A comparative study between responses of isolated bovine and equine digital arteries to vasoactive mediators.

Hemodynamic perturbations, partly resulting from abnormal vasoconstriction of digital vessels, have been implicated in the pathogenesis of bovine and equine laminitis. This study compared the responsiveness of isolated bovine (BDA) and equine (EDA) digital arteries to pharmacological agents that stimulate receptor systems involved in the regulation of normal vessel tone. The role of the endothelium and the short- and longer-term effects of an experimentally induced endothelial damage were also evaluated. Species-related differences were found in the vessel reactivity to all of the receptor agonists tested. In intact BDA, as compared to intact EDA, norepinephrine was a more effective vasoconstrictor, 5-hydroxytryptamine a more effective but less potent vasoconstrictor, isoproterenol a less effective vasodilator and carbamylcholine a less potent vasodilator. In BDA, but not in EDA, the contractile responses to norepinephrine and 5-hydroxytryptamine were enhanced immediately after endothelium removal. However, the contractile reactivity of denuded BDA returned to basal values following overnight incubation. The differences suggest species specificity for the pathophysiology of digital vasomotor tone and function in horses and cattle.

Effects of endotoxin and influence of cyclooxygenase-2 on ß-adrenergic mediated relaxation in isolated equine digital artery.

The effects of endotoxin on ß-adrenergic-mediated relaxation were investigated in the equine digital artery (EDA). Possible involvement of cyclooxygenase-2 (COX-2) in endotoxin-induced effects and basal EDA ß-adrenoceptor functionality was also evaluated. Endothelium-intact (e(+)) and/or -denuded (e(-)) EDA rings were incubated overnight with lipopolysaccharide (LPS), LPS+NS398 (selective COX-2 inhibitor) or NS398 alone. Vessel rings were then mounted in organ baths and relaxant responses to isoproterenol (ISOP) recorded on U44069-induced pre-contraction. Response to ISOP was further evaluated in either incubated or freshly isolated (e(-)) rings acutely exposed to NS398. Fresh and incubated (e(-)) EDAs were also analysed for COX-2 expression by Western blotting. LPS caused endothelium-dependent enhancement of ß-adrenergic mediated relaxation. NS398 did not reverse endotoxin effects, suggesting that COX-2 did not have a mediating role. In the absence of LPS, NS398 significantly increased ISOP-induced relaxation. This finding, together with immunoblot detection of COX-2 in both fresh and incubated (e(-)) vessels, revealed the existence of a constitutive COX-2 exerting tonic inhibitory modulation on EDA ß-adrenergic-mediated relaxation. The results support the possible role of endotoxin in the vascular disturbances associated with equine laminitis. Moreover, the involvement of COX-2 in the physiological regulation of EDA tone warrants further clinical investigation into the efficacy and safety of selective COX-2 inhibitors on digital circulation in horses.

A clonal cell line (BME-UV1) as a possible model to study bovine mammary epithelial metabolism: metabolism and cytotoxicity of aflatoxin B1.

Despite the toxicological risks to which humans and animals are exposed due to the transfer of toxic xenobiotic metabolites into milk of domestic animals, studies on the metabolizing mechanisms occurring in ruminant mammary gland are totally lacking. To investigate the possible biotransformation capabilities of a bovine mammary epithelial cell line (BME-UV1), monolayers were exposed to aflatoxin B1 (AFB1--1.0-8.0 microM). Starting from 4 h of exposure, the hydroxylate metabolite aflatoxin M1 (AFM1) was detected in media by high performance liquid chromatography. AFM1 concentration increased linearly with time for 36-48 h and the percent biotransformation of AFB1 (2-4 microM) at 48 h was about 12-14%. Parallel cytotoxicity assays (neutral red uptake-NRU and MTT assays) were performed to investigate the possible interference of AFB1 cytotoxicity with cellular metabolism. MTT assay (from 24 h of cell exposure) and NRU assay (from 16 h of cell exposure) showed time-dependent and time/concentration-dependent decrease of cell viability, respectively, and the former assay being more successful at revealing cytotoxic effects (NRU: CC50 at 48 h = 12.00 +/- 2.66 microM; MTT: CC50 at 72 h = 20.42 +/- 7.30 microM). The results suggest that BME-UV1 cells express metabolizing enzymes having catalytic activity, thus representing a potential in vitro model for studying biotransformation in bovine mammary gland.

Investigation of the metabolic activity of a bovine mammary epithelial cell line (BME-UV1).

The metabolic activity of a mammary epithelial cell line (BME-UV1) was evaluated on monolayers exposed, in serum free medium, to different concentrations (2-4-8 muM) of aflatoxin B1 (AFB1), a mycotoxin eliminated into milk especially as hydroxylated metabolite aflatoxin M1 (AFM1). After 4, 8, 12, 24 h of treatment, a dose and time dependent production of AFM1 has been detected. As the enzymes involved in the hydroxylation of AFB1 in bovine hepatocytes are mainly CYP1A and CYP3A, the results suggest that BME-UV1 express CYP450 isoenzymes which metabolize AFB1 thus representing a potential model for the investigation of the metabolic activity of bovine mammary epithelial tissue.

Oral and intravenous administration of nimesulide in the horse: rational dosage regimen from pharmacokinetic and pharmacodynamic data.

REASONS FOR PERFORMING STUDY: The selective COX-2-inhibitor nimesulide is used extra-label in equine veterinary practice as an anti-inflammatory agent. However, there are no data on which to base the rational use of the drug in this species. OBJECTIVES: To determine the effective COX selectivity of nimesulide in the horse, and suggest a suitable dosing schedule. METHODS: The pharmacokinetics of nimesulide in the horse after oral administration (1 mg/kg bwt), and oral and i.v. administration (1.5 mg/kg bwt) were investigated, effects of feeding status on bioavailability determined, and plasma protein binding of the drug and its principal metabolites measured. Compartmental and noncompartmental pharmacokinetic analyses were performed. The plasma concentration-time profile was used, together with in vitro literature data on nimesulide inhibition of COX isoforms, to determine the effective COX selectivity of nimesulide in the horse, and suggest a suitable dosing schedule. RESULTS AND CONCLUSIONS: The findings suggest that 1.5 mg/kg bwt may produce adequate clinical effects, and the dosing interval should be 12-24 h depending on condition severity. However, at that dose, the concentration in the animal exceeds the in vitro IC50 for both isoforms, so that COX-1/COX-2 selectivity is lost and side-effects due to COX-1 inhibition are a possibility. Nimesulide should therefore be used with caution in equine clinical practice.

Pharmacokinetics and mammary elimination of imidocarb in sheep and goats.

The pharmacokinetics and mammary excretion of imidocarb dipropionate, a therapeutic/prophylactic agent against a variety of tick-borne hemoparasitic diseases in domestic animals, have been investigated in sheep and goats. A commercial formulation of imidocarb di-propionate was injected i.m. at a single dose of 3 mg/kg of body weight in 7 mature lactating ewes and 8 lactating does in good health. Blood samples were collected for 48 h after administration and milk samples were collected every 12 h for 10 d. A weak cation-exchange solid-phase procedure was used to remove imidocarb from plasma. A hexane/isoamyl alcohol liquid-liquid procedure was adopted to extract the drug from the milk of sheep. The same method was used for goat milk after exposing the matrices to enzymatic digestion. The extracted samples were analyzed by HPLC. The i.m. disposition kinetics of imidocarb in the 2 species showed significant differences in the rate of elimination (0.0075 +/- 0.002 and 0.025 +/- 0.004 L/h in sheep and goats, respectively), being faster in ewes than in does. Nevertheless, a smaller area under the concentration-time curve (12.21 +/- 0.76 and 9.49 +/- 0.54 microg/mL per h in sheep and goats, respectively), a larger volume of distribution (4.18 +/- 0.44 and 7.68 +/- 0.57 L/kg in sheep and goats, respectively), and a longer mean residence time (9.07 +/- 0.77 and 14.75 +/- 2.20 h in sheep and goats, respectively) were found in goats, suggesting a more rapid and effective drug storage in tissues during the first 48 h after the injection. The concentrations of imidocarb in milk of both species were higher than in plasma. However, a fast passage through the blood-milk barrier and a high storage of imidocarb were observed in the milk of ewes, whereas the drug concentrations were not as high nor was the extent of drug penetration from blood to milk as great in the milk of goats (AUC(milk 0-48)/AUC(plasma 0-48) = 2.5 +/- 0.45 and 1.26 +/- 0.27 in sheep and goat, respectively). Despite the differences in pharmacokinetic behavior, and considering the sensitivity of pathogens to imidocarb, the same dosage regimen can be used for clinical efficacy against Babesia spp. infection in both species. In contrast, the differences in depletion of imidocarb residue in milk and the large variability in mammary drug elimination found in goats suggests that great care should be taken in defining the withdrawal time in small ruminant dairy species.

Adrenergic regulation of vascular smooth muscle tone in calf digital artery.

Radioligand binding studies and functional assays on isolated smooth muscle preparations were performed in order to obtain a biochemical and functional characterization of the beta-adrenoceptor (beta-AR) subtypes involved in regulation of the smooth muscle relaxation of the calf's common digital artery. The results indicate that the common digital artery possesses two beta-AR populations (40% beta(1) and 60% beta(2)) and the beta(2)-subtype appears to predominate as far as function is concerned. Only the beta(2)-AR agonists clenbuterol and fenoterol caused dose-related relaxant effects, antagonized by propranolol, when tested in preparations precontracted both with PGF(2alpha) (1.4 x 10(-5) m) and noradrenaline (1.2 x 10(-6) m). In noradrenaline precontracted preparations the beta(1)-AR selective agonists dobutamine and xamoterol caused vasodilation which was not antagonized by (+/-)propranolol. While the functional relaxant effects of dobutamine may be attributed to its potent competitive alpha-AR blocking activity, further investigations are required to explain the effect of xamoterol. The vasodilator effect of (+/-)isoproterenol was irregular. The recorded contractile effects, mainly at dosages greater than 10(-6) m, suggest the loss of drug selectivity for beta-AR and alpha-AR activation. Indirect evidence indicates that the alpha-adrenoceptor (alpha-AR) population in this tissue which produces a strong contraction is functionally dominant over the beta-AR, suggesting limited therapeutic benefit for beta-AR drugs to control blood flow disorders in the calf's distal limb.

Pharmacokinetics of imidocarb dipropionate in horses after intramuscular administration.

The objective of this study was to determine the pharmacokinetic behaviour of imidocarb in horses following a single i.m. injection at the dose commonly administered to treat Babesia caballi infections or to prevent babesiosis. Eight horses were injected i.m. with a single dose of 2.4 mg imidocarb dipropionate/kg bwt and blood, faecal, urine and milk samples were collected. For imidocarb determination, a high-performance liquid chromatographic method (HPLC) was used after weak cation-exchange solid phase, or liquid-liquid, extraction procedures. Twelve hours after treatment, no detectable plasma concentrations were recorded in any of the treated animals. The distribution and elimination patterns of the drug suggested that it is quickly sequestrated in some storage tissues and remains in the body for a long time. Its prolonged presence in the body may confer a reservoir effect to imidocarb in some tissues, therefore making it undetectable in the plasma of animals but sufficient to produce its described therapeutic and prophylactic activities.

Identification of functional alpha-adrenoceptor subtypes in the bovine female genital tract during different phases of the oestrous cycle.

The concentration and functionality of the alpha-adrenoceptor (alpha-AR) subtypes in the genital tract of cyclic heifers were investigated. In each tissue sample, a single class of alpha1-ARs was observed, whereas two distinct classes of alpha2-ARs were discriminated: low-affinity (LA) and high-affinity (HA) alpha2-ARs. Statistical analysis showed the presence of significantly (p < 0.05) higher concentrations of all alpha-AR subtypes in the follicle than in the corpus luteum. No significant differences were found in the ovary or myometrium between the luteal and follicular phases. In the ovary, the density of alpha1-ARs was significantly (p < 0.05) higher than that of alpha2-ARs. By contrast, there were significantly (p < 0.05) more alpha2-ARs than alpha1-ARs in the myometrium. As far as alpha2-ARs are concerned, LA alpha2-ARs were significantly (p<0.05) higher than HA alpha2-ARs in all tested tissues. Competition studies suggested that the rank order of potency of antagonists for alpha1-ARs was prazosin > phentolamine > yohimbine, whereas for alpha2-ARs the order of potency was yohimbine > or = phentolamine>prazosin. Functional assays performed on myometrium showed that noradrenaline, phenylephrine and clonidine elicited concentration-dependent contractions only in dioestrus and pro-oestrus preparations and that clonidine was more effective than phenylephrine as a contractile agent. It appeared that there were no significant modifications in alpha-AR affinity or concentration during the different stages of bovine oestrous cycle, whereas the uterine spontaneous activity and the responsiveness to alpha-adrenergic stimulation was strongly influenced by hormonal levels. The modifications of uterine contractility observed during the oestrous cycle may be related to modifications induced in the transductional mechanisms of alpha-ARs.

Identification and coupling to adenylate cyclase of three different [(3)H]CGP 12177 binding sites in Caco-2 cell membranes.

In the present investigation the identification of beta -adrenoceptor (beta -ARs) subtypes in the Caco-2 cell line was performed using radiometric assays. beta -ARs were measured using increasing concentrations of the highly specific beta -AR antagonist (-)[(3)H]CGP 12177 (0.06-4 nM), whereas the beta(1)- and beta(2)-AR subtypes discriminated through selective binding assays using the highly selective unlabelled antagonists CGP 20712A and ICI 118551. Atypical beta -ARs were measured using an incubation system formed by higher concentrations (0.6-20 nM) of (-)[(3)H]CGP 12177. beta - Atypical binding site concentrations (69 +/- 5 fmol mg ml(-1)of membrane protein) were higher than beta(1)-ARs (7 +/- 1) and beta(2)-ARs (24 +/- 2), respectively. The different beta -AR subtype affinities were characterized by binding inhibition experiments and the adrenergic agonists displaced the radioligand from its specific binding sites in the following order of potency: isoproterenol > clenbuterol > dobutamine > SR 58611A; for antagonists the order of potency was: propranolol approximately = ICI118551 approximately = CGP20712A. For atypical beta -ARs the order was: SR 58611A > clenbuterol > dobutamine > isoproterenol for agonists and propranolol > CGP 20712A > ICI 118551 for antagonists. As far as in vitro functional studies are concerned, beta -AR subtypes were shown to be coupled to adenylyl cyclase as their stimulation produced cAMP in an amount significantly higher than basal values. cAMP production after stimulation with dobutamine, clenbuterol, isoproterenol, and SR 58611A was measured using a cAMP radioassay kit. The order of efficacy suggested that the stimulation of beta(2)-ARs was the most effective in inducing the activation of cell signalling mechanisms. The identification of functional beta -ARs in a cancer cell line represents the first step in the study of the possible adrenergic control of cellular activities (e.g. proliferation and/or differentiation), which could suggest the use of this cancer cell line as a model for the study of cell activity or possibly new therapeutic strategies.

Determination of dexamethasone in milk of dairy cows by immuno-enzymatic assay.

Eight lactating cows received 3 im injections at 24 h intervals of a commercial formulation containing dexamethasone. Each treatment provided 25 microg/kg bw/d of dexamethasone acetate, equivalent to 22.6 mg of dexamethasone. Milk samples were obtained before treatment (5 d), during the treatment period, and for up to 22 milking after the last injection. The concentrations of dexamethasone in the milk samples were determined by a commercial competitive immunoenzymatic assay for corticosteroids (detection limit 0.15 ng dexamethasone/ml). The conventional therapeutic dose of dexamethasone acetate caused milk drug concentrations exceeding the tolerated maximum residue limit (0.3 mg/kg). A withdrawal time of 3-3.5 d for dexamethasone in milk provided sufficient protection for consumer health. The commercial enzyme immunoassay kit employed in this study was sufficiently sensitive, easy to use, and appropriate to monitor the use of dexamethasone in lactating animals.

The relationship between heritability and smoking habits in Crohn's disease. Italian Cooperative Study Group.

OBJECTIVE: In Crohn's disease (CD), the relationship between genetic predisposition and smoking has not been well defined. The aim of this study was to compare the smoking habits at the time of the diagnosis of CD patients having familial occurrence of inflammatory bowel disease (IBD) with those of some control groups. METHODS: In a multicenter study, 136 CD patients with a relative with IBD, 272 healthy controls matched for sex and age, 500 CD patients without familial occurrence of IBD, and 84 ulcerative colitis patients (UC) with familial occurrence of IBD were personally interviewed about their smoking habits. In addition, data for 35 healthy siblings of patients with familial CD were collected by interviewing the patients' relatives. RESULTS: The prevalence of smokers was found significantly higher in CD patients with a family history for IBD than in healthy controls and in familial UC patients (OR 2.28 CI 1.5-3.48 and OR 5.81 CI 3.15-10.75, respectively). No significant difference was found either in the percentage of smokers or in the number of cigarettes smoked per day between familial and sporadic CD patients. Among all siblings of CD patients, 72% of affected siblings and 34% of healthy siblings were smokers, concordant with their relatives. CONCLUSIONS: In CD patients with familial occurrence of IBD, the percentage of smokers is elevated. It is possible that in a genetically predisposed population, smoking could be an important environmental factor in determining CD or expressing this disease instead of UC.

Affinity of isoxsuprine for adrenoreceptors in equine digital artery and implications for vasodilatory action.

We used isolated equine digital arteries to study the vasodilatory mechanism of isoxsuprine, and fowl caecum preparations to investigate the affinity of the drug for beta-adrenoceptors. Isoxsuprine is a potent vasodilator of arterial smooth muscle that has been precontracted by an alpha-adrenoceptor agonist such as noradrenaline (log EC50 = -6.33 [-5.98; -6.68]). The present study indicates that its effect is due to alpha-adrenoceptor blockade since: (1) after a long lasting exposure to cumulative doses of isoxsuprine the vasoconstricting action of noradrenaline cannot be restored; (2) isoxsuprine does not promote relaxation on preparations precontracted by PGF2alpha; (3) isoxsuprine shifts the dose-response curve of noradrenaline to the right; and (4) its affinity (pK(B) = 6.90 [6.60; 7.20]) in this experiment is comparable to that in noradrenaline-precontracted preparations and is 14 times lower than that of the selective alpha1-adrenergic antagonist prazosin [pK(B) = 8.04 (7.40; 8.68]). The affinity of isoxsuprine for beta-adrenoceptors was 100 times lower than that of isoprenaline when tested on fowl caecum. This preparation has a large beta-adrenoceptor and negligible alpha-adrenoceptor population concerned with the control of smooth muscle motility. Our data suggest that the alpha-mediated effect of isoxsuprine on horse arterial smooth muscle is due to higher affinity of the drug for alpha- than beta-adrenoceptors rather than low concentration or functionality of beta-sites at this site. According to these data, pure beta2-agonists seem to be more profitable tools to determine vasodilation of the arterial bed in horses legs.

Investigations on the stereoselective action of isoxsuprine on alpha- and beta-adrenoceptors in equine common digital artery.

The affinity and functional effects of isoxsuprine enantiomers were investigated to determine the enantiospecificity of the beta-agonistic and alpha-blocking effects. Functional assays on isolated smooth muscle preparations from equine common digital artery were performed to determine the apparent affinity (pD(2)) and intrinsic activity (alpha(E)) of (-)erythro-isoxsuprine (alphaS, betaR, gammaR) and (+)erythro-isoxsuprine (alphaR, betaS, gammaS). The affinity of two enantiomers for the different adrenoceptor types was studied by radioligand binding assays on membrane preparations from the same tissue, using (-)[(3)H]CGP12177 and [(3)H]prazosin. On noradrenaline-precontracted artery preparations (-)isoxsuprine was markedly more potent than (+)isoxsuprine in dilating preparations, indicating that the laevorotatory enantiomer has a very high apparent affinity for alpha-adrenoceptors. Binding studies confirmed that (-)isoxsuprine has a higher affinity than (+)isoxsuprine for alpha-adrenoceptors, while the (+) isomer competes for beta-adrenoceptors with an affinity similar to that of propranolol. As described for other beta-phenylethylamines, the two isoxsuprine enantiomers studied have different efficacies for alpha- and beta-adrenoceptors and the effects of the commercially available mixture of stereoisomers therefore depend on the density and functional importance of the adrenoceptor types present in the tissue studied. 1999 Academic Press.

Disposition of antimony and aminosidine combination after multiple subcutaneous injections in dogs.

The disposition of a combination of antimony (Sbv) (12.8 mg/kg) and aminosidine (AM) (10 mg/kg) in 10 healthy Beagle dogs after multiple subcutaneous injections is described. Sbvplasma concentrations were determined by atomic absorption spectrometry, and AM by ion-pair liquid chromatography, using a fluorimetric detector. Sbvreached Cmaxat 60 min, and for about 1 h plasma levels were homogeneously stabilized between 10.78 and 11.76 microgram/mL; by 12 h, Sbvplasma concentrations were close to the detection limit (0.3 microgram/mL). AM Cmaxvalues were recorded after 1 h (30.6+/-3.11 microgram/mL, mean +/- SD), and plasma levels reached values close to the detection limit (0.15 microgram/mL) between 7 and 8 h after injection. Sbvkinetic parameters did not appear modified by the presence of AM. Moreover, repeated injections of the combination did not modify the kinetic behaviour of the two drugs and did not alter the renal function of the animals. The superimposition analysis of the Sbvdata suggests that a twice daily injection of the metal at a dose of 12.8 mg/kg would be sufficient to maintain inhibitory Sbvconcentrations similar to those recorded in humans.

Suitability of the old fowl rectal caecum preparation for investigating the selectivity of beta-adrenergic drugs.

We tested alpha- and beta-adrenergic drugs on isolated strips of fowl rectal caecum from 14- to 16-week-old Warren hens. Basal tone and spontaneous motility were dose-dependently reduced by isoprenaline and all the selective beta-agonists tested (except xamoterol) with the following order of potency: isoprenaline=fenoterol=procaterol=clenbuterol>dobutamine> SR58611A. The results indicate that this tissue preparation consists almost entirely of beta2-adrenoceptors. This preparation may, therefore, be considered a suitable assay for discriminating beta1- from beta2-agonists according to their selectivity.

Differences between longitudinal and circular smooth muscle in beta-adrenergic control of motility of isolated equine ileum.

OBJECTIVE: To identify beta-adrenoceptor subtypes involved in motility inhibition of circular and longitudinal smooth muscle layers of equine ileum. SAMPLE POPULATION: Isolated strips of equine ileum circular smooth muscle and membrane preparations from circular and longitudinal muscle layers. PROCEDURE: Functional assays of circular muscle preparations and radioligand binding assays and measurements of cAMP production in smooth muscle membranes from circular and longitudinal layers. RESULTS: Selective beta-adrenergic agonists exerted inhibitory effects on circular muscle preparations. Binding studies of cell membranes indicated that the density and distribution of 3 beta-adrenoceptor subtypes did not differ between longitudinal and circular muscle layers. Measurement of cAMP production in membrane preparations of longitudinal and circular muscle after selective beta-stimulation confirmed presence of the 3 adenylate cyclase-coupled beta-adrenoceptor subtypes; however, preparations from the 2 layers had differing cAMP production efficacy. CONCLUSIONS: The data may partly explain the differing functional responses between circular and longitudinal muscle preparations. CLINICAL RELEVANCE: Findings support the important role of beta-atypical adrenoceptors in the inhibitory regulation of equine ileum motility.

Controlled trial of oral 5-aminosalicylic acid for the prevention of early relapse in Crohn's disease.

BACKGROUND: Recent data indicate that 5-aminosalicylic acid (5-ASA) is most effective in preventing relapse of Crohn's disease in patients with a short duration of remission before enrollment. AIM: To evaluate the efficacy of oral 5-ASA treatment, started immediately after achieving steroid-induced remission, in preventing clinical relapses of Crohn's disease. METHODS: Patients with active Crohn's disease, achieving remission on steroids, were randomized to oral 5-ASA 3 g/day or placebo, while steroids were tapered over 6 weeks. The trial was terminated after interim analysis showed a slightly higher relapse rate in the 5-ASA group, and the calculated probability of seeing a statistically significant difference by completing the study was minimal. RESULTS: Final analysis included 117 patients (58 taking 5-ASA and 59 taking placebo; follow-up 9.2 +/- 6.5 months). Cumulative relapse rates at 6 and 12 months were 34% and 58% in 5-ASA patients and 31% and 52% in placebo patients, respectively (rate difference +0.095; 95% CI = -0.085 to +0.274). Subgroups analysis showed that 5-ASA was equally ineffective in patients with ileal, colonic or ileocolonic disease. CONCLUSIONS: Contrary to previous results, in our study early introduction of treatment with oral 5-ASA did not prevent relapse in Crohn's disease patients treated with steroids to induce remission.