Takumi Tamayama

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.

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.

Histologic distribution of insulin and glucagon receptors

Insulin and glucagon are the hormonal polypeptides secreted by the B and A cells of the endocrine pancreas, respectively. Their major physiologic effects are regulation of carbohydrate metabolism, but they have opposite effects. Insulin and glucagon have various physiologic roles, in addition to the regulation of carbohydrate metabolism. The physiologic effects of insulin and glucagon on the cell are initiated by the binding of each hormone to receptors on the target cells. Morphologic studies may be useful for relating biochemical, physiologic, and pharmacologic information on the receptors to an anatomic background. Receptor radioautography techniques using radioligands to label specific insulin and glucagon receptors have been successfully applied to many tissues and organs. In this review, current knowledge of the histologic distribution of insulin and glucagon receptors is presented with a brief description of receptor radioautography techniques.

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.

Autoradiographic distribution of radioactivity from 14C-GABA in the mouse

We investigated the distribution of radioactivity from 14C‐labeled γ‐aminobutyric acid (GABA) in the mouse by in vivo autoradiography to clarify the tissues that show GABA uptake and/or GABA binding. Male mice were injected intravenously with 14C‐GABA in both the absence and presence of an excess of unlabeled GABA, baclofen and isoguvacine. Whole‐body autoradiography of 3H‐baclofen, a GABAB receptor agonist was also performed. At short intervals after 14C‐GABA injection ( 3 and 6 minutes), very high radioactivity was detected in the kidney cortex, liver, pineal gland, hypophysis, median eminence of the hypothalamus, and cervical ganglion. The hyaline cartilage and glandular part of the stomach showed moderate radioactivity. In the presence of an excess amount of unlabeled GABA, radioactivity in most of tissues decreased significantly, but no significant difference in radioactivity was observed in the presence of baclofen and isoguvacine, agonists of GABAA and GABAB receptors, respectively. Autoradiography of 3H‐baclofen showed that the kidney had high level of radioactivity, whereas the activity in other tissues and organs was similar or lower than in the blood except for the content of the urinary bladder and the pancreas at 15 minutes after injection. These data indicate that radioactivity from incorporated 14C‐GABA into a variety of cells is much higher than that from bound 14C‐GABA to the receptor sites. Our results suggest that GABA can be quickly localized in many organs of the mouse body after 3 minutes following injection, and GABA may serve multiple functions in those organs. Microsc. Res. Tech. 41:116–126, 2000.

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.

Alopecia of IFN-γ knockout mouse as a model for disturbance of the hair cycle : a unique arrest of the hair cycle at the anagen phase accompanied by mitosis

Interferon-gamma(-/-) (IFN-gamma(-/-)) and IFN-gamma(+/+) C57BL/6 mice (3 weeks of age) completed the production of morphogenesis-derived hair. Around 6 weeks of age, however, most of the IFN-gamma(-/-) but none of the IFN-gamma(+/+) mice began to lose hairs in the dorsal and occipital areas in the absence of inflammatory reactions, and the alopecia was sustained for at least several 10-week periods of observation. A single subcutaneous injection of IFN-gamma to IFN-gamma(-/-) mice at 3, but not 4, 5, or 8 weeks of age could protect all the mice from alopecia, revealing that the lack of IFN-gamma around 3 weeks of age is directly responsible for the alopecia. Histologic features showed that the hair follicles of the IFN-gamma(+/+) mice passed through the anagen (4-5 weeks of age) and catagen/telogen ( approximately 6 weeks of age) phases, whereas those of IFN-gamma(-/-) mice (5 weeks of age or older) stayed in the anagen phase. TUNEL and bromodeoxyuridine experiments suggested that an arrest with unlimited DNA synthesis of the hair cycle in the anagen phase by the lack of IFN-gamma-dependent apoptosis in the midfollicle region and diffuse shedding of previously formed hair induced alopecia in IFN-gamma(-/-) mice.

Histocytochemical study of cell death in mice cortical area after callosotomy

DEMONSTRATION OF A NEW DOPAMINE-SYNTHESIZING CELL GROUP IN THE HUMAN 220 BASAL FOREBRAIN: USING DUAL LABELING IMMUNOHISTOCHEMICAL TECHNIQUE OF TYROSINE HYDROXYLASE AND AROMATIC L-AMINO ACID DECARBOXYLASE KEIKO IKEMOTO’, RYOHACHI ARAI 1, KUNIO KITAHAMAZ, ANNE JOUVET3, AKIYOSHI NISHIMURAJ, KATSUJI NISHI4, TOSHIHIRO MAEDA5, IKUKO NAGATSU’ ‘Dept. of Anatomy, Fujita Heath Univ., School of Medicine, Toyoake, 470-l 101, 21NSERM U480, Univ. Claude Bernard, Lyon, France, 3Lab. d’ Anatomie Pathologique, Hapital Pierre Wertheimer, Lyon, France, Depts. of 4Legal Medicine and 5Anatomy, Shiga Univ. of Medical Science, Otsu, 520-2121 A recent study demonstrated a new dopamine-containing neuron group in the monkey (macacafuscatu) basal forebrain using a dopamine antibody (Ikemoto et al. Neurosci. Lett. 220 (1996) 69-71). We examined whether this neuron group exists in the human homologous area. As dopamine is degraded rapidly in the human post-mortem tissue, immunohistochemical dual labeling of tyrosine hydroxylase (TH: the first step dopamine-synthesizing enzyme) and aromatic L-aminoacid decarboxylase (AADC: the second step dopamine-synthesizing enzyme) was applied. The observation was performed using confocal laser-scanning microscopy. Neurons dually labeled for TH and AADC were found in the anterior olfactory nucleus, olfactory tubercle and the ventral margin of the rostra1 nucleus accumbens. The examination in the basal forebrain of the monkey also gave substantially the same results. These neurons appear to constitute an independent dopaminergic cell group in the primate basal forebrain.