AMPK-activation reduces relaxations attributable to the opening of potassium channels in endothelial and smooth muscle cells of the rat superior mesenteric artery

Abstract

Endothelium-dependent hyperpolarization (EDH) is a major vasodilator mechanism in small arteries, the constriction of which contributes importantly to peripheral resistance and, thus, arterial blood pressure. The present thesis aimed to examine the possible, but hitherto unexplored, involvement of adenosine monophosphate-activated protein kinase (AMPK) in EDH-like relaxations of resistance arteries under physiological and pathological conditions.

In superior mesenteric arteries with endothelium of Sprague-Dawley (SD) rats, acetylcholine induced significant EDH-like relaxations [decreases in isometric tensions during phenylephrine-contraction in preparations incubated with inhibitors of nitric oxide synthases and cyclooxygenases]. The AMPK-activator AICAR significantly inhibited EDH-like relaxations, an effect prevented by compound C (AMPK-inhibitor), but did not affect K+-induced relaxations in preparations without endothelium, suggesting that AICAR acts upstream of the hyperpolarization of vascular smooth muscle cells. Endothelial calcium-permeable transient receptor potential V4 (TRPV4) and calcium-activated potassium (KCa) channels are functionally coupled; AICAR reduced TRPV4-channel currents in endothelial cells of mouse superior mesenteric arteries, suggesting that AICAR interferes with endothelial TRPV4-KCa signaling to attenuate EDH-like relaxations. Another AMPK-activator A769662 also inhibited EDH-like relaxations, confirming that indeed AMPK-activation blunts EDH-like relaxations in rat superior mesenteric arteries. Unlike AICAR, A769662 reduced K+-induced relaxations in rings without endothelium, suggesting that, in addition, it inhibits signaling downstream of smooth muscle hyperpolarization.

Acetylcholine induced similar EDH-like relaxations in superior mesenteric arteries of Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Like in preparations of SD rats, AICAR almost abolished EDH-like relaxations in those of WKY and SHR, but did not affect K+-induced relaxations in rings without endothelium of either strain, demonstrating again that AICAR acts upstream of smooth muscle hyperpolarization. Compared to preparations of SD rats, A769662 exerted a similar inhibitory effect on EDH-like relaxations in WKY arteries, however in SHR arteries, it affected neither EDH-like nor endothelium-independent K+-induced relaxations, which further supports the conclusion that A769662 has a different site of action than AICAR.

While compound C did not affect EDH-like relaxations in WKY mesenteric arteries, it inhibited those in SHR preparations. In WKY arteries, EDH-like relaxations were significantly reduced by TRAM-34 [intermediate-conductance KCa (IKCa)-blocker]; compound C further reduced such relaxations. By contrast, UCL1684 [small-conductance KCa (SKCa)-blocker], in the absence or presence of compound C, had no significant effect. In SHR preparations, TRAM-34 or UCL1684 administered alone inhibited EDH-like relaxations with no further alteration by compound C. These results suggest that in WKY superior mesenteric arteries, IKCa can fully compensate for the absence of SKCa, but not vice versa, while in SHR preparations, neither IKCa nor SKCa compensate for each other’s absence; with such loss of compensation, EDH-like responses are maintained by constitutive AMPK-activity.

Taken in conjunction, the experiments in rat superior mesenteric arteries reported in the present thesis demonstrate that AMPK-activation with AICAR or A769962 blunts EDH-like relaxations under conditions when endothelial nitric oxide production is insufficient (with nitric oxide synthase-inhibition); thus, caution may be required when administering therapeutic agents activating AMPK to patients with endothelial dysfunction. By contrast, the constitutive AMPK-activity favors EDH-like relaxations, suggesting that, various levels of AMPK-activation (constitutive versus additional-stimulation) differently modulate EDH-like relaxations.