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. 2011 May;38(5):328-33.
doi: 10.1111/j.1440-1681.2011.05509.x.

Chronic activation of AMP-activated protein kinase prevents 20-hydroxyeicosatetraenoic acid-induced endothelial dysfunction

Natalie C Ward  1 , Kai Chen, Chunying Li, Kevin D Croft, John F Keaney Jr
Affiliations

Chronic activation of AMP-activated protein kinase prevents 20-hydroxyeicosatetraenoic acid-induced endothelial dysfunction

Natalie C Ward et al. Clin Exp Pharmacol Physiol. 2011 May.

Abstract

1. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor involved in vascular dysfunction and blood pressure regulation. Studies have revealed strong associations between 20-HETE and endothelial dysfunction; however, the signalling mechanisms are largely unknown. Therefore, the aim of the present study was to investigate the effect of 20-HETE on the association between endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (Hsp90). 2. In mouse aortic rings, 20-HETE significantly enhanced the constriction to phenylephrine and inhibited the relaxation to acetylcholine (P=0.05 vs control rings). In mice with chronic AMP-activated protein kinase (AMPK) activation, this protected against the negative effects of 20-HETE (P<0.05). Immunoprecipitation of eNOS in human umbilical vein endothelial cells treated with 20-HETE revealed a decrease in basal and vascular endothelial growth factor-stimulated Hsp90 association with eNOS (P<0.05). Pretreatment of cells with 5'-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR; a chronic activator of AMPK) prevented the loss of Hsp90 association with eNOS following 20-HETE treatment. Treatment with 20-HETE for 24 h induced an increase in eNOS phosphorylation that was not seen following acute treatment (30 min). The increased eNOS phosphorylation was accompanied by transient changes in Akt phosphorylation. 3. In conclusion, 20-HETE impairs eNOS-Hsp90 association, which can be reversed by chronic activation of AMPK. This provides a mechanism for reduced nitric oxide bioactivity and endothelial dysfunction in diseases with elevated 20-HETE levels, such as hypertension.

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Figures

Figure 1
Figure 1
Mouse aortic ring (a) constriction with increasing doses of phenylephrine. 2-way ANOVA with repeated measures analysis, p = 0.05 versus control and p = 0.025 versus AICAR+20-HETE (n=6–9) and (b) relaxation in response to increasing doses of acetylcholine, following pre-constriction with phenylephrine. 2-way ANOVA with repeated measures analysis, p = 0.05 versus control, p = 0.04 versus AICAR and p = 0.03 versus AICAR+20-HETE (n=5).
Figure 2
Figure 2
eNOS immunprecipitation of HUVECs treated with 20-HETE (10μM), under basal and VEGF stimulated conditions (n=3). Fold change where black bars are p-eNOS/eNOS and white bars are Hsp90/eNOS. ANOVA with Dunnett's post-hoc analysis *p<0.05 versus control.
Figure 3
Figure 3
eNOS immunoprecipitation of HUVECs pre-treated with AICAR (1mM, 1hr), followed by 20-HETE (10μM) under (a) basal and (b) VEGF stimulated conditions (n=3). (c) eNOS immunoprecipitation of HUVECs pre-treated with AICAR and/or Compound C (20μM, 1 hr), followed by 20-HETE (10μM). Fold change where black bars are p-eNOS/eNOS and white bars are Hsp90/eNOS.
Figure 3
Figure 3
eNOS immunoprecipitation of HUVECs pre-treated with AICAR (1mM, 1hr), followed by 20-HETE (10μM) under (a) basal and (b) VEGF stimulated conditions (n=3). (c) eNOS immunoprecipitation of HUVECs pre-treated with AICAR and/or Compound C (20μM, 1 hr), followed by 20-HETE (10μM). Fold change where black bars are p-eNOS/eNOS and white bars are Hsp90/eNOS.
Figure 4
Figure 4
Phosphorylation of (a) Akt and (b) eNOS in HUVECs following acute and chronic treatment with 20-HETE (10μM). ANOVA with Dunnett's post-hoc analysis *p<0.05 versus control.
Figure 5
Figure 5
Intracellular ROS production following (a) acute 20-HETE treatment. ANOVA with Dunnett's post-hoc analysis, p < 0.05 versus control (n=4) and (b) chronic 20-HETE treatment. ANOVA with Dunnett's post-hoc analysis (n=4). Extracellular ROS production (c) following acute and chronic 20-HETE treatment. ANOVA with Dunnett's post-hoc analysis (n=4).
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