Figure 1.
Knockout of β2-AR prevent increase of phenylephrine contractile response induced by isoproterenol in aorta.
7-day isoproterenol treatment (ISO) increased the vasoconstrictor response to phenylephrine in isolated thoracic aorta from wild-type (WT) (A) and β1-KO (B) mice. This effect was abolished in β2-KO mice (C). The contraction response is expressed as a % of the contraction to KCl (125 mM). The number of animals used in each group is indicated in parenthesis. Values are presented as the mean ± SEM. Significance was assessed with a 2-way ANOVA: ***p<0.0001 vs. WT; +++p<0.0001 vs. β1KO.
Figure 2.
Role of superoxide anion and NOS in the vascular effect of isoproterenol treatment.
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 vs. WT E+; +p<0.05, ++p<0.01 vs. β1KO E+; #p<0.05 vs. β2KO E+.
Figure 3.
Aortic β-AR subtypes expression.
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.
Figure 4.
Isoproterenol treatment induces β2-AR-Gi-ERK1/2 pathway activation and eNOS uncoupling.
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 vs. WT.
Figure 5.
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.
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 vs. WT ISO; *p<0.05 vs. WT.
Figure 6.
Giα protein activity mediates the vascular oxidative stress induced by isoproterenol.
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 vs. respective basal values for each group; #p<0.05 vs. basal WT value; +p<0.05 vs. ISO-treated WT value.