Effect of nebivolol treatment during pregnancy on the intrauterine fetal growth, mortality and pup postnatal development in the l-NAME-induced hypertensive rats.

The present study was carried out to evaluate the effect of nebivolol vs. bisoprolol treatment on the intrauterine fetal growth, mortality and postnatal development in Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME)-induced hypertensive rats. Hypertension was induced in normotensive pregnant Wistar rats by daily administration of l-NAME (100mg/kg/day, in the drinking water) for the period of pregnancy. After 9 days of l-NAME treatment, rats with systolic and diastolic blood pressure (SBP and DBP) more than 140/90mmHg were considered hypertensive. Then, some of them were treated from day 11 to day 18 of pregnancy with nebivolol (8mg/kg/day) or bisoprolol (10mg/kg/day) via oral gavage. SBP, DBP and heart rate (HR) were re-evaluated by tail cuff method on day 19 of pregnancy and morphometrical or histological studies were performed on day 20. In addition, the mortality and postnatal development of newborn pups were assessed in all groups. The l-NAME administration during pregnancy induced an increase in SBP and DBP while HR did not change. Nebivolol or bisoprolol treatment completely prevented the elevation of SBP and DBP induced by l-NAME with a reduction in HR in pregnant and non-pregnant rats. The intra-uterine fetal growth and the postnatal development of newborn rats in nebivolol-treated hypertensive group were significantly lower vs. control and higher vs. bisoprolol-treated group with a higher mortality in the both types of treatments vs. control rats. The nebivolol and bisoprolol administration produce adverse effects on fetal growth and postnatal development, that limits their therapeutic use in females during pregnancy.

Equine digital veins are more sensitive to superoxide anions than digital arteries.

This work was designed to investigate (i) the effect of superoxide dismutase (SOD) inhibition on endothelial function and (ii) the free radical-induced endothelial dysfunction in equine digital veins (EDVs) and equine digital arteries (EDAs) isolated from healthy horses. EDV and EDA rings were suspended in a 5 ml organ bath containing Krebs solution. After a 60 min equilibration period, EDV and EDA rings were contracted with phenylephrine. Then, cumulative concentration-response curves (CCRCs) to acetylcholine were performed. In both EDVs and EDAs, acetylcholine (1 nM to 10 µM) produced concentration-dependent relaxation. We investigated the influence of SOD inhibition by diethyldithiocarbamate (DETC; 100 µM), a CuZnSOD inhibitor, on EDAs and EDVs relaxant responses to acetylcholine. Acetylcholine -mediated relaxation was impaired by DETC only in EDVs. SOD activity assayed by a xanthine-xanthine oxidase method was higher in EDAs compared with EDVs (P<0.05). CCRCs to acetylcholine established in the presence of pyrogallol (30 µM) or homocysteine (20 µM), two superoxide anions generating systems showed that in both EDVs and EDAs, the acetylcholine-mediated relaxation was significantly impaired by pyrogallol and homocysteine. This impairment was more pronounced in EDVs than in EDAs. Moreover, the pyrogallol-induced impairment of acetylcholine-mediated relaxation was potentiated by DETC to a greater extent in EDVs. We concluded that due to the lower activity of SOD, EDVs are more sensitive to superoxide anions than EDAs. So, any alteration of superoxide anions metabolism is likely to have a more important impact on venous rather than arterial relaxation.

Celiprolol induces ß(3)-adrenoceptors-dependent relaxation in isolated porcine coronary arteries.

In porcine coronary arteries (PCAs), celiprolol, a selective ß(1)-adrenoceptors antagonist, induces vasodilatation by an endothelium- and nitric oxide (NO)-dependent pathway. However, the mechanisms of that vascular effect have not been precisely established. ß(3)-Adrenoceptors have been shown to be involved in the relaxation per se of various vascular beds, including coronary vessels. Thus, we evaluated (i) the presence of ß(3)-adrenoceptors in the PCA and (ii) their role in celiprolol-induced vasodilatation. PCA rings were placed in organ baths and preconstricted with KCl. All experiments were performed in the presence of nadolol (a ß(1)/ß(2)-adrenoceptor antagonist). Cumulative concentration-response curves to SR 58611A and ICI 215001 (2 ß(3)-adrenoceptor agonists) and to celiprolol were constructed. We also used semiquantitative reverse transcription - polymerase chain reaction, which clearly showed the presence of ß(3)-adrenoceptor transcripts. SR 58611A, ICI 215001, and celiprolol induced concentration-dependent relaxations in PCA rings. SR 58611A-induced relaxation was almost abolished after removal of endothelium or pretreatment with L-NAME (a NO synthase inhibitor). The vasorelaxations induced by SR 58611A and celiprolol were inhibited in the presence of SR 59230A and L-748337 (2 selective ß(3)-adrenoceptor antagonists). We showed (i) that PCAs possess functional ß(3)-adrenoceptors mediating endothelium- and NO-dependent relaxation, and (ii) that celiprolol exerts a ß(3)-adrenoceptor agonistic activity in this vascular bed.

Vasorelaxant effect of propentofylline in isolated equine digital veins.

We evaluated the vasorelaxant effect of propentofylline (PPF), a methylxanthine derivative, and its mechanism of action in equine digital veins (EDVs). Cumulative concentration-response curves to PPF (1 nM-300 µM) were recorded in phenylephrine-precontracted EDV rings under different experimental conditions. PPF-induced relaxation was partially inhibited by endothelium removal, but was unaltered by CGS-15943 (an adenosine receptor antagonist; 3 µM). PPF-induced relaxation was partially inhibited in the presence of L-NAME (a nitric oxide (NO) synthase inhibitor; 100 µM), ODQ (an inhibitor of soluble guanylyl cyclase; 30 µM) or Rp-8-Br-PET-cGMP-S (a protein kinase G inhibitor; 3 µM). It was not modified by indomethacin (a non-selective cyclooxygenase (COX) inhibitor; 10 µM), and was slightly potentiated by H-89 (a protein kinase A inhibitor; 2 µM). In endothelium-intact EDVs, PPF-induced relaxation was associated with a 2.4- and 24.1-fold increase in the tissue cGMP and cAMP content respectively. PPF (100 µM) did not shift the concentration-response curve to phenylephrine (1 nM-300 µM) but reduced the maximal effect. To investigate whether PPF can affect cAMP- and cGMP-induced relaxations, relaxation curves to forskolin (an activator of adenylate cyclase) and to sodium nitroprusside (SNP, a NO donor) were recorded in EDV rings pretreated with PPF (100 µM). PPF only slightly potentiated the forskolin-induced relaxation without affecting the SNP-induced relaxation. We demonstrated that PPF-induced relaxation in EDVs is partially endothelium-dependent. The PPF-induced relaxation partially occurred via NO release and both cAMP and cGMP generation, through COX-independent mechanisms but could also result from the inhibition of cAMP-phosphodiesterase activity for the highest concentrations.

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

Circulating autoantibodies directed against the 2nd extracellular loop (EL-2) of ß(1)-adrenoceptors (ß(1)-AABs) have been detected in the serum of patients with various cardiovascular pathologies. ß(1)-AABs induce agonistic, positive inotropic effects via ß(1)-adrenoceptors (ß(1)ARs). In the mammalian heart, ß(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 ß(1)-AAB is dependent on the affinity state of ß(1)AR in isolated ventricular cardiomyocytes of adult Wistar rats. Immunoglobulin G (IgG) containing ß(1)-AAB obtained from animals immunized with a peptide corresponding to the EL-2 of human ß(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 ß(1)-AAB and in cardiomyocytes isolated from immunized rats. The negative effects of preincubation with IgG containing ß(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 ß(1)-AAB in the presence of propranolol and 3-isobutyl-1-methylxanthine. The present study demonstrates that ß(1)-AABs have no agonist/antagonist-like effects upon low-affinity state ß(1)-ARs. This result indicates that ß(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 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.

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's 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 beta-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 (E(m), n, pD(2)), 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).

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.

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.

Impairment of the low-affinity state beta1-adrenoceptor-induced relaxation in spontaneously hypertensive rats.

1 In hypertension, a decrease of the vascular beta-adrenergic relaxation has been described. However, the specific involvement of each beta-adrenoceptor (beta-AR) subtype, in particular the low-affinity state of beta1-AR, has not yet been evaluated. We investigated whether the low-affinity state of beta1-AR-induced relaxation was impaired in Spontaneously Hypertensive Rats (SHR). 2 The relaxant responses to CGP 12177 and cyanopindolol, low-affinity state beta1-AR agonists (with beta1-/beta2-AR antagonistic and partial beta3-AR agonistic properties) were evaluated on thoracic aortic rings isolated from 12-weeks-old Wistar Kyoto rats (WKY) and SHR. 3 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 beta1-, beta2-AR (nadolol) or beta3-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. 4 In SHR, CGP 12177 produced mainly an endothelium and NO-dependent relaxation. This effect was not modified by nadolol, but was strongly reduced by beta3-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. 5 The immunohistochemical analysis revealed an upregulation of beta3-AR in the endothelial layer of SHR aorta, whereas the beta3-AR-induced relaxation was not modified. 6 In conclusion, we demonstrated an impaired low-affinity state of the beta1-AR-induced relaxation and an upregulation of the beta3-AR in hypertension. Some clinical implications of those findings are discussed.