Abstract 536: Metformin Accumulation and Accentuated Ampk Phosphorylation Contribute to Sustained Inhibition of Agonist-Induced Contractility in Endothelium-Denuded Rat Aorta

Abstract

Objective Metformin, a widely used antidiabetic drug, has been shown to exert protective effects in the vasculature by improving endothelium-dependent vasodilation. However, the beneficial effect of metformin toward smooth muscle contraction and the molecular events involved in this process remain unclear. Using endothelium-denuded aorta, the present study has therefore examined the extent of metformin accumulation and the likely changes in AMP-activated protein kinase phosphorylation during smooth muscle contraction induced by G-protein coupled receptor agonists, including phenylephrine (PE) and serotonin (5-HT). Methods Endothelium-denuded rat aortic ring preparations were mounted in organ baths for isometric tension measurements. The contractile responses to cumulative concentrations of PE or 5-HT were determined before and after pretreatments with metformin or AICAR (a direct AMPK activator). The changes in AMPK phosphorylation were determined by immunoblot analysis. Metformin accumulation was determined by LC-MS/MS. Results Concentration dependency studies revealed that pretreatment with 3 mM metformin resulted in significant inhibition of PE- or 5-HT-induced contractility (p < 0.05). In parallel, pretreatment with 1 mM AICAR led to a similar inhibition of agonist-induced contractility (p < 0.05). Notably, the inhibitory effects of metformin on smooth muscle contractility persisted even after the washout of this drug. In contrast, washout of AICAR restored the contractile responses to PE or 5-HT. Pretreatment of aortic rings with 40 μM compound C, an AMPK inhibitor, resulted in significant reversal of metformin and AICAR-mediated decrease in agonist-induced contractility (p < 0.05). An association between metformin/AICAR treatment and AMPK phosphorylation was confirmed by immunoblot analysis. Importantly, metformin treatment resulted in its significant accumulation in the aortic tissue as revealed by LC-MS/MS analysis. Conclusion In conclusion, metformin accumulation contributes to sustained inhibition of arterial smooth muscle contractility via activation of AMPK. The present findings suggest that metformin has the potential to ameliorate chronic hypertension in patients with type 2 diabetes.