Increased Vascular Contractility and Oxidative Stress in β₂-Adrenoceptor Knockout Mice: The Role of NADPH Oxidase

Abstract

Background/Aims: β₂-adrenoceptor (β₂-AR) activation induces smooth muscle relaxation and endothelium-derived nitric oxide (NO) release. However, whether endogenous basal β₂-AR activity controls vascular redox status and NO bioavailability is unclear. Thus, we aimed to evaluate vascular reactivity in mice lacking functional β₂-AR (β₂KO), focusing on the role of NO and superoxide anion. Methods and Results: Isolated thoracic aortas from β₂KO and wild-type mice (WT) were studied. β₂KO aortas exhibited an enhanced contractile response to phenylephrine compared to WT. Endothelial removal and L-NAME incubation increased phenylephrine-induced contraction, abolishing the differences between β₂KO and WT mice. Basal NO availability was reduced in aortas from β₂KO mice. Incubation of β₂KO aortas with superoxide dismutase or NADPH inhibitor apocynin restored the enhanced contractile response to phenylephrine to WT levels. β₂KO aortas exhibited oxidative stress detected by enhanced dihydroethidium fluorescence, which was normalized by apocynin. Protein expression of eNOS was reduced, while p47^phox expression was enhanced in β₂KO aortas. Conclusions: The present results demonstrate for the first time that enhanced NADPH-derived superoxide anion production is associated with reduced NO bioavailability in aortas of β₂KO mice. This study extends the knowledge of the relevance of the endogenous activity of β₂-AR to the maintenance of the vascular physiology.

Isoproterenol Induces Vascular Oxidative Stress and Endothelial Dysfunction via a Giα-Coupled β2-Adrenoceptor Signaling Pathway

“…Rings were maintained at a resting tension of 0.5 g at 37°C at pH 7.4 as previously described [21]. Isometric tension was recorded using an isometric force transducer (Letica TRI 210, Spain) connected to an acquisition system (MP100, BiopacSystems, USA).…”

“…Then, proteins were transferred to polyvinylidene difluoride membranes (Amersham, USA) overnight at 4°C. The transfer used a Mini Trans-Blot Cell system (Bio-Rad, USA) containing 25 mmol/L Tris, 190 mmol/L glycine, 20% methanol, and 0.05% SDS as previously described [21]. After blockade of nonspecific sites with 5% nonfat dry milk, membranes containing total protein extract were incubated overnight at 4°C with the following primary antibodies: anti-Giα-1,2 protein (1∶2,000; Upstate, USA), anti-Giα-3 protein (1∶2,000; Upstate), anti-ERK 1/2 (1∶1,000, Cell Signaling), anti-phospho (Thr202/Tyr204)-ERK1/2 (Cell Signaling; 1∶1,000), anti-p38 MAPK (1∶1,000, Cell Signaling), anti-phospho (Thr180/Tyr182)-p38 MAPK (Cell Signaling; 1∶1,000), anti-α-actin antibody (1∶3,000, Sigma-Aldrich).…”

Adrenoreceptors and nitric oxide in the cardiovascular system

“…In fact, the reduction of basal content of NO enhanced the vasoconstriction rate in the aorta of mice deficient for β 2 -adrenoreceptors. These experiments suggested that β 2 -ARs in vascular tissues are necessary to maintain basal levels of NO, thus concurring to modulate vascular homeostasis (Davel et al, 2012). The lack of functional β 2 -receptors led to an increase of oxidative stress in the aorta of β 2 -KO mice, and a treatment with antioxidant superoxide dismutase was sufficient to limit the vasoconstrictor response to phenylephrine.…”

Apocynin Attenuates Cobalt Chloride-Induced Pheochromocytoma Cell Apoptosis by Inhibiting P38-MAPK/Caspase-3 Pathway

“…Previous studies showed that administration of apocynin in a middle cerebral artery occlusion (MCAO) model produced significant abatement in the infarct volume and reduced the degree of inflammatory disease [29][30][31][32]. They concluded that apocynin possessed a neuroprotective effect.…”