Activation of AMP kinase α1 subunit induces aortic vasorelaxation in mice

AMPK is a ubiquitous serine/threonine protein kinase activated by pathological stimuli, such as oxidative damage, osmotic shock, hypoxia and glucose deprivation, as well as by physiological stimuli such as exercise and muscle contraction, and by hormones including leptin and adiponectin (Hardie et al. 2003). AMPK is activated in response to decreased cellular energy charge (high AMP/ATP ratio) and is involved in regulating carbohydrate and fat metabolism (Hardie et al. 2003; Sambandam & Lopaschuk, 2003). AMPK exists in cells as a heterotrimeric complex composed of a catalytic subunit (α) and two regulatory subunits (α and γ). Two α subunit isoforms exist, α1 and α2, which are unevenly distributed in the tissues. AMPK is expressed both in endothelial cells and in smooth muscle cells. The predominant isoform expressed in vascular endothelial cells is α1 (Zou et al. 2004; Davis et al. 2006). AMPK expression in vascular smooth muscles is different from its expression in striated muscles (Rubin et al. 2005). Both α1 and α2 catalytic subunits are expressed in arterial smooth muscle cells, although their relative proportion differs between different arteries (Rubin et al. 2005; Evans et al. 2006). AMPK can be artificially activated by treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). This nucleoside is taken up by cells, resulting in accumulation of the monophosphorylated derivative 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP) and activation of AMPK (Corton et al. 1995). Treatment of human aortic smooth muscle cells or isolated rabbit aortas with AICAR induces phosphorylation of AMPK and of acetyl-CoA carboxylase, a key target of AMPK, resulting in inhibition of growth factor-induced cell proliferation (Igata et al. 2005). A target of AMPK is endothelial nitric oxide synthase (eNOS), an important modulator of angiogenesis and vascular tone. It has been clearly established that AMPK may associate with and phosphorylate eNOS in cardiomyocytes and endothelial cells (Chen et al. 1999), in association with the heat shock protein 90 (Davis et al. 2006), thus increasing eNOS activity and NO production. Direct activation of AMPK with AICAR stimulates NO synthesis in human aortic endothelial cells (Morrow et al. 2003). Furthermore AMPK can be activated independently in endothelial cells by extracellular nucleotides and adenosine through P2 receptors and adenosine transporters (da Silva et al. 2006). Finally, metabolically challenged endothelium-denuded porcine carotid artery segments exhibit a rapid increase in AMPK activity after metabolic stress associated with the recruitment of signalling pathways that may regulate smooth-muscle contraction (Rubin et al. 2005). However, AICAR failed to relax endothelin-1 precontracted carotid artery rings in this species (Rubin et al. 2005). These data suggest that AMPK may play a complex role in vascular function and remodelling. However, the possible involvement of AMPK in vasorelaxation has not at present been directly shown. In this study we investigated whether pharmacological activation of AMPK by AICAR could induce relaxation of preconstricted mouse aorta and whether this effect was mediated by endothelium and/or NOS activation. Furthermore, AMPK isoform specificity of AICAR-induced relaxation was investigated in AMPKα1 and α2 knock-out mice and their littermate controls (Viollet et al. 2003; Jorgensen et al. 2004). The results show that activation of AMPKα1 induces vasorelaxation of mouse aorta in an endothelium- and NOS-independent manner.