Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs

Myers, M. J., Scott, M. L., Deaver, C. M., Farrell, D. E., Yancy, H. F. Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs. J. vet. Pharmacol. Therap. 33 , 1–8.
The impact of nonsteroidal anti-inflammatory drugs (NSAID) on prostaglandin E₂ (PGE₂) production and cyclooxygenase 2 (COX-2) mRNA expression in bovine whole blood (WB) cultures stimulated by lipopolysaccharide (LPS) was determined, using the blood from six Holstein dairy cattle in various stages of lactation. Peak production of PGE₂ occurred 24 h after LPS stimulation but did not result in detectable concentrations of thromboxane B₂ (TXB₂). The NSAID indomethacin, aspirin, flunixin meglumine, and 4-[5-phenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzene sulfonamide (PTPBS; celecoxib analogue), along with dexamethasone, were all equally effective in reducing the concentration of PGE₂ in the bovine WB culture supernatants. Bradykinin exhibited peak supernatant concentrations 1 h after LPS stimulation. Dexamethasone and the NSAID used in this study were equally effective at inhibiting bradykinin production. Peak induction of COX-2 mRNA occurred 3 h post-LPS stimulation. However, neither dexamethasone nor any of the NSAID used in this study altered COX-2 mRNA concentrations. In contrast, aspirin, flunixin meglumine, and PTPBS reduced tumor necrosis factor-alpha (TNFα) mRNA concentration. These results demonstrate that bovine blood cells respond to NSAID therapy like other mammalian cells with respect to inhibition of PGE₂ production and suppression of TNF mRNA induction, but do not inhibit induction of COX-2 mRNA.     

Induction of Chemoresistance by Transfection of the Full‐Length Canine mdr1 Gene in Canine Cell Lines

Introduction:  In the chemotherapy for treatment of lymphoid tumors in dogs, myelosuppression is a frequently encountered dose‐limiting factor. One possible approach to overcome myelosuppression is induction of chemoresistance in hematopoietic stem cells through expression of the mdr1 gene. A full‐length canine mdr1 cDNA clone was isolated in our laboratory. The present study was carried out to assess whether it confers multidrug resistance in canine cell lines.
Materials and methods:  The full‐length canine mdr1 cDNA was inserted into an expression plasmid vector. A canine mammary tumor cell line (CIPP) and osteosarcoma cell line (OOS) were transfected with the canine mdr1 expression vector. Expression of P‐gp was examined by immunoblotting. Function of ATP‐dependent drug efflux was measured by flow cytometric analysis using Rhodamine 123. Sensitivity to chemotherapeutic drugs was shown by estimation of 50% inhibitory concentrations (IC₅₀) of vincristine or doxorubicin.
Results:  Immunoblotting of the transfected cells revealed a strong band of P‐gp detected by a monoclonal antibody directed to P‐gp. Rhodamine 123 efflux test showed an apparent drug efflux activity in the transfected cells. From the IC₅₀ of the chemotherapeutic agents, the transfected cells showed a remarkable increase (20 to 60‐fold) in the resistance to vincristine or doxorubicin.
Conclusion:  Transfection of canine mdr1 gene induced P‐gp expression and strong drug resistance in canine cell lines.     

Reactivity of Equine Palmar Digital Arteries and Veins to Vasodilating Agents

Palmar digital arteries and veins removed surgically from healthy horses under general anesthesia were cut into 4 mm vascular rings, suspended in tissue baths, and attached to force displacement transducers for continuous measurement of vascular tension. In vitro vascular responses were determined for acetylcholine, acepromazine, isoxsuprine hydrochloride (isoxsuprine), prostaglandin E₂ (PGE₂), and prostaglandin I₂ (prostacyclin). After preconstriction with norepinephrine hydrochloride (norepinephrine), or prostaglandin F₂ alpha (PGF₂ alpha), the concentrations needed to produce 50% maximum relaxation (EC₅₀) and the maximum percentage of relaxation were determined for each drug.
Acetylcholine was the most potent arterial vasodilator (smallest EC₅₀ value) and PGE₂ was the least potent. Prostacyclin was the least potent venodilator (highest EC₅₀ value); there were no differences between acetylcholine, acepromazine, isoxsuprine, and PGE₂. Isoxsuprine produced greater arterial relaxation than all other agents. Isoxsuprine and acepromazine produced significantly greater venous relaxation than did acetylcholine and PGE₂. Prostacyclin produced minimal vasodilation of arteries or veins. Acepromazine and isoxsuprine relaxed the veins significantly more than the arteries. When PGF₂ alpha was used instead of norepinephrine to preconstrict the arteries and veins, the potency and effectiveness of acepromazine and isoxsuprine to produce vasodilation were significantly decreased. Results indicate that acepromazine and isoxsuprine can relax the equine digital vasculature but their effectiveness varies depending on the origin of the constriction.     

Pharmacokinetics and pharmacodynamics of meloxicam in piglets

The pharmacokinetics and pharmacodynamics of meloxicam in piglets (16–23 days old) were studied using a stratified parallel group design. One group ( n  = 13) received 0.4 mg/kg meloxicam intravenously, while the other group ( n  = 12) received physiological saline solution. A carrageenan-sponge model of acute inflammation was used to evaluate the effects of meloxicam. The plasma clearance was low (0.061 L/kg/h), the volume of distribution was low (0.19 L/kg) and the elimination half-life was short (2.7 h). At most time points, the mean concentration of meloxicam in plasma exceeded the concentrations in exudate indicating a limited accumulation of the drug at the site of the inflammation. There were significant differences between the groups in the exudate prostaglandin E₂ (PGE₂) concentration, but the inhibition of PGE₂ in the meloxicam group was limited. The inhibition of thromboxane B₂ (TXB₂) production in serum in the meloxicam group was extensive, but of shorter duration than the PGE₂ inhibition in exudate.     

Characterization of a sterile soft-tissue inflammation model in Thoroughbred horses

This paper describes the use of subcutaneously-placed tissue chambers as a sterile soft-tissue inflammation model in Thoroughbred horses. Acute, nonimmune inflammation was initiated by injecting a sterile lambda carrageenan solution into a tissue chamber. This model was used to study the temporal changes in oxygen and carbon dioxide tensions, pH, bicarbonate, protein, albumin, prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄) concentrations, cell counts and differential counts in tissue fluid from inflamed tissue chambers and control chambers. Skin temperatures over control and inflamed chambers were also compared. Carrageenan-induced inflammation resulted in significant increases in tissue-fluid carbon dioxide tension, leucocyte count, albumin, and PGE₂ and LTB₄ concentrations. It also resulted in a significant decrease in tissue fluid pH and HCO₃- concentration. Inflammation did not result in significant changes in tissue-fluid protein concentration, differential cell counts or skin temperature over the chambers. The use of this type of tissue chamber is wellsuited for studying the pathophysiology of a self-contained, non-immune inflammatory process. The model described in this paper could prove to be very useful in studies of the distribution of anti-inflammatory drugs and the effects of such drugs on various aspects of the inflammatory process.     

Specific binding of dl-cloprostenol and d-cloprostenol to PG F2α receptors in bovine corpus luteum and myometrial cell membranes

Prostaglandin F_2α receptors (PGF_2α Rs) were measured in bovine corpus luteum and myometrial cell membranes using a radiometric method. The inhibition of labelled PGF_2α binding exerted by d-cloprostenol, dl-cloprostenol, PGF_2α and PGE₁ (l0–¹¹ M to 10^–4 M) was evaluated in vitro. Results strongly suggest that cloprostenol binding to PGF_2αRs is stereospecific. d-Cloprostenol and PGF_2α were equipotent, about 150 times more potent than d-cloprostenol (P < 0.05) and approximately 280 times more potent than PGE, (P < 0.05) in inhibiting [3H]PGF_2α binding to corpus luteum cell membranes. Such differences were less evident in myometrial cell membranes, where d-cloprostenol and PGF_2α were about 10 times more potent than d-cloprostenol (P < 0.05) and approximately 95 times more potent than PGE_l (P < 0.05).     

Piroxicam Therapy in 34 Dogs With Transitional Cell Carcinoma of the Urinary Bladder

Thirty-four dogs with histopathologically confirmed, measurable, nonresectable transitional cell carcinoma of the urinary bladder were treated with piroxicam (0.3 mg/kg PO sid) and were evaluated for tumor response and drug toxicity. Dogs were evaluated at the Purdue University Veterinary Teaching Hospital by means of physical examination, thoracic and abdominal radiography, cystography, complete blood count, serum biochemistry profile, and urinalysis. In selected cases, prostaglandin E₂ (PGE₂) concentrations in plasma and in supernatants of stimulated monocytes, and natural killer cell activity were quantified, Dogs were evaluated before therapy and at 28 and 56 days after initiation of therapy. Dogs with stable disease or remission at 56 days remained on the study and were evaluated at 1 to 2 month intervals. Tumor responses were 2 complete remissions, 4 partial remissions, 18 stable diseases. and 10 progressive diseases. The median survival of all dogs was 181 days (range, 28 to 720+ days), with 2 dogs still alive. Piroxicam toxicity consisted of gastrointestinal irritation in 6 dogs and renal papillary necrosis (detected at necropsy) in 2 dogs. Monocyte production of PGE₂ appeared to decrease with therapy in dogs whose tumors were decreasing in size, and increased in dogs with tumor progression. A consistent pattern in natural killer cell activity was not observed. In vitro cytotoxicity assays against 4 canine tumor cell lines revealed no direct antitumor effects of piroxicam. In summary, antitumor activity, which was not likely the result of a direct cytotoxic effect, was observed in dogs with transitional cell carcinoma of the bladder treated with piroxicam.     

Role of Eicosanoids in the Regulation of the Periparturient Period in Cattle

Contents: In this review, the role of eicosanoids in regulation of parturition and the postpartum period was described with special emphasis on the bovine species. The metabolism of arachidonic acid and the production of eicosanoids during the peripartum period was discussed. Prostaglandin E₂ and F_2α (PGE₂, PGF_2α) play an important role in mechanisms controlling parturition. They are involved in luteolysis, uterine contractions and dilation of the cervix. Eicosanoids also seem to influence the loosening processes of the fetal membranes. However, in the literature, conflicting results were found. Many investigations suggested that retained placental membranes could be related to low PGF_2α production and/ or an imbalance of arachidonic acid metabolism in the uterus. The possible role of the lipoxygenase pathway metabolite 15-hydroxy-eicosatetraenoic acid (15-HETE) in expulsion of the fetal membranes was also discussed. As far as the postpartum period is concerned, a relationship between postpartum PGF_2α release and the involution of the uterus was found. Cows with undisturbed uterine involution had higher PGF_2α production than cows with delayed involution. In contrast to the positive effect of PGF_2α on uterine involution, PGE₂ seems to delay the involution processes. Further experiments are necessary in order to study the function of eicosanoids in mechanisms regulating parturition, release of the fetal placental membranes and involution of the uterus.     

In vivo confocal microscopy of the normal equine cornea and limbus

Objective  To describe morphologic features, pachymetry and endothelial cell density of the normal equine cornea and limbus by in vivo confocal microscopy.
Animals studied  Ten horses without ocular disease.
Procedure  The central and peripheral corneas were examined with a modified Heidelberg Retina Tomograph II and Rostock Cornea Module using a combination of automated and manual image acquisition modes. Thickness measurements of various corneal layers were performed and endothelial cell density determined.
Results  Images of the constituent cellular and noncellular elements of the corneal epithelium, stroma, endothelium, and limbus were acquired in all horses. Corneal stromal nerves, the subepithelial nerve plexus, and the sub-basal nerve plexus were visualized. Cells with an appearance characteristic of Langerhans cells and corneal stromal dendritic cells were consistently detected in the corneal basal epithelium and anterior stroma, respectively. Median central total corneal thickness was 835 μm (range 725–920 μm) and median central corneal epithelial thickness was 131 μm (range 115–141 μm). Median central endothelial cell density was 3002 cells per mm² (range 2473–3581 cells per mm²).
Conclusions  In vivo corneal confocal microscopy provides a noninvasive method of assessing normal equine corneal structure at the cellular level and is a precise technique for corneal sublayer pachymetry and cell density measurements. A resident population of presumed Langerhans cells and corneal stromal dendritic cells was detected in the normal equine cornea. The described techniques can be applied to diagnostic evaluation of corneal alternations associated with disease and have broad clinical and research applications in the horse.     

Biomechanical Analysis of the Three-Dimensional Motion Pattern of the Canine Cervical Spine Segment C4–C5

Objective— To study the kinematics of cervical spine segment C₄–C₅ and its association with disc dimensions and the coupled motion (CM) in relation to primary motion (PM).
Study Design— Cadaveric biomechanical study.
Animals— Cadavers of large breed dogs (>20 kg; n=11).
Methods— Spines were freed from muscles. Radiographs were taken orthogonal to the C₄–C₅ disc space and disc thickness, endplate width, and height were measured. Spines were mounted on a simulator for 3-dimensional motion analysis. Data were recorded with an optoelectronic motion analysis system. Range of motion (ROM) and neutral zone (NZ) were determined in the direction of flexion/extension, left/right lateral bending, and left/right axial rotation, as well as the ROM of CM.
Results— ROM in flexion and extension was similar; there was no CM in flexion/extension. Left/right axial rotation and left/right lateral bending were coupled to the same side. CM was 1.72 and 3.56 times the ROM of the PM in lateral bending and axial rotation, respectively. Disc dimensions were positively correlated with body weight. Flexion/extension magnitude was significantly reduced for larger endplates, but axial rotation was not influenced. Lateral bending had no correlation with weight or disc dimensions.
Conclusion— Left/right lateral bending and left/right axial rotation are coupled differently in the C₄–C₅ segment in dogs compared with humans.
Clinical Relevance— The canine C₄–C₅ spinal segment has unique motion coupling patterns that should be considered for dynamic implant designs.     

Comparative study of the action of flunixin meglumine and tolfenamic acid on prostaglandin E2 synthesis in bovine inflammatory exudate

An acute non-immune inflammation model was used to compare the action of two non-steroidal anti-inflammatory drugs, flunixin meglumine and tolfenamic acid, on prostaglandin E₂, (PGE₂) synthesis in bovine inflammatory exudate. The tissue cage model used involves subcutaneous implantation of polypropylene cages and subsequent stimulation by carrageenan injection of the granulation tissue which develops within the cage. Twelve calves were randomly assigned to three groups receiving placebo, flunixin meglumine and tolfenamic acid, respectively. Inflammatory exudate was sampled 30 min after carrageenan injection and at seven subsequent time points. PGE², levels were determined by radioimmunoassay. At each time point post-carrageenan injection, flunixin meglumine inhibited PGE₂, synthesis to a greater extent than tolfenamic acid. At 4, 8,12 and 24 h these differences were statistically significant.     

Influence of vitamin E on mast cell mediator release

Abstract We investigated the influence of vitamin E on mediator activity and release in a canine mastocytoma cell line (C2) as a model for canine atopic dermatitis. Cells were incubated without and with vitamin E (100 µ m ) for 24 h. The histamine and prostaglandin D₂ (PGD₂) release as well as the chymase and tryptase activity were measured. To stimulate the PGD₂ and histamine release, cells were incubated with the wasp venom peptide mastoparan (50 µ m ) for 30 or 45 min. Nonstimulated as well as mastoparan-stimulated histamine and PGD₂ release was reduced significantly in vitamin E-treated cells. The activity of chymase tended to decrease, but the tryptase activity of C2 cells was not influenced by vitamin E. These results indicate that vitamin E decreased the production and release of inflammatory mediators in C2 cells, suggesting that vitamin E might have a possible beneficial effect in inflammatory diseases.     

Pharmacological evaluation of sheep lung strip

Isolated sheep lung parenchymal strips responded to histamine > carbachol > PGF_2a > 5-HT with contractions, and to isoproterenol (Isop), and to large doses of epinephrine (E), norepinephrine (NE) and phenylephrine (PE) with relaxations. PGF_2a-contracted lung strip responded to PGE₁ and PGE₂ with relaxation. The strips which were partially contracted to histamine, PGF_2a, 5-HT and carbachol also responded to isop, E and NE with relaxations. Histamine responses were not modified by metiamide (an H₂-receptor antagonist). Mepyramine and atropine selectively antagonized contractions to histamine and carbachol, respectively. After β-blockade with propranolol, lung strips responded to NE > E > PE > isop with contractions, which were inhibited or reversed by phentolamine and dibenzyline. It is concluded that H₁ receptors are present in sheep peripheral airway smooth muscles, and that a- and β-adrenoceptors mediate contraction and relaxation, respectively, in sheep lung strips.     

Concentrations of thyroid hormones and iodothyronine binding proteins in serum of the koala (Phascolarctos cinereus)

Objective To examine circulating total and free thyroid hormone (T₃ and T₄) concentrations, determine serum iodothyronine binding characteristics and estimate thyroid stimulating hormone (TSH) activity in sera of coastal and inland koalas.
Design A prospective study.
Procedure Koala serum T₃ and T₄ were measured by radioimmunoassay. T₄ binding parameters were determined by radioligand binding and electrophoresis. Koala TSH values were determined by bioassay.
Results Mean total T₄ concentrations were 3.2 ± 2.1 nM although values were significantly higher in inland-dwelling females in comparison to coastal-dwelling males. Free T₄ was 3.3 ± 2.1 pM. Total and free T₃ were 0.4 ± 0.2 nM and 1.4 ± 0.9 pM respectively, although these values were at the lower end of the assay detection limit and should be viewed with reservation. Electrophoresis of [¹²⁵I]-T₄-labelled serum revealed only two proteins of electrophoretic mobility similar to human transthyretin (TTR) and albumin. Scatchard analysis of T₄ binding to serum gave a curvilinear plot, which could be resolved into two binding sites with affinities identical to that of TTR and albumin but both of low concentration. The bioactivity of the TSH present in the sera was measured using a cell line (JP09) transfected with the human TSH receptor. The mean level of stimulation found in the sera corresponded to a bovine TSH activity of less than 10 mU/L.
Conclusion These results suggest that the serum concentrations of free and total thyroid hormones in koalas are low compared to other marsupials and very low compared to eutherian mammals. The mechanism of maintenance of euthyroidism in this species remains to be determined.     

A prospective, randomized comparison of Oxyglobin (HB-200) and packed red blood cell transfusion for canine babesiosis

Objective – To establish the efficacy of Oxyglobin (HB-200) in canine babesiosis and compare it to standard therapy, packed red blood cell transfusion (pRBCT) with respect to improvements in specific parameters of blood gas, acid-base, blood pressure, and subjective evaluations.
Design – Prospective, randomized, clinical trial.
Setting – Onderstepoort Veterinary Academic Hospital.
Animals – Twelve dogs (8–25 kg) naturally infected with Babesia rossi and a hematocrit of 0.1–0.2 L/L (10–20%).
Interventions – Treatment groups were randomized to receive either 20 mL/kg of Oxyglobin or pRBCT over 4 hours via a central venous catheter. Transfusions were followed by lactated Ringer's solution infusion. Rectal temperature, femoral arterial and mixed venous blood sampling, oscillometric blood pressure, and subjective assessment of patient status (habitus), and appetite were performed at time points 0, 1, 4, 8, 24, 48, and 72 hours.
Main Results – Dogs presented with a hypoalbuminemic alkalosis; hyperchloremic, dilutional acidosis; normotensive tachycardia; pyrexia; depression; and anorexia. Both treatments produced similar results, with the exception of significant differences in pH (4 h); PCO₂ (4 h); hemoglobin (8 h, 24 h); mean arterial pressure (48 h); albumin (4 h, 8 h); habitus (8 h, 48 h); and appetite (24 h). Arterial O₂ content was higher for pRBCT than Oxyglobin at 72 hours, but central venous PO₂ did not differ between groups or over time and was consistently subnormal.
Conclusions – Oxyglobin provides similar overall improvements to pRBCT in dogs with anemia from babesiosis, with respect to blood gas, acid-base and blood pressure, although patients receiving packed cells tended to have more rapid normalization of habitus and appetite.     

An Investigation of the Action of Neutral Protamine Hagedorn Human Analogue Insulin in Dogs with Naturally Occurring Diabetes Mellitus

Background: Neutral Protamine Hagedorn human analogue insulin (Humulin N) is commonly used for treatment of canine diabetes mellitus (DM). However, blood glucose and serum insulin concentrations in Humulin N-treated dogs with naturally occurring DM have not been reported.
Objective: To investigate blood glucose and serum insulin concentrations in the clinical setting of client-owned Humulin N-treated dogs with naturally occurring, well-regulated DM.
Animals: Ten client-owned dogs with naturally occurring, well-regulated DM.
Methods: In this clinical study, blood glucose and serum insulin concentrations were measured when dogs received food and insulin (T₀), at approximately every half hour for the next 2 hours, and then approximately every 2 hours for an additional 8 hours. Insulin duration of action was defined as the number of hours from T₀ to the lowest blood glucose concentration and until blood glucose concentration returned to an interpolated value of 70% of basal blood glucose concentration (Glucose_b).
Results: Mean percent of insulin-induced blood glucose suppression was 49.9 ± 17.1% (median, 46%; range, 29–78%). Insulin duration of action ranged from 4 to 10 hours. Blood glucose concentration increased initially and returned to Glucose_b within 0.6–2.2 hours after T₀ in 5 dogs. This initial blood glucose surge then was followed by blood glucose suppression in all 5 dogs.
Conclusions and Clinical Importance: These results suggest that Humulin N administered SC twice daily is an effective mode of treatment for dogs with naturally occurring DM. Postprandial hyperglycemia is present in some well-regulated diabetic dogs treated with Humulin N.     

Effects of Mycobacterial Cell Wall‐DNA Complex (MCC), Alendronate and Pamidronate on Canine Osteosarcoma Cell Lines

Introduction:  MCC, a cell wall composition prepared from Mycobacterium phlei ., inhibits the proliferation and induces apoptosis in a wide range of tumor cells. Bisphosphonates have been reported to inhibit the proliferation of canine osteosarcoma cell lines. In this study, we have determined the activity of MCC alone and in combination with the bisphosphonates alendronate and pamidronate on canine osteosarcoma cell lines.
Methods:  Canine osteosarcoma cell lines D17 and D22 were incubated with different concentrations of MCC (0.01–100 μg/ml) and suboptimal concentrations of alendronate and pamidronate for 72 hours. Cellular proliferation was measured by MTT reduction. Nuclear DNA condensation was determined using with Hoescht 33258 staining, and apoptosis by flow cytometry using active‐caspase‐3/PE and anti‐cleaved‐PARP/FITC antibodies.
Results:  MCC inhibited the proliferation of both canine osteosarcoma D17 and D22 cell lines in a concentration‐dependent manner. The IC₅₀ for D17 cells was 3.9 μg/ml and for D22 cells 44.4 μg/ml. Cells incubated with 100 μg/ml MCC were positive for Hoescht staining, active caspase‐3 and cleaved PARP, indicative of cell death by apoptosis. The addition of alendronate or pamidronate was found to potentiate the apoptosis‐inducing activity of MCC. Maximal activity was observed when 5 μM alendronante or 10 μM pamidronate were used in combination with 100 μg/ml MCC.
Conclusion:  MCC inhibits the proliferation and induces apoptosis in canine osteosarcoma cell lines in vitro . This anticancer activity can be potentiated by the use of alendronate and pamidronate. These data support the development of MCC as a therapeutic agent for the treatment of canine osteosarcoma.     

Functional characterization of the muscarinic receptors involved in endothelium-dependent relaxation in isolated canine uterine artery

Acetylcholine interacts with endothelial muscarinic receptors releasing nitric oxide and causing vasodilatation. To identify the receptor subtype responsible for acetylcholine-induced relaxation in canine uterine artery, the usual organ bath method for in vitro investigation on isolated blood vessels was applied. Using a range of muscarinic receptor antagonists such as atropine (nonselective), pirenzepine (M₁-selective), methoctramine (M₂-selective) and p -fluoro-hexahydro-sila-difenidol ( p -FHHSiD) (M₁/M₃) and determining pA2 value of those antagonists through Shild analysis, we aimed at establishing a precise receptor mechanism underlying acetylcholine-induced relaxation in isolated canine uterine artery. The relaxation of uterine arterial rings in response to acetylcholine in the presence or absence of selective muscarinic receptors antagonists was calculated using concentration response curves. Acetylcholine induced concentration-dependent and endothelium-dependent relaxation of arterial rings precontracted with phenylephrine (pEC₅₀ = 6.90 ± 0.02). Muscarinic receptors antagonists atropine, pirenzepine, methoctramine and p -FHHSiD competitively antagonized the response to acetylcholine and obtained pA₂ values were 9.91 ± 0.06, 6.60 ± 0.04, 6.21 ± 0.08 and 8.05 ± 0.1, respectively. This study showed that acetylcholine induced endothelium-dependent relaxation of canine uterine artery by stimulation of muscarinic receptors localized on the endothelial cells. On the basis of differential antagonist affinity, we suggest that the muscarinic receptors involved in the acetylcholine-induced relaxation of canine uterine artery are predominantly of M₃ subtype.