Kiyoto Kanbara

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.

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.

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.

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.

γ‐Amino‐butyric acid immunoreactivity in intramucosal colonic tumors

Background and Aim:  The level of γ‐amino‐butyric acid (GABA) is reported to be increased in colon cancer. Moreover, data suggests that GABA plays a role in the proliferation or maturation of some types of cells. We examined the expression of GABA in intramucosal colonic tumors to clarify the relation between GABA and the degree of atypia.

Implication for melatonin and its receptor in the spinal deformities of hereditary Lordoscoliotic Rabbits.

Study Design. The association between melatonin system and the spontaneous development of the spinal deformities in the Hereditary Lordoscoliotic Rabbit, the natural animal model for idiopathic scoliosis, was studied. Objectives. To examine the implication for melatonin and its receptor in the spinal deformities of the natural animal model, the Hereditary Lordoscoliotic Rabbit. Summary of Background Data. We previously reported radiologic and histologic studies investigating the etiology of spinal deformities in a breed of Japanese White Rabbit, the Hereditary Lordoscoliotic Rabbit. These animals develop thoracic lordoscoliosis during growth and as such can be used as a model for human idiopathic scoliosis. Although previous studies in chickens have established that pinealectomy produces scoliosis, the cause of the condition is yet to be fully elucidated. Methods. Serum melatonin levels in Hereditary Lordoscoliotic Rabbits were measured by radioimmunoassay and compared with those of Japanese White Rabbits (controls). The expression of melatonin receptor in the rabbit was detected by homology cloning to access the number of the melatonin receptor mRNA in the rabbit spinal cord by quantitative reverse-transcribed polymerase chain reaction. Results. Serum melatonin levels in Hereditary Lordoscoliotic Rabbits were significant higher than those of controls in each period until 20 weeks. We detected the expression of melatonin receptor mRNA in rabbit spinal cord. However, no significant quantitative differences were found in the level of expression of melatonin mRNA in the spinal cord between Hereditary Lordoscoliotic Rabbits and controls. Conclusions. In relation to the present study, we suggest that causes of spinal deformities in the Hereditary Lordoscoliotic Rabbit may be the result of the contribution of melatonin receptors as well as that of altered serum melatonin levels in the Hereditary Lordoscoliotic Rabbit. Further studies will be required to investigate the expression of melatonin receptor in other tissues of the Hereditary Lordoscoliotic Rabbit as well as to delineate the role of melatonin in the pathogenesis of idiopathic scoliosis.

Altered Expression of Sodium Channel Distribution in the Dorsal Root Ganglion after Gradual Elongation of Rat Sciatic Nerves

To elucidate the pathophysiological mechanisms underlying chronic nerve‐stretch injury, we gradually lengthened rat femurs by 15 mm at the rate of 0.5 mm/day (group L, n = 13). The control groups comprised sham‐operated (group S, n = 10) and naive (group N, n = 8) rats. Immediately after the lengthening, we performed a conduction study on their sciatic nerves and harvested samples. Electrophysiological and histological analyses showed mild conduction slowing and axonal degeneration of unmyelinated fibers in group L rats. Altered mRNA expression of the voltage‐gated sodium channels in the dorsal root ganglion was also observed. Tetrodotoxin‐resistant (TTX‐R) sodium‐channel Nav1.8 mRNA expression was significantly decreased and TTX‐R sodium‐channel Nav1.9 mRNA expression showed a tendency to decrease when compared with the mRNA expressions in the control groups. However, tetrodotoxin‐sensitive (TTX‐S) sodium‐channel Nav1.3 mRNA expression remained unaltered. The immunohistochemical alteration of Nav1.8 protein expression was parallel to the results of the mRNA expression. Previous studies involving neuropathic states have suggested that pain/paresthesia is modulated by a subset of sodium channels, including downregulation and/or upregulation of TTX‐R and TTX‐S sodium channels, respectively. Our findings indicate that Nav1.8 downregulation may be one of the pathophysiological mechanisms involved in limb lengthening‐induced neuropathy.

Microautoradiographic studies of glucose uptake in skeletal muscle fibers at rest.

In the present study, differences in glucose uptake by muscle fibers in deep, middle, and superficial regions of the gastrocnemius were studied at rest by 2-deoxyglucose (2-DG) microautoradiography. Expression of the glucose transporter 4 (GLUT-4) protein, an isoform of the glucose transporter family, was analyzed as well. These data were compared with the activity of succinate dehydrogenase, a marker of oxidative metabolism, a-glycerophosphate dehydrogenase, an indicator of the glycolytic capacity, and myofibrillar ATPase. In the deep regions of the muscle, most fibers (86.9%) showed high 2-DG uptake and large amounts of GLUT-4 protein, whereas in the superficial regions, all fibers showed low 2-DG uptake and GLUT-4 expression. In the middle regions, fibers dominated (80.4%) showed low 2-DG uptake and small amounts of GLUT-4 protein. Analysis of metabolic properties revealed that most fibers in the deep region were oxidative and showed the highest 2-DG uptake; in the superficial region, the fibers were anaerobic and showed the lowest 2-DG uptake. In the middle region, most fibers were of the anaerobic and fast twitch type. It is concluded that 2-DG uptake correlates with GLUT-4 expression in the plasma membrane of type I and IIx fibers rather than with oxidative enzyme activity.

Epidermal growth factor binding sites in the mouse exocrine and endocrine pancreas shown by in vivo quantitative microautoradiography and confocal laser scanning microscopy

Microautoradiography at 3, 6 and 15 min after intravenous injection of 125I-EGF was used to investigate the distribution of epidermal growth factor (EGF) binding sites in the pancreas of normal male mice. The autoradiographs were observed by confocal laser microscopy, which allows the quantification of silver grains. The results demonstrated that both endocrine and exocrine pancreatic cells exhibited substantial specific binding of 125I-EGF. The highest level of EGF binding was found in the duct cells of the exocrine pancreas followed by the acinar cells. The cells of the islets of Langerhans also showed substantial specific binding of 125I-EGF though the binding level was lower than that of the exocrine pancreas. In the control experiments, mice were injected with 125I-EGF and various amounts of unlabeled EGF.

GABA B receptor regulates proliferation in the high-grade chondrosarcoma cell line OUMS-27 via apoptotic pathways

BackgroundHigh-grade chondrosarcoma, which has a high incidence of local recurrence and pulmonary metastasis despite surgical resection, is associated with poor prognosis. Therefore, new and effective adjuvant therapies are urgently required for this disease. Gamma-aminobutyric acid (GABA), which acts as a neurotrophic factor during nervous system development, is related to the proliferation and migration of certain cancer cells. The GABAergic system, which is composed of GABA, the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), and GABA receptors, has an important function in nerve growth and development of neural crest. Therefore, the GABAergic system may play important functional roles in the proliferation of chondrosarcoma cells, which are derived from neural crest cells. We examined the anti-tumor effects of the GABAergic system on a chondrosarcoma cell line.MethodsWe evaluated the underlying mechanisms of the anti-tumor effects of the GABAergic system, such as the involvement of different signaling pathways, apoptosis, and cell cycle arrest, in the high-grade chondrosarcoma cell line OUMS-27. In addition, we performed whole-cell patch-clamp recordings for Ca2+ currents and evaluated the changes in intracellular Ca2+ concentration via Ca2+ channels, which are related to the GABAB receptor in high-grade chondrosarcoma cells.ResultsThe GABAB receptor antagonist CGP had anti-tumor effects on high-grade chondrosarcoma cells in a dose-dependent manner. The activities of caspase 3 and caspase 9 were significantly elevated in CGP-treated cells compared to in untreated cells. The activity of caspase 8 did not differ significantly between untreated cells and CGP-treated cells. However, caspase 8 tended to be up-regulated in CGP-treated cells. The GABAB receptor antagonist exhibited anti-tumor effects at the G1/S cell cycle checkpoint and induced apoptosis via dual inhibition of the PI3/Akt/mTOR and MAPK signaling pathways. Furthermore, the changes in intracellular Ca2+ via GABAB receptor-related Ca2+ channels inhibited the proliferation of high-grade chondrosarcoma cells by inducing and modulating apoptotic pathways.ConclusionsThe GABAB receptor antagonist may improve the prognosis of high-grade chondrosarcoma by exerting anti-tumor effects via different signaling pathways, apoptosis, cell cycle arrest, and Ca2+ channels in high-grade chondrosarcoma cells.