Functional Expression of γ–Amino Butyric Acid Transporter 2 in Human and Guinea Pig Airway Epithelium and Smooth Muscle

Zaidi, Sarah; Gallos, George; Yim, Peter D.; Xu, Dingbang; Sonett, Joshua R.; Panettieri, Reynold A.; Gerthoffer, William T.; Emala, Charles W.

doi:10.1165/rcmb.2010-0177oc

Cited by 15 publications

(15 citation statements)

References 28 publications

(61 reference statements)

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“…These conditions were chosen since high external potassium (KCl) is a known membrane depolarizer for both epithelium and airway smooth muscle cells. This depolarization has been shown by our group (24) to stimulate GABA release through GATs in both of these cell types.…”

Section: Methodssupporting

confidence: 63%

“…Although the neurotransmitter ␥-aminobutyric acid (GABA) has classically been perceived as limited to neuronal signaling, recent evidence suggests GABA also plays a novel role as a paracrine signaling molecule in airways. For example, GABA A chloride ion channels, G i -coupled GABA B metabotropic receptors, GABA transporters, and the enzymes that synthesize GABA (glutamic acid decarboxylase) have all been recently identified on both airway epithelium and airway smooth muscle (3,15,16,(22)(23)(24).…”

mentioning

confidence: 99%

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Airway epithelium is a predominant source of endogenous airway GABA and contributes to relaxation of airway smooth muscle tone

Gallos

Townsend

Yim

et al. 2013

American Journal of Physiology-Lung Cellular and Molecular Phys

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Chronic obstructive pulmonary disease and asthma are characterized by hyperreactive airway responses that predispose patients to episodes of acute airway constriction. Recent studies suggest a complex paradigm of GABAergic signaling in airways that involves GABA-mediated relaxation of airway smooth muscle. However, the cellular source of airway GABA and mechanisms regulating its release remain unknown. We questioned whether epithelium is a major source of GABA in the airway and whether the absence of epithelium-derived GABA contributes to greater airway smooth muscle force. Messenger RNA encoding glutamic acid decarboxylase (GAD) 65/67 was quantitatively measured in human airway epithelium and smooth muscle. HPLC quantified GABA levels in guinea pig tracheal ring segments under basal or stimulated conditions with or without epithelium. The role of endogenous GABA in the maintenance of an acetylcholine contraction in human airway and guinea pig airway smooth muscle was assessed in organ baths. A 37.5-fold greater amount of mRNA encoding GAD 67 was detected in human epithelium vs. airway smooth muscle cells. HPLC confirmed that guinea pig airways with intact epithelium have a higher constitutive elution of GABA under basal or KCl-depolarized conditions compared with epithelium-denuded airway rings. Inhibition of GABA transporters significantly suppressed KCl-mediated release of GABA from epithelium-intact airways, but tetrodotoxin was without effect. The presence of intact epithelium had a significant GABAergic-mediated prorelaxant effect on the maintenance of contractile tone. Airway epithelium is a predominant cellular source of endogenous GABA in the airway and contributes significant prorelaxant GABA effects on airway smooth muscle force.

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Section: Methodssupporting

confidence: 63%

mentioning

confidence: 99%

Airway epithelium is a predominant source of endogenous airway GABA and contributes to relaxation of airway smooth muscle tone

Gallos

Townsend

Yim

et al. 2013

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…The cDNA products were then amplified by PCR using a GeneAmp thermal cycler (Applied Biosystems, Foster City, CA). Specific sense and antisense primers for the experiments (Lee et al, 2004(Lee et al, , 2010aMizuta et al, 2008;Neumann et al, 2004;Stepulak et al, 2009;Zaidi et al, 2010) are listed in Table 1. PCR conditions were as follows: initial denaturation at 95°C for 6 min followed by a 30-cycle amplification program consisting of denaturation at 95°C for 45 s, annealing at 55-60°C for 1 min and extension at 72°C for 1 min.…”

Section: Messenger Rna Analysis By Reverse-transcriptase Polymerase Cmentioning

confidence: 99%

“…To test this possibility, we next examined the effects of inhibitors of GABA transport systems. We utilized as inhibitors NNC 711 at 20 lM for GAT1 (Borden et al, 1994a), b-alanine at 300 lM for GAT2 (Christiansen et al, 2007;Zaidi et al, 2010), (S)SNAP-5114 at 10 lM for GAT3 (Borden et al, 1994b), and NNC 05-2090 at 40 lM for BGT1 (Zaidi et al, 2010). It must be noted that these inhibitors are not absolutely specific but only preferential for particular transporters.…”

mentioning

confidence: 99%

Mechanisms of GABA release from human astrocytes

Lee

McGeer

2011

Glia

101

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We have previously demonstrated that human astrocytes are GABAergic cells. Throughout the adult human brain, they express the GABA synthesizing enzyme GAD 67, the GABA metabolizing enzyme GABA-T, and the GABA(A) and GABA(B) receptors. GABA modulates the actions of microglia, indicating an important role for astrocytes beyond that of influencing neurotransmitter function. Here we report on the mechanisms by which astrocytes release GABA. Astrocytes were found to express the mRNA and protein for multiple GABA transporters, and multiple receptors for glutamate, GABA, and glycine. In culture, untreated human astrocytes maintained an intracellular GABA level of 2.32 mM. They exported GABA into the culture medium so that an intracellular-extracellular gradient of 3.64 fold was reached. Inhibitors of the GABA transporters GAT1, GAT2, and GAT3, significantly reduced this export in a Ca(2+)-independent fashion. Intracellular GABA levels were enhanced by treatment with the GABA-T inhibitors gabaculine or vigabatrin. Treatment with glutamate increased GABA release in a concentration-dependent fashion. This was partially inhibited by blockers of N-methyl-D-aspartate and kainate receptors. Conversely, glycine and D-serine, co-agonists of NMDA receptors, enhanced the GABA release. GABA release was accompanied by an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and was reduced by adding the Ca(2+) chelator, BAPTA-AM to the medium. These data indicate that astrocytes continuously synthesize GABA and that there are multiple mechanisms which can mediate its release. Each of these may play a role in the physiological functioning of astrocytes.

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“…glycine) [11], creatine [12], and the organic osmolytes betaine [13] and taurine [14]. BGT1 is expressed in a wide variety of tissues including brain [15,16], liver [16,17,18], kidney [16] and airways [19]. BGT1 expression is stimulated by osmotic cell shrinkage [20,21,22].…”

Section: Introductionmentioning

confidence: 99%

Upregulation of Na⁺,Cl^--Coupled Betaine/ γ-Amino-Butyric Acid Transporter BGT1 by Tau Tubulin Kinase 2

Almilaji

Muñoz

Hosseinzadeh

et al. 2013

Cell Physiol Biochem

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Background/Aims: The serine/threonine kinase Tau-tubulin-kinase 2 (TTBK2) is expressed in various tissues including kidney, liver and brain. Loss of function mutations of TTBK2 lead to autosomal dominant spinocerebellar ataxia type 11 (SCA11). Cell survival is fostered by cellular accumulation of organic osmolytes. Carriers accomplishing cellular accumulation of organic osmolytes include the Na⁺, Cl^--coupled betaine/γ-amino-butyric acid transporter BGT1. The present study explored whether TTBK2 participates in the regulation of BGT1 activity. Methods: Electrogenic transport of GABA was determined in Xenopus oocytes expressing BGT1 with or without wild-type TTBK2, truncated TTBK2[1-450] or kinase inactive mutants TTBK2- KD and TTBK2[1-450]-KD. Results: Coexpression of wild-type TTBK2, but not of TTBK2[1-450], TTBK2-KD or TTBK2[1-450]-KD, increased electrogenic GABA transport. Wildtype TTBK2 increased the maximal transport rate without significantly modifying affinity of the carrier. Coexpression of wild-type TTBK2 significantly delayed the decline of transport following inhibition of carrier insertion with brefeldin A, indicating that wild-type TTBK2 increased carrier stability in the cell membrane. Conclusion: Tau-tubulin-kinase 2 TTBK2 is a powerful stimulator of the osmolyte and GABA transporter BGT1.

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Functional Expression of γ–Amino Butyric Acid Transporter 2 in Human and Guinea Pig Airway Epithelium and Smooth Muscle

Cited by 15 publications

References 28 publications

Airway epithelium is a predominant source of endogenous airway GABA and contributes to relaxation of airway smooth muscle tone

Airway epithelium is a predominant source of endogenous airway GABA and contributes to relaxation of airway smooth muscle tone

Mechanisms of GABA release from human astrocytes

Upregulation of Na⁺,Cl^--Coupled Betaine/ γ-Amino-Butyric Acid Transporter BGT1 by Tau Tubulin Kinase 2

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Functional Expression of γ–Amino Butyric Acid Transporter 2 in Human and Guinea Pig Airway Epithelium and Smooth Muscle

Cited by 15 publications

References 28 publications

Airway epithelium is a predominant source of endogenous airway GABA and contributes to relaxation of airway smooth muscle tone

Airway epithelium is a predominant source of endogenous airway GABA and contributes to relaxation of airway smooth muscle tone

Mechanisms of GABA release from human astrocytes

Upregulation of Na+,Cl--Coupled Betaine/ γ-Amino-Butyric Acid Transporter BGT1 by Tau Tubulin Kinase 2

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Upregulation of Na⁺,Cl^--Coupled Betaine/ γ-Amino-Butyric Acid Transporter BGT1 by Tau Tubulin Kinase 2