Kentaro Maemura

GABA and GABA Receptors in the Central Nervous System and Other Organs

Gamma-aminobutyrate (GABA) is a major inhibitory neurotransmitter in the adult mammalian brain. GABA is also considered to be a multifunctional molecule that has different situational functions in the central nervous system, the peripheral nervous system, and in some nonneuronal tissues. GABA is synthesized primarily from glutamate by glutamate decarboxylase (GAD), but alternative pathways may be important under certain situations. Two types of GAD appear to have significant physiological roles. GABA functions appear to be triggered by binding of GABA to its ionotropic receptors, GABA(A) and GABA(C), which are ligand-gated chloride channels, and its metabotropic receptor, GABA(B). The physiological, pharmacological, and molecular characteristics of GABA(A) receptors are well documented, and diversity in the pharmacologic properties of the receptor subtypes is important clinically. In addition to its role in neural development, GABA appears to be involved in a wide variety of physiological functions in tissues and organs outside the brain.

Therapeutic effect of intracolonically administered nuclear factor kappa B (p65) antisense oligonucleotide on mouse dextran sulphate sodium (DSS)-induced colitis.

Cytokines such as IL‐1, tumour necrosis factor‐alpha (TNF‐α), IL‐6 and IL‐8 are increased in inflamed colonic mucosa after administration of mouse DSS. Nuclear factor κB (NF‐κB) is a transcription factor which regulates the expression of these cytokine genes. The effect of intracolonically administered NF‐κB (p65) antisense phosphorothioate oligonucleotide was examined in mouse DSS‐induced colitis using drinking water containing 5% DSS. When antisense oligonucleotide was given on day 0, the disease activity index (DAI) representing clinical symptoms improved and the histological score decreased; furthermore, IL‐1, IL‐6, and TNF‐α concentrations in rectal mucosa were lower compared with the control group. Clinical and histological improvement was also observed when antisense oligonucleotide was begun on day 2 but not on day 7. In addition, the distribution of antisense oligonucleotides was investigated by confocal laser microscopy. In colonic mucosa, oligonucleotides were predominantly localized to cells in the lamina propria, but also in the epithelium. Western blot analysis using homogenized rectal mucosa showed the decreased expression of NF‐κB p65 in the antisense oligonucleotide‐treated group, although it was increased in the colitis group. These results suggest that intracolonic administration of NF‐κB antisense oligonucleotide may be effective in ulcerative colitis.

Inhibition of dextran sulphate sodium (DSS)-induced colitis in mice by intracolonically administered antibodies against adhesion molecules (endothelial leucocyte adhesion molecule-1 (ELAM-1) or intercellular adhesion molecule-1 (ICAM-1)).

We examined the effect of intracolonic administration of anti‐adhesion molecule antibodies on DSS‐induced colitis in mice. Immunohistochemical staining in mice with colitis showed increased expression of ELAM‐1 and ICAM‐1 on endothelial cells of vessels in the lamina propria and submucosa at sites of inflamed lesions. Intracolonic administration of anti‐ELAM‐1 or anti‐ICAM‐1 antibody decreased bloody stools, anaemia, and histologically evident damage, as well as myeloperoxidase activity and IL‐1β content. We concluded that adhesion molecule expression is important in the development of DSS‐induced colitis in mice and that intracolonic administration of anti‐adhesion molecule antibodies, especially anti‐ELAM‐1 antibody, effectively inhibits the colonic inflammation. Intracolonic administration of anti‐adhesion molecule antibodies may show therapeutic promise in ulcerative colitis.

Expression of the EP4 Prostaglandin E2 Receptor Subtype with Rat Dextran Sodium Sulphate Colitis: Colitis Suppression by a Selective Agonist, ONO-AE1-329

Expression of the EP4 receptor, a prostaglandin (PG)E2 receptor subtype, as well as disease suppression by the administration of a selective EP4 agonist (ONO‐AE1‐329) was investigated in the colorectal mucosa of rats with dextran sodium sulphate (DSS)‐induced colitis. Rats were given drinking water containing 3% DSS for 2 weeks. Expression of EP4 receptor mRNA was barely detectable under normal conditions according to reverse transcription‐polymerase chain reaction (RT‐PCR). By 1 week after the initial administration of DSS, the receptor mRNA was strongly expressed. After ONO‐AE1‐329 was administered intracolonically to rats with DSS colitis for 7 consecutive days, erosion and ulceration decreased. Peripheral white blood cell (WBC) counts became less elevated. Interleukin (IL)‐1β and growth‐regulated gene product/cytokine‐induced neutrophil chemoattractant (GRO/CINC‐1) concentrations in colorectal mucosa were lower than in colitis control group (IL‐1β: 12.8 ± 4.6 and 30.8 ± 6.2 µg/mg protein, P < 0.05; GRO/CINC‐1: 15.5 ± 3.0 and 39.2 ± 5.4 µg/mg protein, P < 0.05), and the expression of the corresponding cytokine mRNA was strongly suppressed. IL‐10 concentration was higher than in control group (14.5 ± 1.7 and 7.9 ± 1.2 µg/mg, P < 0.05), and the mRNA was more strongly expressed. These results suggest that the EP4 receptor is important in colonic inflammation, and that PGE2 suppresses DSS colitis at least partly via the EP4 receptor and the above cytokine changes. Intracolonic administration of selective EP4 agonist might have therapeutic applicability in inflammatory bowel disease such as ulcerative colitis.

A local GABAergic system within rat trigeminal ganglion cells

We investigated the GABAergic system within the Sprague–Dawley rat (2–3‐weeks old) trigeminal ganglion (TG). Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis revealed expression of glutamate decarboxylase (GAD) 65 and GAD67 mRNAs and mRNAs encoding GABAA receptor subunits α1–6, β1–3, γ1–3, and δ. In situ hybridization revealed that GAD65 and GAD67 mRNAs were expressed in neuronal cell bodies but not satellite cells. Immunohistochemical analysis showed that only GAD65 was expressed in all neuronal cell bodies, and approximately 70% of all neurons exhibited GABA immunoreactivity. Satellite cells were strongly immunopositive for GABA. GABAA receptor α1, α5, β2/3 and γ1/2/3 subunit immunoreactivities were observed in the majority of neurons, but no immunoreactivity for α2 was observed. Two types of cells were identified in TG based on cell size and morphology, type A and B. The percentage of cells expressing α3, α4, α6, and δ subunits appeared to be dependent on cell size, as δ and α6 expression were only observed in small (B‐type) neurons. In whole‐cell patch clamp experiments, GABA application induced inward Cl– currents in all neurons examined. The EC50 for GABA varied from 5.3 to 240 µm, and the Hill Coefficient (nH) varied between 0.98 and 2.6 at −60 mV. We found that GABA was released from TG cells by increasing extracellular K+ concentration to 100 mm. We speculate that GABA acts as a nonsynaptically released diffusible neurotransmitter, which may modulate somatic inhibition of neurons within the TG.

Estimation of Mucosal Inflammatory Mediators in Rat DSS-Induced Colitis

In order to investigate the mucosal injury mechanism in UC, we made dextran sulfate sodium (DSS)-induced colitis in rat and examined pathological findings, MPO activity, PGE2 level, and local mRNA expression and secretion of IL-1β, TNF-α, GRO/CINC-1 and IL-10 in DSS colitis mucosa. Moreover, we estimated the correlation between the severity of mucosal damage and changes of these local inflammatory mediators’ values. Neutrophil infiltration was marked and MPO activity was locally increased in proportion to the severity of mucosal damage. The mRNA expression and secretion of IL-1β, GRO/CINC-1 and IL-10 were increased. Especially, the secretions of IL-1β and GRO/CINC-1 were increased in proportion to the severity of mucosal damage. However, those of TNF-α were not increased in the colitis mucosa. An abnormal macrophage function and the presence of macrophage subtypes producing different cytokines would be predicted from our TNF-α data. The lesion was less severe in the colonic mucosa with higher levels of endogenous PGE2, while it was more severe in the colonic mucosa with lower levels of endogenous PGE2, implicating this compound as an inhibitory factor against the development of inflammation in the affected mucosa. Our results suggest that PGE2 might have therapeutic applicability to UC.

A simple method of detecting K-ras point mutations in stool samples for colorectal cancer screening using one-step polymerase chain reaction/restriction fragment length polymorphism analysis

BACKGROUND We examined a technique for detecting point mutations of K-ras codon 12 in stool samples using one-step polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis, in order to determine whether it could be used to screen for colorectal cancer. METHODS DNA was extracted from 200-mg stool specimens of 5 healthy controls and 31 colorectal cancer patients. A 107-base-pair fragment of exon 1 of K-ras was amplified by PCR using mismatched primers. PCR products were digested with Bst NI and analyzed by gel electrophoresis followed by silver staining. Specificity of one-step PCR/RFLP was examined by using synthetic oligonucleotides. The detection limit of K-ras codon 12 mutations was determined by using SW480 and HT29 cells. RESULTS The K-ras gene was successfully amplified from all healthy controls and colorectal cancer patients studied. Mutations of K-ras codon 12 were not detected in any of the healthy controls, but were identified in 13 (41.9%) of the 31 patients with colorectal cancer. Mutations were detectable in all six synthetic mutant DNAs, while none were detected among the wild type. The detection limit of this method was > or = 0.1%. CONCLUSIONS PCR/RFLP analysis could be used in mass screening for colorectal cancer, because it is highly specific, has a low detection limit, and is simpler than conventional methods for detecting genetic abnormalities.

Expression of GABAA and GABAB receptors in rat growth plate chondrocytes : Activation of the GABA receptors promotes proliferation of mouse chondrogenic ATDC5 cells

Our previous study showed the local production of γ-aminobutyrate (GABA) in hypertrophic-zone chondrocytes of the rat tibial growth plate, an important long bone growth site. The aim of this study was to identify the presence of GABA receptors in growth plate chondrocytes by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Chondrocytes expressed both GABAA and GABAB receptor subunit mRNAs as well as the corresponding proteins necessary for the assembly of functional receptors. The GABAA receptor subunits detected included α1–α4, α6, β1–β3, and δ, and both R1 and R2 subunits of GABAB receptors were detected. All receptor subunits were expressed in chondrocytes of the proliferative and hypertrophic zones. These results suggest that GABA is an autocrine/paracrine factor that regulates the physiological state of the growth plate. Subsequent studies with the mouse chondrogenic cell line ATDC5 showed the presence of mRNAs and the corresponding proteins for GABAA receptor α1, β2, and β3 subunits and GABAB receptor R1 and R2 subunits. GABA, muscimol (a GABAA receptor agonist), and baclofen (a GABAB receptor agonist) increased 5-bromodeoxyuridine (BrdU) incorporation into ATDC5 cells. The effect of muscimol was blocked by bicuculline (a GABAA receptor antagonist), and the effect of baclofen was blocked by CGP 35348 (a GABAB receptor antagonist). These results suggest that GABA contributes to the ATDC5 cell proliferation via GABAA and GABAB receptors and these mechanisms may be involved in cartilaginous cell growth.

Proliferative effects of γ-aminobutyric acid on the gastric cancer cell line are associated with extracellular signal-regulated kinase 1/2 activation

Background and Aim:  γ‐Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the adult mammalian brain. However, GABA is found not only in peripheral neuronal tissue, but also in many peripheral non‐neuronal tissues, and is thought to have important physiological functions in addition to neurotransmission. We previously reported that GABA participates in chondrocyte proliferation. In the present study, we investigated the effects of GABA on the proliferation of a gastric cancer cell line, KATO III.

Immunocytochemical demonstration of glucose transporters in epiphyseal growth plate chondrocytes of young rats in correlation with autoradiographic distribution of 2-deoxyglucose in chondrocytes of mice.

The epiphyseal growth plate, where chondrocytes proliferate and differentiate, is the major site for longitudinal bone growth, matrix synthesis and mineralization. Glucose is an important energy source for the metabolism and growth of chondrocytes. The family of facilitative glucose transporters (GLUTs) mediates glucose transport across the plasma membrane in mammalian cells. We used immunocytochemical methods with anti-GLUT antibodies to investigate the localization of GLUTs in chondrocytes of the epiphyseal growth plate in 3 age groups of rats (3, 7, and 28 days after birth). Intense immunoreactivity of GLUT isoforms 1-5 was detected in chondrocytes of 3-day and 7-day old rats, and all GLUTs were localized in the maturation zone of the hypertrophic zone. On postnatal day 28, chondrocytes in the maturation zone showed intense GLUT1, 4 and 5 immunoreactivity, and weak GLUT2 and 3 immunoreactivity. In addition to chondrocytes in the maturation zone, those in the degenerative zone and in the zone of provisional calcification showed strong GLUT4 and 5 immunoreactivity. Autoradiography of bone sections from 4-week old mice injected with 14C-2-deoxyglucose showed high silver grain density within matrix tissue in the reserve and proliferative zones but not around chondrocytes. However, in the hypertrophic zone, silver grain density was high in matrix and chondrocytes. These data indicate that chondrocytes in the hypertrophic zones use glucose as energy source. High levels of GLUT4 expression imply that glucose use in chondrocytes is regulated by insulin. Expression of GLUT5 in chondrocytes suggests that fructose is also used as an energy source.