Yoel Berhane

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, Raynauds 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.

Evaluation of the induction of vasoactive mediators from equine digital vein endothelial cells by endotoxin

OBJECTIVE To determine the effect of endotoxin (lipopolysaccharide [LPS]) on vasoactive mediator production by cultured equine digital vein endothelial cells (EDVECs). SAMPLE POPULATION EDVECs obtained from forelimb digital veins of 7 healthy adult horses. PROCEDURES EDVECs were incubated with or without LPS (1 microg/mL) for 0, 2, 4, 6, 22, and 24 hours. The EDVECs were incubated for 18 hours with LPS (10 pg/mL to 1 microg/mL) with or without ibuprofen, cycloheximide, or L-nitroarginine methyl ester. Medium concentrations of prostacyclin, cyclic guanosine monophosphate, endothelin-1, and thromboxane A(2) were determined. Changes in inducible nitric oxide synthase and cyclooxygenase-2 expression were determined. RESULTS LPS stimulated mean 4.2- and 14.1-fold increases in EDVEC prostacyclin and cyclic guanosine monophosphate production, respectively, after 22 hours. These effects were LPS concentration-dependent (LPS concentrations that induced a response halfway between the maximum response and baseline of 1.50 and 1.22 ng/mL, respectively). The LPS-induced cyclic guanosine monophosphate production was significantly inhibited (to basal concentrations) by L-nitroarginine methyl ester, and prostacyclin production was inhibited by cycloheximide and ibuprofen. Production of thromboxane A(2) by EDVECs was not detected. Endothelin-1 accumulated in the medium, but LPS did not enhance its production. Inducible nitric oxide synthase expression in EDVECs was not detected with the available antibodies, whereas LPS stimulated cyclooxygenase-2 expression in a time- and concentration-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE LPS stimulated vasoactive mediator production by equine endothelial cells, which may play a role in LPS-induced digital hypoperfusion.

Density and binding characteristics of beta-adrenoceptors in the normal and failing equine myocardium

Beta-adrenoceptors are important regulators of cardiac function and their characteristics are known to change in human and canine diseased myocardium. This study aimed to determine the density and subtypes of beta-adrenoceptors in the normal and failing equine ventricular myocardium. Membrane preparations of the left papillary muscles were incubated with increasing concentrations of the nonselective beta-adrenoceptor antagonist [3H]-CGP12177. Saturable and reversible binding of [3H]-CGP12177 to myocardial membranes was demonstrated with Kd values (+/- s.d.) of 0.49 +/- 0.40 and 0.43 +/- 0.22 nmol/l and Bmax values of 93.4 +/- 20.5 and 110.0 +/- 21.2 and fmol/mg protein for normal (n = 19) and heart failure (n = 10) tissues, respectively. Heart failure had no significant effect on the density of ventricular beta-adrenoceptors. The cardiac beta-adrenoceptors were further characterised by studying displacement of [3H]-CGP12177 (0.6 nmol/l) with the beta1-selective antagonists CGP20712A and the beta2-selective antagonist ICI118.551. In normal ventricular muscle, CGP20712A was 26 times more potent than ICI118.551 (Ki values 30.4 +/- 24.8 and 814.1 +/- 485.2 nmol/l, respectively). In heart failure cases, CGP 20712A curves were monophasic with a Ki value of 45.6 +/- 39.7 nmol/l. ICI 118.551 curves were biphasic in 5 horses where 11-31% of the cardiac beta-adrenoceptors had a high affinity for ICI 118.551. These data suggest that the normal equine ventricular myocardium possesses predominately beta1-adrenoceptors, with no evidence for co-existence of a significant population of beta2-adrenoceptors. The density of beta-adrenoceptors did not appear to change in heart failure, but the appearance of receptors with a high affinity for ICI118.551 may suggest that, in some cases, heart failure increases the expression of beta2-adrenoceptors in equine ventricular myocardium. This study provides an insight into the role of the adrenergic system in heart disease in the horse. Further studies in this area are warranted.

Functional role of α2-adrenoceptor subtypes in the cooling-enhanced vasoconstriction of isolated cutaneous digital veins of the horse

Cooling-enhanced contractile responses in cutaneous arteries have been reported to involve the activation of alpha(2C)-adrenoceptors, but their role in cutaneous veins is not clearly understood. The aim was to pharmacologically characterize the subtype of postsynaptic alpha(2)-adrenoceptors in the equine digital vein mediating contraction at two temperatures. The increase in isometric tension of endothelium-denuded equine digital vein in response to UK-14304 was studied in the absence and presence of relatively selective alpha(2)-adrenoceptor antagonists at temperatures of either 30 degrees C (the peripheral digit temperature of horses maintained in a thermoneutral environment) or 22 degrees C. The response to UK-14304 was enhanced by cooling and antagonised by RX-821002 (alpha(2)-adrenoceptor non-selective; 30 degrees C: apparent pK(b)=8.5; 22 degrees C: pK(b)=8.2) and yohimbine (alpha(2)-adrenoceptor non-selective; 30 degrees C: apparent pK(b)=7.2; 22 degrees C: apparent pK(b)=7.4). The response at 30 degrees C was non-surmountably antagonised by BRL-44408 (alpha(2A)-adrenoceptor-selective; apparent pK(b)=8.9) and MK-912 (alpha(2C)-adrenoceptor-selective: apparent pK(b)=9.9). JP-1302 (alpha(2C)-adrenoceptor-selective) surmountably antagonised the response with a low potency (apparent pK(b)=5.6) at 30 degrees C. The response at 22 degrees C was surmountably antagonised by: BRL-44408 (alpha(2A)-adrenoceptor-selective; apparent pK(b)=6.5); MK-912 (alpha(2C)-adrenoceptor-selective; pK(b)=9.7) and JP-1302 (alpha(2C)-adrenoceptor-selective; apparent pK(b)=7.5). ARC-239 (alpha(2B)-adrenoceptor-selective) did not affect the response at either temperature. The apparent potency of the different antagonists and their non-surmountable effect, suggests that the UK-14304 response at 30 degrees C could be mediated by alpha(2A)-adrenoceptors and alpha(2C)-adrenoceptors. By contrast, the alpha(2C)-adrenoceptor appears to be the main alpha(2)-adrenoceptor mediating the augmented response at 22 degrees C. It is unlikely that the alpha(2B)-adrenoceptor subtype contributes at either temperature. These results support the participation of the alpha(2C)-adrenoceptors in the cooling-enhanced response to UK-14304 in this cutaneous vein.

The differential hepatic uptake of chylomicron remnants of different fatty acid composition is not mediated by hepatic lipase

The hypothesis that hepatic lipase mediates the differential hepatic uptake of chylomicron remnants of different fatty acid composition, demonstrated in previous work from our laboratory, was tested by investigating the effect of antibodies to the enzyme on the uptake of remnants enriched with saturated or n-3 polyunsaturated fatty acids by the perfused rat liver. After perfusion of rat livers with polyclonal antibodies to rat hepatic lipase raised in rabbits or with rabbit non-immune serum for 15 min, [3H]oleate-labelled chylomicron remnants, derived from chylomicrons of rats given a bolus of either palm (rich in saturated fatty acids) oil or fish (rich in n-3 polyunsaturated fatty acids) oil, were added. The disappearance of radioactivity from the perfusate during 120 min and its recovery in the liver at the end of the experiments were then measured. Although the rabbit anti-rat hepatic lipase antiserum was shown to inhibit hepatic lipase activity by up to 90%, and to bind extensively to hepatic sinusoidal surfaces when added to the perfusate, radioactivity from remnants of chylomicrons from rats given a bolus of fish oil as compared with palm oil disappeared from the perfusate and appeared in the liver more rapidly in the presence both the antiserum and the non-immune serum, and the differences between the uptake of the two types of remnants were similar. We conclude, therefore, that differential interaction with hepatic lipase is not responsible for the differences in the rate of removal of chylomicron remnants of different fatty acid composition from the blood.

Rho kinase activation and ROS production contributes to the cooling enhanced contraction in cutaneous equine digital veins

A decrease in environmental temperature can directly affect the contractility of cutaneous vasculature, mediated in part by alpha(2)-adrenoceptors. Most of the cellular mechanisms underlying the cooling-enhanced contractility to alpha(2)-adrenoceptor agonists have been reported in cutaneous arteries but little information is available on cutaneous veins. To investigate the cellular mechanisms associated with the cooling-enhanced contraction to UK-14304 (alpha(2)-adrenoceptor agonist), isolated equine digital veins (EDVs) were studied at 30 degrees C and 22 degrees C. The effects of inhibitors were studied on the contractile response to UK-14304 (0.1 microM). The cooling-enhanced responses were inhibited by Rho kinase inhibitors [maximum response to UK-14304 95.2 +/- 8% of response to depolarizing Krebs solution (DKS) in control vessels cooled to 22 degrees C, compared with 31.4 +/- 6% in the presence of fasudil 1 microM and 75.8 +/- 6% with Y-27632 0.1 microM] and the effects of these inhibitors were considerably less at 30 degrees C (control response 56.4 +/- 5% of DKS; 34.9 +/- 6% with fasudil 1 microM and 50.6 +/- 9% with Y-27632 0.1 microM). Furthermore, Western blotting showed that one of the downstream targets for Rho kinase activity, ezrin/radixin/moesin, was phosphorylated after cooling and reduced by fasudil (1 microM) only at 22 degrees C. The activation of protein kinase C contributed to the contractile response, but predominantly at 30 degrees C (maximum response 82.3 +/- 9% of DKS for control; 57.7 +/- 10% in the presence of chelerythrine 10 microM) with no significant effect at 22 degrees C. The reduction of the response at 22 degrees C by antioxidants, rotenone (14% reduction), and tempol (21% reduction) suggested the contribution of reactive oxygen species (ROS). No evidence was obtained to support the participation of tyrosine kinase. These data demonstrate that Rho kinase activation and the production of ROS contributes to the cooling-enhanced contraction in these cutaneous digital veins.

Characterization of agonist-induced endothelium-dependent vasodilatory responses in the vascular bed of the equine digit

The role of endothelium-derived relaxing factors was studied in the regulation of vascular responses in the Krebs perfused equine isolated digit. Perfusion pressure was recorded in response to bolus doses of 5-hydroxytryptamine (6 nmol) alone or co-administered with carbachol (CCh; 0.2 micromol), bradykinin (BK; 0.2 nmol), substance P (SP; 0.2 nmol) or sodium nitroprusside (SNP; 0.2 micromol). N(omega)-Nitro-L-Arginine methyl ester hydrochloride (L-NAME; 300 microm) caused partial but significant inhibition of CCh-induced vasodilatory response, whereas BK and SP-induced responses were resistant to L-NAME. High potassium (K(+), 30 mm) and the cytochrome P-450 (CYP) epoxygenase inhibitor, clotrimazole (10 microm) plus L-NAME (100 microm), completely abolished the CCh, BK and SP-induced vasodilatory responses, whereas the response to SNP was unaffected. In contrast, the L-NAME-resistant proportion of CCh, BK and SP-induced vasodilatory response was not inhibited by the highly selective CYP2C9 inhibitor, sulphaphenazole (10 microm). The cyclo-oxygenase inhibitor, ibuprofen (10 microm) did not affect the CCh, BK and SP-induced responses. These data demonstrate that CCh, BK and SP-induced relaxation in the equine digit involve a combination of the NO and endothelium-derived hyperpolarizing factor (EDHF) pathways. These results do not support the evidence for the involvement of CYP-derived epoxyeicosatrienoic acids and the exact nature of EDHF in the equine digit remains to be established.

The effect of cooling on the contractility of equine digital small lamellar arteries: modulating role of the endothelium

The equine hoof displays thermoregulatory functions, and the blood vessels lying under the hoof wall are temperature sensitive. The aim of this study was to investigate the effect of cooling on the contractile responses to α‐adrenoceptor and 5‐HT receptor stimulation in equine small lamellar arteries using wire myography. The role of the endothelium in the response mediated by 5‐HT was also evaluated. Moderate cooling caused a reduction of the contraction induced by depolarizing Krebs solution (DKS, containing 118 mm KCl) and the maximal contraction caused by UK‐14304 (α2‐adrenoceptor agonist). The potency of methoxamine (α1‐adrenoceptor agonist) was reduced by cooling [pD2 (−log EC50) at 22°C, 5.7 (5.5–6.0) versus 30°C, 5.9 (5.7–6.1)]; however, the efficacy was maintained. The contractions evoked by sumatripan and α‐methyl 5‐HT (5‐HT receptor agonists) were not modified by moderate cooling. In contrast, a cooling‐enhanced contraction was observed in response to 5‐HT [maximum response (Emax) at 22°C, 213.2 ± 13% DKS versus 30°C, 179.9 ± 11% DKS]. Furthermore, this effect was more evident in endothelium‐denuded lamellar arteries (Emax at 22°C, 270.2 ± 26% DKS versus 30°C, 172.2 ± 20% DKS), suggesting a potential modulating role of the endothelium. The l‐NAME/ibuprofen‐resistant relaxation in response to carbachol was reduced by cooling in small lamellar arteries precontracted with 5‐HT but not phenylephrine. Therefore, a moderate reduction of temperature modulates the reactivity of small lamellar arteries by enhancing the 5‐HT‐mediated contraction, but inhibits the α‐adrenoceptor‐mediated response. Furthermore, the endothelium of these blood vessels may play an important role in preventing excessive vasoconstriction in response to 5‐HT and maintaining digital blood flow in cool environmental temperatures.

Experimental Physiology –Research Paper: The effect of cooling on the contractility of equine digital small lamellar arteries: modulating role of the endothelium

The equine hoof displays thermoregulatory functions, and the blood vessels lying under the hoof wall are temperature sensitive. The aim of this study was to investigate the effect of cooling on the contractile responses to α‐adrenoceptor and 5‐HT receptor stimulation in equine small lamellar arteries using wire myography. The role of the endothelium in the response mediated by 5‐HT was also evaluated. Moderate cooling caused a reduction of the contraction induced by depolarizing Krebs solution (DKS, containing 118 mm KCl) and the maximal contraction caused by UK‐14304 (α2‐adrenoceptor agonist). The potency of methoxamine (α1‐adrenoceptor agonist) was reduced by cooling [pD2 (−log EC50) at 22°C, 5.7 (5.5–6.0) versus 30°C, 5.9 (5.7–6.1)]; however, the efficacy was maintained. The contractions evoked by sumatripan and α‐methyl 5‐HT (5‐HT receptor agonists) were not modified by moderate cooling. In contrast, a cooling‐enhanced contraction was observed in response to 5‐HT [maximum response (Emax) at 22°C, 213.2 ± 13% DKS versus 30°C, 179.9 ± 11% DKS]. Furthermore, this effect was more evident in endothelium‐denuded lamellar arteries (Emax at 22°C, 270.2 ± 26% DKS versus 30°C, 172.2 ± 20% DKS), suggesting a potential modulating role of the endothelium. The l‐NAME/ibuprofen‐resistant relaxation in response to carbachol was reduced by cooling in small lamellar arteries precontracted with 5‐HT but not phenylephrine. Therefore, a moderate reduction of temperature modulates the reactivity of small lamellar arteries by enhancing the 5‐HT‐mediated contraction, but inhibits the α‐adrenoceptor‐mediated response. Furthermore, the endothelium of these blood vessels may play an important role in preventing excessive vasoconstriction in response to 5‐HT and maintaining digital blood flow in cool environmental temperatures.

Experimental Physiology -Research Paper: The effect of cooling on the contractility of equine digital small lamellar arteries: modulating role of the endothelium: Effect of cooling in equine small lamellar arteries

The equine hoof displays thermoregulatory functions, and the blood vessels lying under the hoof wall are temperature sensitive. The aim of this study was to investigate the effect of cooling on the contractile responses to α‐adrenoceptor and 5‐HT receptor stimulation in equine small lamellar arteries using wire myography. The role of the endothelium in the response mediated by 5‐HT was also evaluated. Moderate cooling caused a reduction of the contraction induced by depolarizing Krebs solution (DKS, containing 118 mm KCl) and the maximal contraction caused by UK‐14304 (α2‐adrenoceptor agonist). The potency of methoxamine (α1‐adrenoceptor agonist) was reduced by cooling [pD2 (−log EC50) at 22°C, 5.7 (5.5–6.0) versus 30°C, 5.9 (5.7–6.1)]; however, the efficacy was maintained. The contractions evoked by sumatripan and α‐methyl 5‐HT (5‐HT receptor agonists) were not modified by moderate cooling. In contrast, a cooling‐enhanced contraction was observed in response to 5‐HT [maximum response (Emax) at 22°C, 213.2 ± 13% DKS versus 30°C, 179.9 ± 11% DKS]. Furthermore, this effect was more evident in endothelium‐denuded lamellar arteries (Emax at 22°C, 270.2 ± 26% DKS versus 30°C, 172.2 ± 20% DKS), suggesting a potential modulating role of the endothelium. The l‐NAME/ibuprofen‐resistant relaxation in response to carbachol was reduced by cooling in small lamellar arteries precontracted with 5‐HT but not phenylephrine. Therefore, a moderate reduction of temperature modulates the reactivity of small lamellar arteries by enhancing the 5‐HT‐mediated contraction, but inhibits the α‐adrenoceptor‐mediated response. Furthermore, the endothelium of these blood vessels may play an important role in preventing excessive vasoconstriction in response to 5‐HT and maintaining digital blood flow in cool environmental temperatures.