Autoantibodies against cardiac β(1)-adrenoceptor do not affect the low-affinity state β(1)-adrenoceptor-mediated inotropy in rat cardiomyocytes

Circulating autoantibodies directed against the 2nd extracellular loop (EL-2) of β(1)-adrenoceptors (β(1)-AABs) have been detected in the serum of patients with various cardiovascular pathologies. β(1)-AABs induce agonistic, positive inotropic effects via β(1)-adrenoceptors (β(1)ARs). In the mammalian heart, β(1)-AR can exist in 2 distinct activated configurations (the so-called high- and low-affinity states). The aim of the present study was to investigate whether the action of β(1)-AAB is dependent on the affinity state of β(1)AR in isolated ventricular cardiomyocytes of adult Wistar rats. Immunoglobulin G (IgG) containing β(1)-AAB obtained from animals immunized with a peptide corresponding to the EL-2 of human β(1)-AR, caused a dose-dependent increase in cell shortening. Isoproterenol-induced inotropy was significantly reduced in cardiomyocytes that had been preincubated with IgG containing β(1)-AAB and in cardiomyocytes isolated from immunized rats. The negative effects of preincubation with IgG containing β(1)-AAB on the response to isoproterenol was inhibited in the presence of bisoprolol. CGP 12177A and pindolol-induced inotropy was not affected by IgG preincubation or immunization. No detectable inotropic effect of cell shortening was obtained with IgG containing β(1)-AAB in the presence of propranolol and 3-isobutyl-1-methylxanthine. The present study demonstrates that β(1)-AABs have no agonist/antagonist-like effects upon low-affinity state β(1)-ARs. This result indicates that β(1)-AABs recognize and stabilize the high-affinity state, but are unable to stabilize and (or) induce the low-affinity state receptor

Positive influence of AT(1) receptor antagonism upon the impaired celiprolol-induced vasodilatation in aorta from spontaneously hypertensive rats

We evaluated celiprolol-induced vasodilatation in aorta taken from 12-week-old spontaneously hypertensive rats (SHR) and the effect of AT(1) angiotensin II receptor antagonism on the vasodilatory action of celiprolol in Wistar Kyoto (WKY) rats and SHR. In WKY rats, the celiprolol-induced relaxation was greatly decreased in denuded aorta, and completely abolished in intact aorta by Nω-nitro-l-arginine methyl ester (⌊-NAME, 100 microM). In SHR, celiprolol-induced relaxation was reduced compared to WKY rats (Emax (value obtained for the highest concentration, 300 µM)=39.1+ or - 3.78%, n=21 vs. 80.4 + or - 3% in WKY rats, n=10; P<0.0001). Endothelium removal or pre-treatment with ⌊-NAME did not alter celiprolol-induced relaxation in SHR. In both strains, relaxation to celiprolol was decreased in the presence of nadolol (a β1/β2-adrenoceptor antagonist, 10 µM). N-[[3-[(2S)-2-hydroxy-3-[[2-[4-[(phenylsulfonyl)amino] phenyl]ethyl]amino] propoxy]phenyl]methyl]-acetamide (L748337, a β3-adrenoceptor antagonist, 7 µM) had no effect. A 12-day treatment with candesartan cilexetil (an AT(1) receptor antagonist, 0.37 or 1mg/kg/day) reduced systolic blood pressure in both strains, but only improved relaxation to celiprolol in SHR, and only at the highest dose (Emax=64.2+/-3.9%, n=10, P<0.0001 vs. SHR control). In both strains, local aortic AT1 receptor antagonism with candesartan CV11974 (100 µM) had no effect. The endothelial β1/β2 relaxation induced by celiprolol was therefore impaired in SHR aorta and AT1 receptor antagonism improved the response to celiprolol, in conjunction with a reduction in blood pressure. This work highlights the need to analyse the potential benefit of a combination of celiprolol/AT1 receptor antagonist in the treatment of hypertension

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

Antibodies against the second extracellular loop of β1-adrenergic receptors induce endothelial dysfunction in conductance and resistance arteries of the Wistar rat

Autoantibodies against β1-adrenoceptors (β1-ARs) have been detected in the serum of patients with various cardiac diseases; however, the pathological impact of these autoantibodies (β1-AABs) has only been evaluated in cardiac tissue. The purpose of the present study was to evaluate whether β1-AABs have deleterious effects on vascular reactivity in rats. An enzyme-linked immunosorbent assay was used to detect β1-AABs in sera from immunized rats over a period of 1–3 months using the peptidic sequence of the second extracellular loop of human β1-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 β-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 β1-ARs has a deleterious impact on vasorelaxations in the TA and SMA of rats, involving alterations in endothelium-dependent NO signaling pathways

Nonlinear mixed effects models applied to cumulative concentration-response curves

Objectives In experimental pharmacology, drug effect studies currently establish and analyse cumulative concentration–response curves (CCRC) under repeated measurements designs. Usually the CCRC parameters are estimated using the Hill\u27s function in a nonlinear regression for independent data. The two-way analysis of variance is generally used to identify a statistical difference between the responses for two treatments but that analysis does not take into account the nonlinearity of the model and the heteroscedasticity (uneven distribution) of the data. We presently tested the possibility of finding a statistical solution for the nonlinear response in repeated measurements data using the nonlinear mixed effects (nlme) models. Methods Experimental data sets, originating from studies on β-adrenoceptor-induced relaxation in rat thoracic aorta ring, were analysed using the nlme methods. Key findings Comparison with classical methods showed the superiority of the nlme models approach. For each pharmacological parameter (Em, n, pD2), a point estimate, a standard error and a confidence interval are returned by the nlme procedures respecting the assumption of independency and normality of the residuals. Conclusions Using the method presently described, it is now possible to detect significant differences for each pharmacological parameter estimated in different situations, even for designs with small samples size (i.e. at least six complete curves)

Effects of long-term active immunization with the second extracellular loop of human β1- or β3-adrenoceptors in thoracic aorta and mesenteric arteries in Lewis rats

Objective To evaluate whether active immunization producing β1- or β3-antibodies (β1-ABs and β3-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 6 months with peptidic sequences corresponding to the second extracellular loop of β1- and β3-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 β-AR agonists, phenylephrine and KCl. Results The immunizations producing functional β1-ABs and β3-ABs did not affect the SBP. However, in TA from β1-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 β3-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 β1- or β3-peptides, relaxations induced by the various β-agonists were not changed whereas phenylephrine and KCl-mediated contractions were impaired. Conclusions Our study shows that β1- and β3-ABs can affect vascular reactivity. β1-ABs would have a pathogenic action whereas β3-ABs would have a beneficial effect on aorta reactivity. Array ( [0] => public://js/js_NhB8QqEMkIRnGegV_fyHSoTNS4QcuYAxmtYDZC610gE.js.gz : fichier présent sur le disque mais absent dans la base de données [1] => public://js/js_YqvqIXMHR_JA_6L7V5VgwgrhCDVtmWC_wCWsaINFQtk.js : fichier présent sur le disque mais absent dans la base de données [2] => public://js/js_YqvqIXMHR_JA_6L7V5VgwgrhCDVtmWC_wCWsaINFQtk.js.gz : fichier présent sur le disque mais absent dans la base de données [3] => public://js/js_NhB8QqEMkIRnGegV_fyHSoTNS4QcuYAxmtYDZC610gE.js : fichier présent sur le disque mais absent dans la base de données