N. Edward Robinson

Mediators of anaphylaxis but not activated neutrophils augment cholinergic responses of equine small airways

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. 10(6) cells-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 x 10(6) 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.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.

Experimental induction of pulmonary fibrosis in horses with the gammaherpesvirus equine herpesvirus 5.

Gammaherpesviruses (γHV) are implicated in the pathogenesis of pulmonary fibrosis in humans and murine models of lung fibrosis, however there is little direct experimental evidence that such viruses induce lung fibrosis in the natural host. The equine γHV EHV 5 is associated with equine multinodular pulmonary fibrosis (EMPF), a progressive fibrosing lung disease in its natural host, the horse. Experimental reproduction of EMPF has not been attempted to date. We hypothesized that inoculation of EHV 5 isolated from cases of EMPF into the lungs of clinically normal horses would induce lung fibrosis similar to EMPF. Neutralizing antibody titers were measured in the horses before and after inoculation with EHV 5. PCR and virus isolation was used to detect EHV 5 in antemortem blood and BAL samples, and in tissues collected postmortem. Nodular pulmonary fibrosis and induction of myofibroblasts occurred in EHV 5 inoculated horses. Mean lung collagen in EHV 5 inoculated horses (80 µg/mg) was significantly increased compared to control horses (26 µg/mg) (p < 0.5), as was interstitial collagen (32.6% ± 1.2% vs 23% ± 1.4%) (mean ± SEM; p < 0.001). Virus was difficult to detect in infected horses throughout the experiment, although EHV 5 antigen was detected in the lung by immunohistochemistry. We conclude that the γHV EHV 5 can induce lung fibrosis in the horse, and hypothesize that induction of fibrosis occurs while the virus is latent within the lung. This is the first example of a γHV inducing lung fibrosis in the natural host.

Effects of enantiomers of β2-agonists on ACh release and smooth muscle contraction in the trachea

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.

Endotoxin concentrations within the breathing zone of horses are higher in stables than on pasture

Inflammatory airway disease is common in stabled horses, with a prevalence of 17.3% in Michigan pleasure horses. Stable dust is rich in endotoxin, which may induce neutrophilic airway inflammation. Climatological conditions (ambient temperature and relative humidity) may influence endotoxin concentrations in pastures. The aim of this project was to determine if endotoxin levels in the breathing zone of horses in stables were higher than of horses on pasture, and if the endotoxin on pasture was associated with climatological conditions. Endotoxin exposure of six horses that were stabled or on pasture was determined by a Limulus amebocyte lysate assay. Climatological data were obtained from the US National Climatic Data Center. Endotoxin exposure was significantly higher (about 8-fold) in stables than on pasture. On pasture, endotoxin varied widely, despite constant climatological conditions. It was concluded that stabled horses are exposed to higher endotoxin concentrations than horses on pastures. Local endotoxin concentrations may be more important than ambient climatological conditions in determining endotoxin exposure of individual horses.

Mechanism of capsaicin-induced relaxation in equine tracheal smooth muscle

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.The effects of capsaicin and neuropeptides were examined in equine tracheal smooth muscle (TSM). Neither capsaicin nor substance P (SP) contracted TSM. Capsaicin (100 microM) 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 NG-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 alpha-chymotrypsin, alpha-chymotrypsin did not affect the capsaicin-induced relaxation. Capsaicin-induced relaxation was abolished by charybdotoxin, a blocker of large-conductance Ca(2+)-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 Ca(2+)-activated K+ channels. The peptidergic nerves play no important role in the regulation of TSM tone in horse airways.

Immunohistochemical analysis of laryngeal muscles in normal horses and horses with subclinical recurrent laryngeal neuropathy.

We used immunohistochemistry to examine myosin heavy-chain (MyHC)-based fiber-type profiles of the right and left cricoarytenoideus dorsalis (CAD) and arytenoideus transversus (TrA) muscles of six horses without laryngoscopic evidence of recurrent laryngeal neuropathy (RLN). Results showed that CAD and TrA muscles have the same slow, 2a, and 2x fibers as equine limb muscles, but not the faster contracting fibers expressing extraocular and 2B MyHCs found in laryngeal muscles of small mammals. Muscles from three horses showed fiber-type grouping bilaterally in the TrA muscles, but only in the left CAD. Fiber-type grouping suggests that denervation and reinnervation of fibers had occurred, and that these horses had subclinical RLN. There was a virtual elimination of 2x fibers in these muscles, accompanied by a significant increase in the percentage of 2a and slow fibers, and hypertrophy of these fiber types. The results suggest that multiple pathophysiological mechanisms are at work in early RLN, including selective denervation and reinnervation of 2x muscle fibers, corruption of neural impulse traffic that regulates 2x and slow muscle fiber types, and compensatory hypertrophy of remaining fibers. We conclude that horses afflicted with mild RLN are able to remain subclinical by compensatory hypertrophy of surviving muscle fibers.

Pre- and postjunctional effects of inflammatory mediators in horse airways.

In addition to their direct contractile effects, histamine (Hist), serotonin [5-hydroxytryptamine (5-HT)], and leukotriene (LT) D4, in low concentrations, dramatically augment electrical field stimulation (EFS)-induced smooth muscle contractions in equine airways. To determine the mechanism of their action, we studied, in trachealis strips, the effect of these mediators on both cholinergically induced tension and the release of ACh from cholinergic nerves. All three mediators synergistically augmented the contraction of the trachealis that was due to release of endogenous ACh, i.e., EFS-induced contraction. These same mediators caused only a small but parallel shift of the ACh concentration-response curve. Comparison of the mediator effects on the responses to endogenous and exogenous ACh suggested a prejunctional effect. However, release of ACh was augmented only by Hist and 5-HT but not by LTD4. Hist-induced contraction of trachealis was abolished by pyrilamine (H1-receptor antagonist) but not by ranitidine (H2-receptor antagonist), whereas thioperamide (H3-receptor antagonist) shifted the Hist response curve to the left. The augmenting effect of Hist on EFS-induced contraction was abolished by pyrilamine and unaffected by ranitidine or thioperamide. We conclude that inflammatory mediators can increase endogenous cholinergic responses of equine airways via both prejunctional and postjunctional mechanisms. LTD4 acts solely on smooth muscle, whereas 5-HT and Hist additionally act on neuronal receptors to facilitate release of ACh. Excitatory effects of Hist, i.e., direct contractile effect, and augmentation of endogenous cholinergic response are both mediated via H1receptors, whereas the inhibitory H3 receptors partially oppose the direct contractile effect of this mediator.In addition to their direct contractile effects, histamine (Hist), serotonin [5-hydroxytryptamine (5-HT)], and leukotriene (LT) D(4), in low concentrations, dramatically augment electrical field stimulation (EFS)-induced smooth muscle contractions in equine airways. To determine the mechanism of their action, we studied, in trachealis strips, the effect of these mediators on both cholinergically induced tension and the release of ACh from cholinergic nerves. All three mediators synergistically augmented the contraction of the trachealis that was due to release of endogenous ACh, i.e., EFS-induced contraction. These same mediators caused only a small but parallel shift of the ACh concentration-response curve. Comparison of the mediator effects on the responses to endogenous and exogenous ACh suggested a prejunctional effect. However, release of ACh was augmented only by Hist and 5-HT but not by LTD(4). Hist-induced contraction of trachealis was abolished by pyrilamine (H(1)-receptor antagonist) but not by ranitidine (H(2)-receptor antagonist), whereas thioperamide (H(3)-receptor antagonist) shifted the Hist response curve to the left. The augmenting effect of Hist on EFS-induced contraction was abolished by pyrilamine and unaffected by ranitidine or thioperamide. We conclude that inflammatory mediators can increase endogenous cholinergic responses of equine airways via both prejunctional and postjunctional mechanisms. LTD(4) acts solely on smooth muscle, whereas 5-HT and Hist additionally act on neuronal receptors to facilitate release of ACh. Excitatory effects of Hist, i.e., direct contractile effect, and augmentation of endogenous cholinergic response are both mediated via H(1) receptors, whereas the inhibitory H(3) receptors partially oppose the direct contractile effect of this mediator.

Distribution of Venous Remodeling in Exercise-induced Pulmonary Hemorrhage of Horses Follows Reported Blood Flow Distribution in the Equine Lung

Exercise-induced pulmonary hemorrhage (EIPH), which has been reported in humans and a variety of domestic animals following strenuous exercise, is most often documented in racehorses. Remodeling of pulmonary veins (VR) in equine EIPH was recently described, suggesting that it contributes to the pathogenesis of the disease. The cause of VR is unknown. We tested the hypothesis that the development of VR follows pulmonary blood flow distribution, preferentially occurring in the caudodorsal lung region. Furthermore, we hypothesized that VR underpins development of the other lesions of EIPH pathology. The lungs of 10 EIPH-affected horses and 8 controls were randomly sampled for histopathology (2,520 samples) and blindly scored for presence and severity of VR, hemosiderin (H), and interstitial fibrosis (IF). Mean sample score (MSS), mean lesion score, and percent samples with lesions were determined in four dorsal and three ventral lung regions, and the frequency, spatial distribution, and severity of lesions were determined. MSS for VR and H were significantly greater dorsally than ventrally (P < 0.001) and also decreased significantly in the caudocranial direction (P < 0.001). IF decreased only in the caudocranial direction. The percent samples with lesions followed the same distribution as MSS. VR often was accompanied by H; IF never occurred without VR and H. Similarity of the distribution of EIPH lesions and the reported fractal distribution of pulmonary blood flow suggests that VR develops in regions of high blood flow. Further experiments are necessary to determine whether VR is central to the pathogenesis of EIPH.

Prejunctional muscarinic autoreceptors on horse airway cholinergic nerves

Muscarinic autoreceptors on horse airway cholinergic nerves were studied by examining the effects of muscarinic receptor antagonists on electrical field stimulation (EFS)-induced acetylcholine (ACh) release in trachealis preparations. All the antagonists including atropine (non-selective), pirenzepine (M1-selective), AF-DX 116 (M2-selective), and hexahydrosiladifenidol (M3-selective) augmented ACh release concentration-dependently. The augmentation was not due to displacement of ACh molecules from tissue receptors into the bath liquid because incubation with atropine after EFS had no influence on the measured amount of ACh. Hexahydrosiladifenidol was more potent in inhibiting ACh-induced muscle contraction, which is known to be mediated by M3 receptors, than in augmenting ACh release. The maximal ACh release rate in response to the selective antagonists was much less than that following atropine. Furthermore, the concentrations of the selective antagonists required to augment ACh release far exceeded their KdS for M1, M2, or M3 receptors. These observations suggest that the muscarinic autoreceptors on horse airway cholinergic nerves may belong to a novel subtype.

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

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.To evaluate the functional status of neuronal alpha2-adrenoceptors (ARs) and beta2-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 beta2-agonists RR- and RR/SS-formoterol on ACh release from airway cholinergic nerves of horses with RAO. Because SS-formoterol, a distomer of the beta2-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 beta2-ARs are functioning; however, the alpha2-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.