Effects of enantiomers of beta 2-agonists on ACh release and smooth muscle contraction in the trachea

Xiang-Yang Zhang, Feng-Xia Zhu, Michal A. Olszewski, and N. Edward Robinson

Departments of Large Animal Clinical Sciences and Physiology, Michigan State University, East Lansing, Michigan 48824-1314

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The beta 2-agonists currently used as bronchodilators are racemic mixtures of R- and S-enantiomers. In the present study, we examined the effects of enantiomers of the beta 2-agonists albuterol and formoterol on acetylcholine (ACh) release from equine trachealis parasympathetic nerves. ACh release was evoked by electrical field stimulation (20 V, 0.5 ms, 0.5 Hz) and measured by high-performance liquid chromatography coupled with electrochemical detection. We also tested the effects of enantiomers of albuterol and formoterol on equine tracheal smooth muscle (TSM) contraction in response to exogenous ACh. R- and RS-albuterol (10-8 to 10-5 M) and RR- and RR/SS-formoterol (10-8 to 10-5 M) augmented ACh release in a concentration-dependent manner. Beginning at 10-6 M, SS-formoterol significantly increased ACh release, and at 10-5 M, release increased by 71.9 ± 8.7% over baseline. This effect was only observed, however, when the prejunctional muscarinic autoinhibitory effect of ACh was prevented with atropine. Both the RR- and SS-formoterol-induced increases in ACh release were abolished by the beta 2-antagonist ICI-118551 (3 × 10-7 M). The effect of S-albuterol on ACh release was variable, and the mean increase induced by 10-5 M was 30.8 ± 16.1% in the presence of atropine. In the muscle tension study, R- and RS-albuterol and RR- and RR/SS-formoterol (10-8 to 10-5 M) but not the S-enantiomers inhibited TSM contraction. Even though R-enantiomers augment ACh release, they potently inhibit TSM contraction. Because racemic beta 2-agonists are bronchodilators on acute administration, the postjunctional spasmolytic effects of R-enantiomers predominate over the spasmogenic effect evoked via increased ACh release. The S-enantiomers, in contrast, do not inhibit TSM contraction and therefore would not contribute to the observed bronchodilation of the racemate. The S-enantiomers do prejunctionally facilitate ACh release when prejunctional muscarinic autoreceptors are dysfunctional, suggesting a potentially deleterious effect.

acetylcholine; albuterol; formoterol; cholinergic neurotransmission; airway hyperresponsiveness; muscarinic receptor

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

EPIDEMIOLOGIC STUDIES suggest a possible correlation between increased asthma morbidity and mortality and the use of beta 2-agonists in continuous or excessive amounts (18). Clinically, a loss of protective efficacy with a subsequent deterioration of asthma control is observed after regular use of beta 2-agonists (16, 19). Sears et al. (16) and Taylor et al. (19) reported that chronic use of inhaled beta 2-agonists resulted in more exacerbations, a significant decline in baseline lung function, and an increase in airway responsiveness to methacholine in asthmatic subjects but did not alter bronchodilator responsiveness. These results indicated that regular treatment with inhaled beta 2-agonists may be deleterious in the long-term control of asthma but that dysfunction of the receptor on airway smooth muscle was unlikely to be involved in the beta 2-agonist-induced hyperresponsiveness of the asthmatic airway. beta 2-Agonists currently used in the clinic are racemic mixtures of R- and S-enantiomers. Experimental studies in the guinea pig have demonstrated that prolonged use of a racemic mixture (7, 10, 12, 14, 22) or acute administration of the S-enantiomer of beta 2-agonists (12, 14) can induce hyperresponsiveness to inhaled spasmogens or antigens in ovalbumin-sensitized airways. Bilateral vagotomy before application of beta 2-agonists prevents development of airway hyperresponsiveness; however, the mechanism of hyperresponsiveness remains unknown (12, 14).

Activation of beta 2-adrenoceptors (beta 2-ARs) by isoproterenol (Iso) augments acetylcholine (ACh) release from guinea pig (3) and equine (23, 24, 26) airway parasympathetic nerves. It is therefore plausible to hypothesize that the S-enantiomer of beta 2-agonists may possess the ability to increase ACh release from airway parasympathetic nerves and that this augmentation of ACh release may contribute to airway hyperresponsiveness induced by beta 2-agonists. In the present study, we tested the effects of enantiomers of the beta 2-agonists albuterol and formoterol on ACh release evoked by electric field stimulation (EFS) from equine trachealis parasympathetic nerves in both the absence and presence of the muscarinic-autoreceptor blocker atropine. To make a simultaneous comparison of their pre- and postjunctional effects, we also examined the postjunctional effects of enantiomers of albuterol and formoterol on equine tracheal smooth muscle (TSM) contraction induced by exogenous ACh.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Tissue

Tissue was collected from 16 horses (body weight 412.0 ± 11.6 kg) for this study, which was approved by the All-University Committee on Animal Use and Care of Michigan State University (East Lansing). Other investigators also used tissues from the same animals for a variety of studies. The horses had no clinical signs of respiratory disease for several weeks before they were killed by injection of an overdose of pentobarbital sodium through the jugular vein. Postmortem examination revealed that the lungs and airways were normal in gross appearance. A segment of the trachea between the 6th and 30th cartilaginous rings above the carina was quickly collected, immersed in Krebs-Henseleit (KH) solution (composition in mM: 118.4 NaCl, 25.0 NaHCO3, 11.7 dextrose, 4.7 KCl, 2.6 CaCl2 · 2H2O, 1.19 MgSO4 · 7H2O, and 1.16 KH2PO4), and gassed with 95% O2-5% CO2 during the whole experiment.

The trachea was opened longitudinally by dissection of the cartilages in its anterior aspect and was pegged flat on a paraffin block submerged in KH solution. TSM strips with epithelium intact were cut with a template along the fiber direction before they were suspended in tissue baths. The temperature within the baths was maintained at 37°C, and the KH solution was changed every 15 min. Square-wave electrical impulses were produced by a stimulator (S88, Grass Instrument, Quincy, MA) and passed through a stimulus power booster (Stimu-Splitter II, Med-Lab Instruments, Loveland, CO) to electrodes in the tissue baths.

Measurement of EFS-Induced ACh Release

Four tissue strips (each measuring 2 × 15 mm) were cut and tied together at both ends with 3-0 surgical silk thread. Each trachealis strip bundle (wet weight 196.8 ± 4.0 mg; n = 72 strips) was suspended in a 2-ml tissue bath by a pair of parallel platinum wire electrodes built against the wall of the bath in the vertical direction (Radnoti Glass Technology, Monrovia, CA). One end of the tissue strip was secured to the bottom of the bath with a glass tissue holder; the other end was attached to an 8-g weight via a surgical thread that passed over a steel bar above the tissue bath (21). After an ~120-min equilibration period, the tissues were incubated for 60 min with the cholinesterase inhibitor neostigmine (10-6 M) and the sympathetic nerve blocker guanethidine (10-5 M) with or without the muscarinic-autoreceptor antagonist atropine (10-7 M). These agents were present during the remainder of the experiment.

The ACh concentration in the tissue bath liquid was measured by high-performance liquid chromatography coupled with electrochemical detection. The mobile phase contained 100 mM Na2HPO4 (pH = 8.0), and the flow rate was 0.35 ml/min. The samples were filtered through 0.2-µm nylon membrane filters (Acrodiscs 13, Gelman Sciences, Ann Arbor, MI) and injected into the high-performance liquid chromatography column at a volume of 25 µl/injection. An external ACh standard (2.5 pmol in 25 µl) was injected every six samples, and the concentration of ACh in the samples was calculated based on the bracketed calibration. For details of this technique, see Wang et al. (21).

Muscle Tension Study

Strips (2 × 10 mm) tied with 3-0 surgical silk thread on both ends were suspended between platinum ring electrodes in 15-ml tissue baths that contained KH solution. The lower thread was attached to a hook on the electrode unit, whereas the upper thread was connected to a force transducer (Grass model FTO3) that was mounted on a manipulator to adjust tissue length. Tension produced by the tissue was measured by the transducer and recorded on a polygraph (Grass model 7E). EFS (16 Hz, 20 V, 0.5 ms) was applied for 2-3 min at 7- to 10-min intervals to equilibrate the muscle. Passive tension (optimal tissue length) was adjusted for each strip to produce the maximal response to EFS. Equilibration was ended after ~90 min when both baseline tension and the response to EFS were stable. After equilibration, the maximal tension response to a 127 mM KCl-substituted KH solution was recorded in grams, and the tissues were repeatedly washed until the muscle tension returned to baseline.

Study Design

Protocol 1: Effect of R-, RS-, and S-albuterol and RR-, RR/SS-, and SS-formoterol on EFS-induced ACh release. Seven tissue-strip bundles from each horse were used in the absence of the muscarinic-receptor blocker atropine. One horse did not receive enantiomers of beta 2-agonists and served as the control. The remainder received one of the following six treatments: R-, RS-, or S-albuterol or RR-, RR/SS-, or SS-formoterol. EFS (0.5 Hz, 0.5 ms, 20 V) was applied to all the tissues for five 15-min periods, with a 30-min resting interval between consecutive stimuli. During the first EFS, we determined the baseline release of ACh. Enantiomers of albuterol or formoterol (10-8 to 10-5 M) were then added to the baths 10 min before subsequent EFS. To eliminate any ACh that may have been released during the incubation period (21), the tissue baths were drained and refilled with fresh KH solution containing the tested drugs immediately before the beginning of EFS. Tissue bath solution was collected on the completion of each EFS for the measurement of ACh. The tissues were rinsed four times with the KH solution immediately after the collection of samples. At the end of the experiment, the tissues were blotted dry and weighed. This protocol was repeated in the presence of blockade of muscarinic receptors with atropine (10-7 M).

To determine whether enantiomers exerted their effect via beta 2-ARs, we tested the effect of the specific beta 2-AR antagonist ICI-118551 (4, 5) on RR- or SS-formoterol-induced augmentation of ACh release. Five tissue-strip bundles incubated with atropine (10-7 M) were used. One bundle received neither beta 2-agonist nor antagonist and served as the control. The remainder received either RR-formoterol (10-10 to 10-8 M) or SS-formoterol (10-6 to 10-4 M) in the absence or presence of ICI-118551 (3 × 10-7 M). After the first EFS, ICI-118551 was added to the baths 30 min before and RR- or SS-formoterol was added 10 min before subsequent EFS.

Protocol 2: Effect of R-, RS-, and S-albuterol and RR-, RR/SS-, and SS-formoterol on smooth muscle contraction in response to exogenous ACh. Exogenous ACh (10-7 to 10-4 M) was applied to each of the strips to elicit smooth muscle contraction. The first concentration-response curve (priming curve) was not used for data analysis. The subsequent five curves were used to determine the postjunctional effect of enantiomers of albuterol or formoterol (10-8 to 10-5 M) on smooth muscle contraction in response to exogenous ACh. Seven TSM strips were used. One strip served as a time control; the other six each received four concentrations of either R-, RS-, or S-albuterol or RR-, RR/SS-, or SS-formoterol (10-8 to 10-5 M) after the first exogenous ACh-induced contraction. Enantiomer solutions were added to the baths 10 min before subsequent applications of exogenous ACh. Before generation of another concentration-response curve to exogenous ACh, the tissues were washed for 30 min with at least four changes of KH solution.

Drugs

Atropine sulfate, ACh chloride, neostigmine methylsulfate, guanethidine monosulfate (Sigma Chemical, St. Louis, MO), ICI-118551 hydrochloride (Research Biochemicals International, Natick, MA), and enantiomers of albuterol and formoterol (Sepracor, Marlborough, MA) were dissolved and diluted in KH solution. All the drugs were prepared on the day of experiment. The drug solution was pipetted into the tissue bath at 1% of the bath volume. The final concentration of the drugs was expressed as their bath molar concentration.

Data Analysis

The ACh release rate is expressed both in picomoles per gram per minute and as a percentage of the baseline value (first EFS without drug treatment). The contractile responses of the muscle strips were expressed as percentages of their maximal tension responses to a 127 mM KCl-substituted KH solution. Means ± SE for all parameters were calculated. The data were evaluated by repeated-measures analysis of variance and analysis of variance with contrasts by Statview II (Abacus Concepts, Calabasas, CA) for the Macintosh computer. Means were compared by Fisher's (protected) least significant difference test. Significance was accepted at the 0.05 level of probability.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Protocol 1: Effect of R-, RS-, and S-Albuterol and RR-, RR/SS-, and SS-Formoterol on EFS-Induced ACh Release

In the absence of atropine, baseline ACh release, i.e., release during the first EFS period, averaged 1.39 ± 0.08 pmol · g-1 · min-1 (n = 48 strips). In the time-control tissues, ACh release remained constant throughout the five stimulations. R- and RS-albuterol and RR- and RR/SS-formoterol augmented ACh release in a concentration-dependent manner (Fig. 1). The augmentation reached significance at concentrations of 10-7 M for R- and RS-albuterol and 10-8 M for RR- and RR/SS-formoterol. Neither S-albuterol nor SS-formoterol (10-8 to 10-5 M) had any effect on ACh release in the absence of atropine (Fig. 1).


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Fig. 1.   Effects of R-, RS-, and S-albuterol (A) and RR-, RR/SS-, and SS-formoterol (B) at concentrations from 10-8 to 10-5 M on acetylcholine (ACh) release from equine trachealis strips (n = 5) in response to electrical field stimulation (20 V, 0.5 ms, 0.5 Hz) in absence of muscarinic-autoreceptor blocker. * Significantly different from time control.

In the presence of blockade of muscarinic receptors with atropine (10-7 M), baseline ACh release was increased to 5.50 ± 0.35 pmol · g-1 · min-1 (n = 49 strips). Under these conditions, R- and RS-albuterol and RR- and RR/SS-formoterol still augmented ACh release in a concentration-dependent manner. The augmentation reached a plateau at the concentrations of 10-6 M for R- and RS-albuterol and 10-8 M for RR- and RR/SS-formoterol (Fig. 2). Maximal augmentation, i.e., an approximate doubling of ACh release, was of similar magnitude to that induced by activation of beta 2-ARs by Iso (23, 24, 26). Beginning at 10-6 M, SS-formoterol significantly increased ACh release, and at 10-5 M, release increased by 71.9 ± 8.7% over baseline. The effect of S-albuterol on ACh release was variable, and the mean increase induced by 10-5 M was 30.8 ± 16.1% (Fig. 2).


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Fig. 2.   Effects of R-, RS-, and S-albuterol (A) and RR-, RR/SS-, and SS-formoterol (B) at concentrations from 10-8 to 10-5 M on ACh release from equine trachealis strips (n = 6) in response to electrical field stimulation (20 V, 0.5 ms, 0.5 Hz) in presence of muscarinic- autoreceptor blockade with atropine (10-7 M). * Significantly different from time control.

In the presence of atropine, both RR-formoterol (10-10 to 10-8 M) and SS-formoterol (10-6 to 10-4 M) facilitated ACh release in a concentration-dependent manner. The RR- and SS-formoterol-induced augmentation of ACh release was abolished by the beta 2-antagonist ICI-118551 (Fig. 3).


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Fig. 3.   Antagonizing effects of beta 2-antagonist ICI-118551 (3 × 10-7 M) on RR-formoterol (A)- and SS-formoterol (B)-induced augmentation of ACh release from equine trachealis strips (n = 5) in response to electrical field stimulation (20 V, 0.5 ms, 0.5 Hz). * Significantly different from time control.

Protocol 2: Effect of R-, RS-, and S-Albuterol and RR-, RR/SS-, and SS-Formoterol on Smooth Muscle Contraction in Response to Exogenous ACh

Application of exogenous ACh in the tissue bath concentration dependently contracted the TSM. Even though the contractile response to subsequent ACh in the time-control TSM decreased slightly, the difference was not significant (data not shown). R- and RS-albuterol (Fig. 4, A and B) and RR- and RR/SS-formoterol (Fig. 5, A and B) at concentrations of 10-8 to 10-5 M inhibited the contractile responses to exogenous ACh in a concentration-dependent manner. The inhibition became significant at concentrations of 10-7 M for R- and RS-albuterol and 10-8 M for RR- and RR/SS-formoterol. However, S-albuterol (10-8 to 10-5 M ) and SS-formoterol (10-8 to 10-5 M) had no significant effect on exogenous ACh-induced TSM contraction (Figs. 4C and 5C).


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Fig. 4.   Effects of R- (A), RS- (B), and S-albuterol (C) on equine trachealis contractile response (n = 5 strips) to exogenous ACh (10-7 to 10-4 M). Inhibitory effects of R- and RS-albuterol on exogenous ACh-induced contraction became significant at concentration of 10-7 M. S-albuterol had no effect.


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Fig. 5.   Effects of RR- (A), RR/SS- (B), and SS-formoterol (C) on equine trachealis contractile response (n = 5 strips) to exogenous ACh (10-7 to 10-4 M). Inhibitory effects of RR- and RR/SS-formoterol on exogenous ACh-induced contraction became significant at concentration of 10-8 M. SS-formoterol had no significant effect.

    DISCUSSION
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Introduction
Materials & Methods
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beta 2-Agonists are widely used as bronchodilators because of their ability to dilate airways via beta 2-receptors on smooth muscle cells (6). However, there is increasing concern that regular use of beta 2-agonists has a deleterious effect on the control of asthma (1, 16, 18, 19). Although a number of theories have been advanced to explain such an effect, none is well established (1, 15). In the airways of most species, the parasympathetic innervation is the predominant neural pathway (2), and ACh released from cholinergic nerve terminals provides the dominant constrictor mechanism. Zhang and colleagues (23, 24, 26) previously demonstrated that the racemic beta -agonist Iso increases ACh release from equine airway parasympathetic nerves. The Iso-induced augmentation of ACh release is abolished by the beta 2-antagonist ICI-118551 but not by the beta 1-antagonist CGP-20712A, indicating that beta 2-ARs mediate the facilitation of ACh release (23, 24, 26). Activation of beta 2-ARs also facilitates [3H]ACh release from guinea pig tracheal parasympathetic nerves (3). These excitatory beta 2-ARs may have important consequences in the treatment of airway obstructive disease because beta 2-agonists might paradoxically potentiate the release of ACh while relaxing smooth muscle.

The beta 2-agonists used for treatment of airway obstruction currently are the racemic mixture of R- and S-enantiomers. The differential effects of enantiomers on neuromuscular regulation of airway tone have been suspected as one of the possible mechanisms for the beta 2-agonist-induced deleterious effects (15). It has been reported that prolonged exposure to a racemic mixture (7, 10, 22) or acute administration of S-enantiomers (12, 14) of beta 2-agonists can induce hyperresponsiveness of ovalbumin-sensitized guinea pig airways to spasmogens. However, the S-enantiomer no longer elicits airway hyperresponsiveness when given after the vagus nerves are sectioned (12, 14). These observations indicate that induction of airway hyperresponsiveness by the S-enantiomer of beta 2-agonists may involve neurotransmitters released by the vagus nerves. For this reason, we hypothesized that the differential actions of enantiomers of beta 2-agonists prejunctionally on ACh release from airway parasympathetic nerves and postjunctionally on airway smooth muscle contraction may contribute to the beta 2-agonist-induced airway hyperresponsiveness.

The prejunctional autoinhibitory M2 receptors are dysfunctional in ovalbumin-sensitized guinea pig airway parasympathetic nerves after antigen challenge (9) and in virus-infected guinea pigs (8) and also may be dysfunctional in some asthmatic patients (13). Therefore, in the present experiment, we first examined the effects of R-, RS-, and S-albuterol and RR-, RR/SS-, and SS-formoterol on ACh release from airway parasympathetic nerves in both the absence and presence of the muscarinic-autoreceptor blocker atropine (10-7 M). We demonstrated that in the absence of muscarinic blockade, only R- and RS-albuterol and RR- and RR/SS-formoterol, but not S-albuterol and SS-formoterol, augmented ACh release. However, after the blockade of the muscarinic autoinhibitory receptor, SS-formoterol significantly increased EFS-induced ACh release, and a similar, although lesser, trend was noted with S-albuterol. To explain these observations, we considered the response of the muscarinic autoreceptor to ACh. When a weak stimulus such as the S-enantiomer causes only a small increase in ACh release, this increase is totally masked by the negative feedback originating from the muscarinic autoreceptor. Only when this feedback is prevented by atropine is it possible to observe the ACh-augmenting effect of the S-enantiomer. When there is a greater increase in ACh release, such as that induced by the R-enantiomer, the muscarinic autoreceptor cannot totally prevent the increase. Hence the R-enantiomer effect on ACh release is observed even in the absence of autoreceptor blockade by atropine.

Both RR- and SS-formoterol-induced facilitations of ACh release were abolished by the specific beta 2-antagonist ICI-118551, suggesting that facilitation of ACh release by both RR- and SS-enantiomers is via activation of beta 2-ARs. Zhang et al. (25) previously demonstrated that activation of beta 2-ARs facilitates ACh release via the intracellular adenosine 3',5'-cyclic monophosphate pathway. Thus we concluded that the augmenting effect of both the R- and S-enantiomers on ACh release is also mediated via the intracellular adenosine 3',5'-cyclic monophosphate pathway.

To make a simultaneous comparison of their pre- and postjunctional effects, we also tested the effects of enantiomers of both albuterol and formoterol on TSM contraction in response to exogenous ACh. The results revealed that R- and RS-albuterol and RR- and RR/SS-formoterol potently inhibited exogenous ACh-induced smooth muscle contraction, whereas S-albuterol and SS-formoterol had little effect. Johansson et al. (11) also recently reported that R- and RS-albuterol are far more potent than S-albuterol at inhibiting carbachol-induced TSM contraction in guinea pigs.

Because both R- and S-enantiomers of beta 2-agonists augment ACh release after blockade of autoinhibitory muscarinic M2 receptors, it seems likely that the deleterious effects may be a property of both enantiomers. However, to truly evaluate the effects of these enantiomers on airway smooth muscle function, one must consider the combined pre- and postjunctional effects. Even though R-enantiomers prejunctionally augment ACh release, they potently inhibit TSM contraction postjunctionally. Because racemic beta 2-agonists are demonstrably bronchodilatory on acute administration (12), the postjunctional spasmolytic effects of R-enantiomers must predominate over the prejunctional spasmogenic effect that is evoked via increased ACh release. However, in the presence of prejunctional M2-receptor dysfunction, S-enantiomers might enhance rather than inhibit smooth muscle contraction because they increase ACh release but have no effect on smooth muscle.

Metabolism of racemic beta 2-agonists is stereoselective in humans (17, 20), with minimal absorption and preferential and rapid elimination of the R-enantiomer. In contrast, the S-enantiomer of albuterol exhibits a three- to fourfold greater plasma level and a significantly longer duration. This stereoselective metabolism and subsequent disproportionate accumulation of the S-enantiomer of beta 2-agonists may result in the progressive loss of the protective effectiveness of the beta 2-agonist mediated by action of the R-enantiomer on smooth muscle and may thus be responsible for airway hyperresponsiveness. In our present study, S-enantiomers of beta 2-agonists increased release of ACh, an airway spasmogen, from airway parasympathetic nerves in the presence but not in the absence of muscarinic autoinhibitory receptor blockade. These results are consistent with the observation that racemic or S-enantiomer beta 2-agonist-evoked airway hyperresponsiveness can be induced more readily in ovalbumin-sensitized animals in which M2 receptors are dysfunctional than in control animals in which autoinhibitory M2 receptors are functional (12). In the situation of prejunctional M2-receptor dysfunction in airways (8, 9, 13), and combined with the preferential elimination of R-enantiomers (17, 20), S-enantiomer-mediated facilitation of ACh release may become evident and may contribute to the beta 2-agonist-induced airway hyperresponsiveness.

    ACKNOWLEDGEMENTS

We thank Cathy Berney for technical assistance and Victoria Hoelzer-Maddox and MaryEllen Shea for manuscript preparation.

    FOOTNOTES

This work was supported in part by Sepracor Inc. and by an endowment from the Matilda R. Wilson Fund.

Address for reprint requests: Dr. X.-Y. Zhang, Dept. of Large Animal Clinical Sciences, Michigan State Univ., East Lansing, MI 48824-1314.

Received 25 June 1997; accepted in final form 23 September 1997.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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