Marc Gogny

Impairment of the low-affinity state β1-adrenoceptor-induced relaxation in spontaneously hypertensive rats

In hypertension, a decrease of the vascular β‐adrenergic relaxation has been described. However, the specific involvement of each β‐adrenoceptor (β‐AR) subtype, in particular the low‐affinity state of β1‐AR, has not yet been evaluated. We investigated whether the low‐affinity state of β1‐AR‐induced relaxation was impaired in Spontaneously Hypertensive Rats (SHR). The relaxant responses to CGP 12177 and cyanopindolol, low‐affinity state β1‐AR agonists (with β1‐/β2‐AR antagonistic and partial β3‐AR agonistic properties) were evaluated on thoracic aortic rings isolated from 12‐weeks‐old Wistar Kyoto rats (WKY) and SHR. In WKY, CGP 12177 and cyanopindolol produced an endothelium and nitric oxide (NO)‐independent relaxation. CGP 12177‐induced endothelium‐independent relaxation was not modified either by β1‐, β2‐AR (nadolol) or β3‐AR (L‐748337 or SR 59230A) antagonists but was significantly reduced by high concentrations of CGP 20712A (P<0.05). This relaxation was also reduced by adenylyl cyclase inhibitors, SQ 22536 or MDL 12330A. In SHR, CGP 12177 produced mainly an endothelium and NO‐dependent relaxation. This effect was not modified by nadolol, but was strongly reduced by β3‐AR blockade. Endothelium‐independent relaxation to CGP 12177 was not altered by adenylyl cyclase inhibition, but was amplified in preparations from pertussis toxin‐pretreated SHR. The immunohistochemical analysis revealed an upregulation of β3‐AR in the endothelial layer of SHR aorta, whereas the β3‐AR‐induced relaxation was not modified. In conclusion, we demonstrated an impaired low‐affinity state of the β1‐AR‐induced relaxation and an upregulation of the β3‐AR in hypertension. Some clinical implications of those findings are discussed.

β-Adrenoceptor-mediated vascular relaxation in spontaneously hypertensive rats

Abstract Although the impairment of beta-adrenoceptor (β-AR)-induced vascular relaxation to isoprenaline has been extensively described, discrepancy persisted in the literature. In this work, we investigated β-AR-induced relaxation in spontaneously hypertensive and normotensive rats aorta. We attempted to determine β-AR subtypes involved in order to understand the conflicting data regarding the β-AR-induced vasodilation to isoprenaline. Aortic rings isolated from 12-week-old Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were placed in organ baths and constricted with phenylephrine (α 1 -AR agonist). Then, cumulative concentration–relaxation curves (CCRC) to AR agonists were constructed. In intact aortic rings from both strains, isoprenaline (a nonselective β-AR agonist) (0.001–10 μM) induced similar concentration-dependent relaxations. CCRC was shifted to the right and upward in the presence of nadolol (a nonspecific β 1 and β 2 -AR antagonist) (10 μM). After endothelium removal, the response to isoprenaline was partly inhibited in WKY rats, but was strongly inhibited in SHRs. In WKY rats, isoprenaline-induced endothelium-independent relaxation was not modified in the presence of nadolol but was inhibited in the presence of CGP 20712A (low-affinity-state β 1 -AR antagonist). In endothelium-denuded rings, SR 58611A (a preferential β 3 -AR agonist) (0.1–30 μM) produced a very small relaxation in both strains. In WKY rats, CGP 12177 (CGP) (0.1–30 μM) and cyanopindolol (0.01–3 μM) (partial β 3 -AR and low-affinity-state β 1 -AR agonists with β 1 -AR and β 2 -AR antagonistic properties) produced endothelium-independent relaxations. CGP-induced effect was significantly inhibited by CGP 20712A (10 μM) or bupranolol (10 μM) (low-affinity-state β 1 -AR antagonists). In SHRs, similarly to the impaired endothelium-independent relaxation to isoprenaline, endothelium-independent relaxations to CGP and cyanopindolol were greatly blunted. These relaxations were not modified in the presence of CGP 20712A. In endothelium-denuded rings pretreated with pertussis toxin, CGP-induced relaxation was not modified in WKY rats, but was partly restored in SHRs. In conclusion, these results showed, that in 12-week-old SHRs, the endothelium-independent component of the relaxation to isoprenaline was impaired, and this impairment could involve the low-affinity-state β 1 -AR. G i protein overexpression and/or overstimulation may be possible factors that contribute to this alteration in hypertension.

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 Hills 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.

Positive influence of AT1 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 N(omega)-nitro-l-arginine methyl ester (l-NAME, 100 microM). In SHR, celiprolol-induced relaxation was reduced compared to WKY rats (E(max) (value obtained for the highest concentration, 300 microM)=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 l-NAME did not alter celiprolol-induced relaxation in SHR. In both strains, relaxation to celiprolol was decreased in the presence of nadolol (a beta(1)/beta(2)-adrenoceptor antagonist, 10 microM). N-[[3-[(2S)-2-hydroxy-3-[[2-[4-[(phenylsulfonyl)amino] phenyl]ethyl]amino] propoxy]phenyl]methyl]-acetamide (L748337, a beta(3)-adrenoceptor antagonist, 7 microM) 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 (E(max)=64.2+/-3.9%, n=10, P<0.0001 vs. SHR control). In both strains, local aortic AT(1) receptor antagonism with candesartan CV11974 (100 microM) had no effect. The endothelial beta(1)/beta(2) relaxation induced by celiprolol was therefore impaired in SHR aorta and AT(1) 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/AT(1) receptor antagonist in the treatment of hypertension.

Evaluation of the role of superoxide anions in endotoxin-induced impairment of β-adrenoceptor-mediated vasodilation in equine digital veins.

OBJECTIVE To investigate the role of superoxide anions in the lipopolysaccharide (LPS)-induced impairment of beta-adrenoceptor-mediated equine digital vein (EDV) vasodilation. SAMPLE POPULATION EDVs isolated from forelimbs of 24 healthy adult horses. PROCEDURES Endothelium-intact or endothelium-denuded EDV rings were incubated with or without LPS (10 microg/mL) of Escherichia coli (O55:B5) for 4 hours. Cumulative concentration-relaxation curves resulting from administration of isoprenaline, a nonselective beta-adrenoceptor agonist, or from administration of SR 58611A, a selective beta(3)-adrenoceptor agonist, were recorded in phenylephrine-preconstricted EDVs in the absence or the presence of superoxide dismutase (200 U/mL). Isoprenaline-induced relaxation was also evaluated with or without the cyclooxygenase inhibitors indomethacin (10 microM) and NS-398 (10 microM). RESULTS Isoprenaline and SR 58611A induced concentration-dependent relaxation of EDV rings, which was inhibited by LPS exposure. Superoxide dismutase abolished the inhibitory effect of LPS on the isoprenaline- and SR 58611A-mediated relaxation. Pretreatment of the LPS-treated EDVs with indomethacin or NS-398 restored the isoprenaline-mediated relaxation and abolished the LPS-induced impairment to a similar extent as superoxide dismutase. CONCLUSIONS AND CLINICAL RELEVANCE Results supported a role of superoxide anions in the LPS-induced impairment of beta-adrenoceptor-mediated EDV vasodilation. The LPS-induced oxidative stress in EDVs may contribute to vascular dysfunctions associated with laminitis in horses.

Low-affinity state beta1-adrenoceptor-induced vasodilation in SHR

Low-affinity state beta1-adrenoceptor (beta1-AR) was functionally expressed in some blood vessels and was different from beta1, beta2 and beta3-AR. In rat aorta, low-affinity state beta1-AR activation produced an endothelium-independent relaxation which was impaired in spontaneously hypertensive rats (SHRs). In the present work, we investigated whether renin-angiotensin system was involved in this alteration by evaluating the effects of enalapril, an angiotensin converting enzyme (ACE) inhibitor or losartan, an AT1 angiotensin receptor antagonist. Cumulative concentration-response curves to low-affinity state beta1-AR agonists (CGP 12177, cyanopindolol or alprenolol) and to NS 1619, a large conductance Ca2+-activated K+ channels (BK) agonist were performed in denuded aortic rings isolated from control or treated Wistar Kyoto (WKY) rats or SHRs in different experimental conditions. The low-affinity state beta1-AR-mediated aortic vasodilation was impaired in 5 and 12 weeks old SHRs when compared to age-matched WKY. Twelve days enalapril (5 mg/kg/day) or losartan (15 mg/kg/day) treatments reduced systolic blood pressure (SBP) only in 12 weeks old SHRs whereas no significant change was observed in other groups. These treatments improved low-affinity state beta1-AR effect only in SHRs groups. In 12 weeks old WKY rats, CGP 12177-induced relaxation was insensitive to glibenclamide, a K(ATP)+ channel blocker, but was reduced by TEA or iberiotoxin, two large conductance Ca2+-activated K+ channel (BK) blockers. The impairment of NS 1619-induced vasodilation in both 5 and 12 weeks old SHRs was restored by enalapril or losartan. These results suggested that improvement of the low-affinity state beta1-AR-mediated vasodilation in 5 and 12 weeks old SHRs could be attributed to enhanced BK channels-induced hyperpolarization in SHRs independently of lowering of SBP.

Effect of moderate cooling on contractile responses in mouse vas deferens and its relation to calcium

Isolated mouse vas deferens preparations were used to study the effect of temperature on noradrenaline-induced contractions. Preparations were suspended in the organ bath containing Krebs-Henseleit solution for isometric tension recording. Contractile responses to noradrenaline were investigated in the mouse vas deferens after moderate cooling from 37 to 26 or 22° C. A significant increase of the phasic contractions to noradrenaline was observed at 26 or 22°C compared with responses obtained at 37° C (about 12.3 and 35.6% increase at 26 and 22° C, respectively). The secondary noradrenaline-induced sustained contraction was also significantly enhanced after moderate cooling to 26° C. The potentiation of noradrenaline-induced contraction at 26° C remained in a Ca2+-free EGTA (1 mM)-containing solution. However, sustained contraction was suppressed after removal of the calcium from the medium at 37 and 26°C. Contraction to caffeine was significantly enhanced at 22° C compared with 37°C. By contrast, barium chloride-induced contraction of the vas deferens was markedly decreased after moderate cooling to 22° C. In the presence of ouabain (0.1 mM), the noradrenaline-induced peak contraction was significantly increased at 37°C. However, potentiation of the noradrenaline response at 22° C was unaffected by the Na+/K+ pump inhibitor. Noradrenaline-induced peak contractions were depressed in the presence of vanadate (1 mM) and cyclopiazonic acid (10 μM), two Ca2+-ATPase inhibitors, at 37° C and also at 22° C. These results suggest that temperature-induced hyperreactivity is partly due to an increase of the amount of calcium released from intracellular stores. The inhibition of the Na+/K+ pump due to cooling may participate in this effect whereas Ca2+-ATPase inhibition does not seem to be involved.

Effect of temperature reduction on the reactivity of the mouse vas deferens to adrenergic drugs.

1. Dose-response curves for noradrenaline, phenylephrine and clonidine were determined isometrically on the mouse vas deferens at 26 degrees C, 15 degrees C and compared to the one obtained at 37 degrees C. 2. In the presence of noradrenaline, reducing temperature induced an increase of both maximal developed tension and sensitivity to the drug. Reduction by 50% of the extracellular calcium concentration abolished the maximal contraction potentiation. 3. When reducing temperature to 26 degrees C, the maximal contraction was increased and depressed in the presence of phenylephrine and clonidine respectively. 4. The results suggest (a) that cooling increases the reactivity of mouse vas deferens by activation of alpha 1 adrenoceptors and depresses it by activation of alpha 2 adrenoceptors (b) that calcium ions could play an important role in the potentiation of the maximal contraction.

CGP12177-induced haemodynamic and vascular effects in normotensive and hypertensive rats.

CGP12177 is a non-conventional partial agonist, known to have cardiostimulating and vasorelaxant properties related to its agonist action on the low affinity state of the beta(1)-adrenoceptor (beta(1LA)-adrenoceptor). In normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR), CGP12177-induced vasorelaxant effects were analysed in hindquarter vessels to assess modifications in hind limb vascular resistance, and in femoral artery rings. The global haemodynamic effects induced by CGP12177 were also investigated using telemetry in conscious animals. In hindquarters vasculature precontracted with 5-hydroxytryptamine, CGP12177 (0.16 to 475 microg) produced a similar dose-dependent decrease in hindquarters perfusion pressure in both strains. Vasorelaxation was not modified by nadolol, a beta(1) and beta(2)-adrenoreceptor antagonist, nor by L748337, a beta(3)-adrenoceptor antagonist, but was concentration dependently inhibited by bupranolol, a beta(1LA)-adrenoceptor antagonist at high concentrations. In femoral artery rings from WKY rats and SHR, CGP12177 produced a concentration-dependent relaxation, which was unaffected by nitric oxide synthases inhibition but was significantly reduced in the presence of bupranolol. With double cardiac autonomic blockade (atropine plus atenolol) in conscious WKY rats and SHR, CGP12177 greatly increased heart rate with minor changes in mean arterial pressure in both strains. Conversely, in the absence of double cardiac autonomic blockade, the amplitude of CGP12177-induced heart rate increase was less pronounced and had an hypotensive effect. The reduction in tachycardia and the hypotension were significantly greater in SHR compared to WKY rats. In conclusion, in both strains, CGP12177 produced vasodilating effects in hindquarter vessels and femoral arteries that can be attributed to a beta(1LA)-adrenoceptor stimulation. In conscious WKY rats and SHR, CGP12177-induced cardiostimulation and hypotension were not significantly different after baroreflex blockade, but were decreased and increased respectively, in the presence of baroreflex activity.

Loss of nitric oxide release in passively sensitized guinea-pig aorta with purified immunoglobulin G1

Background Vascular hyperresponsiveness can be reproduced by in vitro passive sensitization of isolated aorta with immunoglobulin G1 (IgGl) taken from ovalbumen‐sensitized BFA guinea‐pig.