Modulation of ACh release from airway cholinergic nerves in horses with recurrent airway obstruction

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

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To evaluate the functional status of neuronal alpha 2-adrenoceptors (ARs) and beta 2-ARs on ACh release in horses with recurrent airway obstruction (RAO), we examined the effects of the physiological agonists epinephrine (Epi) and norepinephrine (NE) and the beta 2-agonists RR- and RR/SS-formoterol on ACh release from airway cholinergic nerves of horses with RAO. Because SS-formoterol, a distomer of the beta 2-agonist, increases ACh release from airways of control horses only after the autoinhibitory muscarinic receptors are blocked by atropine, we also tested the hypothesis that if there is an M2-receptor dysfunction in equine RAO, SS-formoterol should increase ACh release even in the absence of atropine. ACh release was evoked by electrical field stimulation and measured by HPLC. Epi and NE caused less inhibition of ACh release in horses with RAO than in control horses. At the catecholamine concentration achieved during exercise (10-7 M), the inhibition induced by Epi and NE was 10.8 ± 13.2 and 3.4 ± 6.8%, respectively, in equine RAO versus 41.0 ± 6.4 and 27.1 ± 5.6%, respectively, in control horses. RR- and RR/SS-formoterol (10-8 to 10-5 M) increased ACh release to a similar magnitude as that in control horses. These results indicate that neuronal beta 2-ARs are functioning; however, the alpha 2-ARs are dysfunctional in the airways of horses with RAO in response to circulating catecholamines. SS-formoterol (10-8 to 10-5 M) facilitated ACh release in horses with RAO even in the absence of atropine. Addition of atropine did not cause significantly more augmentation of ACh release over the effect of SS-formoterol alone. The magnitude of augmentation in horses with RAO in the absence of atropine was similar to that in control horses in the presence of atropine. The latter observations could be explained by neuronal muscarinic-autoreceptor dysfunction in equine RAO.

adrenoceptor; prejunctional; muscarinic receptor; catecholamine; formoterol; enantiomer; autoreceptor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACUTE EXACERBATIONS of recurrent airway obstruction (RAO) of horses are characterized by bronchospasm, inflammation of the tracheobronchial tree, and nonspecific airway hyperresponsiveness (14). The rapid decrease in pulmonary resistance and increase in dynamic compliance after muscarinic blockade with atropine indicates that a large part of the bronchospasm is mediated through cholinergic mechanisms (3).

The release of ACh from equine airway cholinergic nerves is inhibited prejunctionally by an inhibitory alpha 2-adrenoceptor (AR) (20, 22) and an autoinhibitory muscarinic receptor (18). Dysfunction of these receptors may result in an exaggerated release of ACh, an airway spasmogen, and contribute to the tracheobronchial constriction and hyperresponsiveness in horses with RAO. In normal horses, the alpha 2-AR is activated by levels of circulating catecholamines achieved during exercise (16, 22). Wang et al. (19) have previously demonstrated that the inhibitory effect of the alpha 2-AR agonist clonidine on ACh release is lacking in bronchi and less potent in the trachea of horses with RAO. We wanted to know whether the alpha 2-AR dysfunction is reflected in the response to endogenous catecholamines. Therefore, in the first protocol of the present study, we examined the effect of epinephrine (Epi; 10-8 to 10-5 M) and norepinephrine (NE; 10-8 to 10-5 M) on ACh release from airway parasympathetic nerves of horses with RAO.

Zhang and colleagues (21, 22, 24, 25) were the first to report an excitatory beta 2-AR in airway parasympathetic nerves. After their report in the horse, this receptor was subsequently reported in guinea pig (2) and human airway parasympathetic nerves (5). In airway preparations of control horses, activation of beta 2-ARs by isoproterenol, by the racemic mixture of specific beta 2-agonists such as albuterol and formoterol, or by R-enantiomers of beta 2-agonists can increase ACh release in a concentration-dependent manner. The maximal augmentation resulting from activation of beta 2-ARs is approximately twofold (21, 22, 24, 25). To evaluate the neuronal beta 2-AR function in horses with RAO, in the second protocol of our present study, we examined the effect of the racemic mixture of formoterol (RR/SS-enantiomer) and RR-formoterol on ACh release from tracheal parasympathetic nerves of horses with RAO.

The prejunctional muscarinic autoreceptor provides negative feedback on airway cholinergic nerves, and these receptors are dysfunctional in guinea pigs challenged by ovalbumin (9) or infected with parainfluenza virus (8) and in human asthmatic patients (13). However, the functional status of neuronal M2 receptors in equine RAO is still unknown. SS-formoterol, a distomer of the beta 2-AR agonist, increases ACh release in the control horses only in the presence of muscarinic blockade with atropine, i.e., when neuronal M2 receptors are dysfunctional (24). Therefore, in the third protocol of the present study, we tested the hypothesis that if there is a prejunctional M2-receptor dysfunction in horses with RAO, SS-formoterol should increase ACh release even in the absence of muscarinic blockade with atropine.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. The study was approved by the All-University Committee on Animal Use and Care of Michigan State University (East Lansing). Six horses (body wt 481.7 ± 29.0 kg, age 14.5 ± 1.2 yr) with RAO during acute exacerbation were studied. Several days before the experiment, the RAO-susceptible horses were transferred from the pasture to stalls where they were fed dusty hay (6). They remained in the stalls until clinical signs of airway obstruction, such as flared nostrils and forced abdominal effort during expiration, were observed.

Tissue preparation. The animals were euthanized by injection of an overdose of pentobarbital sodium through the jugular vein. Other investigators also used tissues from the same animals for a variety of studies. Postmortem examination revealed that the lungs remained inflated when the chest was opened, and plugs of mucus and exudate were observed in the airways of all the animals. A segment of the trachea between the 16th 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 in its anterior aspect by dissection of the cartilages and was pegged flat on a paraffin block submerged in KH solution. Tracheal smooth muscle strips with the epithelium intact were cut with a template along the fiber direction before being 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 Instruments, 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 electrical field stimulation-induced ACh release. Four tissue strips (each measuring 2 × 15 mm) were cut and tied together at both their ends with 3-0 surgical silk thread. Each trachealis strip bundle (wet weight 217.3 ± 3.3 mg; n = 72 strips) was suspended in a 2-ml tissue bath with 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 (17). 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 and 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 HPLC 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 HPLC column at a volume of 25 µl/injection. An external ACh standard (2.5 pmol in 25 µl) was injected into every six samples, and the concentration of ACh in the samples was calculated based on the bracketed calibration (for details of this technique, see Refs. 17, 22).

Study design. Electrical field stimulation (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. The tested drugs were then added to the baths 10 min before subsequent EFS. To eliminate any ACh that may have been released during the incubation period (17), the tissue baths were drained and refilled with fresh KH solution containing the tested drugs immediately before EFS was begun. The 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 collection of the samples. At the end of the experiment, the tissues were blotted dry and weighed.

Protocol 1: Effects of Epi and NE on EFS-induced ACh release from trachealis cholinergic nerves of horses with RAO. Three tissue-strip bundles from each horse were used in the presence of the muscarinic-receptor blocker atropine. One did not receive tested drug treatment and served as the time control. The second and third tissue-strip bundles each received Epi (10-8 to 10-5 M) and NE (10-8 to 10-5 M). The results of the effects of Epi and NE on ACh release obtained from the horses with RAO were compared with those of control horses in a previously published study from our laboratory (22).

Protocol 2: Effects of RR- and RR/SS-formoterol on EFS-induced ACh release from trachealis cholinergic nerves of horses with RAO. Six tissue-strip bundles from each horse were used. Three tissue-strip bundles were used in the absence of the muscarinic-receptor blocker atropine. One did not receive beta 2-agonists and served as the time control. The remaining two received either RR- or RR/SS-formoterol (10-8 to 10-5 M) after the first EFS. A similar protocol was repeated with the other three tissue-strip bundles in the presence of muscarinic-receptor blockade with atropine (10-7 M). The magnitude of augmentation of ACh release in horses with RAO was compared with that in control horses (24) to evaluate the neuronal beta 2-AR function in horses with RAO.

Protocol 3: Effects of SS-formoterol on EFS-induced ACh release from trachealis cholinergic nerves of horses with RAO. Four tissue-strip bundles from each horse were used. Two tissue-strip bundles were used in the absence of the muscarinic-receptor blocker atropine. One did not receive SS-formoterol and served as the time control. The other received SS-formoterol (10-8 to 10-5 M). A similar protocol was repeated with the other two tissue-strip bundles in the presence of muscarinic-receptor blockade with atropine (10-7 M). The results of the effects of SS-formoterol on ACh release obtained from the horses with RAO were compared with those of control horses (24) to evaluate the neuronal muscarinic function in horses with RAO.

Drugs. Atropine sulfate, acetylcholine chloride, neostigmine methylsulfate, guanethidine monosulfate (all from Sigma, St. Louis, MO), and enantiomers of formoterol (Sepracor, Marlborough, MA) were dissolved and diluted in KH solution. Epinephrine bitartrate and norepinephrine hydrochloride (Sigma) were dissolved and diluted in a 0.1% ascorbate solution. All the drugs were prepared on the day of the 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 results of the present study obtained from horses with RAO were compared with data from historical control horses studied by the same personnel in the same laboratory. The ACh release rate is expressed in both picomoles per gram per minute and a percentage of baseline (first EFS without drug treatment). Means ± SE for all parameters were calculated; n is the number of horses studied except where otherwise specified. The percent inhibition of Epi and NE at each concentration (10-8 to 10-5 M) on ACh release in horses with RAO was compared with that in control horses (22) with unpaired Student's t-test. The other data were evaluated by repeated-measures ANOVA and ANOVA with contrasts with Statview II (Abacus Concepts, Calabasas, CA) for the Macintosh computer. Means were compared by Fisher's (protected) least significant difference test. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the absence of atropine, EFS-induced baseline ACh release averaged 1.55 ± 0.11 pmol · g-1 · min-1 (n = 24 strips). This value was not significantly different from that in the control horses (1.39 ± 0.08 pmol · g-1 · min-1; Ref. 24). In the presence of blockade of muscarinic receptors with atropine (10-7 M), EFS-induced baseline ACh release reached 5.23 ± 0.27 pmol · g-1 · min-1 (n = 48 strips). This rate of ACh release is similar to that in the control horses (5.50 ± 0.35 pmol · g-1 · min-1; Ref. 24).

Protocol 1: Effects of Epi and NE on EFS-induced ACh release from trachealis cholinergic nerves of horses with RAO. In time-control tissues of horses with RAO, ACh release remained constant throughout the five stimulations. Although the maximal inhibition was similar to that in the control horses (22), the response curve to both Epi and NE was shifted to the right in RAO-affected animals. Epi and NE caused a significant inhibition of ACh release only at the concentrations of 10-6 and 10-5 M but not at 10-7 M or lower (Fig. 1). Figure 2 compares the percent inhibition of ACh release by Epi and NE in airways of control horses (22) and horses with RAO. At the catecholamine concentrations achieved during exercise (10-7 M), the inhibition induced by Epi and NE was 10.8 ± 13.2 and 3.4 ± 6.8%, respectively, in RAO-affected horses (n = 6). However, in the control horses, the inhibition was 41.0 ± 6.4 and 27.1 ± 5.6%, respectively (22). The differences were significant (Fig. 2).


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Fig. 1.   Effect of epinephrine and norepinephrine at indicated concentrations on ACh release in response to electrical field stimulation (20 V, 0.5 ms, 0.5 Hz) of trachealis strips from horses with recurrent airway obstruction (RAO; n = 6). * Significantly different from time control.




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Fig. 2.   Comparison of percent inhibition of ACh release with epinephrine (A) and norepinephrine (B) at indicated concentrations in response to electrical field stimulation (20 V, 0.5 ms, 0.5 Hz) of trachealis strips from control horses (n = 6; Ref. 22) and horses with RAO (n = 6). * Significantly different from control horses.

Protocol 2: Effects of RR- and RR/SS-formoterol on EFS-induced ACh release from trachealis cholinergic nerves of horses with RAO. In the absence of atropine, ACh release from time-control tissues remained constant throughout the five stimulations. RR- and RR/SS-formoterol augmented ACh release in a concentration-dependent manner (Fig. 3A). The augmentation reached significance at the concentration of 10-8 M for both RR- and RR/SS-formoterol. At the concentration of 10-6 M, RR- and RR/SS-formoterol increased ACh release to 199.3 ± 24.6 and 187.5 ± 8.8%, respectively, of baseline (n = 6 animals). This magnitude is similar to that in the control horses (181.9 ± 9.1 and 187.7 ± 22.7%, respectively, of baseline; Ref. 24). In the presence of atropine, RR- and RR/SS-formoterol also increased ACh release in a concentration-dependent manner (Fig. 3B). The maximal augmentation, i.e., an approximate doubling of ACh release that was reached at a concentration of 10-7 M for both RR- and RR/SS-formoterol, was of similar magnitude to that in the control horses (24).



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Fig. 3.   Effect of RR- and RR/SS-formoterol at indicated concentrations on ACh release in response to electrical field stimulation (20 V, 0.5 ms, 0.5 Hz) of trachealis strips from horses with RAO (n = 6) in absence (A) and presence (B) of muscarinic-autoreceptor blockade with atropine (10-7 M). * Significantly different from time control.

Protocol 3: Effects of SS-formoterol on EFS-induced ACh release from trachealis cholinergic nerves of horses with RAO. In the control horses, SS-formoterol (10-8 to 10-5 M) had no effect on ACh release in the absence of atropine. However, SS-formoterol dose dependently facilitated ACh release in the presence of atropine, and at the concentration of 10-5 M, SS-formoterol increased the release to 166.1 ± 7.7% of baseline (Fig. 4A; Ref. 24). In the horses with RAO, SS-formoterol (10-8 to 10-5 M) increased ACh release even in the absence of atropine (Fig. 4B). At the concentration of 10-5 M, SS-formoterol increased ACh release to 172.3 ± 13.5% of baseline (n = 6 animals). The magnitude of augmentation in the absence of atropine in horses with RAO (Fig. 4B) was similar to that in the control horses in the presence of atropine (Fig. 4A; Ref. 24). The addition of atropine slightly but not significantly increased ACh release over the effect of SS-formoterol alone (Fig. 4B).



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Fig. 4.   Effect of SS-formoterol at indicated concentrations on ACh release in response to electrical field stimulation (20 V, 0.5 ms, 0.5 Hz) of trachealis strips from control horses (A; n = 6; Ref. 24) and horses with RAO (B; n = 6) in absence and presence of muscarinic-autoreceptor blockade with atropine (10-7 M). * Significantly different from time control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of catecholamines on ACh release from equine airway parasympathetic nerves is mediated by both alpha 2-inhibitory and beta 2-excitatory adrenoceptors, with the former being predominant (22). The sympathoadrenal discharge of the horse increases the mean plasma concentrations of Epi and NE from 9 ± 10-10 and 7 ± 10-10 M, respectively, at rest, to 1.53 ± 10-7 and 1.48 ± 10-7 M, respectively, during exercise (16). In a previous study, Zhang et al. (22) demonstrated that both Epi and NE at concentrations of 10-8 to 10-5 M dose dependently inhibited ACh release from airway parasympathetic nerves of control horses and that these inhibitions are mediated via alpha 2-ARs. In our present study, we demonstrated that at the catcholamine concentration achieved during exercise (10-7 M), the inhibition induced by Epi and NE was significantly less in horses with RAO than in control horses. This decreased inhibitory effect of catecholamines may be due to the increased rate of catecholamine metabolism by the smooth muscle of our tissue preparations in horses with RAO. However, this is unlikely because Wang et. al. (19) from our laboratory demonstrated that clonidine, a pharmacological alpha 2-AR agonist that is inactivated independent of catecholamine metabolic pathways, also possesses less inhibitory effect on ACh release from RAO-affected horses. Because Epi can activate both an inhibitory alpha 2-AR and an excitatory beta 2-AR (22), the decreased inhibition of Epi on ACh release may also be due to either a decreased inhibitory effect of alpha 2-AR, an increased facilitatory effect of beta 2-AR, or a combination of both. However, our results demonstrated that in airway parasympathetic nerves of the RAO-affected horse, activation of beta 2-ARs with both RR- and RR/SS-formoterol (10-8 to 10-5 M) increased ACh release with a similar magnitude as that in control horses (24). This result indicated the absence of prejunctional beta 2-AR dysfunction in airway parasympathetic nerves of horses with RAO. Therefore, attenuated inhibition of Epi on ACh release from RAO-affected animals must be due to the dysfunction of prejunctional inhibitory alpha 2-ARs. Because the inhibitory effect of NE is solely mediated via alpha 2-ARs (22), decreased NE-induced inhibition of ACh release from RAO-affected animals provides additional evidence of prejunctional alpha 2-AR dysfunction. Combining all the above evidence, we conclude that the prejunctional alpha 2-AR is dysfunctional in horses with RAO and that this alpha 2-AR dysfunction is reflected in the response to endogenous catecholamines (16). However, the function of prejunctional beta 2-ARs is normal.

In the control horses, Zhang et al. (24) have previously demonstrated that SS-formoterol, a distomer of the beta 2-agonist, facilitates ACh release in the presence of atropine, which blocks the prejunctional muscarinic autoinhibitory receptor, but not in the absence of atropine when the neuronal muscarinic autoinhibitory receptor is functional (Fig. 4A). Therefore, we hypothesized that if the prejunctional M2 receptors are dysfunctional in equine RAO, SS-formoterol should increase ACh release even in the absence of muscarinic blockade with atropine. In support of this hypothesis, our results revealed that SS-formoterol facilitated ACh release in the absence of atropine and that the magnitude of augmentation in the horses with RAO in the absence of atropine was similar to that in the control horses in the presence of atropine. Furthermore, in the RAO-affected horses, blockade of muscarinic receptors by atropine did not cause significantly more augmentation of ACh release over the effect of SS-formoterol alone (Fig. 4B). These observations suggest that the prejunctional muscarinic autoreceptors in airway parasympathetic nerves of horses with RAO are dysfunctional.

The neuronal autoinhibitory M2 receptors are also dysfunctional in ovalbumin-sensitized guinea pig airway parasympathetic nerves after antigen challenge (9), in virus-infected guinea pigs (8), and in ozone-challenged guinea pigs (15) and also may be dysfunctional in some asthmatic patients (13). However, the pathogenesis of M2-receptor dysfunction is different in the different diseases. In antigen-challenged guinea pigs (12) and human asthmatic patients (4), there is a large influx of eosinophils in the airway. These eosinophils and their major basic protein, an antagonist for M2 receptor (12), are especially associated with airway nerves (4). In virus-infected guinea pigs, viruses have both an indirect leukocyte-dependent effect and a leukocyte-independent effect on M2 receptors (10). In ozone-challenged guinea pigs, leukocytes or mediators are important in the pathogenesis of the loss of M2-receptor function (11). Equine RAO is a delayed hypersensitivity response to inhaled antigen, particularly the thermophilic molds and actinomycetes that grow in damp hay (14), and is characterized by a predominant neutrophilic inflammation with few eosinophils as revealed by bronchoalveolar lavage (7). However, the role of inflammation or mediators in the development of neuronal M2-receptor dysfunction in equine RAO is unknown, and further investigations are needed.

Despite the apparent M2-receptor dysfunction, we noticed that the magnitude of RR- and RR/SS-formoterol-induced augmentation of ACh release was similar in the two groups of animals and that the amount of ACh release evoked by EFS in picomoles per gram per minute in equine RAO was not different from that in the control horses. Why should the neuronal M2-receptor dysfunction be reflected only in the response to SS-formoterol, a weak beta 2-agonist, but not in the response to the potent beta 2-agonists (RR- and RR/SS-formoterol) or EFS-induced baseline ACh release? Previous studies in airway parasympathetic nerves (23) and the myenteric plexus (1) suggest that neuronal M2 receptor-mediated inhibition of ACh release is via at least two different intracellular mechanisms: cAMP-dependent and cAMP-independent pathways, with the latter predominant, whereas the facilitation of ACh release by beta 2-AR agonists involves only cAMP-dependent pathways (23). These two pathways are diagrammed in Fig. 5. If neuronal M2-receptor dysfunction in equine RAO affects the cAMP-dependent but not the cAMP-independent pathway, the dysfunction of the M2 receptors would be reflected primarily in their responses to agents that act via intracellular cAMP. Under this scenario, activation of beta 2-AR by a weak agonist, SS-formoterol, has no effect in control horses because the ACh released during EFS activates the cAMP-dependent autoinhibitory mechanism and prevents the beta 2-AR agonist-induced increase in cAMP (Fig. 5). However, when the cAMP-dependent autoinhibitory pathway is dysfunctional, the weak agonist increases cAMP and facilitates release. Such is the situation in RAO-affected horses and in control horse tissue after atropine treatment. By contrast, when the beta 2-ARs are activated by full beta 2-AR agonists such as RR- and RR/SS-formoterol, the increase in intracellular cAMP cannot be overridden by the relatively weak cAMP-dependent M2-receptor autoinhibitory pathway and ACh release increases. In this situation, M2-mediated inhibitory cAMP-dependent pathway dysfunction is irrelevant because the autoinhibition of cAMP production is trivial in comparison to the major increase in cAMP engendered by the strong beta 2-AR agonist.


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Fig. 5.   Diagrammatic representation of mechanisms regulating release of ACh from parasympathetic nerves. M2 autoreceptor inhibits ACh release by both cAMP-dependent and -independent pathways. The latter, which is predominant, may involve direct coupling to an ion channel. beta 2-Adrenoceptor (beta -AR) is coupled only to a cAMP-dependent pathway. We hypothesize that when M2 receptor is functional, its inhibitory effect (-) on cAMP production prevents augmenting effect (+) of a weak beta 2-agonist, SS-formoterol, on ACh release. In presence of M2 dysfunction (RAO or atropine), SS-formoterol can increase cAMP and facilitate ACh release. Increase in ACh release caused by atropine is mediated primarily via cAMP-independent pathway (channel). (----), Major inhibitory role of ion channel on ACh release.

A similar explanation can be used to explain the absence of a difference in EFS-induced ACh release between tissues from control and RAO-affected horses, the fact that atropine still increases ACh release in such horses, and even the dysfunction of the prejunctional inhibitory alpha 2-AR. If the primary M2-receptor mechanism is cAMP independent and there is dysfunction only in the cAMP-dependent pathway, it will be very difficult to detect differences in release between diseased and control horses, and atropine will still block the activation of the cAMP-independent pathway and therefore increase ACh release (Fig. 5). As with the M2 receptor, the prejunctional inhibitory alpha 2-AR acts primarily via cAMP-independent pathways but to a small degree via cAMP-dependent pathways (26). Dysfunction solely in the latter pathway may explain the reduced inhibition of ACh release by lower concentrations of Epi and NE in RAO-affected animals.

In conclusion, the present study demonstrated in the airway of RAO-affected horses that 1) the prejunctional inhibitory alpha 2-AR is dysfunctional, which may lead to less inhibition of bronchospasm in exercising horses with RAO; 2) neuronal beta 2-AR function is normal; and 3) SS-formoterol-mediated augmentation of ACh release in the absence of atropine could be explained by neuronal M2-receptor dysfunction. Therefore, dysfunction of prejunctional receptors is not a generalized effect but may be restricted to specific pathways.


    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 an endowment from the Matilda R. Wilson Fund and by Sepracor, Inc.

X.-Y. Zhang is a fellow of the American Lung Association of Michigan.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: N. E. Robinson, Dept. of Large Animal Clinical Sciences, Michigan State Univ., East Lansing, MI 48824-1314 (E-mail: Robinson{at}cvm.msu.edu).

Received 21 September 1998; accepted in final form 25 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 276(5):L769-L775
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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