Hana Hayasaki

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.

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.

Inflammation as well as angiogenesis may participate in the pathophysiology of brain radiation necrosis

Radiation necrosis (RN) after intensive radiation therapy is a serious problem. Using human RN specimens, we recently proved that leaky angiogenesis is a major cause of brain edema in RN. In the present study, we investigated the same specimens to speculate on inflammations effect on the pathophysiology of RN. Surgical specimens of symptomatic RN in the brain were retrospectively reviewed by histological and immunohistochemical analyses using hematoxylin and eosin (H&E) staining as well as immunohistochemical staining for VEGF, HIF-1α, CXCL12, CXCR4, GFAP, CD68, hGLUT5, CD45, IL-1α, IL-6 TNF-α and NF-kB. H&E staining demonstrated marked angiogenesis and cell infiltration in the perinecrotic area. The most prominent vasculature was identified as thin-walled leaky angiogenesis, i.e. telangiectasis surrounded by prominent interstitial edema. Two major cell phenotypes infiltrated the perinecrotic area: GFAP-positive reactive astrocytes and CD68/hGLUT5-positive cells (mainly microglias). Immunohistochemistry revealed that CD68/hGLUT5-positive cells and GFAP-positive cells expressed HIF-1α and VEGF, respectively. GFAP-positive cells expressed chemokine CXCL12, and CD68/hGLUT5-positive cells expressed receptor CXCR4. The CD68/hGLUT5-positive cells expressed pro-inflammatory cytokines IL-1α, IL-6 and TNF-α in the perinecrotic area. VEGF caused leaky angiogenesis followed by perilesional edema in RN. GFAP-positive cells expressing CXCL12 might attract CXCR4-expressing CD68/hGLUT5-positive cells into the perinecrotic area. These accumulated CD68/hGLUT5-positive cells expressing pro-inflammatory cytokines seemed to aggravate the RN edema. Both angiogenesis and inflammation might be caused by the regulation of HIF-1α, which is well known as a transactivator of VEGF and of the CXCL12/CXCR4 chemokine axis.

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.

Heterogenous GABAB receptor-mediated pathways are involved in the local GABAergic system of the rat trigeminal ganglion: Possible involvement of KCTD proteins

It is well known that Gamma-aminobutyric acid (GABA) plays an important role in signal transduction in the central nervous system. However, the function of GABA in the peripheral nervous system, including sensory ganglions, is still unclear. In this study we have characterized the expression, cellular distribution, and function of GABA(B) receptor subunits, and the recently discovered GABA(B) auxiliary subunits, K(+) channel tetramerization domain-containing (KCTD) proteins, in rat trigeminal ganglion (TG) neuronal cells, which are devoid of synapses. We found heterogeneous expression of both GABA(B1) and GABA(B2) subunits, and a near-plasma membrane localization of KCTD12. In addition, we found that GABA(B2) subunits correlated with KCTD16. Whole-cell current-clamp recordings showed that responses to the GABA(B) receptor agonist, baclofen, were variable and both increases and decreases in excitability were observed. This correlated with observed differences in voltage-dependent K(+) current responses to baclofen in voltage-clamped TG neuronal cells. The functional diversity of the GABA(B)ergic regulation on the excitability of the TG neuronal cell bodies could be due to the heterogenous expression of KCTD proteins, and subsequent regulation of plasma membrane K(+) channels. Taken together with our previous demonstration of a local GABA(A) receptor-mediated system in rat TG, we provide an updated GABAergic model in the rat TG that incorporates both GABA(A)- and GABA(B)-receptor systems.

Marked regression of liver metastasis by combined therapy of ultrasound-mediated NFkB-decoy transfer and transportal injection of paclitaxel, in mouse

Nuclear factor‐kappaB (NFkB) plays a pivotal role in cancer progression. In this study, we developed a decoy cis‐element oligo‐deoxyribonucleic acid against NFkB‐binding site (NFkB‐decoy), which effectively inhibits NFkB activity, and tested the effect of combined therapy comprising local transfection of NFkB‐decoy into the liver and transportal injection of paclitaxel on cancer growth and metastasis using an orthotopic murine model of colon cancer liver metastasis. For NFkB‐decoy transfection, we employed a novel approach using ultrasound exposure with an echocardiographic contrast agent, Optison. We examined the influence of NFkB‐decoy transfer on susceptibility to paclitaxel in cancer cells and the mechanism involved using several in vitro analysis systems. We then studied the in vivo effect of combined NFkB‐decoy transfer and paclitaxel in preventing cancer progression using a murine model of liver metastasis created by splenic injection of a human colon cancer cell line, HT29. In vitro experiments, including MTT‐assay, fluorescence‐activated cell sorter and cDNA array analysis, revealed that NFkB‐decoy transfer significantly increased the susceptibility of cancer cells to paclitaxel, and that decreased expression of anti‐apoptotic genes along with increased expression of genes relevant to the apoptosis‐promotor may be involved. In vivo experiments showed that local transfection of NFkB‐decoy into the liver followed by portal injection of paclitaxel effectively induced cancer cell apoptosis in the liver metastasis, and significantly prolonged animal survival compared to controls, without notable side effects. In conclusion, a combination of local NFkB‐decoy transfer into the liver and transportal injection of paclitaxel may be a safe and effective new therapy for liver metastasis.

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.