Functional expression of γ-amino butyric acid transporter 2 in human and guinea pig airway epithelium and smooth muscle

Abstract

γ-Amino butyric acid (GABA) is a primary inhibitory neurotransmitter in the central nervous system, and is classically released by fusion of synaptic vesicles with the plasma membrane or by egress via GABA transporters (GATs). Recently, a GABAergic system comprised of GABA(A) and GABA(B) receptors has been identified on airway epithelial and smooth muscle cells that regulate mucus secretion and contractile tone of airway smooth muscle (ASM). In addition, the enzyme that synthesizes GABA, glutamic acid decarboxylase, has been identified in airway epithelial cells; however, the mechanism(s) by which this synthesized GABA is released from epithelial intracellular stores is unknown. We questioned whether any of the four known isoforms of GATs are functionally expressed in ASM or epithelial cells. We detected mRNA and protein expression of GAT2 and -4, and isoforms of glutamic acid decarboxylase in native and cultured human ASM and epithelial cells. In contrast, mRNA encoding vesicular GAT (VGAT), the neuronal GABA transporter, was not detected. Functional inhibition of (3)H-GABA uptake was demonstrated using GAT2 and GAT4/betaine-GABA transporter 1 (BGT1) inhibitors in both human ASM and epithelial cells. These results demonstrate that two isoforms of GATs, but not VGAT, are expressed in both airway epithelial and smooth muscle cells. They also provide a mechanism by which locally synthesized GABA can be released from these cells into the airway to activate GABA(A) channels and GABA(B) receptors, with subsequent autocrine and/or paracrine signaling effects on airway epithelium and ASM.

Figure 1.

Representative gel images of RT-PCR of γ–amino butyric acid (GABA) transporter (GAT) subtypes from RNA from freshly dissected human and guinea pig (GP) tissues and cultured human airway smooth muscle (ASM) and epithelial cells. mRNA for (A) GAT2 and (B) GAT4 is present in native human ASM and epithelium, cultured human ASM and epithelium (A and B, left panels), and native guinea pig ASM and epithelium (A and B, right panels). (C) mRNA for GAT1 is present in native human ASM and epithelium, and may represent contaminating neural structures in these tissues. mRNA for (D) GAT3 and (E) vesicular GAT (VGAT) is not present in human or guinea pig ASM or epithelium. Images are representative of experiments performed on RNA isolated from two to three different native guinea pig or human tissues, or two to three cultured cell flasks. 1, 100 base pair ladder; 2, water, which denotes the negative control (no cDNA input); 3, native human ASM tissue; 4, native human airway epithelial tissue; 5, cultured human ASM cells; 6, cultured human airway epithelial cells; 7, human brain; 8, native guinea pig ASM tissue; 9, native guinea pig airway epithelial tissue; and 10, guinea pig brain. Separate gel images are denoted by a solid demarcating line.

Figure 2.

Representative gel images of RT-PCR of RNA isolated by laser capture microdissection for GAT subtypes 1–4. (A) Human airway epithelium from tracheal rings. (B) Human ASM from tracheal rings. Encoding mRNA for GAT subtypes 2 and 4 were detected in RNA isolated from pure human airway epithelium and smooth muscle cells, whereas mRNA for GAT1 and GAT3 were not. Images are representative of experiments performed on RNA isolated from two to three different native guinea pig or human tissues. HASM, human ASM from tracheal ring; HBr, human brain (positive control); HEpi, human airway epithelium from tracheal ring; MW, 100 base pair ladder. Water denotes the negative control (no cDNA input).

Figure 3.

Representative gel images of RT-PCR of glutamic acid decarboxylase (GAD) isoforms GAD65 and GAD67 from RNA from freshly dissected native human and guinea pig ASM and epithelial tissue, and cultured human ASM and epithelial cells. mRNA for GAD65 is present in guinea pig ASM, whereas mRNA for GAD67 is present in native and cultured human ASM and epithelium. Images are representative of experiments performed on RNA isolated from two to three different native guinea pig or human tissues or two to three cultured cell flasks. 1, 100 base pair ladder; 2, water, which denotes the negative control (no cDNA input); 3, native human ASM; 4, native human airway epithelium; 5, cultured human ASM cells; 6, cultured human epithelial cells; 7, human brain; 8, native guinea pig ASM tissue; 9, native guinea pig airway epithelial tissue; and 10, guinea pig brain. Separate gel images are denoted by a solid demarcating line.

Figure 4.

Representative immunoblot images of GAT and GAD isoforms. (A) GAT4/betaine–GABA transporter (BGT) 1 and (B) GAT2 proteins are present in native guinea pig ASM and epithelium. Guinea pig brain and guinea pig kidney are positive controls for GAT4/BGT1 and GAT2, respectively. (C) GAD protein is present in native guinea pig airway epithelium and smooth muscle and guinea pig brain. GPASM, guinea pig ASM; GPBr, guinea pig brain; GPEpi, guinea pig airway epithelium; GPK, guinea pig kidney. Images are representative of experiments performed on protein isolated from two to three different native guinea pig tissues.

Figure 5.

³H-GABA uptake and release assays. (A–D) Dose–response curves using GAT isoform inhibitors in human airway epithelial and smooth muscle cells. Human airway epithelial cell dose–response curves with (A) NNC 05-2090, a selective GAT4/BGT-1 inhibitor (half maximal [50%] inhibitory concentration or IC₅₀, 39 μM) and (C) β-alanine, a selective GAT2 inhibitor (IC₅₀, 79 μM). Human ASM cell dose–response curves with (B) NNC 05-2090, a selective GAT4/BGT-1 inhibitor (IC₅₀, 51 μM) and (D) β-alanine, a selective GAT2 inhibitor (IC₅₀, 28 μM). Dose–response curves are representative of four experiments. Data are presented as means (±SEM). (E) Effect of GAT2 and GAT4 inhibitors alone or in combination on ³H-GABA uptake in human bronchial epithelial cells. The GAT2 inhibitor, β-alanine, significantly decreases specific ³H-GABA uptake (n = 6; **P < 0.001), and the addition of the GAT4 inhibitor, NNC 05-2090, does not further decrease specific ³H-GABA uptake. Addition of 5 μM NNC 05-2090 alone induced a small but significant decrease in specific ³H-GABA uptake (n = 6; *P < 0.05). (F) Effect of GAT2 and GAT4 inhibitors alone or in combination on ³H-GABA uptake in human ASM cells. The GAT2 inhibitor, β-alanine, significantly decreases specific ³H-GABA uptake (n = 12; **P < 0.001), and the addition of the GAT4 inhibitor, NNC 05-2090, does not further decrease specific ³H-GABA uptake. Data are presented as means (±SEM).

Figure 6.

Effect of elimination of sodium, elimination of chloride, or depolarization with potassium chloride on ³H-GABA uptake. Specific ³H-GABA uptake is significantly decreased in (A) human airway epithelial cells (n = 20; ***P < 0.001) and (B) human ASM cells (n = 8–16; ***P < 0.001) in the absence of sodium or chloride ions. Specific ³H-GABA uptake is significantly decreased in (C) human bronchial epithelial cells (n = 6; **P < 0.01) and (D) human ASM cells (n = 20; *P < 0.05) treated with 80 mM KCl to induce cell membrane depolarization. Data are presented as means (±SEM).

Figure 7.

Effect of cell membrane depolarization on fractional ³H-GABA release by cultured human airway epithelial cells. Fractional release of ³H-GABA by cultured human airway epithelial cells in a basal low–sodium chloride buffer (28 mM) is significantly increased in the presence of potassium gluconate (80 mM) to induce cell membrane depolarization (n = 8; **P < 0.001). GAT blockade by a high concentration (500 μM) of NNC 05-2090, that inhibits all subtypes of GATs, inhibits the action of potassium gluconate (n = 8; **P < 0.001). In the presence of the GAT inhibitor, NNC 05-2090, potassium gluconate does not significantly increase basal fractional release of ³H-GABA (n = 8; ^#P > 0.05). Data are presented as means (±SEM).