Inhibition of Giα protein or ERK1/2 activation reversed hypercontractility to phenylephrine induced by β-AR overactivation in aorta of wild-type, but not in β₂KO mice.

<p>Effect of pertussis toxin (PTx, 4 μM) and PD98,059 (1 μM) on the concentration-response curves to phenylephrine in aortic rings of wild-type (WT) (A, D) and β₂KO (B, E) mice treated for 7 days with vehicle or isoproterenol (ISO). The contraction response is expressed as a % of the contraction to KCl (125 mM). Bar graphs show differences in the area under the concentration-response curve (AUC) in the presence or absence of PTx (C) or PD98,059 (F) in WT and β₂KO mice treated or not with ISO. Values are presented as the mean ± SEM. The number of animals used in each group is indicated in parenthesis. Significance was assessed using a 2-way ANOVA: ⁺p<0.05 <i>vs.</i> WT ISO; *p<0.05 <i>vs.</i> WT.</p

Isoproterenol treatment induces β₂-AR-Gi-ERK1/2 pathway activation and eNOS uncoupling.

<p>Protein expression of Giα-1,2 (A), Giα-3 (B), ERK 1/2 phosphorylated at Thr²⁰² and Tyr²⁰⁴ (C), p38 MAPK phosphorylated at Thr¹⁸⁰ and Tyr¹⁸² (D) and eNOS protein dimerization (E) in aortas from control and 7-day isoproterenol-treated (ISO) wild-type (WT) and β₂KO mice. The top panels in each graph represent typical Western-blot autoradiographs. Giα protein expression was normalized to the α-actin content in each sample, and phosphorylated ERK 1/2 and p38 MAPK were normalized to the total content of ERK 1/2 and p38 MAPK, respectively, and these results were expressed as the fold-change compared to WT aorta. eNOS dimerization was expressed as ratio of dimer:monomer band intensity. The number of animals used in each group is indicated in the bars. Values are presented as the mean ± SEM. Significance was assessed using a 2-way ANOVA: *p<0.05 <i>vs.</i> WT.</p

Role of superoxide anion and NOS in the vascular effect of isoproterenol treatment.

<p>Effect of L-NAME (LN, 100 μM) or superoxide dismutase (SOD, 150 U/mL) on the concentration-response curves to phenylephrine of vehicle- (open symbols) or 7-day isoproterenol-treated (ISO, close symbols) aortic rings from wild-type (A, D), β₁KO (B, E) and β₂KO (C, F) mice. The contraction response is expressed as a % of the contraction to KCl (125 mM). Values are presented at the mean ± SEM. E+ =  intact endothelium. The number of animals used in each group is indicated in parenthesis. Significance was assessed using a 2-way ANOVA: *p<0.05, **p<0.01 <i>vs</i>. WT E+; ⁺p<0.05, ⁺⁺p<0.01 <i>vs.</i> β₁KO E+; ^#p<0.05 <i>vs</i>. β₂KO E+.</p

Giα protein activity mediates the vascular oxidative stress induced by isoproterenol.

<p>Panel A shows representative fluorographs of microscopic sections of thoracic aorta from wild-type (WT) and β₂KO mice treated for 7 days with vehicle or isoproterenol (ISO). Vessels were labeled with the oxidative dye hydroethidine, which produces a red fluorescence when oxidized to ethidium bromide. Panel B shows the densitometric analysis of the ethidium-bromide-positive nuclei evaluated under basal conditions or incubated with apocynin (APO, 30 μM), L-NAME (LN, 100 μM), PTx (4 μM) or superoxide dismutase (SOD, 150 U/mL). The fluorescence signal was evaluated as the intensity of fluorescence per pixel normalized by vessel area. Values are presented as the mean ± SEM. N = 4–7 animals in each group. Significance was assessed using a 2-way ANOVA: *p<0.05 <i>vs</i>. respective basal values for each group; ^#p<0.05 <i>vs</i>. basal WT value; ⁺p<0.05 <i>vs</i>. ISO-treated WT value.</p

Aortic β-AR subtypes expression.

<p>Protein expression of β₁- β₂- and β₃-adrenoceptors (AR) evaluated in the membrane fraction of aortas from WT, β₁KO and β₂KO mice treated for 7 days with vehicle or isoproterenol (ISO). (A) Representative Western-blot autoradiographs for each β-AR subtype in membrane preparations of aorta and positive controls (+C: heart for β₁-AR; skeletal muscle for β₂-AR; adipose tissue for β₃-AR). Densitometric quantification was evaluated for β₁- (B) and β₂-AR (C) but not for β₃-AR, as this subtype was not expressed (n.e.) in the mouse aorta. The number of samples analyzed (pool of 3 aortas in each sample) is indicated in the bar for each group. Values (mean ± SEM) are expressed the fold-change in β-AR expression compared to the WT. Significance was assessed using a 2-way ANOVA.</p

Effect of L-NAME (100 μM) on the concentration—response curve of ACh in aortic rings from SHAM, SHAM-TRA, OVX and OVX-TRA: area under the curve (dAUC %).

<p>* p<0.05 OVX and OVX-TRA vs. SHAM and SHAM-TRA;</p><p>^# p<0.05 OVX-TRA vs. OVX. dAUC are expressed as a percentage of the corresponding AUC for L-NAME aortic rings.</p><p>Effect of L-NAME (100 μM) on the concentration—response curve of ACh in aortic rings from SHAM, SHAM-TRA, OVX and OVX-TRA: area under the curve (dAUC %).</p

Representative images of aortic slices from SHAM, SHAM-TRA, OVX and OVX-TRA rats, in the absence (upper panel) and presence (lower panel) of the superoxide dismutase mimetic, MnTMPyP (A). Quantification of fluorescence intensity emitted by DHE oxidation in aortic slices in the absence and presence of the MnTMPyP as an indicative of reactive oxygen species in SHAM, SHAM-TRA, OVX and OVX-TRA rats (B).

<p>The numbers shown in bars represent the number of animals analyzed in each group. Results are expressed as means±SEM. Two-way ANOVA: *p<0.01 in comparison to SHAM, # p<0.01 in comparison to OVX.</p

Concentration—response curve to acetylcholine (A) and sodium nitroprusside (B) in aortic rings from SHAM, SHAM-TRA, OVX and OVX-TRA.

<p>The numbers shown in the legend represent the number of animals analyzed in each group. Results are expressed as means±SEM. Two-way ANOVA: *p<0.01 in comparison to SHAM, # p<0.01 in comparison to OVX.</p

Anthropometric and Hemodynamic data from SHAM, SHAM-TRA, OVX and OVX-TRA rats.

<p>* p<0.05 OVX and OVX-TRA vs. SHAM and SHAM-TRA;</p><p>^# p<0.05 OVX-TRA vs. OVX,</p><p>^$ p<0.05 OVX-TRA vs. SHAM.</p><p>Anthropometric and Hemodynamic data from SHAM, SHAM-TRA, OVX and OVX-TRA rats.</p

Effect of L-NAME (100 μM) on the concentration—response curve of ACh in aortic rings from SHAM vs SHAM-TRA (A) and OVX vs OVX-TRA (B).

<p>The numbers shown in the graphs represent the number of animals analyzed in each group. Results are expressed as means±SEM. Two-way ANOVA: & p<0.01 in comparison to control; *p<0.01 in comparison to SHAM, # p<0.01 in comparison to OVX.</p