AMPK-activation reduces EDH-type relaxations in rat small arteries

Abstract

Introduction: The role of adenosine monophosphate-activated protein kinase (AMPK) in controlling the vascular tone, especially in small arteries which contribute majorly to the regulation of peripheral resistance and arterial blood pressure, is still unclear. Endothelium-dependent hyperpolarization (EDH) is an important vasodilator signal in small arteries; it is triggered by the opening of endothelial calcium-activated potassium channels (KCa), resulting in the release of potassium ions and hyperpolarization of the endothelial cells. The endothelial hyperpolarization is transmitted to the underlying vascular smooth muscle cells, leading to their relaxation. The present study aimed to examine whether or not AMPK affects EDH-mediated relaxations in small arteries of the rat. Methods: Male Sprague-Dawley rats (12 weeks old) were used. Superior mesenteric arteries were isolated and suspended in conventional organ chambers for isometric tension recording. In some preparations, the endothelium was removed by perfusing the lumen of the arteries with 0.5% Triton X-100. To study EDH-type relaxations, the preparations were incubated with L-NAME (nitric oxide synthase inhibitor; 10-4 M) and indomethacin (cyclooxygenase inhibitor; 10-5 M) during 40 minutes before they were contracted with phenylephrine followed by exposure to vasodilator agents. The activity and protein presence of AMPK were measured by ELISA and Western blotting, respectively. Results: AICAR (10-4 M) and A769662 (10-4 M), two AMPK activators with different structures and binding sites on the enzyme (the active metabolite of the former binds to the γ subunit, leading to conformational change of the complex and exposure of the α subunit while the latter is a β1-selective activator), significantly reduced EDH-type relaxations in response to acetylcholine and those to SKA-31 (KCa channels opener). The inhibitory effects of AICAR and A769662 were prevented by compound C (AMPK inhibitor; 10-5 M). AMPK activity assays confirmed that at 10-4 M, AICAR and A769662 increased the kinase activity in rings with endothelium; however, in rings without endothelium, only A769662 did so. A769662, but not AICAR, significantly inhibited relaxations to potassium ions and levcromakalim (opener of ATP-sensitive potassium channel) in rings without endothelium. The relaxations to the nitric oxide-donor DETA NONOate were not affected by A769662. The protein levels of α subunit and β subunit of AMPK in preparations with endothelium were comparable to those without endothelium. Conclusions: AMPK-activation causes reduction of EDH-type relaxations in rat superior mesenteric arteries. While AICAR appears to reduce the generation of the endothelial signal, A769662 prevents the signaling downstream of hyperpolarization of the vascular smooth muscle cells.