Elsevier

Neuropharmacology

Volume 60, Issues 2–3, February–March 2011, Pages 513-519
Neuropharmacology

An intrinsic GABAergic system in human lymphocytes

https://doi.org/10.1016/j.neuropharm.2010.11.007Get rights and content

Abstract

γ-Amino butyric acid (GABA) is an ubiquitous neurotransmitter in the central nervous system and it is also present in non-neuronal cells. In this study we investigated the presence of neuronal components of the GABAergic system in lymphocytes and its functional significance.

By using RT-PCR we detected mRNA expression of different components of the GABAergic system in resting and mitogen-activated lymphocytes: i) GAD67, an isoform of the enzyme that synthetizes GABA; ii) VIAAT, the vesicular protein involved in GABA storage; iii) GABA transporters (GAT-1 and GAT-2); iv) GABA-T, the enzyme that catabolizes GABA; and v) subunits that conform ionotropic GABA receptors. The presence of VIAAT protein in resting and activated cells was confirmed by immunocytochemistry. The functionality of GABA transporters was evaluated by measuring the uptake of radioactive GABA. The results show that [3H]GABA uptake is 5-fold higher in activated than in resting lymphocytes. To determine if GABA subunits assemble into functional channels, we performed whole-cell recordings in activated lymphocytes. GABA and muscimol, a specific agonist of ionotropic GABA receptors, elicit macroscopic currents in about 10–15% of the cells. Finally, by using [3H]thymidine incorporation assays, we determined that the presence of agonists of GABA receptor during activation inhibits lymphocyte proliferation.

Our results reveal that lymphocytes have a functional GABAergic system, similar to the neuronal one, which may operate as a modulator of T-cell activation. Pharmacological modulation of this system may provide new approaches for regulation of T-cell response.

Research highlights

►Human Lymphocytes have a functional GABAergic system, similar to the neuronal one. ► Lymphocytes express the necessary components to synthesize, store and degradate GABA. ► Lymphocytes also express functional GABA transporters and ionotropic receptor subunits. ► GABA and Muscimol inhibit lymphocyte proliferation.

Introduction

Several reports concerning the presence of typical neurotransmitter systems in non-neuronal cells have displaced the concept that these molecules are exclusively expressed in nervous system. For example, it has been described that lymphocytes possess a complete cholinergic system as well as receptors for serotonin, histamine and dopamine (Akdis and Simons, 2006, De Rosa et al., 2005, Kawashima and Fujii, 2003, Kirillova et al., 2008, Yin et al., 2006). However, the physiological relevance of these extraneuronal systems is not completely understood. It has been proposed that they control cell proliferation, differentiation and cell–cell contact (De Rosa et al., 2009, Gladkevich et al., 2006, Wessler and Kirkpatrick, 2008). GABA, the principal inhibitory neurotransmitter in central nervous system, has been also detected in immune cells like monocytes and macrophages (Stuckey et al., 2005). It has been also postulated that GABA is involved in the progression of autoimmune diseases, such as Type-1 diabetes and multiple sclerosis (Bhat et al., 2010, Bjurstom et al., 2008, Tian et al., 2004).

The concentration of GABA in the brain is controlled by three main events: the synthesis by the enzyme glutamate decarboxilase (GAD), the catabolism by the enzyme GABA transaminase (GABA-T) and the uptake of released GABA by GABA transporters (GATs). At least two isoforms of GAD, GAD65 and GAD67, exist in mammals (Metzeler et al., 2004). Once synthesized, the vesicular inhibitory amino acid transporter (VIAAT) mediates the uptake of GABA into secretory vesicles. After being released, the synaptic action of GABA ends by the recapture of the neurotransmitter by specific high-affinity transporters (GATs). Four different GAT subtypes (GAT-1–3 and BGT-1) have been identified in humans. GAT-1 and GAT-3 are abundantly expressed in CNS and absent from the periphery, whereas BGT-1 and GAT-2 are expressed in human kidney, brain, lung and testis (Christiansen et al., 2007).

Once released, GABA exerts its effects through GABAA, GABAB and GABAC receptors. Whereas GABAA and GABAC receptors are ligand-gated ion channels permeable to chloride ions, GABAB receptors are associated to G-proteins. We here focused on ionotropic GABA receptors. Sixteen subunits (α1-6, β1-3, γ1-3, δ, ɛ, π and θ) can differently combine to form heteropentameric GABAA receptors. Most of the GABAA receptors contain at least α and β subunits. The rho subunits (ρ1–3) combine as homo or heteropentamers to conform GABAC receptors (Birnir and Korpi, 2007, Ong and Kerr, 2000).

Our study shows that lymphocytes contain the necessary components to constitute an independent GABAergic system. Furthermore, we describe the effects of GABA and muscimol on proliferation of mitogen-stimulated lymphocytes.

Section snippets

Isolation and culture of human peripheral lymphocytes

The experiments on human subjects were conducted in accordance with the Declaration of Helsinski. All procedures were carried out with the adequate understanding and written consent of the subjects. Lymphocytes were obtained from healthy volunteers (22–40 years old) essentially as described before (De Rosa et al., 2005). Blood was withdrawn from the antecubital vein using EDTA as anticoagulant. The diluted blood was loaded on 3 ml Ficoll separating solution (Amersham Biosciences, AB, Sweden)

Expression of GAD, VIAAT and GABA-T in human lymphocytes

Using RT-PCR we first explored if GAD isoforms (GAD67 and GAD65), VIAAT and GABA-T are expressed in resting and mitogen-activated lymphocytes. Human peripheral lymphocytes were incubated for 72 h in the absence and presence of PHA (10 μg/ml). No GAD65 mRNA expression was detected in samples of either resting or activated lymphocytes (n = 3). By contrast, mRNAs corresponding to GAD67 and VIAAT were detected in 70–80% of samples corresponding to resting cells and 100% of those of activated

Discussion

Our results reveal the presence of a GABAergic system in human peripheral lymphocytes. We determine GAD and VIAAT mRNA expression, which are indicators of GABA-producing cells. Blood GABA concentration in healthy individuals is around 0.1 μM, however the precise origin of this molecule is not known (Bjork et al., 2001). Our results suggest that peripheral lymphocytes are a source of plasmatic GABA. RT-PCR experiments show that mRNA corresponding to GAD67 is present in lymphocytes, whereas GAD65

Acknowledgements

We thank Dr. B. Gasnier (Institut de Biologie Physico-Chimique, Paris, France) for generously supplying the anti-VIAAT antibody. Leonardo Dionisio is a fellow of the Comisión de Investigaciones Científicas (CIC) from Argentina. The present work was supported by grants from Universidad Nacional del Sur (MCE, CB), Agencia Nacional de Promoción Científica y Tecnológica (CB) and CONICET (CB).

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