Endogenous γ-Aminobutyric Acid Modulates Tonic Guinea Pig Airway Tone and Propofol-induced Airway Smooth Muscle Relaxation

ASTHMA is a chronic inflammatory disease of the airways that predisposes patients to episodes of severe, acute airway constriction. Despite an increasing worldwide prevalence of asthma,1new pharmacologic approaches to treat this disease are limited. Although a great deal is known regarding the mechanisms governing airway smooth muscle contraction,2relatively less is known about mechanisms of airway smooth muscle relaxation. Therefore, greater insights into the mechanisms of endogenous control of airway smooth muscle relaxation are required to identify novel therapeutic strategies.Interestingly, both volatile and intravenous anesthetics have long been recognized as potent bronchodilators,3–15yet the exact mechanisms for this effect are incompletely understood and have been attributed to both neural6,7and direct airway smooth muscle effects.8–10Despite the well known effect of anesthetics (e.g. , propofol) as allosteric potentiators of the action of endogenous γ-aminobutyric acid (GABA) at neuronal GABAAchannels,11it has been a long standing belief that any GABAergic contribution to airway tone was largely mediated by GABAAchannels in the brainstem12or by GABABreceptors on preganglionic cholinergic nerves in the lung.13,14Our recent identification of GABAAchannels expressed directly on airway smooth muscle that contribute to relaxation15raises the novel possibility that a previously unrecognized mechanism of anesthetic mediated airway smooth muscle relaxation could be anesthetics allosterically potentiating the effect of endogenous GABA at airway smooth muscle GABAAchannels. In addition to GABAAchannels, GABABreceptors and glutamic decarboxylase (the enzyme responsible for GABA synthesis) have also recently been identified in airway epithelial16,17and smooth muscle cells,18suggesting that endogenous GABA may have autocrine/paracrine functions in airways. Despite the presence of a complex GABAergic system in the airway, measurements of the endogenous ligand GABA or a tonic effect on airway tone by endogenous GABA has not yet been demonstrated.Taken together, the expression of airway smooth muscle GABAAreceptors facilitating relaxation and the clinical benefit of anesthetics on hyperreactive airway tone led us to question (1) whether endogenous GABA is present in the airway, (2) whether contractile agonists increase airway GABA release (3) whether liberated GABA tonically modulates airway smooth muscle tone, and (4) whether airway smooth muscle GABAAchannels mediate a component of anesthetic (i.e. , propofol)-induced airway smooth muscle relaxation. Defining and harnessing this novel relaxation pathway may identify new therapeutic options for hyperreactive airway disease.Indomethacin, N-vanillyinonanamide (capsaicin analogue), pyrilamine, acetylcholine, γ-aminobutyric acid, o-phtaldialdehyde (OPA), 2-mercaptoethanol (βME), and gabazine were obtained from Sigma (St. Louis, MO). Propofol was obtained from ICN Biomedicals and diluted in dimethyl sulfoxide (DMSO) (Aurora, OH). Tetrodotoxin was obtained from Calbiochem (San Diego, CA).All animal protocols were approved by the Columbia University Animal Care and Use Committee (New York, New York). Male Hartley guinea pigs (approximately 400 g) were deeply anesthetized with intraperitoneal pentobarbital (100 mg/kg). After opening the chest cavity, the entire trachea was surgically removed and promptly placed in cold (4°C) phosphate-buffered saline (PBS). Each trachea was dissected under a dissecting microscope into closed rings composed of two cartilaginous segments from which mucosa and connective tissue were removed. Epithelium was left intact for high pressure liquid chromatography (HPLC) and immunohistochemistry studies but removed for organ bath experiments. Tissues were placed into cold Krebs-Henseleit (KH) buffer (in mM: NaCl 118, KCl 5.6, CaCl20.5, MgSO40.24, NaH2PO41.3, NaHCO325, glucose 5.6, pH 7.4) containing 10 μm indomethacin (DMSO vehicle final concentration in organ baths of 0.01%) to block tone due to endogenous release of prostanoids.To demonstrate that GABA is endogenously present in airways, we used intact guinea pig tracheal ring segments (2 rings per sample) harvested from euthanized guinea pigs. All sample tissues were separately incubated in a 500-μl volume of KH buffer (pH 7.4) on ice for 20 min followed by a 500-μl KH buffer wash. Samples were separated into 3 treatment groups (no treatment, acetylcholine 10 μm and β-ala Neurokinin A (NKA) fragment 4–10 (neurokinin receptor 2 agonist) 10 μm) and incubated for 45 min in a fresh volume of 200 μl of KH buffer (pH 7.4) at 37°C. After this treatment period, a 20-μl aliquot was analyzed for GABA by HPLC with electrochemical detection using a method based on precolumn derivatization of the amino acid with (OPA) and βME.19,20The HPLC system used for analysis consisted of a GBC model LC1150 pump (GBC Scientific Equipment Pty. Ltd., Dandenong, Australia), a Rheodyne model 9725i injector (Rheodyne, Rohnert Park, CA), and an INTRO amperometric detector (Antec, Leyeden, The Netherlands) equipped with a VT-03 (Antec) electrochemical flow cell with glassy carbon working electrode and salt bridge Ag/AgCl reference electrode. Chromatographic separation was achieved on a C18column and a mobile phase consisting of 0.1 M Na2HPO4, 50 mg/L EDTA, and 12% methanol (pH 5.2). Separation and detection were performed at 30°C with the cell potential set to +750 mV. Peak heights were measured by using WinChrom chromatography software (GBC Scientific Equipment). To confirm accurate detection of a GABA peak, generation of standard curves with known concentrations of GABA preceded sample analysis (these standard curves were consistently linear over a range of 25 nm to 1 μm). In addition, an intraexperimental control (reanalyzing a previously processed sample spiked with a known concentration of GABA) was performed to confirm that the subsequent increase in the GABA peak is proportional to the amount of GABA added to the sample. For negative control, KH buffer only was analyzed.To illustrate the localization patterns of endogenous GABA under basal (resting) and procontractile stimulated conditions, guinea pig tracheal rings were harvested as above and were treated with or without 10 μm β-ala NKA fragment 4–10 for 15 min. Tracheal rings were immediately fixed using 4% paraformaldehyde/1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 4 h at 4°C for GABA immunostaining as previously described.18Briefly, tracheal rings were paraffin-embedded, sectioned (5 μm), dewaxed in xylene, and rehydrated in a graded alcohol series to water. Endogenous peroxidase was blocked in 0.3% hydrogen peroxide. Heat-mediated antigen retrieval was performed with 10 mm sodium citrate buffer, pH 6.0 for 30 min. An avidin biotin blocking kit (Vector Laboratories, Peterborough, United Kingdom) was used (in 10% serum in PBS) to block endogenous biotin. Slides were rinsed with PBS and incubated overnight at 4°C in primary antibody against GABA (mouse, MAB316; Chemicon, Temecula, CA) at a concentration of 1:50 in 2% serum in PBS. Tracheal ring sections were also incubated with the same concentration of a mouse isotype IgG antibody (IgG1) (as a negative control) or with primary antibody directed against α-smooth muscle actin (mouse, MAB1522; Chemicon, Temecula, CA) at a concentration of 1:10,000 to identify smooth muscle (positive control). After overnight incubation at 4°C, slides were washed with PBS, and primary antibodies were detected using biotinylated anti-mouse antibodies (Vector Laboratories) at a concentration of 1:100. The antigen antibody complex was then visualized by enzymatic reduction of 3,3-diaminobenzidine tetrahydrochloride. Sections were counterstained with hematoxylin and dried, and cover slides were mounted using Polymount (Polysciences, Warrington, PA).Closed guinea pig tracheal rings were suspended in organ baths as previously described.21Briefly, tissues were attached with silk thread inferiorly to a fixed tissue hook in a water-jacketed (37°C) 2-ml organ bath (Radnoti Glass Technology, Inc., Monrovia, CA) and superiorly to a Grass FT03 force transducer (Grass Telefactor, West Warwick, RI) coupled to a computer via BioPac hardware and Acqknowledge 7.3.3 software (Biopac Systems, Inc., Goleta, CA) for continuous digital recording of muscle force. Tissues were secured such that muscle contraction would align with the vertical plane between the anchoring hook below and transducer above. KH buffer was continuously bubbled with 95% oxygen and 5% carbon dioxide, and tissues were allowed to equilibrate at 1g isotonic force for 1 h with fresh KH buffer changes every 15 min.After equilibration, the capsaicin analog N-vanillylnonanamide (10 μm final) was added to the organ baths containing guinea pig tracheal rings to first activate and then deplete nonadrenergic, noncholinergic nerves. After N-vanillylnonanamide–induced force had returned to baseline (50 min), the KH buffer in the organ baths were changed 6 times to wash out added or liberated mediators. Tracheal rings were then subjected to two cycles of increasing cumulative concentrations of acetylcholine (0.1 μm to 1 mm) with 6 buffer changes and resetting of the resting tension between cycles. The resulting concentration response curves were then used to determine the EC50concentrations of acetylcholine required for each individual ring. Individual tissues have variable sensitivity to contractile agonists (EC50) and variation in the magnitude of contraction (Emax). To avoid bias between treatment groups, tissues were contracted to individually calculated EC50s for acetylcholine and tissues with similar Emaxvalues were randomly assigned to treatments within individual experiments. After preliminary contractile challenges, tissues were subjected to extensive KH buffer changes (8–9 times) and allowed to stabilize at their respective isotonic resting tensions (1.0 g ). To remove confounding effects of other procontractile pathways, each airway ring received a complement of antagonists 20 min before subsequent contractile challenge. The antagonists included pyrilamine (10 μm, H1histamine receptor antagonist), and tetrodotoxin (1 μm; sodium channel blocker negating endogenous neuronal-mediated cholinergic or C-fiber effects).After equilibration, tracheal rings were randomly assigned to one of four groups: ±gabazine with rings at resting tension (1g ) or contracted with an EC50concentration of acetylcholine, which were allowed to achieve a steady-state plateau of increased force (typically 15 min). To determine the functional effect of endogenous GABA on airway smooth muscle GABAAchannels, a single dose of the selective GABAAchannel antagonist gabazine (100 μm) was added to the resting or acetylcholine-precontracted tracheal rings. Changes in muscle force were recorded over 15 min. For all groups, changes in muscle force were analyzed as the percent of change in muscle force from an initial baseline or acetylcholine-induced muscle force measured 15 min after treatments. In a separate set of experiments, to confirm that the effect of gabazine was not attributable to nonspecific actions (a non-GABAAchannel effect), a single dose of the selective GABAAchannel antagonist gabazine (100 μm or 200 μm) was added to acetylcholine-precontracted tracheal rings. After 15 min, treatment groups then received muscimol (200 μm) to reverse the effect of gabazine-induced airway smooth muscle GABAAchannel blockade.To illustrate that functional antagonism of endogenous GABA at the airway smooth muscle GABAAchannel occurs in a dose-dependent fashion, we performed a dose response study using cumulative gabazine concentrations (0–800 μm) administered after an EC50acetylcholine contraction. Changes in muscle force were analyzed as the percent of change in muscle force from an initial acetylcholine EC50contraction measured at 15-min intervals after each treatment. to between gabazine and we also performed a dose response of muscimol (0–800 μm) after a fixed concentration of gabazine (200 μm) that was after an acetylcholine For experiments, changes in muscle force mediated by muscimol were analyzed as the percent of change in muscle force from the gabazine (200 increase in contractile illustrate the of endogenous GABA at GABAAchannels to mediate relaxation of airway smooth we the of an allosteric of GABA to airway smooth muscle after a 1 μm contraction. the guinea pig rings preliminary contractile with the capsaicin analog and After extensive and a resetting of baseline resting tone (1.0 g tissues were randomly assigned to one of groups: control (no vehicle treatment only and treatment. pig tracheal rings were then treated with (1 μm), allowed to a plateau in muscle and then were treated with increasing concentrations and μm) of or vehicle (DMSO) in 6 min In separate contracted guinea pig tracheal rings were with or without μm gabazine 15 min before and with or without increasing concentrations μm) of at For all groups, changes in relaxation were analyzed as a of muscle force from the initial contraction and were to included intraexperimental response curves were using a dose response analysis in software CA), which a to the (1 10 the the initial resting muscle were analyzed by with between are as is conditions, endogenous GABA was at the between airway smooth muscle and the in guinea pig tracheal rings in within the airway smooth muscle was at high not In 15-min to the selective receptor 2 β-ala NKA fragment 4–10 (10 μm) GABA the airway smooth muscle that GABA or release within the airway smooth muscle The of the airway smooth muscle was using an antibody and a control for antibody nonspecific was performed using we airway GABA using GABA has not been previously in airway Therefore, HPLC are first in A of KH buffer is in a of a 1 standard of GABA in KH The GABA peak had a of min and a linear increase in magnitude over a range of GABA standard concentrations not GABA from or stimulated guinea pig rings in 200 μl KH buffer at over a is in tracheal rings in KH buffer for 45 min of but the GABA increased in tracheal rings treated for 45 min with 10 μm β-ala NKA fragment 4–10 or the in GABA 45 min at in 200 μl KH The procontractile acetylcholine and β-ala NKA fragment 4–10 increased GABA to whether antagonism of GABAAchannels in intact guinea pig tracheal rings airway smooth muscle force. pig tracheal rings suspended in organ baths under resting tension ) were treated with which in a increase in muscle force to in organ baths of baseline tone control of baseline tone In μm gabazine was added to the organ baths a contraction with acetylcholine the of airway smooth muscle force was greater increase above acetylcholine force control increase above acetylcholine force separate set of was performed to confirm that effect occurs at endogenous GABAAchannels. force are in 200 μm gabazine treatment of a acetylcholine contraction (EC50) and by 200 μm used μm or 200 μm gabazine a acetylcholine (EC50) contraction and then to reverse both concentrations of gabazine with 200 μm of muscimol (a μm gabazine a acetylcholine contraction in a increase in airway smooth muscle force to control tracheal rings in organ baths increase above acetylcholine force control increase above acetylcholine force μm gabazine-induced increase in muscle force was by 200 μm muscimol increase above acetylcholine to μm gabazine A concentration of 200 μm gabazine the phase of an EC50acetylcholine contraction in an greater increase in airway smooth muscle tension to in organ baths or to the effect of μm gabazine (200 μm gabazine increase above acetylcholine and this increase was by subsequent treatment with 200 μm muscimol increase above acetylcholine to 200 μm gabazine organ bath were performed to demonstrate increasing concentrations of gabazine to μm) administered after an EC50acetylcholine contraction in dose-dependent increase in muscle force and an μm increasing concentrations of the GABAAchannel muscimol (0–800 μm) after a fixed dose of gabazine (200 μm) of the achieved GABAAchannel antagonism in a dose-dependent fashion, at a concentration of μm to treatment of the increase in muscle force of 200 μm required a concentration of muscimol greater 200 μm, that gabazine and muscimol are not potent under increase in muscle force by gabazine the phase of an acetylcholine contraction that endogenous GABA is present and contractile muscle force. To illustrate the endogenous presence and contribution of GABA to muscle were treated with an allosteric at GABAAchannels in the and presence of a pig tracheal rings were contracted with 1 μm and then subjected to increasing concentrations of or μm) or the concentrations of the vehicle for (DMSO) in the or presence of the selective gabazine (5 μm). Propofol μm) had a effect on relaxation to the of an control with effect of the vehicle 10 in each for 20 μm to control, and for 50 or to control To demonstrate that a component of the relaxation was due to allosterically endogenous GABA effects at GABAAchannels, were with μm) in the or presence of μm but with gabazine effect on the contraction in each for to of this study is that endogenous GABA in the guinea pig airway and an in airway smooth muscle tone by facilitating smooth muscle relaxation via of airway smooth muscle GABAAchannels. To the contribution of endogenous GABA in airway tone, we used both as well as functional studies (in organ and we the of GABA by effect against two contractile and receptor 2 In addition, to illustrate that the GABA effects were to GABAAchannels, we performed studies a procontractile effect by which was by is the first that of endogenous GABA are present in the airway, that GABA release and localization changes in response to procontractile and that GABA airway smooth muscle relaxation after smooth muscle anesthetic that are also allosteric at GABAAchannels, which to the effects of endogenous Despite the exact responsible for effect are incompletely has been a belief that any potential GABAergic contribution to airway tone was largely mediated by GABAAchannels in the brainstem12or by on preganglionic cholinergic nerves in the is that a complex airway GABAergic system one which the presence of GABAAchannels not only on airway but also on airway and airway smooth addition, the enzyme responsible for GABA acid is also present in airway airway smooth is that the effect of anesthetic which presence to their is also attributable to an allosteric effect at airway smooth muscle expression of GABABreceptors on nerves in the airways has been known for we recently the expression of GABABreceptors on airway smooth airway to Therefore, in addition to on GABAAchannels in the liberated GABA could release of acetylcholine in the airway but could smooth muscle relaxation by of the which in a to airway smooth muscle we have previously that GABA is in the guinea pig at the between the airway smooth muscle and in the of contractile and only was within the airway smooth muscle with the localization for the which was the airway smooth muscle the study under conditions, GABA immunostaining was the smooth muscle smooth muscle and and between smooth muscle and GABA immunostaining increased over the smooth muscle after to the procontractile selective receptor 2 NKA fragment the possibility that endogenous GABA in of airway smooth muscle HPLC detection and of amino acid has been performed in neuronal has been to airway studies the in GABA analysis by were into in the a we were to identify and GABA from into which intact airway tissues In with we demonstrate that 10 μm acetylcholine or β-ala NKA fragment increased GABA is that or to dissected airway tissues release of To this dissected tracheal rings were washed wash buffer concentrations of GABA were and then the tracheal rings were incubated in a volume of buffer in the or presence of a contractile such that initial buffer concentrations of GABA were and GABA that over 45 min was measured GABA that had into the incubation method not directly the changes in GABA that within the of the airway smooth muscle is an we are the magnitude of change in endogenous airway GABA that airway smooth muscle GABAAchannels are The study not us to determine the of together, and HPLC demonstrate that airway GABA increase after procontractile and the possibility that liberated GABA at airway smooth muscle GABAAchannels may contribute to to the procontractile that of GABA liberated into the airway was not in the present but at 3 in the airway to be The enzyme that GABA acid was recently identified by us in airway smooth muscle and airway addition, of in the airway and may the of GABA in the airway is an of in The study that the contractile agonists acetylcholine and β-ala NKA increase GABA is in the agonists the release of GABA from neuronal cell determine endogenous GABA relaxation via of airway smooth muscle GABAAchannels, we in functional organ bath studies using intact guinea pig airway smooth In or neuronal were by the and by with capsaicin and the functional of the of endogenous GABA to effect via the airway smooth muscle used the GABAAchannel antagonist gabazine is the for vehicle is a of the GABAAchannel for a of effect), is not known to have nonspecific effects at other the GABAAchannel has been used by us in organ bath increased smooth muscle force and had a greater effect under contracted conditions, which with under which we GABA The of the muscimol to reverse effect the of effect at the and that may for a effect of the under contracted is the of the potential under baseline of the GABAAchannel in smooth muscle at resting potential (approximately may of and after an acetylcholine contraction in airway smooth the potential is for and to a that smooth muscle the study not resting potential with resting muscle tonic to high concentrations of neuronal GABAAchannels (as is within the after release of is that an in the for GABAAchannel two of GABAAchannels have been identified in the with and and the that are to concentrations of GABA and tonic GABAAchannels the of an or with a and we have previously that airway smooth muscle GABAAchannels the in addition to expression to and that the of required in to tonic GABAAchannels are also expressed in airway smooth illustrate the functional of endogenous GABA in relaxation of contracted an anesthetic with allosteric effects at the GABAAchannel (i.e. , propofol) was the effect of a ligand at by activate the a to demonstrate a endogenous demonstrate a dose-dependent in relaxation of a contraction by which was by GABAAchannel may that relaxation of airway smooth muscle may mechanisms or that concentrations of gabazine were to demonstrate of we were in the concentrations of gabazine (5 μm) that could be used in studies as concentrations of gabazine (100 μm) increase muscle force by blocking relaxation by endogenous set of yet for from which has been attributed to action on airway cholinergic nerves and less to of phosphate airway smooth muscle studies have smooth muscle effects only at concentrations of μm) above achieved studies in airway tissues from and animal have that contractile from only at concentrations of studies that only a effect at high concentrations have largely on in the and of contraction phosphate in the concentrations of μm) airway smooth muscle relaxation due to to the of concentrations the of of is of in peak concentrations of of achieve 30 μm the concentration in individual tissue is high tissue of by the of has been the effect we demonstrate with occurs at a concentration μm) that is to achieved used for or of the of (1) endogenous airway GABA at in the airway, (2) GABA increasing after procontractile (3) selective of endogenous GABA of airway smooth muscle GABAAchannels resulting in increased muscle (4) GABAAchannel antagonism is greater under contracted resting muscle tone, and allosteric with endogenous relaxation of tissue of a GABAergic system in airway smooth muscle that to the of contractile force.

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