In vitro investigation of the effect of prostaglandins and nonsteroidal anti-inflammatory drugs on contractile activity of the equine smooth muscle of the dorsal colon, ventral colon, and pelvic flexure

OBJECTIVES: To determine the in vitro effect of prostaglandin E2 (PGE2), PGF2alpha, PGI2; and nonsteroidal anti-inflammatory drugs (NSAID; ie, flunixin meglumine, ketoprofen, carprofen, and phenylbutazone) on contractile activity of the equine dorsal colon, ventral colon, and pelvic flexure circular and longitudinal smooth muscle. ANIMALS: 26 healthy horses. PROCEDURE: Tissue collected from the ventral colon, dorsal colon, and pelvic flexure was cut into strips and mounted in a tissue bath system where contractile strength was determined. Incremental doses of PGE2, PGF2alpha,, PGI2, flunixin meglumine, carprofen, ketoprofen, and phenylbutazone were added to the baths, and the contractile activity was recorded for each location and orientation of smooth muscle. RESULTS: In substance P-stimulated tissues, PGE2 and PGF2alpha enhanced contractility in the longitudinal smooth muscle with a decrease or no effect on circular smooth muscle activity. Prostaglandin I2 inhibited the circular smooth muscle response with no effect on the longitudinal muscle. The activity of NSAID was predominantly inhibitory regardless of location or muscle orientation. CONCLUSIONS AND CLINICAL RELEVANCE: In the equine large intestine, exogenous prostaglandins had a variable effect on contractile activity, depending on the location in the colon and orientation of the smooth muscle. The administration of NSAID inhibited contractility, with flunixin meglumine generally inducing the most profound inhibition relative to the other NSAID evaluated in substance P-stimulated smooth muscle of the large intestine. The results of this study indicate that prolonged use of NSAID may potentially predispose horses to develop gastrointestinal tract stasis and subsequent impaction.     

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.     

The effects of cyclo-oxygenase inhibitors on bile-injured and normal equine colon

A potential adverse effect of cyclo-oxygenase (COX) inhibitors (nonsteroidal anti-inflammatory drugs [NSAIDs]) in horses is colitis. In addition, we have previously shown an important role for COX-produced prostanoids in recovery of ischaemic-injured equine jejunum. It was hypothesised that the nonselective COX inhibitor flunixin would retard repair of bile-injured colon by preventing production of reparative prostaglandins, whereas the selective COX-2 inhibitor, etodolac would not inhibit repair as a result of continued COX-1 activity. Segments of the pelvic flexure were exposed to 1.5 mmol/l deoxycholate for 30 min, after which they were recovered for 4 h in Ussing chambers. Contrary to the proposed hypothesis, recovery of bile-injured colonic mucosa was not affected by flunixin or etodolac, despite significantly depressed prostanoid production. However, treatment of control tissue with flunixin led to increases in mucosal permeability, whereas treatment with etodolac had no significant effect. Therefore, although recovery from bile-induced colonic injury maybe independent of COX-elaborated prostanoids, treatment of control tissues with nonselective COX inhibitors may lead to marked increases in permeability. Alternatively, selective inhibition of COX-2 may reduce the incidence of adverse effects in horses requiring NSAID therapy.     

In vitro investigation of the effects of nonsteroidal anti-inflammatory drugs, prostaglandin E2, and prostaglandin F2alpha on contractile activity of the third compartment of the stomach of llamas

OBJECTIVE: To determine the in vitro effect of prostaglandin (PG) E2, PGF2alpha, and the nonsteroidal anti-inflammatory drugs (NSAIDs) indomethacin, ketoprofen, and nabumetone on the contractile strength of the circular smooth muscle layer of the third compartment of the stomach of llamas. SAMPLE POPULATION: Specimens of the third compartment obtained from 5 healthy adult llamas. PROCEDURE: Full-thickness tissue samples were collected from the third compartment immediately after euthanasia. Specimens were cut into strips oriented along the circular muscle layer and mounted in a tissue bath system. Incremental amounts of ketoprofen, nabumetone, indomethacin, PGE2, and PGF2alpha were added, and contractile strength (amplitude of contractions) was recorded. RESULTS: Generally, PGE2 reduced contractile strength of the circular smooth layer of the third compartment, whereas PGF2alpha, increased the strength of contractions. The activity of the NSAIDs was generally excitatory in a concentration-dependent manner, although significant changes were induced only by administration of indomethacin. CONCLUSIONS AND CLINICAL RELEVANCE: On isolated smooth muscle strips of the third compartment of llamas, exogenous PGE2 and PGF2alpha had a variable effect on contractile strength. Administration of the NSAIDs did not inhibit contractility and would not be likely to induce stasis of the third compartment in the absence of an underlying disease process.     

In vitro dose-dependent effects of enrofloxacin on equine articular cartilage.

OBJECTIVE: To determine whether enrofloxacin has detrimental, dose-dependent effects on equine articular cartilage in vitro. ANIMALS: Cartilage explants were developed from 6 healthy horses between 0 and 96 months old. PROCEDURE: Patellar cartilage explants were incubated in 5 concentrations of enrofloxacin (2 microg/ml, 10 microg/ml, 1,000 microg/ml, 10,000 microg/ml, and 50,000 microg/ml) for 72 hours. Proteoglycan synthesis (Na35SO4 incorporation for 24 hours), proteoglycan degradation (Na35SO4 release for 72 hours), endogenous proteoglycan content (dimethylmethlene blue assay), and total protein content were determined. Cartilage explants were evaluated by use of histomorphologic and histomorphometric techniques (toluidine blue stain) for cytologic and matrix characteristics. Quantitative data were analyzed with a one-way ANOVA to compare results among various enrofloxacin concentration groups and the control group. A general linear model was used to determine whether age had an effect. RESULT: Proteoglycan synthesis was excellent in control specimens and in specimens incubated in low concentrations of enrofloxacin (2 microg/ml and 10 microg/ml). High concentrations of enrofloxacin (> 1,000 microg/ml) effectively eliminated proteoglycan synthesis regardless of horse age. Proteoglycan degradation at low concentrations (2 microg/ml and 10 microg/ml) was not different than control. High concentrations of enrofloxacin (> 1,000 microg/ml) caused significant degradation. Different concentrations of enrofloxacin did not affect endogenous proteoglycan. High concentrations of enrofloxacin were associated with a significant increase in number of pyknotic nuclei. CONCLUSION: Concentrations of enrofloxacin that might be achieved following systemic administration did not suppress chondrocyte metabolism in vitro. High concentrations of enrofloxacin (> 1,000 microg/ml) were toxic to chondrocytes.     

In vitro mucolytic activity of recombinant human deoxyribonuclease on equine tracheobronchial mucus

The viscosity of the mucus, its DNA concentration and the size range of the DNA were determined on tracheobronchial samples from 11 horses with lower airway diseases before and after incubation with recombinant human deoxyribonuclease (rhDNase). The horses were divided into two groups on the basis of the cytology of the samples: group A (five horses) with more than 60 per cent neutrophils and group B (six horses) with fewer than 50 per cent neutrophils. The mean mucus viscosity and DNA concentration in the preincubation samples were significantly higher in group A than in group B, and there was a correlation between DNA concentration and mucus viscosity in the preincubation samples from group A. Incubation with rhDNase significantly reduced the viscosity of the samples only in group A.     

In vitro effects of Actinobacillus pleuropneumoniae on inducible nitric oxide synthase and cyclooxygenase-2 in porcine alveolar macrophages

OBJECTIVE: To determine the amount of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) activity in alveolar macrophages in response to Actinobacillus pleuropneumoniae (APP) by determining nitric oxide (NO) and prostaglandin E2 (PGE2) concentrations. SAMPLE POPULATION: Freshly isolated porcine alveolar macrophages. PROCEDURE: Alveolar macrophages were incubated for 48 hours with APP (1 X 10(4) colony-forming units/mL), interleukin-1beta, (IL-1beta; 5 U/mL), tumor necrosis factor-alpha (TNFalpha; 500 U/mL), interferon-gamma (IFN-gamma, 100 U/mL), or lipopolysaccharide (LPS; 10 microg/mL). In a second experiment, alveolar macrophages were incubated with fresh medium (negative control), APP alone, or APP with 1 of the following: IL-1beta, TNF-alpha, or IFN-gamma. In a third experiment, alveolar macrophages were incubated with fresh medium (negative control), LPS (positive control), APP alone, or APP with 1 of the following: an iNOS inhibitor (3.3 microM), a COX-2 inhibitor (10 microM); or both the iNOS and COX-2 inhibitors. Supernatant was obtained at 0, 3, 6, 9, 12, 24, and 48 hours after treatment for determination of NO and PGE2 production. RESULTS: The addition of APP to alveolar macrophages resulted in significant increases in NO and PGE2 production. The addition of APP and IFN-gamma synergistically induced NO production. Inhibition of iNOS and COX-2 decreased NO and PGE2 production, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro activation of alveolar macrophages by APP results in increased production of NO and PGE2. Nitric oxide and PGE2 production appears to be largely dependent on iNOS and COX-2 activity. Pharmacologic modulation of iNOS and COX-2 activity may represent a therapeutic target for pigs with pleuropneumonia.     

The vasomotor effects of 5-hydroxytryptamine on equine basilar arteries In vitro

The vasomotor effects of 5-hydroxytryptamine (5-HT) on isolated equine basilar arteries were studied. 5-HT induced contractions of equine basilar arteries in a concentration-dependent manner, with a pEC₅₀ value (with 95% confidence limits) of 7.35 (7.08–7.62). Similar results were obtained with endothelium-denuded basilar arteries. Contractions were not competitively inhibited by the 5-HT₂ receptor antagonist ketanserin at low concentrations of 5-HT. Conversely, at high concentrations of 5-HT, contractions were inhibited by ketanserin in a concentration-dependent manner, with a pA ₂ value of 8.91 (8.62–9.20). The 5-HT₁ and 5-HT₂ receptor antagonist methiothepin shifted the concentration-response curve of 5-HT downwards and to the right in a concentration-dependent manner. In the presence of 10^-6 mol/L ketanserin, however, methiothepin antagonized 5-HT-induced contractions competitively with a pA ₂ value of 7.95 (7.59–8.31). The 5-HT₃ receptor antagonist MDL 72222 had no effect on 5-HT-induced contractions. The findings of this study indicate that 5-HT₁ and 5-HT₂ receptors are located in equine basilar arterial smooth muscle cells, and that stimulation of these receptors results in contraction.Abbreviations CR concentration ratio - EC₅₀ concentration producing 50% of the maximal response - 5-HT 5-hydroxytryptamine - MDL 72222 1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate - pA ₂ negative logarithm of the molar concentration of antagonist that produces a 2-fold rightward shift of the concentration-response curve - pEC₅₀ negative logarithm of EC₅₀ - PGF₂ prostaglandin F₂     

In vitro interference between equine herpesvirus types 1 and 2

Interference between equine herpesvirus types 1 (EHV-1) and 2 (EHV-2) was studied in equine dermis (ED) monolayer cell cultures and equine lymphocyte cultures. Cell cultures were infected with EHV-2, and after a short incubation period, the cultures were superinfected with EHV-1. At various intervals, different measurements of EHV-1 expression in dually infected cultures, compared with those in cultures infected with EHV-1 alone, were studied. In dually infected ED cell cultures, the EHV-1 cytopathic effect, EHV-1 titer, and EHV-1 enzyme-linked immunosorbent assay antigen titer were maximally reduced to values of 40%, 58.5%, and 54.9%, respectively, at postsuperinfection hour (PSIH) 36. Values of these EHV-1 expressions were subsequently increased at PSIH 48. However, thymidine kinase activity was reduced to a maximum of 67.3% reduction at PSIH 48. In dually infected lymphocyte cultures, the EHV-1 titer, EHV-1 infective centers, EHV-1 enzyme-linked immunosorbent assay antigen titer, and thymidine kinase activity were maximally reduced to values of 77.4%, 78.7%, 98.3%, and 72.9%, respectively, at PSIH 24. These reductions of EHV-1 expressions were completely abrogated at PSIH 48 to 72. In both cell culture systems, a marked interference of EHV-1 by EHV-2 was observed; this was transient in the lymphocyte cultures, but was more prolonged in ED cell cultures. This interference appeared not to be interferon mediated. The multiplication of EHV-2 in the dually infected ED cell cultures appeared unaffected.     

Evaluation of nitric oxide as an inhibitory neurotransmitter in the equine ventral colon

OBJECTIVE: To determine the role of nitric oxide and an apamin-sensitive nonadrenergic noncholingeric inhibitory transmitter on contractility of the ventral colon of horses. SAMPLE POPULATION: Strips of the circular and longitudinal muscle layers and taenia of the ventral colon from 14 horses. PROCEDURE: Muscle strips were suspended in tissue baths and attached to force transducers. Contractile activity of circular, longitudinal, and taenia muscle strips in response to electrical field stimulation was measured after addition of apamin and a nitric oxide inhibitor, N-nitro-L-arginine methyl ester (L-NAME). RESULTS: Electrical field stimulation reduced contractile activity in the circular muscle layer and taenia but not the longitudinal muscle layer. Addition of L-NAME significantly reduced inhibitory contractile activity at all frequencies for the circular muscle layer, whereas a significant effect was evident for the taenia only at the highest frequency. The combination of L-NAME and apamin resulted in a significant reduction in inhibition of the taenia at all frequencies but for circular muscle only at lower frequencies. CONCLUSIONS AND CLINICAL RELEVANCE: Nitric oxide and an apamin-sensitive neurotransmitter appear to mediate a component of inhibitory transmission in the circular muscle and taenia, but not the longitudinal muscle layer, of the equine ventral colon. Nitric oxide has a role in regulating contractile activity of the equine ventral colon, and nitric oxide synthase inhibitors may be useful in horses with ileus of the large colon.     

In vitro anion transport alterations and apoptosis induced by phenylbutazone in the right dorsal colon of ponies

OBJECTIVES: To study the functional and structural responses of the right dorsal colon (RDC) of ponies to phenylbutazone (PBZ) in vitro at a concentration that could be achieved in vivo. ANIMALS: 8 adult ponies. PROCEDURE: Short circuit current and conductance were measured in mucosa from the RDC. Tissues incubated with and without HCO3- were exposed to PBZ, bumetanide, or indomethacin. Bidirectional Cl- fluxes were determined. After a baseline flux period, prostaglandin E2 (PGE2) was added to the serosal surfaces and a second flux period followed. Light and transmission electron microscopy were performed. RESULTS: Baseline short circuit current was diminished significantly by PBZ and indomethacin, and increased significantly after addictions of PGE2. After PGE2 was added, Cl- secretion increased significantly in tissues in HCO3- -free solutions and solutions with anti-inflammatory drugs, compared with corresponding baseline measurements and with control tissues exposed to PGE2. Bumetanide did not affect baseline short circuit current and Cl- fluxes. The predominant histologic change was apoptosis of surface epithelial cells treated with PBZ and to a lesser extent in those treated with indomethacin. CONCLUSIONS AND CLINICAL RELEVANCE: Prostaglandin-induced Cl- secretion appeared to involve a transporter that might also secrete HCO3-. Both PBZ and indomethacin altered ion transport in RDC and caused apoptosis; PBZ can damage mucosa through a mechanism that could be important in vivo. The clinically harmful effect of PBZ on equine RDC in vivo could be mediated through its effects on Cl- and HCO3- secretion.     

In vitro effects and in vivo efficacy of a novel cyclooxygenase-2 inhibitor in cats with lipopolysaccharide-induced pyrexia

OBJECTIVE: To determine cyclooxygenase (COX)-2 selectivity, pharmacokinetic properties, and in vivo efficacy of firocoxib (ML-1,785,713) in cats. ANIMALS: 5 healthy male and 14 healthy female domestic shorthair cats. PROCEDURE: Selectivity of firocoxib for inhibiting COX-2 was determined by comparing the potency for inhibiting COX-1 with that of COX-2 in feline blood. Pharmacokinetic properties were determined after i.v. (2 mg/kg) and oral (3 mg/kg) administration in male cats. In vivo efficacy was evaluated in female cats with lipopolysaccharide (LPS)-induced pyrexia with administration of firocoxib 1 or 14 hours before LPS challenge. RESULTS: Blood concentrations resulting in 50% inhibition of COX-1 and COX-2 activity in vitro were 75 +/- 2 microM and 0.13 +/- 0.03 microM, respectively, and selectivity for inhibiting COX-2 relative to COX-1 was 58. Firocoxib had moderate to high oral bioavailability (54% to 70%), low plasma clearance (4.7 to 5.8 mL/min/kg), and an elimination half-life of 8.7 to 12.2 hours. Firocoxib at doses from 0.75 to 3 mg/kg was efficacious in attenuating fever when administered to cats 1 or 14 hours before LPS challenge. CONCLUSIONS AND CLINICAL RELEVANCE: Firocoxib is a potent COX-2 inhibitor and is the only selective COX-2 inhibitor described for use in cats to date. It is effective in attenuating febrile responses in cats when administered 14 hours before LPS challenge, suggesting it would be suitable for once-a-day dosing. Because selective COX-2 inhibitors have an improved therapeutic index relative to nonselective nonsteroidal anti-inflammatory drugs in humans, firocoxib has the potential to be a safe, effective anti-inflammatory agent for cats.     

The effects of furosemide and pentoxifylline on the flow properties of equine erythrocytes:In vitro studies

The effects of various concentrations of furosemide and pentoxifylline on equine RBCin vitro were evaluated to facilitate better understanding of the potential effects of these drugs on blood flow properties. Furosemide induced increased mean cell volume (MCV), increased RBC potassium concentration, increased whole blood viscosity, and decreased the RBC filtrability. These data indicate that furosemide may block the RBC membrane transport pathways resulting in potassium and water retention. The increase in size and the resultant decrease in the surface-area-to-volume ratio may have caused the impaired RBC filtrability and increased blood viscosity. Pentoxifylline improved RBC filtrability without changing the RBC size or the potassium or chloride concentrations, suggesting that pentoxifylline may increase the deformability of the RBC membrane. The study indicated that pentoxifylline has potential therapeutic applications for improving microvascular blood flow but that furosemide may have adverse effects on blood flow.Abbreviations cP centipoise - EIPH exercise-induced pulmonary haemorrhage - MCV mean cell volume - Na-K-2Cl sodium potassium 2-chloride - PCV packed cell volume - RBC red blood cell     

Trials of serotonin, substance P and alpha 2-adrenergic receptor effects on the equine large colon

Some support for both serotonin and substance P as biotransmitters in the equine large colon is presented. Close arterial injections of small quantities of each of these were effective, respectively, in stimulating marked increases in coordinated pressure peaks in the vicinity of the pelvic flexure, with modest (serotonin) or marked (substance P) increase in colon artery blood flow. Both the acaricide, amitraz, and the tranquilizer xylazine appeared to act on the equine large colon via yohimbine-sensitive alpha 2-adrenergic receptors.     

In vitro effects of hydrochloric and lactic acids on bioelectric properties of equine gastric squamous mucosa

REASONS FOR PERFORMING STUDY: Volatile fatty acids, byproducts of carbohydrate fermentation by resident bacteria, have been implicated in causing nonglandular (NG) gastric ulcers. Lactic acid (LA), also produced by stomach bacteria, may cause gastric ulcers when exposed to the equine NG mucosa. OBJECTIVES: To investigate the in vitro effects of LA on equine NG mucosa bioelectric properties, sodium transport and tissue resistance. METHODS: Gastric tissues obtained from 13 mature horses were studied in Ussing chambers. Short-circuit current (Isc) and potential difference (PD) were measured, and electrical resistance (R) and conductance (G) calculated for tissues after addition of HCl and LA (5, 10, 20 and 40 mmol/l) in normal Ringer's solution (NRS). RESULTS: Mucosa exposed to HCl or LA (5, 10 and 20 mmol/l) in NRS (pH 1.5 and to a lesser extent pH 4.0) had a significant decrease in Isc and PD. Mucosa exposed to a high concentration of LA (40 mmol/l) in NRS (LRS) at pH 1.5 showed an increased G, but this increase was not significant. Values returned to baseline after solutions were returned to pH 7.0. Histological changes were consistent with HCl-induced (pH <4.0) acid damage. CONCLUSIONS: HCl induced alteration in bioelectric properties of equine NG mucosa whereas addition of LRS did not, other than those changes seen with HCl alone.