Effects of WEB 2086, an antagonist to the receptor for platelet-activating factor (PAF), on PAF-induced responses in the horse.

Platelet-activating factor (PAF) causes oedema and neutrophil accumulation when injected into the skin of normal horses. PAF is also known to induce aggregation of horse platelets in vitro. The selective PAF receptor antagonist WEB 2086 has now been used to determine whether these effects are mediated by PAF receptor activation. Addition of WEB 2086 to equine platelets in vitro inhibited PAF-induced aggregation in a competitive reversible manner (pA2 = 7.14). Inhibition of in vivo inflammatory responses to PAF occurred after local administration of WEB 2086: wheal formation induced by 0.1 micrograms PAF/site was reduced by 1-10 micrograms WEB 2086/site. PAF (1 micrograms/site)-induced neutrophil accumulation was also inhibited by co-administration of 10 micrograms WEB 2086/site. Systemic administration of WEB 2086 (3 mg/kg iv) to 4 normal ponies inhibited PAF-induced wheal formation and ex vivo platelet aggregation. At 30 min after drug administration the concentration of PAF required to produce a half maximal aggregation response was increased 496 +/- 137 fold. At 6 h the degree of inhibition was markedly reduced and responses had returned to pre-treatment values by 24 h. PAF-induced increases in cutaneous vascular permeability were also reduced by 80% as early as 30 min after iv administration of WEB 2086 in these animals, the inhibition persisting for at least 6 h. These results suggest that in vitro and in vivo responses to PAF in the horse are mediated via activation to PAF receptors. WEB 2086 will therefore be a useful agent for studying the role of PAF in the pathogenesis of equine inflammatory conditions.     

A comparison of the actions of platelet activating factor (PAF) antagonists WEB 2170 and WEB 2086 in the horse

Foster, A.P., Cunningham, F.M., Andrews, M.J., Lees, P. A comparison of platelet activating factor (PAF) antagonists WEB 2170 and WEB 2086 in the horse. J. vet. Pharmacol. Therap. 16, 477–487.
The effects of the selective platelet activating factor (PAF) receptor antagonist WEB 2170 on PAF-induced responses in equine cells and tissues have been examined and compared with those of WEB 2086. In initial experiments WEB 2170 was shown to inhibit in vitro platelet aggregation in a dose-dependent, competitive reversible manner (pA₂= 7.21). Co-administration of the antagonists with either PAF or histamine also inhibited PAF, but not histamine, induced wheal formation and PAF-induced neutrophil accumulation in vivo in equine skin. Intravenous (i.v.) administration of both drugs at a dose of 0.1 mg/kg blocked PAF-induced ex vivo platelet aggregation. The inhibition produced by WEB 2170 was greater and, at 30 min, this drug also reduced the slope and maximal response. Wheal formation in the skin was significantly inhibited for up to 6 h by WEB 2170 administered i.v., the reduction being more prolonged than that obtained with WEB 2086. Neutrophil accumulation in the skin was also significantly reduced for up to 24 h by WEB 2170, whilst no significant inhibition was produced by WEB 2086. These results demonstrate that WEB 2170, like WEB 2086, is an effective antagonist of PAF in the horse. Moreover, when given i.v., WEB 2170 appears to be a more potent PAF inhibitor than WEB 2086.     

Functional and biochemical characterization of immunologically derived equine platelet-activating factor

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets pre-incubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after base-catalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.     

Actions of the novel gastrointestinal pro kinetic agent cisapride on equine bowel motility

The effect of cisapride was evaluated on the normal fasting bowel motility of four ponies with chronically implanted electromechanical transducers. Cisapride was infused over 60-min periods at 0.05 mg/kg (n = 4), 0.1 mg/kg (n = 5) and 0.25 mg/kg (n = 5). It produced marked and prolonged increases in electrical and mechanical activity at all sites examined. In the stomach there was increased total contraction activity with increased contraction amplitude and a slight reduction in rate. In the small intestine there was an increase in irregular (phase II) activity with an increase in number and amplitude of contractions and a decrease in the number of regular (phase III) activity fronts. There was a decrease in the number of phase III fronts that spread distally from the jejunum to the ileum. The phase II activity was coordinated temporally with prolonged activity in the stomach. Cisapride increased electrical and contractile activity in the left dorsal colon with increased contraction amplitude and an increase in electrical activity in the small colon. In the stomach and small intestine cisapride produced dose-dependent increases in activity but in the left dorsal and small colon the intermediate dose (0.1 mg/kg) produced the largest and most consistent responses. Side-effects observed were increased bowel sounds and frequency of defaecation, a slight increase in heart rate and transient signs of discomfort at the highest (0.25 mg/kg) dose rate.     

Biphasic disruption of fasting equine gut motility by dopamine – a preliminary study

Dopamine was infused intravenously (1, 5 and 10 micrograms/kg/min) for 60 min in three fasted ponies. A dose-dependent increase in heart rate occurred that was rapid in onset and termination at the start and end of the infusions, respectively. Dose-dependent changes in gastric and small intestinal motility were observed. An initial marked inhibition of gastric contraction amplitude was followed by a secondary prolonged period of activity. At the same time the small intestine showed a prolonged period of irregular activity (phase II) and a marked increase in the interval between successive phase IIIs. The left dorsal colon and small colon exhibited variable responses. The normal fasting motility pattern was therefore disrupted by dopamine biphasically, an initial inhibition of the stomach being followed by a period of increased activity in the stomach and small intestine which resembled the postprandial motility pattern. Although the cardiovascular effects of dopamine were transient, the increases in gastrointestinal motility persisted long after the infusion was terminated.     

Protective effects of WEB 2086 (PAF antagonist) and ketoprofen (NSAID) on PAF-induced changes in the morphological ultrastructure of blood platelets in calves

The ultrastructure of bovine platelets was examined by transmission electron microscopy without any pretreatment (control), and after WEB 2086 (a triazolodiazepine) or ketoprofen (NSAID) pretreatment, followed by PAF infusion. The blood platelet count was also investigated. The group of calves that received WEB 2086 pretreatment before platelet-activating factor (PAF) infusion did not show a decreased number of platelets. However, in the other group, with ketoprofen pretreatment before PAF infusion, there was a rapid decrease from 1 to 3 min, while from 5 min the number of platelets recovered to the normal value. Electron microscopy revealed that pretreatment with WEB 2086 followed by PAF infusion did not alter the morphological ultrastructure of bovine platelets, except that the microtubules were briefly modified from 1 until 3 min after PAF challenge. After ketoprofen pretreatment, bovine platelets kept their regular shape, the number of dense bodies was not significantly altered, the number of mitochondria was maintained from 5 min after PAF infusion, giant platelets were not observed and the Golgi apparatus was rarely visible. Thus pretreatment with WEB 2086 and ketoprofen before PAF infusion had a protective activity on the ultrastructure of bovine platelets and, in cattle, pretreatment with WEB 2086 and ketoprofen before PAF challenge prevented the thrombocytopenia induced by PAF.     

In vitro evaluation of the effect of the opioid antagonist N-methylnaltrexone on motility of the equine jejunum and pelvic flexure

REASONS FOR PERFORMING STUDY: Although potent analgesics, opioids decrease intestinal activity, leading to ileus in many species. N-methylnaltrexone (MNTX), an opioid antagonist which does not cross the blood-brain barrier and antagonises the morphine effect on the intestine, directly stimulates motility and restores function without affecting analgesic properties. While its use has been reported in human subjects, there is no information with regard to its usage in the horse. OBJECTIVES: To determine whether MNTX has an effect on contractile activity of the equine jejunum and pelvic flexure. METHODS: Using circular smooth muscle strips obtained from 8 mature horses, increasing concentrations of MNTX were added to tissue baths in the range of 1 x 10(-9) to 1 x 10(-5) mol/l, and contractile responses were recorded for 3 mins. Data were analysed using a repeated measures ANOVA to determine whether there was a significant drug effect compared to baseline activity. Data were analysed between the jejunum and pelvic flexure using a Mann-Whitney U test. Statistical significance was established as P < 0.05. RESULTS: The administration of MNTX significantly increased the contractile frequency and amplitude at all concentrations relative to baseline (P < 0.0001) for the jejunum. The response was greatest at 1 x 10(-7) mol/l (P = 0.0005), with a mean difference from baseline of 115.12 g/cm2. The highest concentration evaluated (1 x 10(-5) mol/l) had a mean contractile strength of 69.76 g/cm2, which was significantly greater than baseline activity (P = 0.04). A significant increase in contractile activity for the colon was detected at 3 x 10(-7) mol/l and all subsequent concentrations (P < 0.04). Unlike the jejunum, the contractile activity of the pelvic flexure increased progressively with the addition of each subsequent concentration. CONCLUSIONS: N-methylnaltrexone has a direct effect on circular smooth muscle of the equine jejunum and pelvic flexure resulting in an increase in contractile activity. POTENTIAL RELEVANCE: N-methylnaltrexone could potentially be used in conjunction with morphine to provide potent and effective analgesia without compromising intestinal function. Further in vivo investigations are required to determine whether this agent antagonises morphine's effect on motility.     

Involvement of platelet activating factor in the endotoxin-induced effects on gastrointestinal electrical activity and some haematological parameters in the conscious miniature pig

In conscious miniature pigs the influence of intravenous dose of lipopolysaccharide (LPS), 10 microg/kg over 10 min, with and without pretreatment with a platelet activating factor (PAF) antagonist, SAH 63-675 10 mg/kg, on gastrointestinal electrical activity, arterial pressure and clinical and haematological parameters was studied. Dose of LPS provoked mild clinical signs and hypotension, which were prevented by PAF antagonism. The LPS induced leukocytosis and increase in mature neutrophils, however, were PAF independent. Pretreatment with the PAF antagonist attenuated the LPS-provoked inhibition of electrical activity in the antrum, jejunum, ileum and caecum. These results suggest a beneficial effect of PAF antagonism in porcine endotoxaemia.     

Motility of the equine gastrointestinal tract: Physiology and pharmacotherapy

Normal gastrointestinal (GI) motility patterns are necessary to maintain transit of ingesta and to facilitate digestion and absorption of nutrients. Disorders of the equine GI tract are frequently encountered by the equine practitioner and these disorders are often associated with an interruption in normal intestinal motility patterns, thus complicating treatment of the primary disease. Consequently, numerous treatments have been investigated in horses to facilitate the return of normal intestinal motility. The purpose of this article is to provide a brief review of the anatomy and physiology of the GI tract in the horse and review medications available to the equine veterinarian that may potentially promote intestinal motility.     

In vitro inhibitory effect of SR 27417, a potent platelet-activating factor (PAF) receptor antagonist, on the PAF-induced bovine platelet aggregation

The in vitro inhibitory effect of SR 27417, an antagonist of the platelet-activating factor (PAF) receptor, on PAF-induced platelet aggregation was studied in blood collected from seven healthy Friesien calves. Inhibitory effects of SR 27417 were determined at thirteen different concentrations (0.1-400 nM) by using the dose-response curves of PAF on calf platelet aggregation. In the presence of SR 27417, the maximal slopes of aggregation (%/min) induced by low and high concentrations of PAF were significantly different from the control values obtained without an antagonist at p < or = 0.05 and p < or = 0.01 respectively. In vitro PAF-induced calf platelet aggregation was dose-dependently inhibited by SR 27417. The drug inhibited PAF-induced platelet aggregation in a competitive reversible manner (pA2 = 10.46 +/- 2.36 mol x L(-1)). In conclusion, the results of our study showed that addition of SR 27417 to bovine platelet in vitro inhibits PAF-induced platelet aggregation.     

Passage rate of digesta through the equine gastrointestinal tract: A review

Passage rate through the equine gastrointestinal tract is best described by mean retention time (MRT). Transit times of digesta differ greatly between the different parts of the gastrointestinal tract. Passage through the stomach and small intestine is rapid (5 h on average), whereas a longer retention time is recorded in the cecum and colon (35 h on average).Important considerations in estimating MRT have been the use of a solute phase and a fluid phase marker and calculations applied, especially when sampling frequency is reduced.A number of animal and feed related factors influence MRT: body weight, pregnancy and lactation may increase passage rate, whereas the impact of exercise, the most important animal related factor, will depend on the exercise type and the fluid/particle ratio.Information on the effect of the main feed related factors is lacking. Smaller particles and feed with a higher water-holding capacity move slower through the gut, but reduced fibre length, increased feeding level and increased forage/concentrate ratio will accelerate passage rate. Feeding frequency seems not to affect passage rate.Sometimes, conclusions are contradictory as effect of animals, feeds and methods are confusing.     

Reduced endotoxin-induced production of tumor necrosis factor activity by equine peritoneal macrophages exposed to the dual inhibitor of arachidonic acid metabolism, SK & F 86002.

The purpose of this study was to determine if a structurally novel dual inhibitor of arachidonic acid metabolism, SK & F 86002, would inhibit the endotoxin-induced production of tumor necrosis factor (TNF) activity by equine peritoneal macrophages. Equine peritoneal macrophages were variously pretreated for 0, 0.5 and 2 h with SK & F 86002 at 10(-9) to 10(-4) molar final concentrations or were left untreated. Then, the macrophages were cultured in vitro in the presence of endotoxin (5 ng/mL). Supernatant media were collected after 4 h and stored at -70 degrees C until assayed for TNF activity and immunoreactive thromboxane B2 (iTxB2). Macrophage supernatant TNF activities were estimated by an in vitro cytotoxicity bioassay using the murine fibrosarcoma cell line, WEHI 164 clone 13. Concentrations of iTxB2 were quantitated by radioimmunoassay. Coincubation of macrophages with SK & F 86002 significantly decreased the subsequent supernatant TNF activity. Concentrations of SK & F 86002 from 10(-7) to 10(-4) molar effectively reduced TNF production when added to macrophages 0 and 0.5 h prior to endotoxin. After 2 h of preincubation, SK & F 86002 significantly reduced supernatant TNF activity at 10(-5) and 10(-4) M concentrations. Supernatant concentrations of iTxB2 were reduced when SK & F 86002 was added at 10(-6) to 10(-4) M concentrations, 0 and 0.5 h prior to endotoxin, and at all concentrations (10(-9) to 10(-4)) when preincubated with macrophages for 2 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endoscopic observations of the effect of number of spasmolytic and analgesic drugs on equine caecal motility

Movement of ingesta within the equine caecum has been observed endoscopically through a caecal fistula (Dyce and Hartman, 1973), but the observations were made unrelated to diet and in some cases on ponies from which food had been withheld for 24 hours. Strong movements of muscle bands, termed haustral formation, and eruption of caecal body contents and discharge of these contents into the caecal base were reported in the study. Also seen endoscopically was constriction of the walls of the caecal base dividing that region into cranial and caudal compartments. These findings were confirmed by Sellers et al (1982), whilst implanting an inflatable catheter into the caecum through a fistula.     

Effect of reduced gastrointestinal motility on the regulation of gastrointestinal flora and the pathogenesis of coli enterotoxinemia in market swine]

Opium tincture and Spasmentral were applied to piglets early after weaning and reduced their gastro-intestinal motility, which, however, caused only very minor changes in quantitative germ flora composition in those first days. Short-time suppression of gastro-intestinal motility obviously does not result in detrimental consequences to the organism as a whole, since there seem to be several factors which are involved in the control and regulation of the intestinal germ flora. Impairment of gastro-intestinal motility appeared to be of no importance to the pathogenesis of coli-enterotoxaemia, as it was not followed by higher incidence of the disease.     

Freezing equine semen: the effect of combinations of semen extenders and glycerol on post-thaw motility

Objective   We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility.
Design   A randomised 2 × 3 block design was used.
Procedure   Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy in fresh and post-thaw semen.
Results   There was a significant difference between the two extenders in the motility of spermatozoa after cryopreservation (48.9% for INRA 96; 38.6% for EZ Mixin OF; P < 0.0001). Glycerol at 4% in freezing medium yielded the highest post-thaw motility, significantly better than 2% ( P < 0.05). Three of four stallions had significantly higher post-thaw motility using INRA 96 relative to EZ Mixin OF ( P < 0.01), and two of four stallions had significantly higher post-thaw motility using 4% glycerol ( P < 0.05). The combination of INRA 96 and 4% glycerol in freezing medium gave the highest average post-thaw motility of 51.5%.
Conclusion   In this study, INRA 96 combined with 4% glycerol yielded an average recovery of progressively motile sperm consistently above the 35% target.     

Role of platelet-activating factor in alveolar septal injury associated with experimentally induced pneumonic pasteurellosis in calves

OBJECTIVE: To determine whether platelet-activating factor (PAF) is involved in acute lung microvascular injury associated with pneumonic pasteurellosis in calves. ANIMALS: 15 healthy 2- to 4-week-old male Holstein calves. PROCEDURE: Calves were anesthetized and inoculated intrabronchially with saline (0.9% NaCl) solution (n = 5) or 1x10(9) Pasteurella haemolytica organisms (n = 10). Of the 10 calves inoculated with P haemolytica, 5 also were treated with WEB 2086, a potent inhibitor of PAF, and 5 were treated with vehicle. Blood and bronchoalveolar lavage samples were collected before and 1, 2, 4, and 6 hours after inoculation of P. haemolytica. Blood samples were analyzed to evaluate total number and differential counts of leukocytes, dilute whole-blood leukocyte deformability, size of neutrophils, and neutrophil CD11b expression. Bronchoalveolar lavage samples were analyzed for total number and differential counts of nucleated cells, total protein concentration, and hemoglobin concentration. Size and gross and histologic appearance of lung lesions also was determined. RESULTS: Treatment of calves with WEB 2086 reduced size of lung lesions, attenuated the increase in microvascular permeability, and reduced neutrophil infiltration in the first 4 hours after inoculation. Treatment with WEB 2086 also attenuated a decrease in leukocyte deformability, increase in size of neutrophils, and CD11b expression by circulating neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: It appears that PAF is a major mediator for altered lung microvascular permeability and activation of circulating neutrophils in the first 4 hours after onset of pneumonic pasteurellosis in calves.     

The effect of prostaglandin E1 on motility of the equine gut

Prostaglandin E1 was infused intravenously (25, 50 and 75 ng/kg/min) in three ponies. Changes in gastrointestinal mechanical and electrical activity were recorded from chronically implanted strain-gauge force transducers and electrodes. Dose-dependent responses were obtained: there were significant decreases in electrical spiking activity in the stomach, left large colon and small colon, with a corresponding decrease of activity in the left dorsal colon mechanogram. The small intestine was also affected, showing a decrease in both contraction rate and amplitude, which was more marked in the proximal jejunum than in the ileum. There was an association between these changes in gastrointestinal activity and the presence of discomfort and diminished gut sounds.     

Effects of housing,feeding and use on equine health with emphasis on respiratory and gastrointestinal diseases

In a random population of Swiss horses 54% suffered from a subclinical to moderate COPD. Cause of a COPD is a hypersensitivity of the respiratory tract to spores of fungi and thermophil actinomyces. Teeth problems are strongly associated with the type of diet and the feeding regime. Problems of the teeth belong to the most often treated equine diseases by large animal practitioners. Racehorses are the population of horses most often affected by gastric ulcers with an ulcer prevalence between 63 and 90%. In contrast, a much lower prevalence (37%) of stomach ulcers is seen in pleasure horses and the degree of ulceration is less severe. Large amounts of concentrated high-energy feeds, small rations of forage and a low feeding frequency per day as well as the use of spoiled food can contribute to the development of colics.     

Influence of technical parameters on the in vitro motility of equine neutrophils in the presence of streptococcal culture supernatant

To identify the influence of technical factors on the in vitro motility of equine neutrophils towards streptococcus culture supernatant in an under-agarose assay, we studied the changes in eight cell migration parameters. The distances the phagocytes travelled by directed, random and spontaneous migration increased with incubation time, cell concentration and the gelatin and serum contents of the migration plates. The contribution of chemotaxis to the phagocyte migrations, however, decreased simultaneously. The directed and random, though not the spontaneous, migrations of the phagocytes increased also when the chemoattractant wells were placed closer to the cell wells but so did the influence of the chemokinetic activity of the bacterial culture supernatant on phagocyte motility. In contrast, preincubation of migration plates with the chemoattractant, the agarose content of the migration plates and contamination of the granulocytes with non-migrating, mononuclear cells did not substantially affect the in vitro migrations of the neutrophils. The changes in the in vitro motility of the equine neutrophils by these technical factors were, in general, comparable to those reported for human cells attracted by a variety of host-and bacteria-derived chemoattractants.