ca(2+)/calmodulin-nNOS, as previously reported, and is, in part, associated with the pathogenesis of amyotrophic lateral sclerosis (ALS). In the present study, we investigated how mitochondrial uptake of Ca(2+) is involved in GNT in spinal motor neurons. Acute excitotoxicity induced by exposure to 0.5 mM glutamate for 5 min was found in both motor and nonmotor neurons in cultured spinal cords from rat embryos and was dependent on extracellular Ca(2+) and on N-methyl-D-aspartate (NMDA) receptor activation. Mitochondrial uncouplers markedly blocked acute excitotoxicity, and membrane-permeable superoxide dismutase mimics attenuated acute excitotoxicity induced by glutamate and NMDA but not by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) or kainate. Fluorimetric analysis showed that mitochondrial Ca(2+) was elevated promptly with subsequent accumulation of reactive oxygen species (ROS) in the mitochondria. An NMDA receptor antagonist and a mitochondrial uncoupler eliminated the increase in fluorescence of mitochondrial Ca(2+) and ROS indicators. These data indicate that acute excitotoxicity in spinal neurons is mediated by mitochondrial Ca(2+) overload and ROS generation through the activation of NMDA receptors. This mechanism is different from that of chronic GNT.

Requirement of neural activity for the maintenance of dopaminergic neurons in rat midbrain slice cultures.

Chronic treatment of organotypic midbrain slice cultures with L-type Ca(2+) channel blocker nicardipine (3-10 microM) or verapamil (10 microM) for 18 days resulted in a drastic decrease in the number of dopaminergic neurons. A voltage-dependent Na(+) channel blocker tetrodotoxin (1 microM) was also effective in decreasing the number of dopaminergic neurons. Concurrent application of forskolin (20 microM) or dibutyryl cyclic AMP (1 mM) counteracted the effects of nicardipine and tetrodotoxin. These results suggest that spontaneous neuronal activity within midbrain slice cultures, causing Ca(2+) influx through L-type Ca(2+) channels that maintains intracellular cyclic AMP levels, is required for the maintenance of dopaminergic neurons.

Protective effects of ifenprodil against glutamate-induced neurotoxicity in cultured retinal neurons.

PURPOSE: To examine the effects of ifenprodil on glutamate-induced neurotoxicity in cultured retinal neurons. METHODS: Primary cultures obtained from the fetal rat retina (gestation day 17-19) were used for the experiment. Neurotoxicity effects on retinal cultures were quantitatively assessed by the trypan blue exclusion method. The cells were exposed briefly (10 min) to excitatory amino acids (EAA, 1 mM) and then were incubated for 1 h in an EAA-free medium. Ifenprodil (10 mM) was added for the 10-min exposure to EAA and the subsequent 60-min incubation in an EAA-free medium. RESULTS: Ifenprodil dose-dependently prevented cell death induced by glutamate or NMDA, but did not affect that induced by kainate. The protective effects of ifenprodil against glutamate neurotoxicity were significantly reduced by spermidine, a polyamine modulatory site agonist, but not by glycine, a strychnine-insensitive glycine site agonist. CONCLUSION: The findings suggest that ifenprodil protected the cultured retinal cells we used in this study against glutamate neurotoxicity by its inhibitory action on the polyamine modulatory site of the NMDA receptor.

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.

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.

FGF-20, a novel neurotrophic factor, preferentially expressed in the substantia nigra pars compacta of rat brain.

We have isolated cDNA encoding a novel FGF (212 amino acids) from rat brain. Because this is the 20th documented member of the FGF family, we tentatively term it FGF-20. Among FGF family members, FGF-20 is most similar to FGF-9 and FGF-16 (70 and 62% amino acid identity, respectively). Human FGF-20 gene was found in the human genomic sequence mapped to the 8p21.3-p22 region. Human FGF-20 is highly identical to rat FGF-20 (95% amino acid identity). FGF-20 mRNA was preferentially expressed in rat brain among the adult major tissues examined. The localization of FGF-20 mRNA in rat brain was also examined by in situ hybridization. FGF-20 mRNA was preferentially expressed in the substantia nigra pars compacta. To examine the biological activity of FGF-20, recombinant rat FGF-20 was produced by insect cells infected with recombinant baculovirus containing rat FGF-20 cDNA. Recombinant rat FGF-20 enhanced the survival of midbrain dopaminergic neurons. The present results indicate that FGF-20 is a novel neurotrophic factor preferentially expressed in the substantia nigra pars compacta of rat brain.