Relaxation of equine tracheal muscle in vitro by different adrenoceptor drugs

Strips of tracheal smooth muscle from 12 horses were contracted by carbachol in tissue baths under isometric conditions. This contraction (≅50% of maximum: EC₅₀) was relaxed completely with adrenoceptor drugs. The only exception was clenbuterol, where the degree of relaxation was ≅90%. In all horses the EC₅₀-value for isoprenaline (mean 1.6 × 10⁻⁸M) was less than that for adrenaline (mean 9.6 × 10^{− 8}M) and noradrenaline (mean 1. 8 × 10_{- 6}M). The potency ratio was 1 < 6 < 110 which indicates that the β₂-subtype dominates among the β-adrenoceptors of equine airways. All preparations were also very sensitive to the specific and potent β₂-receptor agonists clenbuterol (mean 5.7 × 10^{− 9}M) and procaterol (mean 3.6 × 10⁻¹⁰M). No differences in EC₅₀-values due to age, sex and breed were observed in this material. The standard deviation of the mean EC₅₀-values seems to be larger for the specific β₂-adrenoceptor agonists than for the unspecific. A reason for this could be differences in the pattern of the β-adrenoceptor population.     

Response of equine airway smooth muscle to acetylcholine and electrical stimulation in vitro

Smooth muscle strips from the midcervical portion of the trachea and bronchial smooth muscle strips from third-generation airways of horses were placed in tissue baths, and isometric contractile force was measured. Active force was measured in response to electrical stimulation and exogenous acetylcholine. Square-wave electrical stimuli were applied at various voltages (10, 12, 15, 18, 20, 25 V), frequencies (3, 5, 10, 15, 20, 25, 30 Hz), and pulse durations (0.2, 0.5, 1.0, 1.5, 2.0 ms). Isometric contractile force increased as voltage, frequency, and pulse duration increased. Maximal contractile response to electrical stimulation was obtained at 18 V, 25 Hz, and 0.5 ms. Atropine (10(-6)M) or tetrodotoxin (3 x 10(-6)M) blocked the contraction, indicating that the contractile response was attributable to the release of neurotransmitter from cholinergic nerves. Cumulative concentration-response curves to acetylcholine (10(-9)M through 10(-4)M) were determined. Isometric contractile force increased as acetylcholine concentration increased. There was a significant (P less than 0.05) difference in the 50% effective dose for acetylcholine in tracheal smooth muscle and bronchial smooth muscle. The mean (+/- SD) contractile response to maximal electrical stimulus was 89% (+/- 7.4%) of that in response to 10(-4)M acetylcholine in tracheal smooth muscle and was 68% (+/- 10.4%) of the response to 10(-4)M acetylcholine in bronchial smooth muscle.     

Participation of H1-receptors in histamine-induced contraction and relaxation of horse coronary artery in vitro.

The mechanisms of histamine-induced contraction and relaxation were investigated in rings isolated from a middle part of the left descending coronary arteries of horses. Intact and endothelium-denuded preparations were compared. Rings of horse coronary arteries contracted in response to histamine in a concentration dependent manner, but some of them relaxed with lower concentrations and contracted with higher concentrations. Removal of the endothelium abolished the relaxation and potentiated the contraction. The pD2 values were 4.70 +/- 0.08 in the rings with intact endothelium and 4.95 +/- 0.08 in endothelium-denuded rings. Histamine-induced contractions in intact and denuded preparations were not affected by an H2-antagonist, cimetidine, but were inhibited by an H1-antagonist, diphenhydramine in non-competitive manner in the rings with endothelium and in competitive manner in denuded rings. After precontraction with PGF2 alpha or norepinephrine, histamine relaxed preparations with intact endothelium (pD2 value, 7.80 +/- 0.11), although histamine-induced relaxations were not observed in denuded preparations. The relaxation was competitively inhibited by diphenhydramine. Relaxing response was significantly attenuated by methylene blue, quinacrine, L-nitro-arginine, gossypol and AA861 but not by indomethacin. These results suggest that the histamine-induced contraction and relaxation in horse coronary arteries are mediated mainly by H1-receptors in the smooth muscle and endothelium, respectively, and H1-receptor activation of endothelial cells may liberate vasodilator substances.     

Isoproterenol- and salbutamol-induced relaxation of acetylcholine- and histamine-induced contraction of equine trachealis muscle in vitro

Strips of trachealis muscle were dissected from the midcervical portion of the trachea of horses that were free of respiratory tract disease, and the overlying epithelium and mucosa were removed. Muscle strips were suspended in tissue baths that were filled with Krebs-bicarbonate solution, aerated with 5% CO2 in oxygen and maintained at 37 C. Isometric tension was continuously recorded. The increase in active isometric tension was concentration dependent when acetylcholine (10(-9) to 10(-4) M) or histamine (10(-9) to 10(-4) M) was added to the tissue baths in 0.5-logarithmic increments. When the tissues were contracted with acetylcholine (3.1 x 10(-6) M) or histamine (10(-4) M), the decrease in active isometric tension was concentration dependent when isoproterenol (10(-9) to 10(-4) M) or salbutamol (10(-9) to 10(-4) M) was added to the tissue baths in 0.5-logarithmic increments. There was no difference between the response to isoproterenol and salbutamol when tissues from the same horses were compared whether the tissues were contracted in response to acetylcholine (3.1 x 10(-6) M) or histamine (10(-4) M). Relaxation was antagonized by 10(-6) M propranolol. The degree of relaxation obtained in these muscle strips was considerably less than that reported from other species' tracheal muscle strips that had the epithelium and mucosa intact. We concluded that equine tracheal smooth muscle contains beta-adrenoceptors that can be stimulated by either a mixed beta-1, beta-2 agonist or a selective beta-2 agonist.     

A comparison between clenbuterol, salbutamol and terbutaline in relation to receptor binding and in vitro relaxation of equine tracheal muscle

Beta₂-adrenoceptor agonists are used as bronchodilators in both humans and horses. Of these drugs, clenbuterol is the one most frequently used when treating chronic obstructive pulmonary disease in the horse, while salbutamol and terbutaline are used in the treatment of human asthma. Little is known of the properties of the latter two drugs in equine medicine.
  We have compared salbutamol and terbutaline with clenbuterol in relation to their ability to relax muscle strips from equine tracheal muscle, pre- contracted with 40 n m carbachol, in tissue chambers. The affinities of these drugs to the β₂-adrenoceptors in homogenates of the same muscle tissue were also examined. These experiments were performed with radioligand binding studies using the very potent β-adrenoceptor antagonist ¹²⁵I-cyanopindolol.
  The three drugs were almost equipotent in relaxing the muscle strips. The EC₅₀-values for salbutamol, terbutaline and clenbuterol were 5.6, 13.8 and 2.1 n m , respectively, and all three drugs relaxed the preparations completely. In the competitive binding study, however, the K_d-value of clenbuterol was much lower (24 n m ) than that of salbutamol and terbutaline (1100 n m and 3900 n m , respectively). The amount of receptors bound at the EC₅₀-value of clenbuterol was 8% compared to less than 1% for salbutamol and terbutaline. This indicates a lower intrinsic efficacy of clenbuterol than of the other two drugs. The β-adrenoceptor density was 45 ± 14.3 fmol/mg protein (mean ± SD) and the K_d-value of ¹²⁵I-cyanopindolol was 11.4 ± 3.3 p m .     

Effects of verapamil on the smooth muscle of the horse urinary tract

The effects of verapamil, a calcium antagonist agent, were studied on smooth muscle preparations of the lower urinary tract of horses. Verapamil (2 X 10(-4) to 2 X 10(-8) M) relaxed the ureter, urethra and urinary bladder preparations contracted by potassium (127 mM), L-noradrenaline (2 X 10(-5) M), histamine (2 X 10(-5) M) and acetylcholine (2 X 10(-5) M). These results allow the conclusion that verapamil has a dose-dependent relaxing effect on smooth muscle of the lower urinary tract.     

Effects of 8-epi-PGF2α on isolated bronchial smooth muscle of healthy and heaves-affected horses

8-Epi-PGF2alpha, a prostaglandin-like compound generated by oxidative stress, has been shown to be an in vitro bronchoconstrictor in airways from healthy laboratory animals and healthy humans, but it has never been studied in diseased airways. Here, the bronchoconstrictive capacity of 8-epi-PGF2alpha on isolated bronchial rings (BR) of healthy and heaves-affected horses was evaluated by comparing the maximal effect and the potency of 8-epi-PGF2alpha to those of (1) acetylcholine (ACh), (2) its stereoisomer PGF2alpha and (3) its synthetic receptor agonist, U46619. Furthermore, the potential capacity of 8-epi-PGF2alpha to enhance the cholinergic (ACh) responsiveness of bronchial smooth muscle was investigated. 8-Epi-PGF2alpha contracted BR with a rank order of efficacy of Ach > U44619 > PGF2alpha > 8-epi-PGF2alpha in both healthy and heaves-affected horses. The contractile maximal response elicited by 8-epi-PGF2alpha was significantly smaller than that elicited by the other drugs, but was significantly higher in BR from heaves-affected horses than in those sampled in healthy horses, whilst pD2 values were similar. A subthreshold concentration of 8-epi-PGF2alpha (10-7 M) did not induce in vitro cholinergic hyper-responsiveness in BR of either healthy or heaves-affected horses. In conclusion, it has been demonstrated that 8-epi-PGF2alpha is an in vitro bronchoconstrictor of minor importance in healthy horses, but whose efficacy is significantly increased in heaves-affected horses.     

Evaluation of clinical signs of disease, bronchoalveolar and tracheal wash analysis, and arterial blood gas tensions in 13 horses with chronic obstructive pulmonary disease treated with prednisone, methyl sulfonmethane, and clenbuterol hydrochloride.

We evaluated the efficacy of 3 treatments for chronic obstructive pulmonary disease in horses: prednisone (400 mg/horse, PO, daily; n = 7), methyl sulfonmethane (10 g/horse, PO, q 12 h; n = 6), and clenbuterol hydrochloride (0.4 mg/horse, PO, q 12 h; n = 7). A fourth group acted as controls (n = 6) and was not treated. The treatment period lasted 10 days. Each horse was a member of 2 different groups for 10 days, separated by an 18-day interval of no treatment. All horses were housed together in an outdoor pen without bedding. Horses were fed alfalfa/grass hay mix ad libitum from a large feeder. The same batch of hay was fed throughout the study. Multiple physical and laboratory variables were monitored prior to, during, and at the end of each 10-day trial period. Changes in lung sounds, respiratory effort, degree of anal movement, nasal discharge, temperature, respiratory rate, or heart rate were not significant. Changes in arterial blood gas tensions, tracheal wash or bronchoalveolar lavage cytologic findings, or phagocyte function were not significant. All horses were tachypneic and most were tachycardic. The median value for PaO2 was below normal for all horses. All tracheal wash and most bronchoalveolar lavage cytologic findings represented a suppurative response. Negative linear correlation was observed between PaO2 and degree of respiratory effort in these horses (eg, as PaO2 decreased, the degree of respiratory effort increased).     

Palmar digital vessel relaxation in healthy horses and in horses given carbohydrate

OBJECTIVE: To compare in vitro smooth muscle relaxation of palmar digital vessels from healthy horses with those from horses in the prodromal stage of experimentally (carbohydrate) induced laminitis. ANIMALS: 16 adult horses. PROCEDURE: Segments of palmar digital vessels were obtained from 5 healthy horses and 6 horses given carbohydrate. Vascular rings from the palmar digital artery and vein were suspended in individual organ baths containing buffer solution and indomethacin; isometric tension was recorded, and contraction and relaxation were compared. Smooth muscle contraction in response to cumulative addition of phenylephrine was recorded in the absence and presence of 1 microM NG-nitro-L-arginine methyl ester (L -NAME). After wash out, vascular rings were preconstricted with phenylephrine (0.3 microM), and cumulative endothelium-dependent (acetylcholine-induced) and independent (nitroprusside-induced) smooth muscle relaxations were recorded in the absence or presence of L -NAME. RESULTS: Phenylephrine increased vascular smooth muscle tone in ring preparations of palmar digital arteries and veins. Addition of acetylcholine or nitroprusside induced relaxation of palmar digital artery and vein ring preparations. Use of L-NAME (1 microM) significantly reduced maximal relaxation induced by acetylcholine, but not by nitroprusside. Maximal relaxation induced by acetylcholine, but not by nitroprusside, was reduced in vascular rings prepared from carbohydrate-overloaded horses. CONCLUSION AND CLINICAL RELEVANCE: Reduced endothelium-dependent relaxation of palmar digital vessels may have a role in the pathophysiology of acute laminitis after carbohydrate overload in horses.     

Influence of clenbuterol on mucociliary transport in healthy horses and horses with chronic obstructive pulmonary disease

The mucociliary tracheal transport rate and clearance index were measured in 16 healthy horses and 16 horses suffering from chronic obstructive pulmonary disease. A marker, Indian ink in syrup, was placed in the mucus layer of the interior wall of the trachea, just in front of the carina, and its displacement was measured after 30 and 60 minutes. The same procedure was repeated 30 minutes after the intravenous administration of 0.8 microgram clenbuterol/kg bodyweight. In the diseased horses the mucociliary transport rate was significantly lower than in the healthy horses. After clenbuterol administration there was a significant increase in the transport rate and a similar increase in the clearance index. Sixty and 90 minutes after clenbuterol there was a significant increase of mucus secretion in both the healthy and diseased horses. Measurements in five healthy horses and five diseased horses on two different days gave similar results, showing that the technique provided reproducible data.     

Intratracheal clenbuterol in the horse: its pharmacological efficacy and analytical detection

Clenbuterol, a beta2 agonist/antagonist, is the only bronchodilator approved by the US Food and Drug Administration for use in horses. The Association of Racing Commissioners International classifies clenbuterol as a class 3 agent, and, as such, its identification in post-race samples may lead to sanctions. Anecdotal reports suggest that clenbuterol may have been administered by intratracheal (IT) injection to obtain beneficial effects and avoid post-race detection. The objectives of this study were (1) to measure the pharmacological efficacy of IT dose of clenbuterol and (2) to determine the analytical findings in urine in the presence and absence of furosemide. When administered intratracheally (90 microg/horse) to horses suffering from chronic obstructive pulmonary disease (COPD), clenbuterol had effects that were not significantly different from those of saline. In parallel experiments using a behavior chamber, no significant effects of IT clenbuterol on heart rate or spontaneous locomotor activity were observed. Clenbuterol concentrations in the urine were also measured after IT dose in the presence and absence of furosemide. Four horses were administered i.v. furosemide (5 mg/kg), and four horses were administered saline (5 mL). Two hours later, all horses were administrated clenbuterol (IT, 90 microg), and the furosemide-treated horses received a second dose of furosemide (2.5 mg/kg, i.v.). Three hours after clenbuterol dose (1 h after hypothetical 'post-time'), the mean specific gravity of urine samples from furosemide-treated horses was 1.024, well above the 1.010 concentration at which furosemide is considered to interfere with drug detection. There was no interference by furosemide with 'enhanced' ELISA screening of clenbuterol equivalents in extracted and concentrated samples. Similarly, furosemide had no effect on mass spectral identification or quantification of clenbuterol in these samples. These results suggest that the IT dose of clenbuterol (90 microg) is, in pharmacological terms, indistinguishable from the dose of saline, and that, using extracted samples, clenbuterol dose is readily detectable at 3 h after dosing. Furthermore, concomitant dose of furosemide does not interfere with detection or confirmation of clenbuterol.     

In vitro effects of erythromycin, lidocaine, and metoclopramide on smooth muscle from the pyloric antrum, proximal portion of the duodenum, and middle portion of the jejunum of horses

OBJECTIVE: To evaluate effects of erythromycin, lidocaine, and metoclopramide on smooth muscle of the pyloric antrum (PA), proximal portion of the duodenum (PD), and middle portion of the jejunum (MJ) of horses. Sample Population-Strips of smooth muscle from 7 horses. PROCEDURE: Isolated muscle strips were suspended in a bath and attached to isometric force transducers. Once stable spontaneous contractions were observed, agents were added. Isometric stress responses were compared with the amplitude of spontaneous contractions. RESULTS: A single dose of erythromycin to the PA increased contractile amplitude (CA) for the longitudinal smooth muscle (mean +/- SEM, 76+/-16 g/cm2) but decreased CA for circular smooth muscle (-79+/-23 g/cm2). The inhibitory effect was decreased by tetrodotoxin, N(G)-nitro-L-arginine methyl ester, and a vasoactive intestinal peptide antagonist. Erythromycin increased CA for the MJ, which was maximal at 10(-4)M (171+/-36 g/cm2). Lidocaine increased CA for the PD, which was maximal at 10(-4) M (60+/-5 g/cm2). Metoclopramide increased the CA, which was maximal at 10(-4) M for the PA (75+/-26 g/cm2), PD (279+/-33 g/cm2), and MJ (456+/-59 g/cm2). CONCLUSIONS: Regional differences in responses to erythromycin, lidocaine, and metoclopramide were evident in the gastrointestinal tract of horses. Metoclopramide increased CA in all tissues used, whereas erythromycin inhibited CA in circular smooth muscle but stimulated CA in longitudinal smooth muscle from the PA. Inhibition is caused by stimulation of inhibitory nerves and is mediated, in part, by nitric oxide and vasoactive intestinal peptide.     

Can an external device prevent dorsal displacement of the soft palate during strenuous exercise?

REASONS FOR PERFORMING STUDY: Dorsal displacement of the soft palate (DDSP) is a common condition in racehorses for which various surgical treatments are often performed. In light of recent findings that suggested the position of the larynx may influence the occurrence of DDSP, we investigated whether a noninvasive mean of affecting the position of the larynx could be effective in the management of DDSP. HYPOTHESIS: An external device (laryngohyoid support; LHS) positioning the larynx in a more rostral and dorsal location and preventing caudal displacement of the basihyoid bone would be effective in preventing DDSP during strenuous exercise. METHODS: Ten horses were exercised on a high-speed treadmill under 4 different treatment conditions: control (n = 10); control with external device (n = 10); after bilateral resection of thyrohyoid (TH) muscles (n = 7); and after bilateral resection of TH muscles with external device (n = 7). Two trials were performed randomly for each of the 4 conditions. In Trial 1, videoendoscopic images of the upper airway, pharyngeal and tracheal static pressures, and arterial blood gases were collected. In Trial 2, airflow measurement combined with mask and tracheal static pressure was obtained, and upper airway impedance calculated. The trials allowed calculation of airway impedance and respiratory frequency, and assessment of ventilation using arterial PO2 and PCO2. RESULTS: Under control conditions, none of the 10 horses developed DDSP. There was no statistically significant effect from the LHS on airway impedance or respiratory frequency, nor on arterial PO2 and PCO2. Seven of the 10 horses developed DDSP during exercise after resection of the TH muscles. None of these 7 horses continued to experience DDSP during exercise with the external device. In the latter group and condition, the LHS significantly improved inspiratory and expiratory flow and impedance. CONCLUSIONS: The LHS helped prevent experimentally induced DDSP at exercise, probably by statically positioning the larynx in a more rostral and dorsal position. POTENTIAL RELEVANCE: Field studies are required to investigate whether the LHS can successfully prevent DDSP in horses with naturally occurring disease.     

Effect of chronic clenbuterol administration and exercise training on immune function in horses

Effects of longitudinal exercise training and acute intensive exercise (simulated race test) on immune function have not been reported in horses. Clenbuterol, a beta2-adrenergic agonist, is used to manage inflammatory airway disease in horses. This study investigated the interaction of 8 wk of exercise training with or without 12 wk of clenbuterol administration in horses. Twenty-three untrained standardbred mares (10 +/- 3 yr, Mean +/- SE) were used and divided into four experimental groups. Horses given clenbuterol plus exercise (CLENEX; n = 6) and clenbuterol alone (CLEN; n = 6) received 2.4 microg/kg BW of clenbuterol twice daily (in an average volume of 20 mL) on a schedule of 5 d on and 2 d off for 12 wk. The CLENEX group was also aerobically trained 3 d/wk. Mares given exercise alone (EX; n = 5) were aerobically trained for 3 d/wk, and the control group (CON; n = 6) remained sedentary. Both EX and CON horses were administered similar volumes (approximately 20 mL) of molasses twice daily. A simulated race test (SRT) resulted in an elevation in lymphocyte number postexercise (P < 0.05). There was no significant difference after acute exercise in either monocyte or granulocyte number. Acute exercise resulted in a decrease (P < 0.05) in the percentage of CD4+ and an increase (P < 0.05) in the percentage of CD8+ cells. The SRT resulted in a decreased lymphoproliferative response to pokeweed mitogen (P < 0.05). A SRT had no effect on antibody production in response to equine influenza vaccine. The EX group demonstrated greater cortisol concentrations at rest and at all other time points postexercise after completing the training regimen compared with CLENEX horses (P < 0.05). Preexercise (SRT) peripheral blood monocyte number was lower in CLENEX horses than in other treatment groups (P < 0.05). Clenbuterol and exercise training did not significantly affect post-SRT changes in leukocyte numbers. Exercise training resulted in a decrease (P < 0.05) in the percentage of CD8+ cells post-SRT compared with other groups, but the percentage of CD4+ cells was not altered by either clenbuterol or exercise conditioning. Lymphocyte proliferative response was not affected by clenbuterol or exercise treatment. Horses demonstrated responses to bouts of acute exercise as noted with other species, namely humans and rodents.     

Adrenergic, cholinergic, and nonadrenergic-noncholinergic intrinsic innervation of the jejunum in horses.

OBJECTIVE: To determine the major neurotransmitters that regulate contractile activity in the jejunum of horses. SAMPLE POPULATION: Jejunal specimens from 65 horses without gastrointestinal tract lesions. PROCEDURE: Jejunal smooth muscle strips, oriented in the plane of the circular or longitudinal muscular layer, were suspended isometrically in muscle baths. Neurotransmitter release was induced by electrical field stimulation (EFS) delivered at 30 and 70 V intensities and at various frequencies on muscle strips maintained at low or high muscle tone. To detect residual nonadrenergic-noncholinergic neurotransmission, the response of muscle to EFS in the presence of adrenergic and cholinergic blockade was compared with the response in the presence of tetrodotoxin. RESULTS: Atropine (ATR) decreased the contractile response of muscle strips to EFS under most conditions. However, ATR increased the contractile response of high-tone circular muscle. Adrenergic blockade generally increased the muscle responses to 30 V EFS and in high-tone longitudinal muscle but decreased contractile responses in high-tone circular muscle. Tetrodotoxin significantly altered the responses to EFS, compared with adrenergic and cholinergic receptor blockade. CONCLUSIONS: Acetylcholine and norepinephrine appear to be important neurotransmitters regulating smooth muscle contractility in the equine jejunum. They induce contraction and relaxation, respectively, in most muscle preparations, although they may cause opposite effects under certain conditions. In addition, nonadrenergic-noncholinergic excitatory and inhibitory influences were detected. CLINICAL RELEVANCE: Acetylcholine or norepinephrine release within the myenteric plexus of horses may alter gastrointestinal motility.     

Polysaccharide storage myopathy in Cob Normand draft horses

Gluteus medius muscle was sampled from 53 Cob Normand horses for histologic evaluation. Twenty horses (38%) exhibited amylase-resistant material in myocytes consistent with polysaccharide storage myopathy. Diameter of affected type II fibers was increased (67.7 +/- 21.4 microm) compared with normal ones (57.3 +/- 19.7 microm). Two groups were distinguished by quantitative study. The first group (n = 14; 26%) was characterized by a low percentage of fibers (m = 0.98%) containing aggregates occurring singly or in perifascicular clusters without myopathic changes. The second group (n = 6; 11%) was characterized by a high percentage (m = 18.1%) of fibers containing aggregates scattered in biopsy with chronic myopathic changes. Re-biopsy of 4 horses showed an increase with time in the number of aggregate-containing fibers for horses of the first group only. In 1 necropsied horse, aggregates were observed in a wide range of muscles including smooth muscles. Ultrastructurally, granular material was found interspersed among arrays of filamentous material.     

Calcium sensitivity of force production and myofibrillar ATPase activity in muscles from Thoroughbreds with recurrent exertional rhabdomyolysis

OBJECTIVE: To determine whether the basis for recurrent exertional rhabdomyolysis (RER) in Thoroughbreds lies in an alteration in the activation and regulation of the myofibrillar contractile apparatus by ionized calcium. ANIMALS: 4 Thoroughbred mares with RER and 4 clinically normal (control) Thoroughbreds. PROCEDURES: Single chemically-skinned type-I (slow-twitch) and type-II (fast-twitch) muscle fibers were obtained from punch biopsy specimens, mounted to a force transducer, and the tensions that developed in response to a series of calcium concentrations were measured. In addition, myofibril preparations were isolated from muscle biopsy specimens and the maximal myofibrillar ATPase activity, as well as its sensitivity to ionized calcium, were measured. RESULTS: Equine type-I muscle fibers were more readily activated by calcium than were type-II muscle fibers. However, there was no difference between the type-II fibers of RER-affected and control horses in terms of calcium sensitivity of force production. There was also no difference between muscle myofibril preparations from RER-affected and control horses in calcium sensitivity of myofibrillar ATPase activity. CONCLUSIONS AND CLINICAL RELEVANCE: An alteration in myofibrillar calcium sensitivity is not a basis for pathologic contracture development in muscles from RER-affected horses. Recurrent exertional rhabdomyolysis in Thoroughbreds may represent a novel heritable defect in the regulation of muscle excitation-contraction coupling or myoplasmic calcium concentration.     

Equine recurrent airway obstruction does not alter airway muscarinic acetylcholine receptor expression and subtype distribution

In recurrent airway obstruction (RAO) or heaves, bronchospasm has been attributed to enhanced cholinergic activity. However, the expression and function of muscarinic acetylcholine receptors (mAChR) and their signaling components are not yet known. Thus, we examined the expression, subtype distribution and postreceptor signaling pathways of mAChR in the peripheral lung, bronchial and tracheal epithelia with the underlying smooth muscle from nine horses with RAO and 11 healthy control horses. In RAO horses, no significant segment-dependent alteration in mAChR density and subtype distribution (assessed by [N-methyl-3H]-scopolamine binding; ([3H]-NMS)), was found, except a trend in receptor down-regulation in some peripheral parts of the lung. The total number of high mAChR binding sites (assessed by carbachol-displacement experiments in the presence or absence of guanosine 5'-triphosphate) was not changed in RAO, suggesting that the functional coupling of mAChR to the corresponding G-proteins is intact. The M2-mediated inhibition of adenylate cyclase (AC) as well as the M3-receptor-G(q/11)-phospholipase C (PLC) activity was not different between RAO and control airway tissues. In conclusion, in equine RAO airways, mAChR expression and function were not altered, and thus appear not to account for the enhanced cholinergic activity in RAO.     

Clenbuterol plasma concentrations after repeated oral administration and its effects on cardio-respiratory and blood lactate responses to exercise in healthy Standardbred horses

To evaluate the effects of clenbuterol on cardio-respiratory parameters and blood lactate relation to exercise tolerance, experimental horses performed standardized exercise tests on a high-speed treadmill before and after administration of the drug. Clenbuterol was administered in feed to six healthy Standardbreds at a dose rate of 0.8 micrograms/kg b.wt twice daily for 5.5 days. Each horse was tested twice, without and with a respiratory mask, during two consecutive days. One week elapsed between the baseline tests without drug and the tests with clenbuterol treatment (each horse served as its own control). The results show an unchanged heart rate response to exercise 2 h after the last clenbuterol administration. The blood lactate response and the arterial oxygen tension during exercise did not differ before and after drug treatment. The oxygen uptake as well as pulmonary ventilation relative to the work load performed was essentially unaffected. The arterial pH during exercise was significantly increased (P less than 0.05) following clenbuterol treatment. Plasma levels of clenbuterol were maximal 2 h post-administration with values between 0.45 and 0.75 ng/ml. The plasma half-life of elimination was 10.4 h (+/- 2.25 SD). In conclusion, clenbuterol did not cause any major effects on the cardio-respiratory and blood lactate parameters studied in healthy horses performing submaximal exercise tolerance tests.     

Morphological changes in the small intestinal smooth muscle layers of horses suffering from small intestinal strangulation. Is there a basis for predisposition for reduced contractility?

Reasons for performing study: Intestinal strangulation often leads to enterectomy after which ileus can develop. This has prompted research to look into possible pathophysiological processes triggering equine ileus. However, morphological changes of the small intestinal smooth muscle in relation to equine colic have not yet been studied. Objectives: The presence of some smooth muscle proteins was morphologically assessed and quantified in control and colic horses. In addition, the up‐ or down‐regulation of heat shock proteins (HSP20 and HSP27) influencing the contractility of smooth muscles was studied. Methods: Cranial resection margins of 18 strangulated small intestinal samples were collected. Small intestinal control samples were collected from 11 horses subjected to euthanasia for other than gastrointestinal‐related reasons. Formaldehyde‐fixed tissue was paraffin‐embedded and processed for conventional staining and immunohistochemistry. Snap‐frozen full‐thickness biopsies were collected for western blot analyses. Results: Evaluating the muscle layer microscopically, colic samples showed significantly more signs of degradation than controls (P = 0.026) of which vacuolar degeneration was most prominent (P = 0.009). In colic samples, myosin protein levels were decreased (P = 0.022) whereas desmin (P = 0.049) and HSP20 protein levels (P = 0.005) were elevated. Conclusions: In colic samples, microscopic lesions at the level of the muscle layer indicate a stress response. In addition, modified amounts of structural proteins such as myosin and desmin together with increased HSP20 levels could perhaps provide a basis for explaining the malfunctioning of the intestinal muscle layer. Potential relevance: Post operative ileus, following small intestinal strangulation and resection, could be related in part to a dysfunctional muscle layer. In addition to microscopic signs of degeneration, myosin and HSP20 were affected. Pharmacological interventions might alter HSP20 expressions and thus serve a protective effect.