title: Metformin exaggerates phenylephrine-induced AMPK phosphorylation independent of CaMKKβ and attenuates contractile response in endothelium-denuded rat aorta.
abstract: Metformin, a widely prescribed antidiabetic drug, has been shown to reduce the risk of cardiovascular disease, including hypertension. Its beneficial effect toward improved vasodilation results from its ability to activate AMPK and enhance nitric oxide formation in the endothelium. To date, metformin regulation of AMPK has not been fully studied in intact arterial smooth muscle, especially during contraction evoked by G protein-coupled receptor (GPCR) agonists. In the present study, ex vivo incubation of endothelium-denuded rat aortic rings with 3mM metformin for 2h resulted in significant accumulation of metformin (∼ 600 pmoles/mg tissue), as revealed by LC-MS/MS MRM analysis. However, metformin did not show significant increase in AMPK phosphorylation under these conditions. Exposure of aortic rings to a GPCR agonist (e.g., phenylephrine) resulted in enhanced AMPK phosphorylation by ∼ 2.5-fold. Importantly, in metformin-treated aortic rings, phenylephrine challenge showed an exaggerated increase in AMPK phosphorylation by ∼ 9.7-fold, which was associated with an increase in AMP/ATP ratio. Pretreatment with compound C (AMPK inhibitor) prevented AMPK phosphorylation induced by phenylephrine alone and also that induced by phenylephrine after metformin treatment. However, pretreatment with STO-609 (CaMKKβ inhibitor) diminished AMPK phosphorylation induced by phenylephrine alone but not that induced by phenylephrine after metformin treatment. Furthermore, attenuation of phenylephrine-induced contraction (observed after metformin treatment) was prevented by AMPK inhibition but not by CaMKKβ inhibition. Together, these findings suggest that, upon endothelial damage in the vessel wall, metformin uptake by the underlying vascular smooth muscle would accentuate AMPK phosphorylation by GPCR agonists independent of CaMKKβ to promote vasorelaxation.
authors.affiliation: Center for Pharmacy and Experimental Therapeutics, University of Georgia College of Pharmacy, 1120 15th Street, HM-1200 Georgia Regents University Campus, Augusta, GA 30912-2450, USA; Charlie Norwood VA Medical Center, Augusta, GA, USA.
authors.forename: Rajkumar
authors.initials: R
authors.lastname: Pyla
authors.affiliation: Center for Pharmacy and Experimental Therapeutics, University of Georgia College of Pharmacy, 1120 15th Street, HM-1200 Georgia Regents University Campus, Augusta, GA 30912-2450, USA; Charlie Norwood VA Medical Center, Augusta, GA, USA.
authors.forename: Islam
authors.initials: I
authors.lastname: Osman
authors.affiliation: Center for Pharmacy and Experimental Therapeutics, University of Georgia College of Pharmacy, 1120 15th Street, HM-1200 Georgia Regents University Campus, Augusta, GA 30912-2450, USA; Charlie Norwood VA Medical Center, Augusta, GA, USA.
authors.forename: Prahalathan
authors.initials: P
authors.lastname: Pichavaram
authors.affiliation: Center for Pharmacy and Experimental Therapeutics, University of Georgia College of Pharmacy, 1120 15th Street, HM-1200 Georgia Regents University Campus, Augusta, GA 30912-2450, USA; Charlie Norwood VA Medical Center, Augusta, GA, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Hansen
authors.affiliation: Center for Pharmacy and Experimental Therapeutics, University of Georgia College of Pharmacy, 1120 15th Street, HM-1200 Georgia Regents University Campus, Augusta, GA 30912-2450, USA; Charlie Norwood VA Medical Center, Augusta, GA, USA; Vascular Biology Center, Department of Pharmacology and Toxicology, Georgia Regents University, Augusta, GA, USA; Department of Medicine, Pennsylvania State University College of Medicine, Hershey, PA, USA. Electronic address: lsegar@gru.edu.
authors.forename: Lakshman
authors.initials: L
authors.lastname: Segar
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000201:enzymology*; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008687:Metformin / Q000494:pharmacology*; D009924:Organ Culture Techniques; D010656:Phenylephrine / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology
chemical_list: D010656:Phenylephrine; D008687:Metformin; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D055372:AMP-Activated Protein Kinases
keywords: AMPK; CaMKKβ; Metformin; Phenylephrine; Vascular smooth muscle