Endogenous gamma-aminobutyric acid modulates tonic guinea pig airway tone and propofol-induced airway smooth muscle relaxation

Abstract

Background: Emerging evidence indicates that an endogenous autocrine/paracrine system involving gamma-aminobutyric acid (GABA) is present in airways. GABAA channels, GABAB receptors, and the enzyme that synthesizes GABA have been identified in airway epithelium and smooth muscle. However, the endogenous ligand itself, GABA, has not been measured in airway tissues. The authors sought to demonstrate that GABA is released in response to contractile agonists and tonically contributes a prorelaxant component to contracted airway smooth muscle.

Methods: The amount and cellular localization of GABA in upper guinea pig airways under resting and contracted tone was determined by high pressure liquid chromatography and immunohistochemistry, respectively. The contribution that endogenous GABA imparts on the maintenance of airway smooth muscle acetylcholine-induced contraction was assessed in intact guinea pig airway tracheal rings using selective GABAA antagonism (gabazine) under resting or acetylcholine-contracted conditions. The ability of an allosteric agent (propofol) to relax a substance P-induced relaxation in an endogenous GABA-dependent manner was assessed.

Results: GABA levels increased and localized to airway smooth muscle after contractile stimuli in guinea pig upper airways. Acetylcholine-contracted guinea pig tracheal rings exhibited an increase in contracted force upon addition of the GABAA antagonist gabazine that was subsequently reversed by the addition of the GABAA agonist muscimol. Propofol dose-dependently relaxed a substance P contraction that was blocked by gabazine.

Conclusion: These studies demonstrate that GABA is endogenously present and increases after contractile stimuli in guinea pig upper airways and that endogenous GABA contributes a tonic prorelaxant component in the maintenance of airway smooth muscle tone.

Figure 1. Immunohistochemical staining for γ-aminobutyric acid (GABA) in guinea pig tracheal rings

Representative immunohistochemical GABA staining in guinea pig tracheal rings. (A) Untreated tracheal segment illustrates limited staining for GABA (brown) in airway smooth muscle (ASM), with the majority of staining localized to the interface between the ASM and adjacent epithelium (epi). (B) Following 15 min treatment with a neurokinin 2 agonist (10uM β-ala neurokinin A fragment 4–10) a dramatic increase in GABA staining occurs over airway smooth muscle. (C) Immunohistochemical staining for α–actin confirms the identity of the airway smooth muscle layer. (D) Antibody negative control: Isotype specific (IgG₁) negative control antibody for the mouse anti-GABA antibody used in panels A and B reveals no staining in airway smooth muscle.

Figure 2. High pressure liquid chromatography (HPLC) detection of γ-aminobutyric acid (GABA) in buffer eluates from guinea pig tracheal rings

Panel A: Representative chromatograms of GABA eluted from guinea pig tracheal rings. Negative control (buffer only); Panel B: Positive control (buffer spiked with 1pmol GABA): indicated peak at 15 min retention time linearly increased with standard GABA concentrations of 0.5–10 pmoles; Panel C: Superimposed chromatograms of GABA eluted from untreated guinea pig tracheal ring, guinea pig tracheal ring treated with β-ala neurokinin A (10uM), and guinea pig tracheal ring treated with acetylcholine (10uM). Eluted airway GABA levels are detectable by HPLC under baseline (unstimulated) conditions, and increase following treatment (tx) with pro-contractile stimuli. Panel D: Endogenous GABA levels normalized to tissue weight and expressed as percentage of the control group (no treatment). Eluted GABA levels demonstrate a significant increase above baseline levels following treatment with 10uM acetylcholine or 10uM β-ala neurokinin A fragment 4–10 (n=6). ** p<0.01

Figure 2. High pressure liquid chromatography (HPLC) detection of γ-aminobutyric acid (GABA) in buffer eluates from guinea pig tracheal rings

Panel A: Representative chromatograms of GABA eluted from guinea pig tracheal rings. Negative control (buffer only); Panel B: Positive control (buffer spiked with 1pmol GABA): indicated peak at 15 min retention time linearly increased with standard GABA concentrations of 0.5–10 pmoles; Panel C: Superimposed chromatograms of GABA eluted from untreated guinea pig tracheal ring, guinea pig tracheal ring treated with β-ala neurokinin A (10uM), and guinea pig tracheal ring treated with acetylcholine (10uM). Eluted airway GABA levels are detectable by HPLC under baseline (unstimulated) conditions, and increase following treatment (tx) with pro-contractile stimuli. Panel D: Endogenous GABA levels normalized to tissue weight and expressed as percentage of the control group (no treatment). Eluted GABA levels demonstrate a significant increase above baseline levels following treatment with 10uM acetylcholine or 10uM β-ala neurokinin A fragment 4–10 (n=6). ** p<0.01

Figure 3. The effect of selective γ-aminobutyric acid channel subtype A (GABA_A) antagonism on airway smooth muscle force under baseline or contracted (EC₅₀ acetylcholine) conditions

100uM gabazine effect on airway smooth muscle force under baseline or acetylcholine (EC₅₀)-contracted conditions. Blockade of endogenous GABA_A channels results in a functional increase in muscle force compared to time controls. (n=7) *** p < 0.001 compared to time controls; $$$ p < 0.001 compared to baseline tension.

Figure 4. Following an EC₅₀ acetylcholine contraction, selective γ-aminobutyric acid subtype A channel (GABA_A) antagonism increases airway smooth muscle force that is reversed by the GABA_A channel agonist muscimol

Representative tracings in force/time of guinea pig tracheal rings contracted with an EC₅₀ concentration of acetylcholine and treated with; Panel A: 200uM gabazine followed 15 min later by 200uM muscimol; Panel B: 200uM gabazine; or Panel C: nothing. Blockade of GABA_A channels with gabazine results in a sustained increase in muscle force which is reversed by the selective GABA_A agonist muscimol. Panel D: Muscle force in guinea pig tracheal rings contracted to an EC₅₀ with acetylcholine and then subjected to nothing, 100uM gabazine or 200uM gabazine followed by 200uM muscimol. 100uM gabazine significantly increases muscle force compared to time control (n=8) (* p<0.05 compared to untreated time control), and this effect is reversed by 200uM muscimol (n=8) (### p<0.001 compared to 100uM gabazine). Panel E: 200uM gabazine significantly increases muscle force compared to time control (n=8) (*** p<0.001 compare to untreated time control) and this effect is significantly attenuated by 200uM muscimol (n=7) (+++ p<0.001 compared to 200uM gabazine).

Figure 4. Following an EC₅₀ acetylcholine contraction, selective γ-aminobutyric acid subtype A channel (GABA_A) antagonism increases airway smooth muscle force that is reversed by the GABA_A channel agonist muscimol

Representative tracings in force/time of guinea pig tracheal rings contracted with an EC₅₀ concentration of acetylcholine and treated with; Panel A: 200uM gabazine followed 15 min later by 200uM muscimol; Panel B: 200uM gabazine; or Panel C: nothing. Blockade of GABA_A channels with gabazine results in a sustained increase in muscle force which is reversed by the selective GABA_A agonist muscimol. Panel D: Muscle force in guinea pig tracheal rings contracted to an EC₅₀ with acetylcholine and then subjected to nothing, 100uM gabazine or 200uM gabazine followed by 200uM muscimol. 100uM gabazine significantly increases muscle force compared to time control (n=8) (* p<0.05 compared to untreated time control), and this effect is reversed by 200uM muscimol (n=8) (### p<0.001 compared to 100uM gabazine). Panel E: 200uM gabazine significantly increases muscle force compared to time control (n=8) (*** p<0.001 compare to untreated time control) and this effect is significantly attenuated by 200uM muscimol (n=7) (+++ p<0.001 compared to 200uM gabazine).

Figure 5. Gabazine-induced increase in muscle force following an EC₅₀ acetylcholine contraction and antagonism of gabazine’s effect by theγ-aminobutyric acid channel subtype A channel (GABA_A) agonist muscimol are dose dependent

Panel A: Cumulatively increasing concentrations of gabazine (from 0 to 800uM) result in a significant increase in muscle force above a sustained EC₅₀ acetylcholine contraction with an IC₅₀ of 163uM. (n = 12) (** p<0.01 compared to time controls (no gabazine treatment); and *** p<0.001 compared to time controls). Panel B: Cumulatively increasing concentrations of the GABA_A channel agonist muscimol (0–800uM) following a fixed dose of gabazine (200uM) demonstrates significant reversal of the achieved GABA_A channel antagonism in a dose dependent fashion. (n = 4–8) (* p<0.05, ** p<0.01, and *** p<0.001 compared to no treatment (0uM Muscimol)).

Figure 5. Gabazine-induced increase in muscle force following an EC₅₀ acetylcholine contraction and antagonism of gabazine’s effect by theγ-aminobutyric acid channel subtype A channel (GABA_A) agonist muscimol are dose dependent

Panel A: Cumulatively increasing concentrations of gabazine (from 0 to 800uM) result in a significant increase in muscle force above a sustained EC₅₀ acetylcholine contraction with an IC₅₀ of 163uM. (n = 12) (** p<0.01 compared to time controls (no gabazine treatment); and *** p<0.001 compared to time controls). Panel B: Cumulatively increasing concentrations of the GABA_A channel agonist muscimol (0–800uM) following a fixed dose of gabazine (200uM) demonstrates significant reversal of the achieved GABA_A channel antagonism in a dose dependent fashion. (n = 4–8) (* p<0.05, ** p<0.01, and *** p<0.001 compared to no treatment (0uM Muscimol)).

Figure 6. Relaxation of a substance P-induced contraction is enhanced by the selective positive allosteric effect of propofol on the γ-aminobutyric acid subtype A channel (GABA_A)

Panel A: Cumulatively increasing concentration response curves expressed as a percent of initial force following a substance P contraction for propofol (■), Dimethyl sulfoxide (DMSO) vehicle (▼;) or time control (▲). Relaxation of guinea pig tracheal rings contracted with 1uM substance P was significantly enhanced by propofol (≥ 20uM). * p < 0.05 compared to time control; *** p < 0.001 compared to time control. Panel B: Cumulatively increasing concentration response curves expressed as a percent of initial force following a substance P contraction for propofol (■) or with 5uM gabazine pretreatment (▲) before propofol. Partial but significant reversal of propofol-induced relaxation occurred with low dose gabazine (5uM) pretreatment (at 50uM and 100uM propofol ** p < 0.01 and *** p < 0.001 respectively).. Gabazine alone (◆) was not different from untreated time controls (▼;).

Figure 6. Relaxation of a substance P-induced contraction is enhanced by the selective positive allosteric effect of propofol on the γ-aminobutyric acid subtype A channel (GABA_A)

Panel A: Cumulatively increasing concentration response curves expressed as a percent of initial force following a substance P contraction for propofol (■), Dimethyl sulfoxide (DMSO) vehicle (▼;) or time control (▲). Relaxation of guinea pig tracheal rings contracted with 1uM substance P was significantly enhanced by propofol (≥ 20uM). * p < 0.05 compared to time control; *** p < 0.001 compared to time control. Panel B: Cumulatively increasing concentration response curves expressed as a percent of initial force following a substance P contraction for propofol (■) or with 5uM gabazine pretreatment (▲) before propofol. Partial but significant reversal of propofol-induced relaxation occurred with low dose gabazine (5uM) pretreatment (at 50uM and 100uM propofol ** p < 0.01 and *** p < 0.001 respectively).. Gabazine alone (◆) was not different from untreated time controls (▼;).