AMP-activated kinase relaxes agonist induced contractions in the mouse aorta via effects on PKC signaling and inhibits NO-induced relaxation

Abstract

Adenosine monophosphate activated kinase (AMPK), a regulator of cellular metabolism, has been shown to relax arterial smooth muscle via endothelium-dependent and independent mechanisms. We have examined the role of AMPK in different smooth muscles using the activating compound, 5-amino-4-imidazolecarboxamide riboside-1-β-d-ribofuranoside (AICAR). Isolated preparations of mouse aorta, saphenous artery, ileum and urinary bladder were compared. AICAR produced a reversible dose-dependent relaxation in aortic rings pre-incubated with AICAR and activated with phenylephrine. Less prominent relaxation was noted in the other tissues. This difference in sensitivity to AICAR was not due to differences in the expression levels of AMPK α1 mRNA. In the aorta, AICAR had a greater effect on contractions induced by phenylephrine, compared to high-K⁺ induced contractions. Contractions of the aorta in response to the protein kinase C activator PDBu were prominently inhibited by AICAR. The AICAR relaxation observed in the aorta was not prevented by the NOS inhibitor L-NAME, Indomethacin or endothelium removal. Nitric oxide (NO) mediated relaxations in aortic preparations induced by acetylcholine or sodium nitroprusside (SNP) were attenuated by AICAR. In conclusion, AMPK induced relaxation of smooth muscle is tissue-dependent and most prominent in large elastic arteries. The smooth muscle relaxation is NO-independent and occurs downstream of PKC activation and is associated with attenuated relaxant responses to NO.

Introduction

Adenosine-monophosphate-activated-kinase (AMPK) is a widely expressed serine-threonine protein kinase that functions as a cellular energy sensor (Hardie, 2011). It consists of a catalytic α subunit, expressed as α1 and α2 isoforms, and regulatory (β, γ) subunits. It is activated by phosphorylation by kinases primarily acting on a threonine-residue on the α-subunit (Hawley et al., 1996). This phosphorylation is favored by AMP interaction, which also opposes dephosphorylation and inactivation (Cheung et al., 2000). These effects of AMP are antagonized by ATP, thus rendering AMPK activity responsive to changing [AMP]/[ATP] ratios. AMPK has numerous targets in pathways involved in energy metabolism and growth regulation and several proposed physiological functions. In general, AMPK activation results in inhibition of ATP-consuming, anabolic, metabolic pathways, while stimulating pathways that generate ATP (Hardie, 2011). In addition to the physiological activators of AMPK, a range of pharmacological compounds, including the nucleoside compound 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), activate the enzyme (Corton et al., 1995).

In skeletal muscle, AMPK has a role in glucose uptake and has been proposed as a potential target for treating insulin resistance (Kraegen et al., 2009). The detailed functions of AMPK in smooth muscle tissue are largely unknown. AMPK is expressed in smooth muscle and in endothelial cells (Dagher et al., 1999, Rubin et al., 2005). In rat pulmonary arteries, AMPK is activated during hypoxia (Robertson et al., 2008) and AMPK is suggested to couple altered mitochondrial respiration to hypoxic pulmonary vasoconstriction (Evans, 2006). In contrast, AMPK activation is associated with vasodilation in systemic vascular beds. AMPK activates eNOS in vascular endothelium during hypoxia (Nagata and Hirata, 2010). AMPK dilates rat resistance arteries (Bradley et al., 2010) and has been reported to lower blood pressure in vivo (Foley et al., 1989, Bosselaar et al., 2011). In spontaneously hypertensive rats, AMPK activation causes endothelium-NO-dependent vasorelaxation (Ford and Rush, 2011, Ford et al., 2012). In a more long-term perspective, AMPK inhibits proliferation and hypertrophy of vascular smooth muscle (Nagata et al., 2004, Igata et al., 2005, Ferri, 2012). AMPK also has been shown to relax vascular smooth muscle in mouse aorta independently of the endothelium (Goirand et al., 2007). Mechanisms for this effect are unknown and direct actions on the myosin light chain kinase (Horman et al., 2008) or small G protein signaling (Wang et al., 2011) may be involved.

The effects of AMPK activation, and AICAR, have been primarily studied in isolated arteries activated with receptor agonists. Information on other smooth muscle tissues and different modes of activation is not available. The relative contribution of endothelial effects and effects from direct action on smooth muscle are not resolved and the mechanisms for the relaxant effects in smooth muscle are unknown. We have therefore addressed the following questions: (1) Is the relaxant effect of AICAR also observed in a broader range of smooth muscle tissues?, (2) Does AICAR relax smooth muscle via effects on receptor activation or on downstream cellular signaling pathways?, and (3) What are the effects of AICAR on endothelial mediated relaxation in the arteries?