No abstract

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 GABA(A) receptor subunits alpha1-6, beta1-3, gamma1-3, and delta. 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. GABA(A) receptor alpha1, alpha5, beta2/3 and gamma1/2/3 subunit immunoreactivities were observed in the majority of neurons, but no immunoreactivity for alpha2 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 alpha3, alpha4, alpha6, and delta subunits appeared to be dependent on cell size, as delta and alpha6 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 microm, 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.

Our previous study showed the local production of gamma-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 GABA(A) and GABA(B) receptor subunit mRNAs as well as the corresponding proteins necessary for the assembly of functional receptors. The GABA(A) receptor subunits detected included alpha1-alpha4, alpha6, beta1-beta3, and delta, and both R1 and R2 subunits of GABA(B) 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 GABA(A) receptor alpha1, beta2, and beta3 subunits and GABA(B) receptor R1 and R2 subunits. GABA, muscimol (a GABA(A) receptor agonist), and baclofen (a GABA(B) receptor agonist) increased 5-bromodeoxyuridine (BrdU) incorporation into ATDC5 cells. The effect of muscimol was blocked by bicuculline (a GABA(A) receptor antagonist), and the effect of baclofen was blocked by CGP 35348 (a GABA(B) receptor antagonist). These results suggest that GABA contributes to the ATDC5 cell proliferation via GABA(A) and GABA(B) receptors and these mechanisms may be involved in cartilaginous cell growth.

GABA may be useful as a tumor marker in combination with other tumor markers such as CEA and CA19-9.

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.