Involvement of AMP-activated protein kinase (AMPK) in calcium regulation in healthy and atherosclerotic blood vessels

Marie-Ann Ewart1, Clare McKinney1, Susan Currie2, Simon Kennedy1. 1University of Glasgow, Glasgow, UK, 2University of Strathclyde, Glasgow, UK.

AMPK plays a central role in cellular energy homeostasis (Hardie et al, 1997) and has been shown to have a critical physiological role in the cardiovascular system (Nagata et al, 2010). Furthermore, the mechanism of action of many drugs commonly used to treat cardiovascular and diabetic disease such as statins, metformin and the thiazolidinediones require AMPK for their actions (Ewart et al, 2011). To date, investigation of the vasculoprotective role of AMPK has primarily focused on the endothelium, while the AMPK cascade in vascular smooth muscle (VSM) remains relatively poorly understood. This study focused on determining a role for AMPK activation in VSM from healthy male C57/BL-6 mice and age matched male mice deficient in apolipoprotein E (ApoE-/-), which are hypercholesterolaemic and plaque prone. Using wire myography, we have demonstrated that activation of AMPK using cumulative doses of the activators 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) or 6,7-Dihydro-4-hydroxy-3-(2’-hydroxy [1,1’-biphenyl]-4-yl)-6-oxo-thieno [2,3-b]pyridine-5-carbonitrile (A769662) induced relaxation of pre-constricted C57/BL-6 mouse aorta Maximal Relaxations. Statistical analysis was performed on dose response curves using 2-way ANOVA. Student’s t-tests were applied to maximal relaxation and contraction values, and data was expressed as mean±SEM. Relaxations were not altered between endothelium denuded or intact vessels, but were significantly inhibited by pre-incubation with 40µM AMPK inhibitor 6-[4-(2-Piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine (Compound C, 30min, 37oC) EC50. In addition, pre-incubation with 2mM AICAR (45min, 37oC) reduced aortic contraction to 30nM 9,11-Dideoxy-9α,11α-methanoepoxy prostaglandin F2a (U46619) by 56±7.6% (n = 5, p<0.005 vs control). Ca2+ release from the sarcoplasmic reticulum (SR) in aortic VSM is governed mainly by IP3 receptors (IP3R) and Ca2+ re-uptake is achieved via the sarco/endoplasmic reticulum ATPase (SERCA). In order to determine if AMPK-induced relaxation was facilitated via the function of these proteins, vessels were pre-incubated (30min, 37oC) with 3µM SERCA blocker thapsigargin (TG) or 60µM IP3R receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB) prior to AMPK activation. Inhibition of SERCA significantly reduced the relaxant effect of A769662 on aorta from C57/BL-6 mice Maximal relaxation. Appropriate solvent controls were carried out where required; dimethyl sulphoxide for A769662 and Compound C, ethanol for U46619, TG and 2-APB. Basal AMPK phosphorylation levels were found to be increased in aortic smooth muscle from ApoE-/- mice, while AICAR-induced relaxation was decreased in comparison to healthy controls EC50. These combined data suggest that AMPK is a mediator of VSM relaxation, and that the function of AMPK or its downstream targets are altered in atherosclerosis.

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2. Nagata D. & Hirata Y.(2010) Hypertens. Res. 33:22-28.

3. Ewart M-A. & Kennedy S.(2011) Pharmacol.Ther.131(2):242-253.