Characterization of the in vitro responses of equine cecal longitudinal smooth muscle to endothelin-1

OBJECTIVE: To characterize the in vitro response of equine cecal longitudinal smooth muscle (CLSM) to endothelin (ET)-1 and assess the role of ETA and ETB receptors in those ET-1-induced responses. ANIMALS: 36 horses without gastrointestinal tract disease. PROCEDURE: To determine cumulative concentration-response relationships, CLSM strips were suspended in tissue baths containing graded concentrations of ET-1 (10(-9) to 10(-6)M) with or without BQ-123 (ETA receptor antagonist); with or without IRL-1038 (ETB receptor antagonist); or with both antagonists at concentrations of 10(-9), 10(-7), and 10(-5)M. To determine the percentage change in baseline tension of CLSM, the areas under the curve during the 3-minute periods before and after addition of each dose were compared. Also, the effects of ET-1 and a combination of selective ETA and ETB receptor antagonists on electrically evoked contractions were studied. RESULTS: ET-1 caused sustained increases in CLSM tension in a concentration-dependent manner. Contractile responses to ET-1 were not significantly inhibited by either BQ-123 or IRL-1038 alone at any concentration; however, responses were significantly inhibited by exposure to the antagonists together at a concentration of 10(-5)M. Electrical field stimulation did not change the spontaneous contractile activity of CLSM and did not significantly alter the tissue response to ET-1, BQ-123, or IRL-1038. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that ET-1 has a contractile effect on equine CLSM that is mediated via ETA and ETB receptors. In vitro spontaneous contractions of equine CLSM apparently originate in the smooth muscle and not the enteric nervous system.     

Muscarinic receptor subtypes in equine tracheal smooth muscle

Selective muscarinic receptor antagonists were used to identify muscarinic receptor subtypes in equine trachealis strips. The M₁ receptor antagonist pirenzepine (10^–7 mol/L to 3 × 10^–5 mol/L) and the M₃ receptor antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 10^–9 mol/L to 3 × 10^–7 mol/L³) dose dependently inhibited the contractile responses to electrical field stimulation (EFS) and exogenous acetylcholine (ACh). Schild plots yielded a pA₂ value for pirenzepine vs ACh of 6.75 ± 0.09, which is consistent with the affinity for M₂ or M₃ receptors, and a pA₂ value for 4-DAMP vs ACh of 8.47 ± 0.09, which is in agreement with the affinity for M₃ receptors. The M₂ receptor antagonist gallamine (10^–5 mol/L and 10^–4 mol/L) did not affect the response of trachealis to exogenous ACh and low-frequency EFS (0.1–2 Hz) but decreased the responses to high-frequency EFS (4–16 Hz). These results suggest that the muscarinic receptors mediating contractions induced by ACh in equine tracheal smooth muscle are of the M₃ subtype. The lack of an increase in the response to EFS following gallamine suggests that functional prejunctional inhibitory M₂ receptors are not present on the cholinergic nerves innervating equine tracheal smooth muscle.     

Characterization of alpha-adrenoceptor subtypes in smooth muscle of equine ileum

OBJECTIVE: To determine the concentration and binding characteristics of alpha-adrenoceptor subtypes in smooth muscle cell membranes of equine ileum. SAMPLE POPULATION: Segments of longitudinal and circular smooth muscle from the ileum of 8 male and 8 female adult horses. PROCEDURE: Distribution of alpha-adrenoceptor subtypes was assessed by use of radioligand binding assays incorporating [3H]-prazosin and [3H]-rauwolscine, highly selective alpha1- and alpha2-adrenoceptor antagonists, respectively. Characterization of adrenoceptor subtypes was performed by use of binding inhibition assays. RESULTS: On the basis of binding affinity for specific radioligands, low- and high-affinity alpha1- and alpha2-adrenoceptors were detected. Concentration of low-affinity alpha2-adrenoceptors was significantly greater in male horses, compared with females. Competition studies confirmed the specificity of the radioligands used in the binding assays. Alpha1-adrenoceptors of both subtypes in male and female horses had a higher affinity for prazosin than phentolamine, whereas yohimbine did not compete with the radioligand for binding. For alpha2-adrenoceptors regardless of subtype, potency of inhibition elicited by each drug varied between sexes. In males, yohimbine was a more potent inhibitor than phentolamine, which was more potent than prazosin. In females, yohimbine was more potent than prazosin, which was more potent than phentolamine. CONCLUSIONS AND CLINICAL RELEVANCE: High- and low-affinity alpha1- and alpha2-adrenoceptors were detected in smooth muscle of equine ileum. Because alpha-adrenoceptor subtypes, particularly alpha2-adrenoceptors, are involved in the regulation of gastrointestinal tract function, characterization of these receptors may represent the basis for development of new therapeutic strategies for the control of gastrointestinal disturbances in horses.     

In vitro investigation of the interaction between nitric oxide and cyclo-oxygenase activity in equine ventral colon smooth muscle

The objective of this study was to determine if a correlation exists between the presence of nitric oxide and prostaglandin release in the equine ventral colon smooth muscle, since this relationship may accentuate the inflammatory process during intestinal injury. Tissue was collected from the ventral colon, cut into muscle strips oriented along the circular, longitudinal and taenial layers, and mounted in a tissue bath system. Samples of the bath fluid were collected before, following electrical field stimulation (EFS), and following EFS in the presence of L-NAME, a nitric oxide synthase inhibitor. Muscle strips were also obtained following systemic administration of a cyclo-oxygnease inhibitor and samples were collected using the previously described protocol. Concentrations of prostaglandins were determined in the fluid samples using an ELISA. Electrical field stimulated release of nitric oxide produced a significant increase in prostaglandin production which did not occur in the presence of L-NAME. Systemic administration of flunixin meglumine reduced prostaglandin levels at all sampling periods, although a small increase was present following EFS. The results of this study support the hypothesis that there is a correlation between the release of nitric oxide and the production of prostaglandins in the smooth muscle of the large colon. This association between nitric oxide and prostaglandin may act as an important regulatory mechanism for various physiological mechanisms, such as vascular smooth muscle tone, and may contribute to amplified tissue injury when the induced forms of both enzymes are activated during an inflammatory insult. This suggests that the use and development of COX2 and iNOS inhibitors may help attenuate the inflammatory response following intestinal injury.     

Actions of histamine and other substances on the airway smooth muscle of swine (in vitro)

Isolated swine tracheal and bronchial smooth muscle strips contracted to carbachol and histamine. 5-HT, PGF₂ and bradykinin were inactive. Isoprenaline (a -adrenoceptor agonist), PGE₁, PGE₂, bradykinin and 4-methylhistamine (4-MeH: a specific H₂-receptor agonist) produced relaxation of carbachol contracted trachea and bronchi. Mepyramine (a specific H₁-antagonist) blocked histamine-induced contractions. After H₁-blockade, histamine induced relaxation of carbachol contracted trachea and bronchi. Metiamide (an H₂-receptor antagonist) antagonizes relaxation to 4-MeH and histamine, but not to isoprenaline. The results of this investigation showed the presence of both H₁- and H₂-histamine receptors mediating constriction and relaxation respectively in the airways of swine.     

Effect of microcurrent electrical tissue stimulation on equine tenocytes in culture

OBJECTIVE: To determine effects of microcurrent electrical tissue stimulation (METS) on equine tenocytes cultured from the superficial digital flexor tendon (SDFT). SAMPLE POPULATION: SDFTs were collected from 20 horses at slaughter. PROCEDURE: Tenocytes were isolated following outgrowth from explants and grown in 48-well plates. Four methods of delivering current to the tenocytes with a METS device were tested. Once the optimal method was selected, current consisting of 0 (negative control), 0.05, 0.1, 0.5, 1.0, or 1.5 mA was applied to cells (8 wells/current intensity) once daily for 8 minutes. Cells were treated for 1, 2, or 3 days. Cell proliferation, DNA content, protein content, and apoptosis rate were determined. RESULTS: Application of microcurrent of moderate intensity increased cell proliferation and DNA content, with greater increases with multiple versus single application. Application of microcurrent of moderate intensity once or twice increased protein content, but application 3 times decreased protein content. Application of current a single time did not significantly alter apoptosis rate; however, application twice or 3 times resulted in significant increases in apoptosis rate, and there were significant linear (second order) correlations between current intensity and apoptosis rate when current was applied twice or 3 times. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the present study indicate that microcurrent affects the behavior of equine tenocytes in culture, but that effects may be negative or positive depending on current intensity and number of applications. Therefore, results are far from conclusive with respect to the suitability of using METS to promote tendon healing in horses.     

In vitro model of equine muscle regeneration

Equine satellite cells are responsible for muscle healing and regeneration in the mature horse. We describe the in vitro cell culture conditions required for clonal populations of equine satellite cells to undergo both proliferation and differentiation. Our hypothesis is that these in vitro conditions model regeneration of muscle and can be used to evaluate potential therapeutics. In this study, 2 areas of satellite cell response were tested: proliferation of clones induced by growth factors, and fusion induced by culture conditions. Equine satellite cell clones showed differences in their response to growth factors as well as accumulation of cellular protein concentrations. Equine satellite cells proliferate in response to both human and bovine FGF. IGF-1, a powerful mitogen of other satellite cell culture systems, was not as effective for inducing equine satellite cell proliferation. Protein concentrations were also measured in satellite cell cultures. Clones differed in cellular protein produced depending on growth conditions. Conditions inducing differentiation into myotubes was also determined for a 96 well assay and can be used to study the final stage of functioning muscle production. This in vitro model is the first step in identifying potential therapeutics to speed wound healing and promote muscle regeneration in horses.     

Distribution of the neurokinin-1 receptor in equine intestinal smooth muscle

REASON FOR PERFORMING STUDY: Tachykinins have profound effects on equine intestinal motility, but the distribution of the neurokinin receptors (NKRs) through which they act is unknown. This study reports the distribution of one of these receptors, the neurokinin-1 receptor (NK1R), in smooth muscle throughout the equine intestinal tract. OBJECTIVES: To quantify the distribution of the NK1R, based upon mRNA expression, in smooth muscle of different regions of the equine intestinal tract. METHODS: Nine regions of the intestinal tract were sampled in 5 mature horses. Total RNA was isolated from smooth muscle and reverse transcribed; NK1R mRNA was then quantified using real-time PCR. RESULTS: NK1R mRNA was found at all levels of the sampled intestinal tract. The smooth muscle of the proximal small intestine and the ventral colon exhibited the highest level of NK1R mRNA expression in the equine intestinal tract. CONCLUSIONS: Tachykinins probably affect intestinal contractility and propulsion in the proximal small intestine and in the ventral colon.     

Relationship of equine housing to large airway inflammation

The pasture, as compared to stall confinement, has been considered a more desirable environment for maintaining the health of the respiratory system in the horse. This conclusion is based on reports which showed that ventilation in most barns was generally poor, and that horses bedded on straw and fed hay were exposed to many forms of respirable debris. In this study, six normal horses were evaluated for evidence of airway mucosal inflammation after being housed on pasture or stabled in a barn for one month. The response of the horses' airways was measured by assigning scores for the degree of tracheal mucosal secretions that were observed by endoscopic visualization. Cytological examination of transtracheal wash secretions was also performed, as well as histologic evaluation of tracheobronchial tissues obtained by a transendoscopic epithelial biopsy technique. Samples were collected at three time points; the initial collection occurred after the horses were housed on pasture for one month. The horses were subsequently moved to a barn for an equal length of time and samples were obtained at the end of this period. The horses were then returned to their original pasture and final sampling was performed after they were housed in this environment for two months. There were no significant changes in any of the parameters evaluated, regardless of the environment in which the horses were maintained. These findings indicate that housing horses in a barn for four weeks does not cause tracheobronchial mucosal inflammation in a manner that could be detected using the methods employed in this study.     

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.     

Epithelium- and mucosa-dependent relaxation and contraction of normal equine trachealis muscle in vitro

Strips of trachealis muscle were dissected from the mid-cervical portion of the trachea from horses that were free of respiratory tract disease. The epithelium and mucosa were removed from one group of tissues and were left intact in a second group of tissues. Each tissue was suspended in a bath filled with Krebs-bicarbonate solution that was aerated with 5% CO2 in oxygen and maintained at 37 degrees C. Isometric tension was continuously recorded. The contractile response to square-wave electrical stimulations increased as frequency (3, 5, 10, 15, 20, 25, and 30 Hz), voltage (10, 15, 18, and 25 V), and pulse duration (0.2, 0.5, 1.0, 1.5, and 2.0 ms) increased in tissues with the epithelium and mucosa intact. A stimulus of 18 V, 20 Hz, and 0.5 ms induced maximal contraction. Atropine (10(-6) M) abolished the response to 18 V and 0.5 ms at all frequencies. The increase in active isometric tension was concentration dependent when acetylcholine (10(-9) to 10(-4) M) was added to the baths in 0.5-logarithmic increments. Tissues that were contracted in response to acetylcholine (10(-5) M) had a concentration-dependent decrease in active isometric tension when isoproterenol was added to the baths in 0.5-logarithmic increments (10(-9) to 10(-4) M). The contraction and relaxation curves were qualitatively similar, but quantitatively different in tissues with and without the epithelium and mucosa. Removing the epithelium and mucosa increased the contractile response to acetylcholine at bath concentrations of 3.1 x 10(-7) M and 10(-6) M. The presence of epithelium and mucosa enhanced the magnitude of isoproterenol-induced relaxations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of the ether-a-go-go (ERG) potassium channel in smooth muscle of the equine gastrointestinal tract and influence on activity of jejunal smooth muscle

OBJECTIVE: To determine whether ether-a-go-go (ERG) potassium channels are expressed in equine gastrointestinal smooth muscle, whether ERG channel antagonists affect jejunal muscle contraction in vitro, and whether plasma cisapride concentrations in horses administered treatment for postoperative ileus (POI) are consistent with ERG channels as drug targets. SAMPLE POPULATION: Samples of intestinal smooth muscle obtained from 8 horses free of gastrointestinal tract disease and plasma samples obtained from 3 horses administered cisapride for treatment of POI. PROCEDURE: Membranes were prepared from the seromuscular layer of the duodenum, jejunum, ileum, cecum, large colon, and small colon. Immunoblotting was used to identify the ERG channel protein. Isolated jejunal muscle strips were used for isometric stress response to ERG channel blockers that included E-4031, MK-499, clofilium, and cisapride. Plasma concentrations of cisapride were determined in 3 horses administered cisapride for treatment of POI after small intestinal surgery. RESULTS: Immunoblotting identified ERG protein in all analyzed segments of the intestinal tract in all horses. The selective ERG antagonist E-4031 caused a concentration-dependent increase in jejunal contraction. Clofilium, MK-499, and cisapride also increased jejunal contraction at concentrations consistent with ERG channel block; effects of E-4031 and cisapride were not additive. Peak plasma cisapride concentrations in treated horses were consistent with ERG block as a mechanism of drug action. CONCLUSIONS AND CLINICAL RELEVANCE: The ERG potassium channels modulate motility of intestinal muscles in horses and may be a target for drugs. This finding may influence development of new prokinetic agents and impact treatment of horses with POI.     

In vitro effects of lidocaine on the contractility of equine jejunal smooth muscle challenged by ischaemia‐reperfusion injury

Reasons for performing study: Post operative ileus (POI) in horses is a severe complication after colic surgery. A commonly used prokinetic drug is lidocaine, which has been shown to have stimulatory effects on intestinal motility. The cellular mechanisms through which lidocaine affects smooth muscle activity are not yet known. Objectives: To examine the effects of lidocaine on smooth muscle in vitro and identify mechanisms by which it may affect the contractility of intestinal smooth muscle. Hypothesis: Ischaemia and reperfusion associated with intestinal strangulation can cause smooth muscle injury. Consequently, muscle cell functionality and contractile performance is decreased. Lidocaine can improve basic cell functions and thereby muscle cell contractility especially in ischaemia‐reperfusion‐challenged smooth muscle. Methods: To examine the effects of lidocaine on smooth muscle function directly, isometric force performance was measured in vitro in noninjured and in vivo ischaemia‐reperfusion injured smooth muscle tissues. Dose‐dependent response of lidocaine was measured in both samples. To assess membrane permeability as a marker of basic cell function, release of creatine kinase (CK) was measured by in vitro incubations. Results: Lidocaine‐stimulated contractility of ischaemia‐reperfusion injured smooth muscle was more pronounced than that of noninjured smooth muscle. A 3‐phasic dose‐dependency was observed with an initial recovery of contractility especially in ischaemia‐reperfusion injured smooth muscle followed by a plateau phase where contractility was maintained over a broad concentration range. CK release was decreased by lidocaine. Conclusion: Lidocaine may improve smooth muscle contractility and basic cell function by cellular repair mechanisms which are still unknown. Improving contractility of smooth muscle after ischaemia‐reperfusion injury is essential in recovery of propulsive intestinal motility. Potential relevance: Characterisation of the cellular mechanisms of effects of lidocaine, especially on ischaemia‐reperfusion injured smooth muscle, may lead to improved treatment strategies for horses with POI.     

Response of heifer mammary gland macrophages and neutrophils to interferon-gamma stimulation in vitro.

The phagocytic and killing abilities of heifer mammary gland macrophages (M phi) and neutrophils were evaluated after exposure to recombinant bovine interferon-gamma (rBoIFN-gamma) stimulation in vitro. Macrophages or neutrophils were cultured for 2 h with 0, 10(2), 10(4) and 10(5) units rBoIFN-gamma/mL. Phagocytosis assays were performed by incubation with Staphylococcus aureus at a leukocyte:bacteria ratio of 1:10. After 45 min, cells were stained with acridine-orange and phagocytic and killing abilities were determined. Although rBoIFN-gamma had no effect on M phi phagocytic activity, neutrophil phagocytic activity after incubation in 10(4) units rBoIFN-gamma (41.62%) was significantly higher than 0 (25.24%) or 10(2) units rBoIFN-gamma (24.73%). Neutrophil and M Phi killing abilities were not affected by any dose of rBoIFN-gamma. Results suggested that rBoIFN-gamma promoted neutrophil phagocytic activity, but did not affect neutrophil killing or overall M phi function in vitro.     

山羊皱胃平滑肌细胞的体外分离与培养

近年来,随着奶产业的迅速发展,奶牛疾病的发病率逐年升高.皱胃变位是一种多发于围产期的代谢性疾病,严重影响奶牛的生产性能,给奶产业造成了严重的经济损失.皱胃动力不足和气体积聚是引起皱胃变位的两个先决条件[1].平滑肌是皱胃动力的基本单位.因此,分离培养皱胃平滑肌细胞,可为研究反刍动物皱胃疾病的发病机理提供试验材料.本试验在参照血管平滑肌细胞培养方法的基础上,成功培养出皱胃平滑肌细胞.报告如下.