Assessment of endothelium-dependent vasodilation in equine digital resistance vessels

Haemodynamic disturbances leading to ischaemia and reperfusion injury of the digit are thought to be involved in the pathophysiology of acute equine laminitis. Identification of physiological regulators of blood flow through the equine digit is important in identifying factors, which may predispose animals to laminitis. A method was developed to assess endothelium-dependent responses of the isolated Krebs-perfused equine digit by co-administration of 5-hydroxytryptamine (5-HT) with vasodilator agents, carbachol (CCh), bradykinin (BK) and substance P (SP). Bolus co-administration of CCh (0.02-2 micromol), BK and SP (0.02-0.2 nmol), caused inhibition of the 5-HT pressor response by 50-60%. The vasodilator responses were abolished by the detergent, CHAPS, indicating endothelium dependency; whereas vasoconstrictor responses to 5-HT were potentiated. CCh-induced relaxation was significantly reduced by the nitric oxide synthase inhibitor L-NAME (79.7 +/- 3.4% inhibition), whereas a large proportion of BK and SP-induced relaxation remained (34.1 +/- 6.3% and 33.6 +/- 5.3% inhibition). L-NAME potentiated vasoconstrictor responses to 5-HT. In conclusion, this study demonstrates that endothelium-derived NO modulates the response to vasoconstrictors such as 5-HT and is likely to be an important regulator of blood flow in the digital resistance vascular bed. Other factor(s) released by the endothelium are also important in regulating blood flow, whose identity remains to be established.     

Characterisation of the response of equine digital arteries and veins to substance P

Substance P (SP), a potent vasodilator, has been detected in equine digital sensory-motor nerves. The aim of the study was to characterise the functional responses of equine digital blood vessels to exogenous SP. Pre-constricted equine digital arteries (EDA) and veins (EDV) vasodilated in a biphasic, endothelium- and concentration-dependent manner to SP. A nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME; 300 microm) inhibited both phases of the relaxation response curve of EDAs to SP by >70%. In EDVs, the first relaxant phase to SP was largely L-NAME-resistant, whereas the second phase was inhibited by 60%. Both L-NAME and a cyclo-oxygenase inhibitor (ibuprofen; 10 microm) were required to inhibit EDV relaxation to SP by > or =80%. Experiments determining the receptor mediated responses to physiological concentrations of SP (1 nm) revealed that the relaxant responses of both EDA and EDV were inhibited by a neurokinin-1 (NK1) receptor antagonist (CP-96 345; 10 nm). In conclusion, SP is an endothelium-dependent vasodilator of both EDA and EDV. NO is the predominant pathway activated in EDA, whereas both prostacyclin and NO pathways are involved in EDVs. NK1 receptors appear to mediate responses to low concentrations of SP.     

Vaso-reactivity of isolated bovine intra-mammary artery to endogenous prostanoids and nitric oxide

The modulatory role of locally produced cyclooxygenase products and endothelium-derived nitric oxide in controlling vascular tone was investigated in bovine intra-mammary artery. Vascular reactivity initiated by vasoactive compounds, endothelin-1 (ET-1), bradykinin (BK), and substance P (SP) was measured isometrically in an isolated tissue bath. The effects of a cyclooxygenase inhibitor, indomethacin (10⁻⁵ M) and an inhibitor of nitric oxide production, Nω-Nitro L-Arginine (L-NNA: 3 x 10⁻⁴ M) were determined during agonistmediated responses. Indomethacin alone markedly enhanced vascular contraction produced by ET-1, while L-NNA did not. Inhibition of endothelium-derived nitric oxide synthesis by L-NNA, however, significantly attenuated BK-and SP-induced vascular relaxations, whereas indomethacin had slight influence. The potentiation between indomethacin and L-NNA in regulating vasomotor tone was not observed in this vascular bed. Thus, it appeared that both the cyclooxygenase and endothelium-derived nitric oxide pathways participated in modifying vascular reactivity. Domination of one pathway over the other depended upon the agonist used to stimulate vascular tissue.     

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.     

In vitro and in vivo studies of homocysteine in equine tissues: implications for the pathophysiology of laminitis

REASONS FOR PERFORMING STUDY: Elevated plasma homocysteine (HCy) concentration is a risk factor for cardiovascular diseases associated with endothelial dysfunction, including the human digital ischaemic disease, Raynaud's phenomenon. HYPOTHESIS: HCy causes dysfunction of equine vascular endothelium and elevated plasma concentrations predispose to laminitis. OBJECTIVES: To determine 1) the concentration of HCy in vitro, which inhibits equine vascular endothelial cell function and 2) any association between risk of laminitis and plasma HCy concentration. METHODS: Endothelial function was studied by measuring endothelium-dependent vasodilatory responses of the equine isolated perfused digit and basal nitric oxide (NO) production by cultured equine digital vein endothelial cells (EDVECs). Total plasma HCy (tHCy) concentrations were measured in samples collected in the winter and spring from normal ponies and ponies predisposed to laminitis. RESULTS: HCy (10 and 100 micromol/l) inhibited endothelial function and, at concentrations above 100 micromol/l, inhibited NO production by EDVECs. Plasma tHCy concentration ranged from 13 to 14.7 micromol/l. There was no effect of season or disease status on the concentration measured. CONCLUSIONS: In vitro, HCy was shown to interfere with endothelial cell function at physiologically relevant concentrations. No evidence was found for an association between risk of laminitis and high plasma concentrations of HCy. POTENTIAL RELEVANCE: Elevated plasma HCy concentrations could adversely affect endothelial cell function and mangement regimens that lead to increases in plasma HCy concentration should be avoided in ponies predisposed to laminitis.     

Bradykinin stimulates prostaglandin E2 production and cyclooxygenase activity in equine nonglandular and glandular gastric mucosa in vitro

REASONS FOR PERFORMING STUDY: There are few data available regarding regulation of prostaglandin (PG) generation by equine gastric mucosae and the role of the cyclooxygenase (COX) isoforms in their production. OBJECTIVES: To: 1) characterise and quantify PGE2 output in vitro; 2) examine the sensitivity of PGE2 production to exogenous bradykinin (BK) exposure; 3) determine the contribution of the COX-1 and COX-2 pathways to basal and BK-stimulated PGE2 production; and 4) measure if BK influences electrogenic ion transport in equine gastric mucosae in vitro. METHODS: Full thickness gastric sheets were obtained from horses at post mortem, stripped of muscle layers and mounted in Ussing chambers. Tissues were exposed to bradykinin (BK, 0.1 micromol/l) either alone, or following pretreatment with a selective COX-2 inhibitor (NS-398, 1 micromol/l) or a nonselective COX inhibitor (piroxicam, 1 micromol/l), or were untreated. RESULTS: BK administration increased PGE2 output from the basolateral but not the apical faces of both tissue types. Piroxicam, but not NS-398, reduced basolateral PGE2 release below control levels in both tissue types. Both piroxicam and NS-398 pretreatment inhibited BK-stimulated PGE2 release. In separate experiments, BK was without effect upon electrophysiological parameters of tissues mounted in Ussing chambers. CONCLUSIONS: PGE2 is produced by the nonglandular and glandular equine gastric mucosae in vitro. Significantly more PGE2 is released basolaterally than apically. BK stimulated the production of PGE2 from the basolateral side of both tissue types. These findings suggest that COX-1 is a significant pathway for basal PGE2 production from the basolateral faces of both nonglandular and glandular equine gastric mucosae in vitro.     

Mechanisms of NO-resistant relaxation induced by acetylcholine in rabbit renal arteries

The effects of K+ channel blockers and P2Y receptor agonist/antagonist on the vasorelaxation mediated by endothelium-derived hyperpolarizing factor (EDHF) were investigated in the rabbit renal artery. Acetylcholine (ACh, 1 nM-10 microM) induced endothelium-dependent relaxation of arterial rings precontracted with norepinephrine (NE, 1 microM) in a concentration-dependent manner. NG-nitro-L-arginine (L-NAME. 0.1 mM), an inhibitor of NO synthase, partially inhibited the ACh-induced endothelium-dependent relaxation. The ACh-induced relaxation was only partially inhibited by L-NAME whereas combined addition of L-NAME and 30 mM KCl completely inhibited the relaxation. The ACh-induced relaxation observed in the presence of L-NAME was significantly reduced by a combination of iberiotoxin (0.1 microM) and apamin (1 microM), and almost completely blocked by 4-aminopyridine (5 mM). The ACh-induced relaxation was antagonized by P2Y receptor antagonist, cibacron blue (10 and 100 microM) in a concentration-dependent manner. Furthermore, ADPbetaS, a potent P2Y agonist, induced the endothelium-dependent relaxation, and this relaxation was markedly reduced by either the combination of iberiotoxin and apamin or by cibacron blue alone. In conclusion, ACh may activate the release of ATP from endothelial cells which in turn activates a P2Y receptor on the endothelial cells followed by a release of EDHF, resulting in a vasorelaxation via a mechanism that involves activation of both the voltage-gated K+ channels and the Ca2+-activated K+ channels. EY WORDS: ATP, K+ channel, rabbit renal artery.     

Role of nitrergic nerves in the regulation of motility of the omasum and abomasum in healthy sheep <Emphasis Type="Italic">(Ovis aries)</Emphasis>

We examined the physiological role of nitrergic nerves in the regulation of omasal and abomasal motility in conscious healthy sheep and omasal muscle specimens. Nitric oxide (NO)-donor, S-nitroso-acethyl-dl-penicillamine (SNAP, 3-30 nmol/kg per min, i.v.) significantly inhibited omasal electromyographic (EMG) activity, whereas it did not alter EMG activity in the abomasal antrum. However, NO synthase inhibitor, Nomega-nitro-l-arginine-methyl ester (L-NAME, 0.3-3.0 micromol/kg per min, i.v.) did not alter EMG activity of the omasum and abomasum. In the in vitro experiments, SNAP application (6-200 micromol/l) significantly inhibited bethanechol (10 micromol/l)-induced contraction of longitudinal and circular muscles of the omasum. L-NAME application (0.03-3.0 mmol/l) enhanced electric field stimulation-induced contractions of the circular muscles. The results suggest that the omasal muscles are responsive to exogenous NO and that nitrergic nerves innervate the circular muscle layer of the omasum, however, nitrergic nerves are not or scarcely involved the physiological regulation of omasal and possibly abomasal motility in healthy sheep.     

Disturbances in ex vivo vascular smooth muscle responses following exposure to Pasteurella haemolytica vaccines

Weekley, L.B., Eyre, P. Disturbances in ex vivo vascular smooth muscle responses following exposure to Pasturella haemolytica vaccines. J. vet. Pharmacol. Therap. 16 , 446–453.
Rats were vaccinated with saline (control) or one of the two commercially available Pasteurella haemolytica vaccines Presponse or Precon-PH. Animals were killed 3 days later and thoracic aorta removed for evaluation of the ex vivo biophysical responses to carbachol (CCh). In some experiments, vascular endothelium was mechanically removed. Vaccination of rats impairs the endothelial-dependent relaxation to CCh. In vessels with endothelium removed, the contractile response to CCh is converted into a relaxation following vaccination. Treatment of endothelial-denuded vascular rings ex vivo with methylene blue, a guanylate cyclase inhibitor, reduced the vaccination effect. Treatment of vascular rings with the superoxide dismutase inhibitor diethyl-dithiocarbamate, impairs the relaxant reponse of de-endothelialized vessels to CCh in Presponse vaccinated rats while enhancing the relaxation response of vessels from Precon-PH vaccinated rats. De-endothelialized vessels from vaccinated rats, but not control rats, relaxed in the presence of 7V-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide synthetase. Furthermore, in the presence of L-NMMA, the relaxant response to CCh is significantly enhanced by Precon-PH but not Presponse. The normal relaxant response to hydrogen peroxide is converted into a contraction following vaccination. Results suggest that exposure to commercially available P. haemolytica vaccines alters vascular smooth muscle reactivity to CCh and that several independent pathways may be altered.     

Role of endothelium and nitric oxide in modulating in vitro responses of colonic arterial and venous rings to vasodilatory neuropeptides in horses

The objective of this study was to determine and compare the in vitro responses of equine large colon arterial and venous rings to vasodilatory neuropeptides; calcitonin gene-related peptide (CGRP); substance P (SP); vasoactive intestinal polypeptide (VIP); and acetylcholine (ACh), a standard nonpeptide endothelium-dependent vasodilator. Responses of vessel rings to graded concentrations (10(-11) M to 10(-5) M) of each drug were determined in endothelium-intact, denuded, and Nomega-nitro-L-arginine methyl ester (L-NAME, 10(-5) M)-treated rings that were pre-contracted with norepinephrine. Percentage maximal relaxation (PMR), defined as the % decrease from the contracted state, was determined. Because all rings did not relax at least 50%, EC50 values could not be consistently calculated. Arterial rings with intact endothelium were more sensitive to CGRP, compared with VIP and SP, and venous rings of all conditions were more sensitive to VIP than CGRP or SP. Overall, arteries had a greater PMR for ACh compared with SP and VIP. Intact and L-NAME treated arteries had a greater PMR than denuded arteries; there were no differences in PMR of intact and L-NAME treated arteries. Veins had a greater PMR for VIP than CGRP, SP, or ACh. Calcitonin gene-related peptide caused greater relaxation in intact arteries, whereas VIP causes greater relaxation in veins. Arterial relaxation was dependent upon the presence of intact endothelium. The response of veins to VIP among the conditions tested was not different, suggesting VIP has direct actions on venous smooth muscle. These neuropeptides modulate vasomotor tone via vasorelaxation in colonic arteries and veins.     

Homeostasis of intracellular Ca2+ in equine chondrocytes: response to hypotonic shock

REASONS FOR PERFORMING STUDY: Ca2+ homeostasis in articular chondrocytes affects synthesis and degradation of the cartilage matrix, as well as other cellular functions, thereby contributing to joint integrity. Although it will be affected by mechanical loading, the sensitivity of intracellular Ca2+ concentration ([Ca2+]i) in equine articular chondrocytes to many stimuli remains unknown. HYPOTHESIS: An improved understanding of Ca2+ homeostasis in equine articular chondrocytes, and how it is altered during joint loading and pathology, will be important in understanding how joints respond to mechanical loads. METHODS: [Ca2+]i was determined using the fluorophore fura-2. We examined the effects of hypotonic shock, a perturbation experienced in vivo during mechanical loading cycles. We used inhibitors of Ca2+ transporters to ascertain the important factors in Ca2+ homeostasis. RESULTS: Under isotonic conditions, [Ca2+]i was 148 +/- 23 nmol/l, increasing by 216 +/- 66 nmol/l in response to reduction in extracellular osmolality of 50%. Resting [Ca2+]i, and the increase following hypotonic shock, were decreased by Ca2+ removal; they were both elevated when extracellular [Ca2+] ([Ca2+]o) was raised or following Na+ removal. The hypotonicity-induced rise in [Ca2+]i was inhibited by exposure of cells to gadolinium (Gd3+; 10 micromol/l), an inhibitor of mechanosensitive channels. [Ca2+]i was also elevated following treatment of cells with thapsigargin (10 micromol/l), an inhibitor of the Ca2+ pump of intracellular stores. CONCLUSIONS: A model is presented which interprets these findings in relation to Ca2+ homeostasis in equine articular chondrocytes, including the presence of mechanosensitive channels allowing Ca2+ entry, a Na+/Ca2+ exchanger for removal of intracellular Ca2+ and intracellular stores sensitive to thapsigargin. POTENTIAL RELEVANCE: A more complete understanding of Ca2+ homeostasis in equine chondrocytes may allow development of future therapeutic regimes to ameliorate joint disease.     

Characterization of bradykinin-induced endothelium-independent contraction in equine basilar artery

We investigated the effect of bradykinin (BK) on isolated equine basilar arterial rings with and without endothelium. BK induced concentration-dependent contraction of resting arterial rings and no relaxation when the rings were precontracted by prostaglandin F_2α. The maximal response and pD₂ value were 161.2 ± 28.1% (to 60 m m KCl-induced contraction) and 8.24 ± 0.25 respectively. The cumulative concentration–response curve for BK was not shifted to the right by des-Arg⁹-[Leu⁸]-BK (a B₁-receptor antagonist), HOE140 (a B₂-receptor antagonist) or NPC567 (another B₂-receptor antagonist). In four of six basilar arteries, NPC567 induced concentration-dependent contraction. Indomethacin (a cyclooxygenase inhibitor), nordihydroguaiaretic acid (a lipoxygenase inhibitor), quinacrine (a phospholipase A₂ inhibitor), tetrodotoxin (a selective blocker of Na⁺ channels), guanethidine (a nor-adrenergic neuron blocking drug), phentolamine (an α-adrenoceptor antagonist), Nω-nitro- l -arginine ( l -NNA, a nitric oxide (NO) synthase inhibitor) and endothelial denudation did not affect the BK-induced contraction. l -NNA and indomethacin induced contraction and relaxation under resting vascular tone respectively. These results suggest that endothelial cells are not involved in BK-induced contraction and that the contraction is not mediated via activation of known B₁ and B₂ receptors. Arachidonic acid metabolites and neurotransmitters like norepinephrine and NO might not play a role in BK-induced contraction in equine basilar artery.     

Vasodilatory effect of pentoxifylline in isolated equine digital veins

The direct vasodilatory action of pentoxifylline (1-(5-oxohexyl)-3,7-dimethylxanthine) and its signalling pathway was evaluated in equine digital veins. Cumulative concentration-response curves to pentoxifylline (1 nM to 300 μM) were recorded in phenylephrine-precontracted equine digital vein rings under different experimental conditions. Relaxation to pentoxifylline was partially inhibited by endothelium removal, but was unaltered by CGS-15943 (a non-xanthine adenosine receptor antagonist; 3 μM). Nitric oxide synthase (NOS), soluble guanylate cyclase and cyclooxygenase (COX) inhibitors (Nω-nitro-L-arginine methyl ester (100 μM), ODQ (30 μM) and indomethacin (10 μM), respectively) significantly reduced the maximum relaxation induced by pentoxifylline. Moreover, pentoxifylline-induced relaxation was strongly reduced by Rp-8-Br-PET-cyclic guanosine monophosphate-S (a protein kinase G inhibitor; 3 μM), but remained unaffected by H-89 (a protein kinase A inhibitor; 2 μM). Pentoxifylline-induced relaxation was associated with a 3.4-fold increase in tissue cGMP content. To investigate whether pentoxifylline can affect cAMP- and cGMP-mediated relaxations, curves to forskolin, to sodium nitroprusside (SNP) and 8-bromo-cGMP were also recorded in endothelium-denuded equine digital vein rings pretreated with pentoxifylline (10 and 100 μM). Pentoxifylline only potentiated the SNP-mediated relaxation at the highest concentration (100 μM). Thus, pentoxifylline relaxed equine digital veins via endothelium-dependent and endothelium-independent components. The effect was mediated through both the NOS and COX pathways and could also result from inhibition of cGMP specific-phosphodiesterase activity at the highest concentrations used.     

Inhibition of endogenous nitric oxide production influences ovine hindlimb metabolism independently of insulin concentrations

The hindlimb arteriovenous difference (AVD) model was used to determine whether 30 mg/ kg of the nitric oxide synthase (NOS) inhibitor L-NGnitroarginine methyl ester (hydrochloride; L-NAME) inhibited ovine NO synthesis and influenced muscle metabolism. Eight Border Leicester x Merino cross lambs (50 to 55 kg BW) were infused with saline (control) or saline containing L-NAME via an indwelling jugular vein catheter in a balanced randomized crossover design with 3 d between treatments. The abdominal aorta and deep femoral vein were catheterized for assessment of AVD of hind limb metabolism. Arterial hematocrit and insulin concentration and both arterial and venous concentrations of nitrate/nitrite (NOx), glucose, lactate, NEFA, and urea were determined. Infusion of L-NAME decreased arterial NOx concentrations (P = 0.049), indicating inhibition of systemic NO synthesis. Treatment had no effect on arterial (3.5 vs. 3.6 +/- 0.19 mmol/L for control and L-NAME lambs, respectively; P = 0.39) or venous (3.3 vs. 3.4 +/- 0.16 mmol/L, P = 0.55) plasma glucose concentrations or on glucose AVD (0.19 vs. 0.27 +/- 0.065 mmol/L, P = 0.20). There was an interaction (P = 0.038) between time and treatment, such that L-NAME initially increased the AVD of glucose (up to 180 m) divergent from control lambs. The response was then decreased before a possible inflection beyond 240 min. Infusion of L-NAME increased hindlimb venous NEFA (222 vs. 272 +/- 13.2 micromol/L, P = 0.007) and NEFA AVD (79.4 vs. -13.3 +/- 31.5 micromol/L, P = 0.018). These metabolic changes were independent of plasma insulin concentrations, which were not affected by L-NAME infusion (25.3 vs. 27.8 +/- 3.62 mU/L, P = 0.85). The increase in hindlimb lipolysis after L-NAME infusion does not seem to be due to increased lipolysis of plasma triacylglycerol because circulating arterial (155 vs. 142 +/- 20.8 micromol/L, P = 0.58), venous (154 vs. 140 +/- 20.5 micromol/L, P = 0.50), and AVD (1.0 vs. 2.9 +/- 3.17 micromol/L, P = 0.38) triacylglycerol concentrations were unaffected by L-NAME infusion. In conclusion, these data indicate that infusion of 30 mg of L-NAME/kg inhibits NO synthesis, which in turn influences fat and carbohydrate metabolism in the ovine hindlimb independently of plasma insulin concentrations.     

Effects of nitric oxide donor and nitric oxide synthase inhibitor on ruminal contractions in conscious sheep

The present study was planned to evaluate a role of nitric oxide (NO) in the regulation of regular ruminal contractions in conscious sheep. Intravenous infusion of S-nitroso-acetyl-DL-penicillamine (SNAP) at doses of 3-30 nmol kg(-1) min(-1)for 30 minutes inhibited both the amplitude and frequency of ruminal contractions in a dose-dependent manner. However, intravenous infusion of Nomega-nitro-L-arginine-methyl ester (L-NAME) at doses of 0.3-3.0 micromol kg(-1) min(-1)did not alter the basal tone of intraruminal pressure and the amplitude of ruminal contractions. The frequency of contractions was slightly inhibited by L-NAME infusion at 1.0 micromol kg(-1)min(-1). The effects of L-NAME were abolished by simultaneous infusion of L -arginine at 30 micromol kg(-1) min(-1). These results suggest that exogenous NO can diminish the ruminal contractions, while endogenous NO is not involved in the regulatory mechanism of basal tone and regular phasic contractions of the rumen in healthy sheep.     

Endothelin mediated contraction of equine laminar veins

REASONS FOR PERFORMING STUDY: Endothelin-1 (ET-1) may be a key mediator in the pathogenesis of laminitis, but endothelin-mediated responses in the venous microcirculation of the equine foot have yet to be fully characterised. OBJECTIVES: To characterise the response of equine laminar veins to ET-1 and evaluate the ET-1 receptor subtypes that mediate this response. METHODS: Small veins (150-500 microns) draining the equine digital laminae from healthy horses and ponies subjected to euthanasia at an abattoir were investigated using wire myography. Concentration response curves were constructed for ET-1 in the presence of ETA (BQ123) and ETB (BQ788) receptor antagonists, and L-NAME, a nitric oxide synthase blocker. The selective ETB receptor agonist BQ3020 was investigated alone and following incubation with L-NAME, with or without BQ788. RESULTS: Endothelin-1 contraction of laminar veins was significantly inhibited by BQ123 but not by BQ788. In the presence of L-NAME, sensitivity of laminar veins to ET-1 was enhanced 4-fold, and further addition of BQ788 did not alter this increased sensitivity. BQ3020 induced no venoconstriction; however, in the presence of L-NAME, it caused contraction of veins with approximately 30% of the efficacy of ET-1. The action of BQ3020 in the presence of L-NAME was abolished by BQ788. CONCLUSIONS: Both ETA and ETB receptors are involved in the net tonic response to ET-1 in normal laminar veins. A population of ETB receptors may be present on the vascular endothelium and on smooth muscle of laminar veins, and the action of ET-1 at these 2 sites is likely to be approximately equal and opposite. POTENTIAL RELEVANCE: Our results clarify the function of the ET-1 receptor subtypes in laminar veins from healthy horses. Further study of ET-1 receptors in laminitic horses is therefore warranted.     

Effect of acute sublethal endotoxaemia on in vitro digital vascular reactivity in horses

Endotoxaemia is a syndrome linked to the development of equine laminitis; however, the relationship between them is uncertain. The aim of this experiment was to evaluate the effect of an experimental acute sublethal endotoxaemia model on in vitro equine palmar digital vascular reactivity. Rings of arteries and veins of each forelimb were obtained from 11 clinically healthy horses submitted to two surgical procedures, 3 weeks apart. Before the second surgery, 0.25 microg/kg of lipopolysaccharide from Escherichia coli O55:B5 in saline, was administered i.v. in 30 min. After 3 h, the vessels were harvested and submitted to in vitro vascular reactivity experiments and histopathology. The response to depolarizing Krebs solution (DKS, 40 mm), phenylephrine (PHE), acetylcholine (ACh) and sodium nitroprusside (SNP) were evaluated. All horses showed colic pain and watery diarrhoea, tachycardia, tachypnea, hyperthermia and leucopenia. Concentration-response curve (CRC) to PHE was shifted to the left in arteries rings from endotoxemic horses without any effect on vein rings. The CRC to ACh was shifted to the right with a reduction in the maximal response. The response to SNP and DKS was similar between groups. There was no evidence of histopathological effects. The increased response to PHE in digital arteries together with a reduction of the endothelium-dependent response to ACh in arteries and veins, confirm the existing reports where endotoxaemia was found to modify the digital vascular reactivity during the acute phase. As the digital endothelial function is impaired, there may be an increased potential to develop a digital prothrombotic state with a reduced vasodilatory capacity.     

Pulmonary arterial pressure and electrocardiograms in broiler chickens infused intravenously with L-NAME,an inhibitor of nitric oxide synthase,or sodium nitroprusside (SNP), a nitric oxide donor

1. Broilers were divided at 42 to 44 d of age into a Control group (n=30) and a Treatment group (n=30). The mean pulmonary arterial pressure (mPAP) and electrocardiogram (ECG) leads II and aV(F) were measured 1, 2 and 4 h after an intravenous injection of 0.9% saline (Control group) or Nomega-nitro-L-arginine methyl esther (L-NAME), an inhibitor of nitric oxide synthase and thus an inhibitor of endothelial nitric oxide (NO) production (Treatment group). 2. At 1 and 2 h but not 4 h post-injection, L-NAME significantly increased the mPAP and the amplitudes of the ECG S-wave and RS-wave leads II and aVF when compared with Control values. 3. The correlation coefficients between the mPAP and the ECG S-wave and RS-wave amplitudes for lead II within the Treatment group were -0.848 and -0.553 at 1 h and -0.798 and -0.512 at 2 h, respectively. The corresponding coefficients for lead aVF were -0.735, -0.596, -0.663 and -0.724, respectively. 4. After suitable mPAP and ECG values had been recorded at each time interval, sodium nitroprusside (SNP), which acts as a short-lived NO donor molecule, was injected intravenously via a right-cardiac catheter. Within 5 min after the SNP injection, the mPAP and the ECG lead II S-wave and RS-wave amplitudes were transiently reduced to levels that, at 1 and 2 h after L-NAME injection, did not differ from Control values. Within 10 min after the SNP injection, all values returned to the levels previously induced by L-NAME. 5. These results demonstrate that L-NAME increased the myocardial contractility and PAP, whereas SNP transiently reversed the effects of L-NAME on myocardial contractility and PAP. It appears likely from these results that the pulmonary vascular endothelium releases NO that in turn reduces the pulmonary vascular resistance or attenuates myocardial contractility in broiler chickens.     

Role of Endothelium and Nitric Oxide in the Response of Equine Colonic Arterial Rings to Vasoconstrictor Agents

Objective- To determine the in vitro contractile responses of equine colonic arteries to angiotensin II, histamine, serotonin, norepinephrine, prostaglandin F_2α, vasopressin, and a thromboxane-B₂-analogue. Study Design- The tension generated in colonic arterial rings placed in organ baths with oxygenated Tyrode's solution at 37°C after exposure to the previously mentioned chemical agents was measured using force-transducers interfaced with a polygraph. Sample Population- Large colon arterial rings collected from eight horses. Methods- The rings were allowed to equilibrate for 45 minutes after applying 2 g tension. Bath solution was replaced and tension reapplied at 15-minute intervals. Cumulative-concentration-responses were determined for concentrations ranging from 10^-8M to 10^-4M on three vessel groups namely endothelium intact, endothelium denuded, and L-NAME treated. The maximal response for each vessel was considered as 100%; responses to lower concentrations were calculated as a percentage of the maximum. The EC₅₀ value was determined for each concentration-response relationship of each agent. Results- Vessels with denuded endothelium or those incubated with L-NAME had greater contractile responses. Angiotensin, histamine, serotonin, and norepinephrine produced greater maximal responses than the other agents. Endothelium denuded rings had lower EC₅₀ values. Responses to norepinephrine and serotonin were affected less by denudation. Conclusion- Endothelium plays an important role in modulating responses of colonic arterial rings to contractile agents. Endothelium-derived vasodilators, other than nitric oxide, may modulate contractile responses of equine colonic arteries. Clinical Relevance- Endothelial damage associated with colonic vovulus may be a major factor for sustained reduced perfusion after surgical correction.     

Differential inhibition of equine neutrophil function by phosphodiesterase inhibitors

Neutrophils are recruited to the lungs of horses with chronic obstructive pulmonary disease (COPD) and exhibit increased activity after antigen challenge, which may contribute to inflammation and lung damage. Inhibition of phosphodiesterase isoenzymes (PDEs) has been shown to attenuate human neutrophil functions including superoxide production, leukotriene (LT)B4 biosynthesis, enzyme and chemokine release. As equine neutrophils contain predominantly the isoenzyme, PDE4, the present study was undertaken to investigate the effects of rolipram, a PDE4 inhibitor, on equine neutrophil function. For comparison, the effects of the nonselective PDE inhibitor, theophylline, were examined. Cells from both normal horses and COPD horses in remission were used. Superoxide production was significantly inhibited by both rolipram [32.2 +/- 2.6 vs. 10.1 +/- 1.1 nmol/10(6) cells and 49.8 +/- 6.8 vs. 22.7 +/- 2.2 nmol/10(6) cells for normal and COPD susceptible horses, respectively, in response to 10(-7) M human recombinant (hr) C5a] and theophylline (19.0 +/- 0.6 vs. 10.2 +/- 0.6 nmol/10(6) cells and 24.3 +/- 2.1 vs. 10.7 +/- 0.9 nmol/10(6) cells for normal and COPD susceptible horses, respectively, in response to 10(-7) M C5a). However, superoxide production induced by serum treated zymosan was inhibited only by theophylline (10(-3) M). Neither hrC5a- nor platelet activating factor (PAF)-induced neutrophil adherence to fibronectin coated plastic was reduced by rolipram (10(-5) M). These results demonstrate that the effects of PDE inhibitors on equine neutrophils are both stimulus and function dependent. The PDE4 inhibitors may reduce neutrophil activation in vivo in horses with COPD.