Effects of long-term active immunization with the second extracellular loop of human ß1- or ß3-adrenoceptors in thoracic aorta and mesenteric arteries in Lewis rats.

OBJECTIVE: To evaluate whether active immunization producing ß1- or ß3-antibodies (ß1-ABs and ß3-ABs) detected in sera of patients with dilated cardiomyopathies has deleterious effects on vascular reactivity in Lewis rat thoracic aorta (TA) and small mesenteric arteries (SMA). DESIGN AND METHOD: Lewis rats were immunized for 6months with peptidic sequences corresponding to the second extracellular loop of ß1- and ß3-adrenoceptors (ARs). During the immunization, systolic blood pressure (SBP) was monitored using the tail cuff method. The vascular reactivity of immunized rats was assessed by ex vivo studies on SMA and TA using various ß-AR agonists, phenylephrine and KCl. RESULTS: The immunizations producing functional ß1-ABs and ß3-ABs did not affect the SBP. However, in TA from ß1-AR-immunized rats, the relaxations mediated by dobutamine and salbutamol were significantly impaired in comparison with adjuvant rats whereas nebivolol-induced relaxation was not modified. Moreover, phenylephrine and KCl-mediated contractions were enhanced in these rats. In contrast, immunization with ß3-AR peptide led to the increase of relaxations induced by dobutamine in TA but did not change those induced by salbutamol and nebivolol. Surprisingly, in SMA from both rats immunized with ß1- or ß3-peptides, relaxations induced by the various ß-agonists were not changed whereas phenylephrine and KCl-mediated contractions were impaired. CONCLUSIONS: Our study shows that ß1- and ß3-ABs can affect vascular reactivity. ß1-ABs would have a pathogenic action whereas ß3-ABs would have a beneficial effect on aorta reactivity.

Venous Valvular Distribution in the Thoracic and Pelvic Limbs of the Horse.

Dysfunction of venous valves can lead to hemodynamic disorders causing venous stasis, which would favour the occurrence of equine laminitis. However, very few studies have investigated venous valves in the horse digit. The purpose of this study was to compare valvular density between thoracic and pelvic limbs and to study the relationship between valvular density of veins and their location, diameter and wall thickness. After dissection, valvular density was calculated based on the number of valves counted in the principal veins of 7 thoracic and 7 pelvic limbs from 7 horses. Our results showed that the valvular density was higher in thoracic limbs, which probably reflects the adaptation to the consequences of hydrostatic pressure. The superficial veins have a higher valvular density that would prevent the varicose risk in the horse. The lower valvular density in the thick veins can be explained by the high density of the smooth muscular cells contained, which would cause an important vasoconstriction via the sympathetic nervous system. The veins with a large diameter also have a lower valvular density; these veins are not exposed to important changes in hydrostatic pressure. Other valvular characteristics may also be involved in the vascular disorders that may be related to the pathophysiology of laminitis.

Cardiac effects of long-term active immunization with the second extracellular loop of human ß1- and/or ß3-adrenoceptors in Lewis rats.

ß1- and ß3-adrenoceptor (AR) auto-antibodies were detected in patients with dilated cardiomyopathy. Many studies have shown that ß1-AR auto-antibodies with partial agonist-like effect play an important role in the pathogenesis of this disease. Moreover, a recent study carried out in our laboratory has shown that ß3-AR antibodies (ß3-ABs), produced in rats, were able to reduce cardiomyocyte contractility via ß3-AR activation. The aims of this study were (1) to investigate, in isolated cardiomyocytes from rabbit, the role of Gi proteins in the ß3-ABs-induced cardiac negative inotropy, (2) to determine whether ß3-ABs may exhibit ß3-AR antagonistic property which is characteristic of partial agonists, and (3) to determine whether long-term active immunization producing both ß1-ABs and/or ß3-ABs leads to the development of cardiac dysfunction in Lewis rats. Lewis rats were immunized for 6 months with peptidic sequences corresponding to the second extracellular loop of human ß3-AR and/or ß1-AR. Agonistic effect of ß3-ABs was evaluated on electrically field-stimulated isolated cardiomyocytes from adult rabbit by measuring the cell shortening. Echocardiography and ex vivo isolated perfused heart studies were conducted on immunized rats. Finally, ß-AR expression was quantified by immunofluorescence and RT-qPCR. SR58611A (10 nM), a preferential ß3-AR agonist, and purified ß3-ABs (25 µg/ml) induced a decrease in cell shortening (-39.71±4.9% (n=10) and -17.06±3.9% (n=10) respectively). This effect was significantly inhibited when the cardiomyocytes were preincubated with pertussis toxin (0.3 µg/ml), a Gi protein inhibitor (p<0.05). In addition, SR58611A-mediated negative inotropic effect was decreased when cardiomyocytes were preincubated with ß3-ABs (p<0.0001). Echocardiography revealed a decrease in the fractional shortening and ejection fraction in rats immunized against ß1-AR and both ß1- and ß3-AR. However, the study on isolated heart showed a decrease of the isoproterenol-induced lusitropic and inotropic effects in the 3 groups of immunized rats. These systolic and diastolic dysfunctions are correlated with a decrease in the expression of ß1-ARs and an increase of ß3-ARs in rats immunized against the ß1-AR and an increase of both ß3-AR and ß1-AR in rats immunized against the ß3-AR. For the first time, these results showed that ß3-ABs had a ß3-AR partial agonist-like activity which might play a role in the pathogenesis of cardiac dysfunction.

[Cardiovascular effects of beta1 and beta3-adrenergic receptor autoantibodies in Lewis rat]. / Effets cardiovasculaires des auto-anticorps anti-récepteurs béta1 et béta3-adrénergiques chez le rat Lewis.

PURPOSE: To analyze vascular reactivity changes in response to immunization protocols with antigens corresponding to the second extracellular loop of -ß3 and -ß1 and 3 adrenergic receptors (AR). METHODS: Lewis rats were immunized for 3months with peptidic sequences corresponding to the second extracellular loop of ß3-AR or ß1 and 3-AR. Specific ß3-AR antibodies were characterized by Elisa and purified using "Proteus Protein G" kit. Their functionality were tested in rabbit isolated ventricular cardiomyocytes. Aortic and mesenteric artery rings isolated from control or immunized rats were mounted in organ baths and precontracted with phenylephrine. Then, relaxant curves were established. RESULTS: SR58611A (10nM), a preferential ß3-AR agonist and purified ß3-AR antibodies (25µg/mL) induced a decrease of cell shortening (-39.56±4.4% [n=11] and -18.45±3.9% [n=10] respectively) in isolated cardiomyocytes. This decrease was significantly inhibited when the cardiomyocytes were pre-incubated with the L-748337 (1µM), a selective ß3-AR antagonist (P<0.05). In contrast with what was observed in rats immunized against the ß1-AR, vasorelaxations induced by acetylcholine and SR58611A in both aorta and mesenteric arteries were unaltered in rats immunized against the ß3-AR and ß1 and 3-AR. CONCLUSION: These results show, for the first time, that ß3-AR antibodies induced a ß3-AR agonist-like activity. They would not have a vascular pathogenic action but would offset the endothelial dysfunction caused by ß1-AR antibodies.

Antibodies against the second extracellular loop of ß1-adrenergic receptors induce endothelial dysfunction in conductance and resistance arteries of the Wistar rat.

Autoantibodies against ß1-adrenoceptors (ß1-ARs) have been detected in the serum of patients with various cardiac diseases; however, the pathological impact of these autoantibodies (ß1-AABs) has only been evaluated in cardiac tissue. The purpose of the present study was to evaluate whether ß1-AABs have deleterious effects on vascular reactivity in rats. An enzyme-linked immunosorbent assay was used to detect ß1-AABs in sera from immunized rats over a period of 1-3 months using the peptidic sequence of the second extracellular loop of human ß1-AR. Functional studies were performed in thoracic aortic (TA) and small mesenteric artery (SMA) rings from immunized rats. Following pre-contraction with phenylephrine (0.3 µM and 3 µM for the TA and SMA respectively), cumulative concentration-response curves (CCRCs) to various ß-AR agonists (isoproterenol, dobutamine, salbutamol, SR 58611A), acetylcholine, A23187, and sodium nitroprusside (SNP) were then plotted. The relaxations induced by dobutamine, SR 58611A, and acetylcholine were significantly impaired, but salbutamol-induced relaxations were not affected, in both vessels from immunized rats. A significant impairment of isoproterenol-induced relaxation was only observed in SMA. CCRCs to SNP were not modified in either of the vessels. A23187-induced relaxation was impaired in immunized rats. Following pretreatment with L-arginine, vasorelaxation to acetylcholine and SR 58611A was restored in immunized rats. This study demonstrates that immunization against the second extracellular loop of ß1-ARs has a deleterious impact on vasorelaxations in the TA and SMA of rats, involving alterations in endothelium-dependent NO signaling pathways.

Gastrointestinal hemodynamics in dogs with nonfood induced atopic dermatitis.

BACKGROUND: Canine atopic dermatitis can be a result of exposure to aeroallergens or trophallergens. Hemodynamic alterations occur in dogs with food hypersensitivity. HYPOTHESIS/OBJECTIVES: To evaluate if hemodynamic alterations occur in dogs with NFICAD with lowered resistance to diastolic flow at fasting, after feeding, or both. ANIMALS: Ten healthy dogs and 22 dogs with NFICAD were included from the hospital population. METHODS: Blinded prospective study. Peak systolic velocity (PSV), end diastolic velocity (EDV), mean velocity (MV), pulsatility index (PI), resistive index (RI) and PSV/EDV ratio were measured at fasting for both arteries (cranial mesenteric artery [CMA], celiac artery [CA]) and at 40 minutes after feeding in CMA and at 60 minutes in CA. The results were analyzed statistically with a mixed model. RESULTS: There was no difference detected between groups of dogs for any variable except EDV during fasting (P = .01). CONCLUSIONS AND CLINICAL IMPORTANCE: There is no decrease in resistance in NFICAD to diastolic flow. This observation could be explained by the absence intestinal inflammation in NFICAD.

[Antibodies against the second extracellular loop of beta1-adrenergic receptor induce aortic endothelial dysfunction in Wistar rat]. / Les anticorps dirigés contre la deuxième boucle extracellulaire des récepteurs adrénergiques ß(1) induisent une dysfonction endothéliale dans l'aorte de rat Wistar.

PURPOSE: To evaluate the effect of active immunization with a peptide corresponding to the second extracellular loop of the human beta1-adrenoceptors (ß(1)-AR) on the reactivity of Wistar rat isolated aorta. METHODS: Nine-week-old Wistar rats were actively immunized for 3months with a peptide corresponding to the second extracellular loop of the human ß(1)-AR. Specific immunoglobulins G (IgG) were characterized by Elisa and the bicinchonic acid protein assay and their functionality were tested in isolated ventricular cardiomyocytes (IVC) from control rats. Aortic rings isolated from control or immunized rats were mounted in organ baths. Then, contractile curves to phenylephrine (1nM to 300µM) and relaxant curves to acetylcholine (1nM to 100µM) and isoprenaline (1nM to 30µM) were established. RESULTS: Cell shortening increased dose-dependently in rat IVC superfused with IgG containing ß(1)-AR antibodies (10 or 25µg/mL). Isoprenaline-induced positive inotropy was strongly reduced in IgG containing ß(1)-AR antibodies preincubated (3h) IVC. Phenylephrine-and acetylcholine-induced aortic responses were greatly inhibited in immunized rats compared to control ones. However, active immunization did not influence the isoprenaline-mediated relaxation. CONCLUSIONS: The present work confirms that ß(1)-AR antibodies directed against the second extracellular loop of ß(1)-AR induce a positive inotropic effect in adult rat IVC. Moreover, we demonstrated, for the first time, that 3-month immunization with ß(1)-AR peptide was associated with altered aortic endothelial function without change in the ß-AR-mediated vasorelaxation.

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.

In vitro comparison of myometrial contractility induced by aglepristone-oxytocin and aglepristone-PGF2alpha combinations at different stages of the estrus cycle in the bitch.

The aim of this in vitro study was to compare the uterokinetic activity of oxytocin and dinoprost, the natural PGF2α, with or without aglepristone, in canine myometrial fibers. Thirty-three bitches were allocated into one of four groups, depending on their estrous stage and whether or not they had received a treatment with aglepristone (metestrus aglepristone, n = 5; metestrus without treatment, n = 9; anestrus aglepristone, n = 9; anestrus without treatment, n = 10). After hysterectomy, longitudinal and circular uterine strips were mounted in organ baths. Oxytocin or PGF2α (10 nmol/l to 10 micromol/l) were applied non-cumulatively. A linear mixed effects models theory was used to compare the fiber effect, the aglepristone effect, and the treatment effect, from the area under the curves calculated from the contractile effect/concentration curves for each drug. Oxytocin and PGF2α induced concentration-dependent myometrial contractions in longitudinal (LF) and circular myometrial fibers (CF), indicating the presence of functional contractile oxytocin- and PGF2α-receptors in metestrus and anestrus. The contractile response to oxytocin was greater in LF than in CF in all of the groups; the response to PGF2α was greater in LF than in CF in non-treated bitches in anestrus and in treated bitches in metestrus. These results suggest that there is a difference in sensitivity or a heterogeneous distribution of oxytocin and PGF2α-receptors in the myometrial layers, which is independent of hormonal impregnation. The contractile response to oxytocin and PGF2α was significantly increased after aglepristone treatment in LF during metestrus, suggesting that the progesterone withdrawal induced by aglepristone has a role to play. The longitudinal myometrial layer also appeared to be the target for the two drugs at this stage. This study provides new information about canine uterine contractile activity, notably the differing behavior of myometrial CF and LF; in vivo studies are required to test the use of a combination of aglepristone and oxytocin in the treatment of canine pyometra.

BIS response to tamponade and dobutamine in swine varies with hypnotic/opiate ratio.

OBJECTIVES: This study in swine assessed BIS stability in response to decreases and increases in cardiac output under two propofol/remifentanil dosage combinations, both producing the same depth of surgical anaesthesia. METHODS: Eight anaesthetized-paralyzed ventilated adult swine were studied using a random-order cross-over design. Four received a P low/R high combination (P, 8.4+/-0.9 mg/kg/h; and R, 0.54+/-0.02 microg/kg/min) and then a P high/R low combination (P, 26.7+/-2.1mg/kg/h; and R, 0.34+/-0.01 microg/kg/min). The other four had these two combinations in the reverse order. Under each P/R combination, and after a 60-minutes steady state, a 15-minute stable cardiac tamponade was induced by intrapericardial gelatine infusion. Then, after returning to pre tamponade condition, a 15 minutes period with dobutamine was allowed. RESULTS: Tamponade induced falls in average mean arterial pressure (MAP) (from 79+/-18 to 47+/-9 mm Hg; p<0.05) and cardiac output (Qc) (from 1.90+/-0.46 l/min to 1.20+/-0.38 l/min, p<0.05). Conversely, dobutamine increased both MAP and Qc (p<0.05). During each type of hemodynamic challenges, changes in anaesthesia depth as assessed by BIS differed dramatically between the two drug combinations, despite observing the same percent change in P and R effect-site concentration. With P high/R low and tamponade, BIS fell from 65+/-5 to 29+/-10 (p<0.05); dobutamine produced opposite effects. With P low/R high, in contrast, BIS was not influenced by either of the hemodynamic challenges. CONCLUSION: Conversely to a high propofol/low remifentanil combination, a low propofol/high remifentanil combination allows constant anaesthetic depth in the face of haemodynamic challenges.

Effect of short-term treatment with meloxicam and pimobendan on the renal function in healthy beagle dogs.

The aim of the study was to investigate the renal function in clinically normal dogs receiving meloxicam and pimobendan alone or in combination. Ten adult female beagle dogs were administered the treatment for 7 days in a randomized crossover trial (control/meloxicam/pimobendan/meloxicam and pimobendan). Renal function was assessed by blood urea, creatinine, sodium, potassium and chloride concentrations and by glomerular filtration rate, measured by means of renal scintigraphy [renal uptake of (99m)Tc-diethylenetriaminepentacetic acid (DTPA)] and plasma clearance of (99m)Tc-DTPA. As compared with the control group, renal uptake and plasma clearance of (99m)Tc-DTPA were not significantly modified after a 7-day period of treatment with meloxicam or pimobendan alone, or meloxicam and pimobendan in combination. Furthermore, urea, creatinine, sodium, potassium and chloride levels in the serum of the dogs during the 7-day period treatment were not significantly modified in relation to the treatments. It was therefore concluded that meloxicam and pimobendan alone or in combination did not alter renal function in healthy dogs.

Effect of tepoxalin on renal function in healthy dogs receiving an angiotensin-converting enzyme inhibitor.

The objective of this study was to investigate renal function in clinically normal dogs receiving tepoxalin, a nonsteroidal inflammatory drug, either in association with or without an angiotensin-converting enzyme inhibitor (ACEI). Ten adult female Beagle dogs were used in the three phases of the study. The dogs were administered the drugs once daily for 7 days (experiment 1: placebo/tepoxalin/tepoxalin and benazepril; experiment 2: enalapril/tepoxalin and enalapril) or for 28 days (experiment 3: tepoxalin and benazepril together). Renal function was assessed by measurement of glomerular filtration rate (GFR) by renal scintigraphy [(renal uptake of 99mTc-diethylenetriaminepentacetic acid (DTPA)] and plasma clearance of 99mTc-DTPA. Compared with the placebo group, renal uptake and plasma clearance of 99mTc-DTPA were not significantly modified after a 7-day period of treatment with tepoxalin or enalapril alone, tepoxalin and benazepril or tepoxalin and enalapril together. No significant change was obtained in GFR after a 28-day period of dosing with tepoxalin and benazepril together. Therefore, it was concluded that tepoxalin did not alter renal function in healthy Beagle dogs receiving ACEI.