Biomedical subjects

Keiji Wada

Publications and source records attributed to Keiji Wada.

At least 19 recordsLinked to original sources

PubMed2003

Bombesin-like peptide receptor gene expression, regulation, and function in fetal murine lung.

Bombesin-peptide (BLP) immunoreactivity occurs at high levels in fetal lung. Previous studies showed that bombesin promotes fetal lung development. To test the hypothesis that such effects are mediated by known mammalian bombesin receptors [gastrin-releasing peptide (GRP)/bombesin-preferring receptor (GRPR), neuromedin B (NMB) receptor (NMBR), and the orphan bombesin receptor subtype-3 (BRS-3)], we analyzed the ontogeny of GRPR, NMBR, and BRS-3 gene expression in mouse lung. We examined the regulation of these three genes by dexamethasone and bombesin, which modulate lung development. Using incorporation of [3H]thymidine and [3H]choline, we then assessed whether GRP, NMB, and Leu8-phyllolitorin modulate lung growth and maturation in fetal lung explants. GRPR gene expression was detected predominantly in utero, whereas NMBR and BRS-3 genes were expressed from embryonic days 13-16 and on multiple postnatal days. All three mRNAs are present in airway epithelium and mesenchymal cells but occur in different relative patterns. These genes were regulated differently. Dexamethasone and bombesin increased GRPR mRNA, bombesin downregulated NMBR, and neither agent affected BRS-3. GRP increased incorporation of [3H]thymidine and [3H]choline in explants, whereas NMB induced cell proliferation and Leu8-phyllolitorin yielded variable results. Cumulative data suggest the involvement of multiple BLP receptors, including novel molecules, and argue against simple functional redundancy within this gene family during lung development.

Amino Acid Sequence↗

PubMed2003

Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron.

Mammalian neuronal cells abundantly express a deubiquitylating enzyme, ubiquitin carboxy-terminal hydrolase 1 (UCH L1). Mutations in UCH L1 are linked to Parkinson's disease as well as gracile axonal dystrophy (gad) in mice. In contrast to the UCH L3 isozyme that is universally expressed in all tissues, UCH L1 is expressed exclusively in neurons and testis/ovary. We found that UCH L1 associates and colocalizes with monoubiquitin and elongates ubiquitin half-life. The gad mouse, in which the function of UCH L1 is lost, exhibited a reduced level of monoubiquitin in neurons. In contrast, overexpression of UCH L1 caused an increase in the level of ubiquitin in both cultured cells and mice. These data suggest that UCH L1, with avidity and affinity for ubiquitin, insures ubiquitin stability within neurons. This study is the first to show the function of UCH L1 in vivo.

Animals↗

PubMed2003

D-cycloserine for the treatment of ataxia in spinocerebellar degeneration.

We studied the effects of D-cycloserine, a partial NMDA receptor allosteric agonist, on ataxia in patients with spinocerebellar degeneration. Fifteen Japanese ataxic patients enrolled in a 14-day single-blind trial of D-cycloserine (daily oral dose of 50 mg) following a 14-day single-blind placebo phase. At the end of the D-cycloserine administration, there was a significant reduction in the posture, gait and total score of the international cooperative ataxia rating scale and in the time for walking and speech tasks. D-Cycloserine was well-tolerated and no adverse effect was observed. D-Cycloserine may have therapeutic efficacy for spinocerebellar ataxia.

Adult↗

PubMed2003

Bombesin-like peptides and mast cell responses: relevance to bronchopulmonary dysplasia?

Bombesin-like peptides (BLPs) are elevated in newborns who later develop bronchopulmonary dysplasia (BPD). In baboon models, anti-BLP blocking antibodies abrogate BPD. We now demonstrate hyperplasia of both neuroendocrine cells and mast cells in lungs of baboons with BPD, compared with non-BPD controls or BLP antibody-treated BPD baboons. To determine whether BLPs are proinflammatory, bombesin was administered intratracheally to mice. Forty-eight hours later, we observed increased numbers of lung mast cells. We analyzed murine mast cells for BLP receptor gene expression, and identified mRNAs encoding bombesin receptor subtype 3 and neuromedin-B receptor (NMB-R), but not gastrin-releasing peptide receptor. Only NMB-R-null mice accumulated fewer lung mast cells after bombesin treatment. Bombesin, gastrin-releasing peptide, NMB, and a bombesin receptor subtype 3-specific ligand induced mast cell proliferation and chemotaxis in vitro. These observations support a role for multiple BLPs in promoting mast cell responses, suggesting a mechanistic link between BLPs and chronic inflammatory lung diseases.

Animals↗

PubMed2003

Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase L1 variants.

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.

Aldehydes↗

PubMed2003

Blockade of bombesin-like peptide receptors impairs inhibitory avoidance learning in mice.

Several studies reported that peripheral administration of bombesin (BN) and gastrin-releasing peptide (GRP) improved some forms of memory performance. In the present study, we examined the role of endogenous BN-like peptide(s) for the acquisition of inhibitory avoidance learning in mice using BN-like peptide receptor antagonists. An administration of [Leu(13)-(psi-CH(2)NH)-Leu(14)]BN (antagonizes GRP-R>neuromedin B receptor (NMB-R)) impaired the performance of inhibitory avoidance learning in all doses (16, 32, 64 nmol/kg). While the effect was somewhat lesser than [Leu(13)-(psi-CH(2)NH)-Leu(14)]BN, BIM23127 (antagonizes NMB-R>GRP-R) also impaired performance in a moderate dose (32 nmol/kg). These results showed that endogenous BN-like peptides have some role(s) for the modulation of learning and memory, and suggest that NMB/NMB-R pathway may also be involved in the memory acquisition and modulation as well as GRP/GRP-R pathway.

Animals↗

PubMed2003

Bilobalide, a sesquiterpene trilactone from Ginkgo biloba, is an antagonist at recombinant alpha1beta2gamma2L GABA(A) receptors.

The sesquiterpene trilactone bilobalide is one of the active constituents of the 50:1 Ginkgo biloba leaf extract widely used to enhance memory and learning. Bilobalide was found to antagonise the direct action of gamma-aminobutyric acid (GABA) on recombinant alpha(1)beta(2)gamma(2L) GABA(A) receptors. The effect of bilobalide on the direct action of GABA at alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes using two-electrode voltage-clamp method was evaluated and compared with the effects of the classical GABA(A) receptor competitive antagonist bicuculline and noncompetitive antagonist picrotoxinin. Bilobalide (IC(50)=4.6+/-0.5 microM) was almost as potent as bicuculline and pictrotoxinin (IC(50)=2.0+/-0.1 and 2.4+/-0.5 microM, respectively) at alpha(1)beta(2)gamma(2L) GABA(A) receptors against 40 microM GABA (GABA EC(50)). While bilobalide and picrotoxinin were clearly noncompetitive antagonists, the potency of bilobalide decreased at high GABA concentrations suggesting a component of competitive antagonism.

Animals↗

PubMed2003

Expression and function of bradykinin receptors in microglia.

Expression of bradykinin (BK) receptors and their cellular function were investigated in microglia. Microglial cells were isolated from mixed cultures of cerebrocortical cells from postnatal day 3 Wistar rats. Reverse transcription-PCR (RT-PCR) showed that rat primary microglia express mRNAs for the type 2 bradykinin (B(2)) receptor subtype but not the type 1 (B(1)) receptor subtype under our experimental condition. However, the expression of B(1) receptor was greatly up-regulated after the treatment of microglia with BK for 24 hours. The expression of B(2) receptor in microglia was further confirmed by immunocytochemistry. Membrane currents were measured using whole-cell recording under voltage-clamp conditions. In 14% of patched cells (12/85 cells), BK (100-200 nM) induced an outward current at the holding potential of -20 mV, with oscillations in 2 cases. The BK-induced outward current was transient and desensitized rapidly. TEA inhibited the BK-induced outward current in a dose-dependent manner. These results suggest that microglia express B(2) receptors and presumably increase the intracellular Ca(2+) concentration via inositol trisphosphate with the subsequent activation of Ca(2+)-dependent K(+) channels. Our data provide the first evidence that microglia express functional BK receptors and support the idea that microglia play an important role in CNS inflammatory responses.

Animals↗

PubMed2003

Stress-induced impairment of inhibitory avoidance learning in female neuromedin B receptor-deficient mice.

Neuromedin B (NMB) is a mammalian bombesin (BN)-like peptide that exerts its function via the neuromedin B receptor (NMB-R). The NMB/NMB-R system is involved in stress response, and therefore we examined behavioral properties in female mice lacking NMB-R using a restraint-induced stress paradigm. Thirty minutes of restraint in a wire mesh cage constituted a sufficient stress stimulus for mice as evidenced by elevated blood glucose concentrations in stressed wild-type and NMB-R-deficient mice. Using a one-trial passive avoidance test, stressed NMB-R-deficient mice exhibited a marked reduction in memory performance. NMB-R-deficient mice exhibited elevated spontaneous activity in a novel environment compared to non-stressed mutant mice after 30-min stress, and a similar difference was also observed between stressed/non-stressed wild-type mice. An elevated plus maze test showed that the stress stimulus had no effect on anxiety in either wild-type or NMB-R-deficient mice. Furthermore, pain response of wild-type and NMB-R-deficient mice induced by electric foot shock was not affected under either stressed or non-stressed conditions. These results indicate that impaired memory performance in stressed NMB-R-deficient mice is not a consequence of changes in spontaneous activity, anxiety, or pain response, and suggest that the NMB/NMB-R pathway may play a role in regulating the stress response via the neural system that controls learning and memory.

Animals↗

PubMed2003

Bombesin receptor subtype-3 modulates plasma insulin concentration.

Mice lacking a functional bombesin receptor subtype-3 (BRS-3) develop mild obesity. However, the origin of obesity in BRS-3 knockout (KO) mice remains unclear. We used a strain-crossing strategy to investigate the physiological role of the BRS-3 pathway. We crossed female heterozygous BRS-3 KO mice (X-/X) and male KK-Ay mice (Ay/+) to obtain BRS-3 KO/KK-Ay hybrid animals. In X-/Y:Ay/+ mice, plasma insulin concentrations were significantly higher, and on the oral glucose tolerance test, the additional secretion of insulin was impaired compared to other genotypes. Our results indicate that the BRS-3 pathway contributes to the regulation of plasma insulin concentrations.

Animals↗

PubMed2003

Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathways.

Human serotonin 5A (5-HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5-HT5A receptors-expressing cells, accumulation of cAMP by forskolin was inhibited by 5-HT as reported previously. Pertussis toxin-sensitive inhibition of ADP-ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine-sensitive Ca2+ mobilization. On the other hand, 5-HT-induced outward currents were observed using the patch-clamp technique in whole-cell configuration. The 5-HT-induced outward current was observed in 84% of the patched 5-HT5A receptor-expressing cells and was concentration-dependent. The 5-HT-induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5-HT-induced current was significantly attenuated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5-HT-induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5-HT-induced current. IP3 showed a transient increase after application of 5-HT in 5-HT5A receptor-expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP-ribosyl cyclase activity, 5-HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3-sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function.

ADP-ribosyl Cyclase↗

PubMed2002

Identification of an axotomy-induced glycosylated protein, AIGP1, possibly involved in cell death triggered by endoplasmic reticulum-Golgi stress.

We developed a new method, designated N-linked glycosylation signal (NGS) differential display (DD)-PCR, that enables the identification of genes encoding N-linked glycosylated molecules that exhibit varying patterns of expression. Using this innovative technique, we identified an N-linked glycosylated 11-transmembrane domain protein that is upregulated in response to axotomy. Expression levels increased 3 d after axotomy, reached maximal levels at approximately postoperative days 5-7, and then gradually decreased through day 20. The protein was termed axotomy-induced glycosylated/Golgi-complex protein 1 (AIGP1). AIGP1 immunoreactivity is specifically localized in neurons, with subcellular localization within the Golgi, indicating that AIGP1 is a resident Golgi protein. Moreover, AIGP1 gene expression in cultured neurons is specifically induced by the endoplasmic reticulum (ER)-Golgi stressors tunicamycin and brefeldin A. We observed that the frequency of cell death is increased by AIGP1 overexpression and that the corresponding region of the protein implicated in the activity involves the large eighth and ninth transmembrane loops. Our results suggest that AIGP1 gene activation and protein accumulation in the Golgi complex in response to axotomy-induced ER-Golgi stress may contribute to signaling during programmed cell death in damaged neurons.

Animals↗

PubMed2002

Microglia-Müller glia cell interactions control neurotrophic factor production during light-induced retinal degeneration.

Activation of microglia commonly occurs in response to a wide variety of pathological stimuli including trauma, axotomy, ischemia, and degeneration in the CNS. In the retina, prolonged or high-intensity exposure to visible light leads to photoreceptor cell apoptosis. In such a light-reared retina, we found that activated microglia invade the degenerating photoreceptor layer and alter expression of neurotrophic factors such as nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), and glial cell line-derived neurotrophic factor (GDNF). Because these neurotrophic factors modulate secondary trophic factor expression in Müller glial cells, microglia-Müller glia cell interaction may contribute to protection of photoreceptors or increase photoreceptor apoptosis. In the present study, we demonstrate the possibility that such functional glia-glia interactions constitute the key mechanism by which microglia-derived NGF, brain-derived neurotrophic factor (BDNF), and CNTF indirectly influence photoreceptor survival, although the receptors for these neurotrophic factors are absent from photoreceptors, by modulating basic fibroblast growth factor (bFGF) and GDNF production and release from Müller glia. These observations suggest that microglia regulate the microglia-Müller glia-photoreceptor network that serves as a trophic factor-controlling system during retinal degeneration.

Animals↗

PubMed2002

A bitter diterpenoid furanolactone columbin from Calumbae Radix inhibits azoxymethane-induced rat colon carcinogenesis.

The modifying effect of dietary administration of a diterpenoid furanolactone columbin isolated from the crude drug Calumbae Radix (the root of Jateorhiza columba MIERS, Menispermacea) on azoxymethane (AOM)-induced was investigated in male F344 rats. Animals were initiated with AOM (three weekly subcutaneous injections of 15 mg/kg body weight) to induce colonic neoplasms. They were fed the experimental diets mixed with columbin (4, 20, and 100 ppm) for 4 weeks, starting 1 week before the first dosing of AOM and thereafter maintained on the basal diet without columbin. Additional experimental groups included the AOM alone group, the columbin alone group (100 ppm in diet for 4 weeks), and the untreated control group. Dietary feeding of columbin (4, 20, and 100 ppm) during the initiation phase of AOM-induced colon carcinogenesis reduced the incidence and multiplicity of colonic adenocarcinoma and the inhibition by feeding of 20 ppm (incidence: 20%, P=0.0242 and multiplicity: 0.20+/-0.40, P<0.02) and 100 ppm (incidence: 10%, P=0.0029 and multiplicity: 0.10+/-0.30, P<0.002) columbin was significant when compared with the AOM alone group (incidence: 55% and multiplicity: 0.55+/-0.50). Also, columbin administration in diet lowered the number of argyrophilic nucleolar organizer regions protein per nucleus in non-lesional colonic crypts and the blood polyamine content, which are reflected in cell proliferation activity. These results indicate chemopreventive ability of dietary columbin against chemically induced colon tumorigenesis when fed during the initiation phase, providing a scientific basis for chemopreventive ability of columbin against human colon cancer.

Animals↗

PubMed2002

Restraint stress impaired maternal behavior in female mice lacking the neuromedin B receptor (NMB-R) gene.

Neuromedin B (NMB) is a bombesin-like peptide that exerts its function via NMB receptor (NMB-R). The NMB/NMB-R pathway is involved in the regulation of a wide variety of behaviors, such as spontaneous activity, feeding, and anxiety-related behavior. In the current study, we assessed the effects of stress on maternal behavior in female NMB-R-deficient mice. Non-stressed NMB-R-deficient and wild-type mice showed normal maternal behavior. However, immediately after undergoing restraint-induced stress (30 min) both genotypes of mice exhibited severely decreased maternal behaviors. Furthermore, 30 min after stress induction, maternal behavior in wild-type mice recovered to near normal levels whereas that of NMB-R-deficient mice remained significantly lower. These results indicate that NMB-R-deficient mice suffer more severely from stress and suggest that dysfunction in the NMB/NMB-R pathway may constitute one of the risk factors of stress vulnerability.

Animals↗

PubMed2002

Decreased marble burying behavior in female mice lacking neuromedin-B receptor (NMB-R) implies the involvement of NMB/NMB-R in 5-HT neuron function.

Neuromedin B (NMB) is a mammalian bombesin-like peptide distributed widely in the central nervous system. This peptide exerts its function via the NMB receptor (NMB-R). Female NMB-R-deficient mice were used to study the role that NMB/NMB-R may play in 5-HT neuron function since this relationship was suggested in previous in vitro studies. As 5-HT neurons are thought to modulate marble burying behavior, a role for NMB-R in this behavior was assessed. Relative to wild-type mice, NMB-R-deficient mice showed decreased marble burying behavior. However, depletion of 5-HT by treatment with p-chlorophenylalanine (p-CPA) increased burying behavior in NMB-R-deficient mice suggesting that increased levels of 5-HT in the brain cause a decrease in burying behavior in NMB-R-deficient mice. While HPLC analysis showed that 5-HT content in the whole brain does not differ between NMB-R-deficient and wild-type mice, an immunohistochemical analysis of brain sections showed that 5-HT expression in the dorsal raphe (DR) nucleus is elevated in NMB-R-deficient mice. Furthermore, a quantitative RT-PCR analysis revealed that 5-HT(1A)-receptor gene expression is downregulated in NMB-R-deficient mice at the whole brain level. These behavioral and biological results suggest that NMB/NMB-R may modulate 5-HT neuronal activity by affecting DR function.

Animals↗

PubMed2002

Inhibition of glial glutamate transporter GLT-1 augments brain edema after transient focal cerebral ischemia in mice.

Excessively released glutamate is neurotoxic. Glutamate transporters maintain the extracellular level of glutamate by uptake into glia or neurons. We examined the role of GLT-1, a glial glutamate transporter, in brain damage resulting from transient focal ischemia in mice. Heterozygous gene deletion of GLT-1 significantly augmented brain swelling resulting from 1 h of middle cerebral artery occlusion and 24 h reperfusion. In addition, this gene deletion significantly increased brain water contents in ischemic hemisphere at 6 h after reperfusion. Moreover, intraperitoneal injection of dihydrokainate (10 mg/kg), a specific inhibitor of GLT-1, augmented brain swelling. These data suggest that GLT-1 limits brain edema resulting from ischemia.

Animals↗

PubMed2002

Bombesin and its family of peptides: prospects for the treatment of obesity.

Bombesin, its family of bombesin-like peptides, and many other peptides/hormones modulate biological and behavioral functions in animals. Among the wide variety of functions influenced by bombesin/bombesin-like peptides, the most prominent may be their role in feeding-related behavior. Over many years, intensive psychopharmacological studies have addressed the mechanisms by which these peptides induce feeding suppression, and the results suggest the applicability of bombesin/bombesin-like peptides for the treatment of eating disorders and/or obesity in humans. Recent studies using gene-knockout mice also shed new light on the relationship between bombesin/bombesin-like peptides and feeding behavior. In addition, genetic analyses of the possible links between bombesin/bombesin-like peptides/receptors and human obesity have also been undertaken. Here, we briefly review the literature pertaining to the relationship between bombesin/bombesin-like peptides and feeding behavior-with particular attention to human subjects-and discuss the pharmacotherapeutic potential of bombesin/bombesin-like peptides with regard to obesity.

Animals↗