Nobuo Katsube

Evidence that Glyceraldehyde‐3‐Phosphate Dehydrogenase Is Involved in Age‐Induced Apoptosis in Mature Cerebellar Neurons in Culture

Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin‐D and cycloheximide. The level of a 38‐kDa protein in the particulate fraction is markedly increased during age‐induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age‐induced increment of the 38‐kDa particulate protein is suppressed by actinomycin‐D and cycloheximide. N‐terminal microsequencing of the 38‐kDa protein revealed sequence identity with glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age‐induced expression of the particulate 38‐kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age‐induced neuronal death and the 38‐kDa protein overexpression. Moreover, the age‐induced expression of the 38‐kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin‐D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age‐induced apoptosis of cerebellar neurons.

Involvement of Cathepsin E in Exogenous Antigen Processing in Primary Cultured Murine Microglia

We have attempted to elucidate an involvement of cathepsin E (CE) in major histocompatibility complex class II-mediated antigen presentation by microglia. In primary cultured murine microglia, CE was localized mainly in early endosomes and its expression level was markedly increased upon stimulation with interferon-γ. Pepstatin A, a specific inhibitor of aspartic proteases, significantly inhibited interleukin-2 production from an OVA-(266–281)-specific T helper cell hybridomas upon stimulation with native OVA presented by interferon-γ-treated microglia. However, pepstatin A failed to inhibit the presentation of OVA-(266–281) peptide. The possible involvement of CE in the processing of native OVA into antigenic peptide was further substantiated by that digested fragments of native OVA by CE could be recognized by OVA-specific Th cells. Cathepsin D also degraded native OVA into antigenic peptide, whereas microglia prepared from cathepsin D-deficient mice retained an ability for antigen presentation. On the other hand, the requirement for cysteine proteases such as cathepsins S and B in the processing of invariant chain (Ii) was confirmed by immunoblot analyses in the presence of their specific inhibitors. In conclusion, CE is required for the generation of an antigenic epitope from OVA but not for the processing of Ii in microglia.

Attenuation of a delayed increase in the extracellular glutamate level in the peri-infarct area following focal cerebral ischemia by a novel agent ONO-2506.

A novel agent, ONO-2506 [(R)-(-)-2-propyloctanoic acid, ONO Pharmaceutical Co. Ltd.] was previously shown to mitigate delayed infarct expansion through inhibition of the enhanced production of S-100beta, while inducing a prompt symptomatic improvement that attained a significant level as early as 24h after drug administration. To elucidate the mechanism underlying the prompt symptomatic improvement, the present study aimed to examine whether ONO-2506 modulates the level of extracellular glutamate ([Glu]e) in the rat subjected to transient middle cerebral artery occlusion (tMCAO). In this model, it had been shown that ONO-2506 reduces the infarct volume, improves the neurological deficits, and enhances the mRNA expression of glial glutamate transporters (GLT-1 and GLAST). The [Glu]e levels in the ischemic cortices were continuously measured using intracerebral microdialysis. The alterations in the [Glu]e levels in the sham-operated and tMCAO-operated groups with or without drug administration were compared. In the tMCAO groups, the [Glu]e level increased during tMCAO to a similar extent, returned to normal on reperfusion, and increased again around 5h. In the saline-treated group, however, the [Glu]e level further increased from 15 h on to reach about 280% of the normal level at 24h. This secondary increase in the [Glu]e level in the late phase of reperfusion was prevented by ONO-2506. The intracerebral infusion of glutamate transporter inhibitor, l-trans-pyrrolidine-2,4-dicarboxylic acid, at 24h after tMCAO induced an increase in the [Glu]e level, which was marked in both the sham-operated and ONO-2506-treated groups, but much less pronounced in the saline-treated group. The above results suggest that functional modulation of activated astrocytes by pharmacological agents like ONO-2506 may inhibit the secondary rise of [Glu]e level in the late phase of reperfusion, leading to amelioration of delayed infarct expansion and neurological deficits.

Arginine-vasopressin fragment 4–9 stimulates the acetylcholine release in hippocampus of freely-moving rats

We examined the effects of arginine-vasopressin (AVP) C-terminal fragment 4-9, which facilitates learning and memory, on the extracellular acetylcholine (ACh) release in hippocampus of freely-moving rats using the microdialysis technique. Following administration of AVP4-9, p-Glu-Asn-Cys[Cys]-Pro-Arg-Gly-NH2, through the dialysis probe into the hippocampus, ACh levels in dialysates from the hippocampus increased markedly in dose and time dependent manner at 2-2.5 and 2.5-3 hr. AVP1-9, the parent peptide, has a similar enhancing effect on ACh release as AVP4-9. Stimulated ACh release by AVP4-9 was significantly inhibited by V1-selective receptor antagonist ([1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-(O-methyl)-tyrosine]AVP), but not by V2-selective antagonist ([1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-D-Ile, 4-Ile]AVP). From these observations, it is demonstrated that AVP4-9 stimulates the ACh release in rat hippocampus via mediating V1-like vasopressin receptors.

L-Cysteine based N-type calcium channel blockers: structure-activity relationships of the C-terminal lipophilic moiety, and oral analgesic efficacy in rat pain models.

This study was performed to determine the structure-activity relationships (SAR) of L-cysteine based N-type calcium channel blockers. Basic nitrogen was introduced into the C-terminal lipophilic moiety of L-cysteine with a view toward improvement of its physicochemical properties. L-Cysteine derivative 9 was found to be a potent and selective N-type calcium channel blocker with IC(50) of 0.33 microM in calcium influx assay using IMR-32 cells and was 15-fold selective for N-type calcium channels over L-type channels. Compound 9 showed improved oral analgesic efficacy in the rat formalin induced pain model and the rat chronic constriction injury (CCI) model, which is one of the most reliable models of chronic neuropathic pain, without any significant effect on blood pressure or neurological behavior.

The Effects of Op-1206 α-cd on Walking Dysfunction in the Rat Neuropathic Intermittent Claudication Model

IV prostaglandin E1 improves clinical symptoms in patients with spinal canal stenosis. In the present study, we assessed the effects of OP-1206 &agr;-CD, an orally active prostaglandin E1 analog, on walking dysfunction in the rat neuropathic intermittent claudication model. To induce spinal stenosis, two pieces of silicon rubber were placed in the lumbar (L4-6) epidural space in rats. Postsurgical walking function was measured using a treadmill apparatus. Spinal cord blood flow (SCBF) and skin blood flow (SKBF) were measured using a laser-Doppler flowmeter. OP-1206 &agr;-CD was administered orally bid for 11 days from postoperative Day 3. In Control nontreated rats, a significant walking dysfunction was observed from Day 1 after the induction of spinal stenosis and persisted for 14 days when compared with the Sham-Operated group. On postoperative Day 15, SCBF revealed a significant reduction in the territory of spinal stenosis, although SKBF was not affected. OP-1206 &agr;-CD significantly improved walking dysfunction on postoperative Days 5 (300 &mgr;g/kg), 7 (150 and 300 &mgr;g/kg), and 14 (150 and 300 &mgr;g/kg) when compared with the Vehicle-Treated group. On postoperative Day 15, the decrease in SCBF was significantly (150 and 300 &mgr;g/kg) improved by OP-1206 &agr;-CD treatment, albeit SKBF remained unaffected. These data show that oral treatment with OP-1206 &agr;-CD is effective in improving walking dysfunction induced by spinal canal stenosis, and this therapeutic effect is likely mediated by improved SCBF at the territory of spinal stenosis.

Structure–Activity Study of l-Cysteine-Based N-Type Calcium Channel Blockers: Optimization of N- and C-Terminal Substituents

Synthesis and structure-activity relationship (SAR) studies of L-cysteine-based N-type calcium channel blockers are described. In the course of exploring SAR of the N- and C-terminal substituents, the L-cysteine derivative was found to be a potent N-type calcium channel blocker with an IC(50) value of 0.14 microM on IMR-32 assay. Compound showed 12-fold selectivity for N-type over L-type calcium channels on AtT-20 assay.

Structure-activity study and analgesic efficacy of amino acid derivatives as N-type calcium channel blockers.

The synthesis and structure-activity relationship (SAR) study of a novel series of N-type calcium channel blockers are described. L-Cysteine derivative 2a was found to be a potent and selective N-type calcium channel blocker with IC(50) 0.63 microM on IMR-32 assay. Compound 2a showed analgesic efficacy in the rat formalin-induced pain model by intrathecal and oral administration.

Structure-activity study of L-amino acid-based N-type calcium channel blockers.

Synthesis and structure-activity relationship (SAR) study of L-amino acid-based N-type calcium channel blockers are described. The compounds synthesized were evaluated for inhibitory activity against both N-type and L-type calcium channels focusing on selectivity to reduce cardiovascular side effects due to blocking of L-type calcium channels. In the course of screening of our compound library, N-(t-butoxycarbonyl)-L-aspartic acid derivative 1a was identified as an initial lead compound for a new series of N-type calcium channel blockers, which inhibited calcium influx into IMR-32 human neuroblastoma cells with an IC(50) of 3.4 microM. Compound 1a also exhibited blockade of N-type calcium channel current in electrophysiological experiment using IMR-32 cells (34% inhibition at 10 microM, n=3). As a consequence of conversion of amino acid residue of 1a, compound 12a, that include N-(t-butoxycarbonyl)-L-cysteine, was found to be a potent N-type calcium channel blocker with an IC(50) of 0.61 microM. Thus, L-cysteine was selected as a potential structural motif for further modification. Optimization of C- and N-terminals of L-cysteine using S-cyclohexylmethyl-L-cysteine as a central scaffold led to potent and selective N-type calcium channel blocker 21f, which showed improved inhibitory potency (IC(50) 0.12 microM) and 12-fold selectivity for N-type calcium channels over L-type channels.

Effects of OP-1206 α-CD on walking dysfunction in the rat neuropathic intermittent claudication model: comparison with nifedipine, ticlopidine and cilostazol

The systemic treatment effects of OP-1206 alpha-CD (17S-20-dimethyl-trans-delta 2-PGE1 alpha-cyclodextrin clathrate), a prostaglandin E1 (PGE1) analogue, on walking dysfunction, spinal cord blood flow (SCBF) and skin blood flow (SKBF) were assessed in the rat neuropathic intermittent claudication (IC) model in comparison with nifedipine (dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylate), ticlopidine (5-[(2-chlorophenyl)methyl]-4,5,6,7-tetrahydrothieno[3,2-C]pyridine hydrochloride) and cilostazol (6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)-butoxy]-3,4-dihydro-2(1H)-quinolinone). Two pieces of silicone rubber strips were placed in the lumbar (L4 and L6) epidural space in rats. After surgery, walking function was measured using a treadmill apparatus. SCBF and SKBF were measured using a laser-Doppler flow meter. Drugs were administered orally twice a day for 11 days from day 3 post-surgery. Treatment with OP-1206 alpha-CD significantly improved walking dysfunction on days 5, 7 and 14, and improved SCBF on day 14 post-surgery. SKBF remained unaffected. Treatment with nifedipine, ticlopidine or cilostazol had no significant effects on any of the parameters measured in this model. These data suggest that the therapeutic effect of OP-1206 alpha-CD is primarily mediated by the improved local SCBF at the territory of spinal stenosis and not due to improvement of peripheral perfusion and/or antiplatelet activity.