In vitro pharmacologic effect of two endothelin-1 antagonists on equine colonic arteries and veins

OBJECTIVE: To evaluate the effectiveness of 2 potential endothelin (ET)-1 antagonists in blocking the contractile responses of equine colonic vessels to increasing concentrations of ET-1. SAMPLE POPULATION: Mesenteric vessels from 6 clinically healthy horses. PROCEDURE: Colonic vessels (arterial and venous rings) were placed in organ baths with oxygenated Tyrode solution at 37 C. Each was attached to a force transducer interfaced with a polygraph, and 2 g of tension was applied and equilibrated for 45 minutes. Then, B-1 (PD 142893) and B-2 (PD 145065) ET-1 antagonists were tested. One ring from each vessel type was used as a control for determining concentration-response relationships of ET-1 (10(-10) to 10(-6)M). Three rings of each vessel type were incubated with 3 concentrations of each antagonist (10(-7), 10(-6), and 10(-5) M) for 30 minutes before ET induced contractions were determined. The maximum contractile response and pA2 values were determined. RESULTS: Vessels contracted in a concentration-dependent manner to ET-1. Arteries responded slowly but reached greater contractions. Veins responded immediately with sustained contractions. Both antagonists inhibited contractions in a concentration-dependent manner with significant differences at 10(-6) and 10(-5)M for arteries and 10(-5) M for veins. Complete blockade of contractions was observed with B-2 (10(-5)M). The pA2 values for B-1 were 8.26 and 6.82 for arteries and veins, respectively, whereas they were 8.25 and 7.21 for B-2. CONCLUSION AND CLINICAL RELEVANCE: Both antagonists effectively blocked ET-1-induced contractions of equine colonic vessels. Because B-2 is water soluble and caused complete blockade at 10(-5) M, it appears to be the preferred antagonist.     

Finite element analysis of wall stress in the equine pulmonary artery

Reasons for performing study: Arterial calcification is found frequently in the pulmonary artery of racehorses, but the aetiology is unknown. Calcification might be associated with increased wall stress due to arterial geometry (shape) and exercise‐induced hypertension. Hypothesis: High wall stress levels are found in the regions associated with calcified lesion formation, exacerbated as transluminal pressure increases to levels associated with exercise. Methods: The pulmonary arteries of 5 horses, unaffected by calcification, were dissected and pressurised to resting and exercising physiological transluminal pressures and scanned with MRI. Arterial geometries were reconstructed to form 3D computer models and finite element analyses performed. Wall stress levels were measured in 4 regions of interest: the arterial trunk and bifurcation, the wall ipsilateral and contralateral to the bifurcation. Measurements were made for arterial transluminal pressures of 25, 50 and 100 mmHg. Results: High wall stress levels were consistently found at the pulmonary artery bifurcation and wall ipsilateral to the bifurcation, where calcified lesions typically form. Lower wall stress levels were found along the trunk and the wall contralateral to the bifurcation where lesions are less frequently found. Wall stress levels increased 5‐fold over a 4‐fold increase in pressure. The wall stress levels ranged 10 kPa in the wall of the branch contralateral to the bifurcation at 25 mmHg to 400 kPa in the bifurcation at 100 mmHg. Conclusions: Wall stress from arterial geometry and increased pulmonary artery transluminal pressure are factors that may be associated with calcification of the equine pulmonary artery. Potential relevance: Arterial calcification may increase the risk of arterial wall failure in racing horses.     

Characterization and comparison of the responses of equine digital arteries and veins to endothelin-1

OBJECTIVE: To compare the responses of equine digital arteries (EDAs) and equine digital veins (EDVs) to endothelin-1 (ET-1) and determine the role of the endothelium and type of receptors involved in the modulation and mediation of those responses, respectively. SAMPLE POPULATION: 5 to 9 palmar digital vessels/experiment from 28 healthy horses. PROCEDURE: Rings of dissected vessels were mounted under tension between force transducer wires in organ baths containing Krebs-Henseleit solution at 30 degrees C. Responses of EDAs and EDVs (with intact [+e] or denuded [-e] endothelium) to cumulative concentrations of ET-1 (10(-10) to 3 X 10(-7) M) were compared. For (+e)EDAs and (+e)EDVs precontracted with a thromboxane-mimetic (U44069; 10(-8) M) and (-e)EDAs and (-e)EDVs, responses to an ETB receptor agonist (S6c; 10(-10) to 3 X 10(-7) M) were evaluated. Responses to ET-1 (10(-7) M) in (-e)EDAs and (-e)EDVs were evaluated after incubation with an ETA receptor antagonist (BQ-123; 3 X 10(-7) M), an ETB receptor antagonist (BQ-788; 3 X 10(-7) M), or vehicle solution. RESULTS: Endothelin-1 induced a concentration-dependent contraction of endothelium-intact and -denuded EDAs and EDVs; EDVs were more sensitive. Neither vessel type relaxed in response to S6c, although 2 of the (-e)EDAs contracted mildly. Whereas BQ-123 inhibited the (-e)EDA and (-e)EDV responses to ET-1, BQ-788 had no effect. CONCLUSIONS AND CLINICAL RELEVANCE: Endothelin-1 induced digital vasoconstriction (marked constriction in veins). This action was unaffected by endothelium and mediated predominantly by ETA receptors. These findings suggest ET-1 can induce selective digital venoconstriction.     

内皮素-1与肉鸡肺动脉高压综合征

肉鸡肺动脉高压综合征（pulmonary hypertension syndrome，PHS）亦称肉鸡腹水综合征（ascites syndrome，AS），过去也被称为“高海拔病”（high altitude disease，HAD）、“心衰综合征”（heart failure syndrome，HFS）等，是快速生长肉鸡的一种常见非传染性疾病，以明显的腹水和心、肺功能衰竭为特征。与猝死综合征（sudden death syndrome，SDS）和生长障碍综合征（growth retardation syndrome，GRS）一起被称为当前肉鸡业所面临的三大新病。发病后多以死亡告终，是严重危害养禽业发展的禽类疾病之一。     

Characteristics of the in vitro hypoxic pulmonary vasoconstrictor response in isolated equine and bovine pulmonary arterial rings

OBJECTIVE: Hypoxaemia accompanies dorsal recumbency in the horse and frequently complicates general anaesthesia. The physiology associated with this phenomenon is poorly understood. One possible cause of poor tolerance to dorsal recumbency is an absent or reduced response to hypoxic pulmonary vasoconstriction (HPV). This study compared the HPV response in isolated pulmonary artery vessels from equivalent regions of equine and bovine lung. ANIMALS: Equine and bovine, in vitro study. MATERIALS AND METHODS: Equine and bovine pulmonary arteries were removed from the lungs of euthanased horses and cattle. Measurements of isometric tension were made on isolated rings of pulmonary vessels at 37 degrees C in a Krebs' saline solution. Hypoxia was induced by bubbling with a nominally 0% O(2) gas mixture. RESULTS: A significant HPV response was observed above a baseline tension induced by phenylephrine (PE; 0.3 microm) or 5-hydroxytryptamine (5-HT; 0.1 microm). The HPV response in equine pulmonary vessels was approximately 33% less than the response observed in equivalent bovine vessels (equine 196 +/- 20%versus bovine 290 +/- 32%; p < 0.05). Removal of the endothelium (by rubbing the luminal surface) significantly reduced but did not abolish the HPV response. Incubation with the nitric oxide (NO) synthase inhibitor N-nitro-l-arginine methyl ester (L-NAME; 100 microm), or COX-1/COX-2 inhibitor indomethacin (10 microm) markedly attenuated the HPV response in equine vessels. CONCLUSIONS: These results suggest that a significant HPV response exists in isolated equine pulmonary vessels; a component of this response requires a functional endothelium. Inhibition of cyclooxygenase and NO synthase attenuated the response, suggesting the involvement of a COX product and/or NO in mediating this effect either directly or indirectly. Alternatively, a non-COX related action of the nonsteroidal anti-inflammatory drug, indomethacin, may be involved.     

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.     

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

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

Comparison of 2 endothelin-receptor antagonists on in vitro responses of equine palmar digital arterial and venous rings to endothelin-1

The goals of this study were to determine the concentration-response (C-R) relationship of endothelin-1 (ET-1), compare 2 ET-receptor antagonists and determine the antagonist concentrations that block the vasomotor effects of ET-1, and compare the effectiveness of ET-1 and previously studied vasoconstrictors in equine palmar digital arterial and venous rings in vitro. Vessel rings from 8 nonlaminitic horses were placed in Tyrode's solution, 1 side fixed to the floor of an organ bath and the other side fixed to a force-displacement transducer. Two separate studies were conducted: (I) incubation with a single ET-receptor antagonist (PD142893 or PD145065 at a concentration of 10(-7), 10(-6), or 10(-5) M), followed by determination of an ET-1 C-R curve (using concentrations of 10(-10) to 10(-6) M) for medial vessel rings; and (II) comparison of ET-1 with norepinephrine and histamine (10(-10) to 10(-6) M) and comparison of contractile responses of medial and lateral vessel rings. In study I, ET-1 administration caused pronounced and sustained concentration-dependent contraction of vessel rings; these contractile responses were decreased by 10(-5) M PD142893 and were completely blocked by 10(-5) M PD145065. Venous rings had greater apparent maximum contraction in response to ET-1 than arterial rings. In study II, the relative sensitivity of norepinephrine was found to be equivalent to that of ET-1, whereas that of histamine was lower. No significant differences were observed between responses of medial versus lateral vessel rings. Thus, ET-1 is a potent vasoconstrictor of equine palmar digital arteries and veins, and the ET-receptor antagonist PD145065 is more effective than PD142893 in inhibiting these contractile effects in vitro.     

Hypothermic storage of equine isolated hepatocytes

The aim of the study was to establish the optimal methods for hypothermic storage of equine isolated hepatocytes. Viability of equine isolated hepatocytes after hypothermic storage was dependent on the type of storage medium as well as on the cell density in the storage suspension and the preservation period. Hepatocytes stored at 4 degrees C in Hanks' Balanced Salt Solution (HBSS) and Williams' Medium E (WE) for 24 h showed very low viability, numerous cell membrane blebs, very low attachment rate (11.9 +/- 6.5% and 34.8 +/- 19.1%, respectively) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction rate (6.4 +/- 3.9% and 25.1 +/- 14.8%, respectively). In contrast, hepatocytes stored in University of Wisconsin Solution (UW) after 24 h of storage at a density of 12.5 x 10(6) cells/ml showed high viability (over 70%), typical and intact morphology, high cell attachment rates and MTT reduction. Our findings clearly demonstrate that UW is a good preservation solution for equine isolated hepatocytes. Hepatocytes harvested from slaughterhouse organs can be stored at 4 degrees C in UW at a density of 12.5 x 10(6) cells/ml for at least 24 h without significant decrease in functional integrity.     

Contractile responses to histamine of isolated pulmonary artery strips from healthy and heartworm-infected dogs

Pulmonary artery strips from control and Dirofilaria immitis-infected dogs were tested for isometric contractile response to histamine, carbamylcholine, and norepinephrine. The strips from D immitis-positive dogs showed diminished response to histamine; a smaller percentage of the strips contracted, and the magnitude of the contraction was less than the response of the control strips to the highest concentration used. Carbamylcholine caused virtually no contractile effect in either group. Norepinephrine contracted all strips, even those not responding to histamine. A role for histamine in the pathogenesis of the pulmonary arterial lesions associated with dirofilariasis is presented.     

The action of low dose endotoxin on equine bowel motility

Post operative ileus (POI) is a common and serious complication of colic surgery in the horse. There is a high correlation between the incidence of POI and the presence of ischaemic bowel, suggesting a role for endotoxin. 0.1 micrograms/kg endotoxin was administered intravenously to six ponies with chronically implanted gastrointestinal electromechanical recording devices. It produced profound disruption of normal fasting bowel motility patterns, with an inhibition of gastric contraction amplitude and rate, left dorsal colon contraction product and small colon spike rate. In the small intestine an increase in abnormally arranged regular activity and a decrease in irregular activity was observed. There was no significant prolongation in stomach to anus transit time as assessed by the passage of plastic spheres. The bowel motility patterns induced by endotoxin could be mimicked by the intravenous infusion of PGE2 and less potently by PGI2 (prostacyclin), but not by PGF2 alpha. This study provides evidence that systematic endotoxin present in clinical cases of colic may play a role in the pathogenesis of equine ileus. The acute effects of endotoxin on bowel motility appear to be mediated indirectly by prostaglandins, and the inhibitory effects may be mediated mainly by PGE2.     

Serum and bronchoalveolar lavage fluid endothelin-1 concentrations as diagnostic biomarkers of canine idiopathic pulmonary fibrosis

Background: Diagnosis of canine idiopathic pulmonary fibrosis (IPF) is challenging. Endothelin‐1 (ET1) is a biomarker of IPF in humans, but whether ET1 can detect and differentiate IPF from other canine respiratory diseases is unknown. Objective: To evaluate whether measurement of the concentration of ET1 in serum and bronchoalveolar lavage fluid (BALF) can be used to distinguish canine IPF from chronic bronchitis (CB) and eosinophilic bronchopneumopathy (EBP). Animals: Twelve dogs with IPF, 10 dogs with CB, 6 dogs with EBP, 13 privately owned healthy West Highland White Terriers (WHWT), and 9 healthy Beagle dogs. Methods: Prospective, case control study. ET1 concentration was determined by ELISA in serum and in BALF. Results: No significant difference in serum ET1 concentration was detected between healthy Beagle dogs and WHWT. Serum ET1 concentration was higher in dogs with IPF (median interquartile range; 2.32 pg/mL, 2.05–3.38) than healthy Beagle dogs (1.28, 1.07–1.53; P < .001), healthy WHWT (1.56, 1.25–1.85; P < .001), dogs with EBP (0.94 0.68–1.01; P = .001), and dogs with CB (1.54 0.74–1.82; P = .005). BALF ET1 concentration was below the detection limit in healthy WHWT and in dogs with CB, whereas it was measurable in all dogs with IPF. A cut‐off serum concentration of 1.8 pg/mL had a sensitivity of 100% and a specificity of 81.2% for detection of IPF, with an area under the receiver operating characteristic curve of 0.818. Conclusions and Clinical Importance: Serum ET1 can differentiate dogs with IPF from dogs with EBP or CB. ET1 can be detected in BALF of dogs with IPF.     

Mechanical properties of the isolated equine trachea

In order to study the in vitro mechanical properties of the equine trachea submitted to the compressive pressures observed in vivo, the pressure-volume relationship was determined in intra- and extra-thoracic tracheal segments taken post mortem from 29 healthy horses (one to 15 years old; 352 to 651 kg). At the same time, the cross-sectional lumen area (X-SA) at the mid-point of the segment was measured using a slit-lamp transillumination and photographic measurement by endoscopy. The tracheal specific compliance (Cs) as well as the relative changes in X-SA and in the sagittal and transverse diameters, for intraluminal pressures from 5 to - 5 kPa, were calculated. The extrathoracic tracheal Cs was 0.060 +/- 0.002 kPa-1 and, at an intraluminal pressure of - 5 kPa, X-SA was reduced to about 73 per cent of its resting value. The intrathoracic segments were more compliant and, at similar compressive pressure, their X-SA was more reduced. These data show that the equine tracheal compliance is high and suggest that the increase in pulmonary resistance observed during strenuous exercise may be partly explained by a partial tracheal collapse.     

Direct stimulation of the oxidative activity of isolated equine neutrophils by TNF-alpha and IL-1beta

The capacity of the two cytokines TNF-alpha and IL-1beta to directly stimulate the oxidative activity of polymorphonuclear neutrophils remains debated. The purpose of this study was to verify if a direct stimulation of equine neutrophils by TNF-alpha and IL-1beta was possible. Equine neutrophils were isolated from blood by discontinuous density gradient centrifugation. The cell viability after isolation was >98%. The neutrophils were used at 1.25 x 10(6) cells by assay, immediately after isolation. The oxidative activity of neutrophils was measured by luminol- or lucigenin-enhanced chemiluminescence (CL), and the CL was recorded for 60 min. TNF-alpha and IL-1beta were used at concentrations ranging from 0.001 to 100 ng (0.0017-167 ng ml(-1)) for 1.25 x 10(6) neutrophils, and added to the cells just before the CL measurement. Both cytokines highly stimulated the lucigenin-enhanced CL of equine neutrophils in a dose-dependent manner. TNF-alpha was already active at 0.001 ng and IL-1beta at 0.01 ng. The CL response obtained with TNF-alpha was maximal after 5 min and more pronounced with luminol than with lucigenin. With IL-1beta, the luminol-enhanced CL response of neutrophils was short-lived and inversely proportional to the cytokine concentration: the CL response returned to baseline after 12 min, and became even lower than the baseline value for 10 and 100 ng IL-1beta. As luminol (but not lucigenin) enters the cell, we hypothesized that a rapid intracellular consumption of the luminol molecules occurred, explaining the rapid and intense CL response. The choice of the CL enhancer used in previous CL studies of neutrophils stimulation by cytokines could perhaps explain that controversial results were reported. In conclusion, we demonstrated a direct activation of the oxidative activity of equine neutrophils by TNF-alpha and IL-1beta, which was dose-dependent and obtained with very low doses equivalent to the plasma concentrations measured for both cytokines in equine septic shock. TNF-alpha and IL-1beta can thus aggravate neutrophils oxidative activity during septic shock in horses.     

A cooperative action of endothelin-1 with prostaglandin F(2alpha) on luteal function in the cow

Prostaglandin F(2alpha) (PGF(2alpha)) is the primary luteolysin in the cow, and luteal endothelin-1 (ET-1) interacts with PGF(2alpha) during the process of luteolysis. In contrast, a developing corpus luteum (CL) is refractory to exogenous administration of PGF(2alpha). Thus, the present study was aimed to investigate the functional relationship between ET-1 and PGF(2alpha) in the mid-CL (PGF(2alpha)-sensitive) and early-CL (PGF(2alpha)-refractory). In the mid-CL model, cows (n = 6/treatment) were assigned to receive one of five types of treatments on day 10 of the estrous cycle: (1) an injection of saline; control, (2) a 500 microg of PGF(2alpha) analogue (sufficient dose to induce luteolytis); full-PG, (3) an intraluteal injection of 0.25 mg ET-1; ET-1, (4) a 125 micro g of PGF(2alpha) (insufficient dose to induce luteolytis); 1/4PG or (5) an intraluteal injection of 0.25 mg ET-1 after administration of a insufficient dose of PGF(2alpha) analogue; 1/4PG/ET. In the early-CL model, cows were assigned to receive one of two types of treatments on day 5 of the estrous cycle: (1) a sufficient dose of PGF(2alpha) analogue; PG (n = 5) or (2) an intraluteal injection ET-1 after a sufficient dose of PGF(2alpha); PG/ET (n = 7). In the mid-CL model, 1/4PG/ET resulted in a rapid reduction of progesterone (P) concentrations similar to that in full-PG from the next day. However, the levels of P in 1/4PG/ET (1.5-2.5 ng/ml) kept significantly higher than that in full-PG (< 0.5 ng/ml). ET-1 or 1/4PG did not decrease plasma P concentrations (4-6 ng/ml). The plasma ET-1 levels increased with the full-PG administration. In the early-CL model, both treatments had no effect on plasma P increase and ET-1 levels. The overall results indicate that the intraluteal ET-1 injection after administration of insufficient dose of PGF(2alpha) induces the depression of P secretion in vivo during the mid luteal phase in the cow, supporting the concept that ET-1 is one of a local mediator of functional luteolysis in the cow. The result further indicates that the early-CL is not only PG-refractory but also ET-1-refractory.     

Equine pulmonary and systemic haemodynamic responses to endothelin-1 and a selective ET(A) receptor antagonist

Based on previous in vitro studies, we hypothesised that endothelin (ET) would induce vasoconstriction in the pulmonary circululation of the horse and that this action would be mediated via ET(A) receptors. Pulmonary and systemic haemodynamic responses to endothelin-1 (ET-1), a potent vasoactive endogenous peptide, were investigated in 6 conscious, nonsedated horses at rest. Bolus i.v. injections of exogenous ET-1 (0.1, 0.2 and 0.4 microg/kg bwt) caused significant increases in pulmonary (PAP) and carotid (CAP) artery pressures, with peak increases of 79% and 51% for mean PAP and CAP, respectively. The effect of ET-1 on PAP and CAP was rapid and transient for PAP (-10 min) but prolonged for CAP (up to 60 min). ET-1 significantly decreased cardiac output by up to 35% and significantly increased systemic vascular resistance (SVR) by up to 104%. Pulmonary vascular resistance (PVR) showed a trend (P>0.05) to increase with 0.2 and 0.4 microg/kg bwt ET-1. Infusion of a selective ET(A) receptor antagonist (TBC11251) completely inhibited the responses to a subsequent bolus of 0.2 microg/kg bwt ET-1. We conclude that exogenous ET-1 exerts a potent vasoconstrictive action on the pulmonary and systemic circulations of the horse. These effects appear to be mediated largely through ET(A) receptors in both circulations. Endothelin may play a role in hypertensive conditions in the horse.     

Purification and biochemical characterization of equine pulmonary surfactant protein D

OBJECTIVE: To characterize surfactant protein D (SP-D) isolated from bronchoalveolar lavage fluid (BALF) of healthy horses. SAMPLE POPULATION: BALF from 10 Thoroughbreds (5 males, 5 females; 26 to 40 months old) without history or clinical signs of respiratory tract disease. PROCEDURE: BALF was obtained and centrifuged at 33,000 X g. The supernatant was applied to a mannose-Sepharose 6B affinity column in the presence of calcium, and the bound protein fraction was analyzed by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis; amino acid composition was determined and partial sequencing was done. Phospholipid binding and liposome aggregation assay were performed, using purified proteins. RESULTS: The protein isolated by use of mannose affinity matrices was SP-D. It bound carbohydrates and phosphatidylinositol, which are the characteristic features of SP-D isolated from other animal species. Amino acid analysis and partial primary sequence of the isolated protein indicated high homology with rat and human SP-D. Furthermore, immunoblot analysis indicated that equine SP-D reacted with human and rat SP-D-specific antibodies. CONCLUSION AND CLINICAL RELEVANCE: SP-D exists in equine lungs; its measurement may be useful in evaluating equine lung disease.