| dc.description.abstract | Background, Rationale and General Purpose: Several seminal observations suggest that AMPK mediates vascular tone: 1) in endothelial cell culture and in vitro isolated protein experiments, activation of AMPK stimulates nitric oxide (NO) production via phosphorylation of endothelial nitric oxide synthase (eNOS), 2) stimuli associated with AMPK activation relax isolated vascular smooth muscle preparations from healthy animals, and 3) acute activation of AMPK in vivo induces hypotension in normotensive animals, an effect that could be indicative of reduced vascular tone. Together these findings prompt the logical hypothesis that acute activation of AMPK induces relaxation that is both endothelium-, NO-dependent and also vascular smooth muscle dependent; however the direct effects of AMPK activation on the regulation of vascular tone in the context of intact healthy arteries in vitro or in situ have not been tested. AMPK activation is dysregulated in essential hypertension and aging, conditions both characterized by vasomotor dysfunction. The integrity of AMPK-mediated vasomotor effects has not been evaluated in any model of vascular dysfunction or in the presence of AMPK dysregulation, and so it is unknown if or to what extent, activation of AMPK alters vascular tone in vessels with these impairments. The mechanisms of AMPK-mediated vasomotor effects have also not been delineating in healthy or dysfunctional arteries. Studying basic vascular signalling mechanisms in both healthy and dysfunctional models is important for understanding physiological function and regulation of vascular tissue, as well as to understand vascular pathology and aid in the development of therapeutic interventions. Collectively these considerations present compelling reasons to investigate the role of AMPK in vasomotor function in health and disease. The unifying purpose of this thesis was therefore to investigate the role of AMP-activated protein kinase in regulating vascular tone in arteries of healthy, hypertensive and aged rats.
Experimental Approach and Main Findings: The global objective of the thesis is satisfied by four main studies that utilize a combination of in vitro isolated artery preparations to assess vasomotor function, biochemical analyses and in vivo hemodynamic assessments. In Study 1, we characterize the basic nature of the vasomotor response generated acutely by the pharmacological AMPK activator AICAR in vitro in isolated aorta of normotensive (Wistar-Kyoto rats; WKY) and hypertensive rats (Spontaneously Hypertensive rats; SHR), and the mechanisms mediating these responses. In these experiments, acute activation of AMPK using AICAR induced dose-dependent relaxation of isolated, precontracted arteries from WKY and SHR that was dependent in part on both the endothelium and vascular smooth muscle, and vasorelaxation to AICAR was enhanced in aortic rings of SHR versus those of WKY. In WKY, the endothelium-dependent component of relaxation to AICAR was solely NO-mediated, while in SHR it was dependent on both elevated NO-bioactivity and blunted COX-dependent contraction. In Study 2, we investigate the mechanisms responsible for AMPK-mediated inhibition of endothelium- and cyclooxygenase-dependent vasocontraction in aorta from WKY and SHR (a response enhanced in arteries of hypertensive rats that contributes to vasomotor dysfunction). Pre-activation of AMPK blunted endothelium-dependent contractions to acetylcholine in isolated, non-precontracted WKY and SHR aortic rings. The mechanisms accounting for this effect of AICAR were endothelium-specific, occurring via inhibition of the ACh-stimulated production/release of 6-keto-prostaglandin F1α, the major product of prostacyclin, which is the key prostanoid responsible for endothelium-dependent contractions in aorta of WKY and SHR. AMPK activation had no effect on vascular smooth muscle responsiveness to TP-receptor agonists, ruling out a contribution of vascular smooth muscle mechanisms. In Study 3, we examine responses and mechanisms associated with acute pharmacological AMPK activation on vascular tone of isolated mesenteric resistance arteries in vitro, and on in vivo hemodynamics in WKY and SHR. These experiments revealed that administration of AICAR acutely in vivo acutely reduced blood pressure by ~70mmHg in SHR and this effect was partly NO-dependent. In contrast, AICAR had no effect on blood pressure in WKY. Activation of AMPK also produced vasodilation of isolated, precontracted WKY and SHR resistance mesenteric arteries in vitro, and this was dependent on NO to a greater extent in SHR than in WKY. Together, the parallel reductions in blood pressure in vivo and relaxation of isolated arteries in vitro support reduced vascular resistance as a potential explanation for the in vivo blood pressure effects. Finally, Study 4 characterizes the basic vasodilatory responses to acute AMPK activation and mechanisms associated with these responses in aorta from aged animals and their young counterparts (male Sprague Dawley rats) to glean insight using an additional model of vasomotor dysfunction. In this study, acute activation of AMPK using AICAR generates relaxation in a dose-dependent manner that is partly endothelium-, NO-dependent and partly reliant on vascular smooth muscle in precontracted aorta of both young and aged rats. Similar to the findings of Study 1 in SHR versus WKY, vasodilatory response to AICAR were also enhanced in dysfunctional aorta of aged rats versus healthy aorta of young animals. Other agents shown to activate AMPK in other tissues and models, the anti-diabetic drug metformin and the polyphenol resveratrol, generated varying amounts of relaxation in vascular smooth muscle of young and aged aortic rings. These effects were only associated with AMPK activation in rings treated with metformin but not resveratrol.
Conclusions and Perspectives: These findings are the first to characterize the vasomotor responses generated by acutely activating AMPK in intact arteries of any hypertensive or aging model, and to delineate mechanisms mediating these responses in healthy and dysfunctional vessels. Despite vasomotor dysfunction and dysregulated AMPK activity in arteries of hypertensive and aged rats, acute AMPK activation still generates robust relaxation responses via endothelium- and NO-dependent relaxation, inhibition of enhanced endothelium-dependent contractions in SHR, and direct relaxation of the vascular smooth muscle; effects that would aid in reversing the dysfunctional characteristics of arteries from these animals, and may recommend AMPK as a useful therapeutic target for interventions aimed at improving vasomotor function. Future studies will be necessary to reveal whether AMPK plays a role in generating acute changes in vessel tone induced by AMPK-activating physiological stimuli in situ (i.e. such as shear stress during exercise). Together these data continue to support AMPK as a novel regulator of vascular tone, yield valuable, novel, mechanistic insight into AMPK-mediated regulation of vasomotor function in arteries during health, disease and aging, and highlight the need for continued investigation into a vasoregulatory function for AMPK in health and disease. | en |
