Elsevier

Biochemical Pharmacology

Volume 92, Issue 2, 15 November 2014, Pages 266-279
Biochemical Pharmacology

Metformin exaggerates phenylephrine-induced AMPK phosphorylation independent of CaMKKβ and attenuates contractile response in endothelium-denuded rat aorta

https://doi.org/10.1016/j.bcp.2014.08.024Get rights and content

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 3 mM metformin for 2 h 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.

Graphical abstract

Metformin inhibits phenylephrine (PE)-induced smooth muscle contraction through exaggerated AMPK phosphorylation (independent of CaMKKβ), which would occur via ↑AMP/ATP ratio upon coordinated inhibition of ATP synthesis and increase in ATP consumption.

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Introduction

Metformin, a widely prescribed antidiabetic drug, has been shown to reduce the risk of cardiovascular disease [1]. With regard to hypertension, clinical studies reveal its blood pressure-lowering effects in some but not in all subjects with varying co-morbid conditions [cited in [2], [3]]. Studies with different animal models demonstrate that its antihypertensive effects are attributed to endothelium-dependent and endothelium-independent relaxation of the underlying vascular smooth muscle in the vessel wall [2], [4], [5], [6], [7]. Although the molecular mechanism of endothelium-dependent vasodilation by metformin has been extensively studied [8], [9], [10], it remains unclear as to how metformin regulates agonist-induced contractions in intact arterial smooth muscle.

In vascular endothelium, metformin activates AMP-activated protein kinase (AMPK), which phosphorylates endothelial nitric oxide synthase thereby increasing the production of nitric oxide, a potent vasodilator [10], [11]. In endothelium-denuded vessels, metabolic stress and 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) have been shown to activate AMPK [12], [13], [14], [15]. Importantly, activation of AMPK results in diminished myosin light chain kinase activity thereby attenuating vascular smooth muscle contraction [16]. Although metformin has been shown to inhibit vascular smooth muscle contraction [17], the intermediary role of AMPK remains unclear especially during agonist-induced contractions. For instance, metformin does not activate AMPK in endothelium-denuded porcine carotid artery [12], but it induces AMPK phosphorylation in porcine and rat aortic smooth muscle cells in culture [17], [18]. Hence, it is critically important to further investigate metformin regulation of contractile function and AMPK phosphorylation in intact arterial smooth muscle ex vivo.

AMPK is a heterotrimeric protein consisting of a catalytic α subunit and β and γ regulatory subunits [19]. Its activity is regulated by increases in adenosine diphosphate (ADP) and/or adenosine monophosphate (AMP) levels. In addition, increase in AMPK activity occurs through phosphorylation of Thr172 residue, inhibition of dephosphorylation of phosphorylated AMPK, and allosteric activation. The major upstream kinases that phosphorylate AMPK include liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKβ) [19], [20].

Metformin has been shown to inhibit mitochondrial respiratory chain complex 1 [21], [22], thereby diminishing ATP/ADP ratio [22] in hepatocytes. However, it does not affect AMP level or AMP/ATP ratio in skeletal muscle cells [23]. To date, metformin regulation of nucleotide levels has not been examined in vascular smooth muscle during agonist-induced contractions. Previously, we and several other investigators have shown that, in vascular smooth muscle cells, vasoconstrictors evoke a rise in cytosolic free Ca2+ [24], [25] that would facilitate the activation of CaMKKβ [20]. Accordingly, vasoactive peptides (e.g., GPCR agonists such as vasopressin, angiotensin II, and endothelin-1) promote an increase in AMPK phosphorylation through an intermediary activation of CaMKKβ in rat aortic smooth muscle cells [16]. The objectives of the present study are to determine: (i) the effects of metformin and/or phenylephrine on nucleotide levels and AMPK phosphorylation; and (ii) the intermediary role of CaMKKβ toward AMPK phosphorylation in intact arterial smooth muscle during isometric contractions.

Using endothelium-denuded rat aortic rings, we performed isometric tension measurements to determine how metformin regulates phenylephrine-induced smooth muscle contraction. Under the same conditions, we performed LC–MS/MS MRM analysis to determine the changes in nucleotide levels. In addition, we performed immunoblot analysis to determine metformin and/or phenylephrine regulation of AMPK phosphorylation before and after treatment with compound C (AMPK inhibitor) or STO-609 (CaMKKβ inhibitor). To determine whether metformin regulation of contraction and AMPK phosphorylation occurs in a reversible manner, select studies included post-treatment washout protocols. To determine whether metformin treatment results in its uptake by smooth muscle, aortic tissue lysates were subjected to LC–MS/MS MRM analysis. The present findings demonstrate that metformin exaggerates phenylephrine-induced AMPK phosphorylation (independent of CaMKKβ), which is associated with diminished smooth muscle contraction.

Section snippets

Materials

Phenylephrine hydrochloride, serotonin (5-hydroxytryptamine hydrochloride), and acetylcholine chloride were purchased from Sigma–Aldrich (St. Louis, MO). Metformin hydrochloride, AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside or acadesine), compound C (or dorsomorphin dihydrochloride), and STO-609 acetate were purchased from Tocris Bioscience (Minneapolis, MN). Phenformin hydrochloride and l-NMMA (l-NG-monomethyl arginine acetate) were purchased from Cayman Chemical (Ann Arbor, MI).

Long-term metformin treatment, at 100 μM or 1 mM concentration, inhibits PE-induced smooth muscle contractility in rat aorta ex vivo

Previous studies have shown that oral administration of metformin for up to 11 weeks results in diminished vascular reactivity in rats [32]. In particular, the maximal contractile response to norepinephrine is diminished in the mesenteric arteries (with or without endothelium) isolated from metformin-treated rats [32]. In the present study, we examined whether ex vivo incubation of endothelium-denuded aorta with metformin for an extended period of time (18 h) alters vascular smooth muscle

Discussion

The principal findings of the present study using ex vivo endothelium-denuded rat aorta include: (i) metformin accumulation in intact arterial smooth muscle; (ii) a modest but significant increase in AMPK phosphorylation by phenylephrine but not by metformin per se; (iii) an exaggerated increase in AMPK phosphorylation with an associated elevation of AMP/ATP ratio by phenylephrine after metformin treatment; (iv) compound C (AMPK inhibitor) inhibition of AMPK phosphorylation induced by

Acknowledgements

This work was supported by the National Heart, Lung, and Blood Institute/National Institutes of Health Grant (R01-HL-097090), University of Georgia Research Foundation, and University of Georgia RC Wilson Pharmacy Fund to L. Segar.

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