Neutrophilic inflammation in small airways (SA) and bronchospasm mediated via muscarinic receptors are features of chronic obstructive pulmonary disease in horses (COPD). Histamine, serotonin, and leukotrienes (LTs) are reported to be involved in the exacerbation of COPD, and currently, histamine has been shown to increase tension response to electrical field simulation (EFS) in equine SA. We tested the effects of these mediators and the effects of activated neutrophils on the cholinergic responses in SA. Histamine, serotonin, and LTD4 had a synergistic effect on EFS responses and only an additive effect on the tension response to exogenous ACh or methacholine. Atropine and TTX entirely eliminated the EFS-induced tension response in the presence of all three inflammatory mediators, indicating that augmentation of the EFS response applies only to the endogenous cholinergic response. Neutrophils isolated from control and COPD-affected horses were activated by zymosan, producing 18.1 ± 2.3 and 25.0 ± 2.3 nmol superoxide ⋅ 106cells−1 ⋅ 30 min−1, respectively. However, in contrast to the profound effect of mediators, incubation of SA for over 1 h in a suspension of up to 30 × 106 zymosan-treated neutrophils/ml did not significantly affect EFS responses of SA isolated from either control or COPD-affected horses. We conclude that in equine SA 1) the endogenous cholinergic responses are subject to strong facilitation by inflammatory mediators; 2) activated neutrophils do not affect cholinergic responses in SA; and 3) in acute bouts of equine COPD, histamine, LTD4, and serotonin (mediators primarily associated with type I allergic reaction) rather than mediators derived from neutrophils most likely contribute to increased cholinergic airway tone.

The β2-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 β2-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 β2-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 β2-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.

No abstract

To evaluate the functional status of neuronal α2-adrenoceptors (ARs) and β2-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 β2-agonists RR- and RR/ SS-formoterol on ACh release from airway cholinergic nerves of horses with RAO. Because SS-formoterol, a distomer of the β2-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 β2-ARs are functioning; however, the α2-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.

The effects of capsaicin and neuropeptides were examined in equine tracheal smooth muscle (TSM). Neither capsaicin nor substance P (SP) contracted TSM. Capsaicin (100 μM) elicited relaxation in TSM contracted with methacholine. This relaxation was not mimicked by SP or calcitonin gene-related peptide (CGRP). Relaxation was not attenuated by removal of the epithelium or by pretreatment of tissue with meclofenamate or the nitric oxide (NO) synthase inhibitor N G-nitro-l-arginine. Previous exposure of TSM to capsaicin did not eliminate the relaxation responses to subsequent capsaicin. Although vasoactive intestinal peptide (VIP) elicited marked relaxation that was attenuated by α-chymotrypsin, α-chymotrypsin did not affect the capsaicin-induced relaxation. Capsaicin-induced relaxation was abolished by charybdotoxin, a blocker of large-conductance Ca2+-activated K+ channels. These results indicate that capsaicin-induced equine TSM relaxation is not mediated either by neuropeptides such as SP or CGRP released from capsaicin-sensitive sensory nerves or by prostanoids, NO, or VIP. Relaxation is due to the effect of capsaicin on large-conductance Ca2+-activated K+ channels. The peptidergic nerves play no important role in the regulation of TSM tone in horse airways.