Altered acetylcholine, bradykinin and cutaneous pressure-induced vasodilation in mice lacking the TREK1 potassium channel: the endothelial link

Abstract

The TWIK related K+ channel TREK1 is an important member of the class of two-pore-domain K+ channels. It is a background K+ channel and is regulated by hormones, neurotransmitters, intracellular pH and mechanical stretch. This work shows that TREK1 is present both in mesenteric resistance arteries and in skin microvessels. It is particularly well expressed in endothelial cells. Deletion of TREK1 in mice leads to an important alteration in vasodilation of mesenteric arteries induced by acetylcholine and bradykinin. Iontophoretic delivery of acetylcholine and bradykinin in the skin of TREK1+/+ and TREK1-/- mice also shows the important role of TREK1 in cutaneous endothelium-dependent vasodilation. The vasodilator response to local pressure application is also markedly decreased in TREK1-/- mice, mimicking the decreased response to pressure observed in diabetes. Deletion of TREK1 is associated with a marked alteration in the efficacy of the G-protein-coupled receptor-associated cascade producing NO that leads to major endothelial dysfunction.

Figure 1

Vasodilation and contraction in mesenteric resistance arteries of TREK1^+/+ mice and TREK1^−/− mice. (A) TREK1 protein localization in mesenteric artery. TREK1 immunoreactivity is shown in green, the endothelial cell marker CD31 in red or both in yellow merged. The double staining indicates that TREK1 channels are localized to endothelial cells in the mesenteric artery. (B) Myogenic tone determined in mesenteric resistance arteries isolated from TREK1^−/− mice (n=10) and TREK1^+/+ mice (n=10). (C) Flow-induced dilation in arteries isolated from TREK1^−/− mice (n=7) and TREK1^+/+ mice (n=7). (D) Percentage of contraction in response to increasing concentrations of phenylephrine in mesenteric resistance arteries isolated from TREK1^−/− mice (n=10) and TREK1^+/+ mice (n=8). (E) Percentage of vasodilation in response to increasing concentrations of SNP in mesenteric resistance arteries isolated from TREK1^−/− mice (n=7) and TREK1^+/+ mice (n=6). (F,G) Percentage of vasodilation in response to increasing concentrations of acetylcholine in mesenteric resistance arteries isolated from TREK1^+/+ mice (F, n=10) and TREK1^−/− mice (G, n=10), with (dashed line) and without (100 μM) L-NAME. (H) Percentage of vasodilation in response to increasing concentrations of bradykinin in mesenteric resistance arteries isolated from TREK1^−/− mice (n=4) and TREK1^+/+ mice (n=4). (I) Percentage of vasodilation in response to increasing concentrations of the Ca²⁺ ionophore A23187 in mesenteric resistance arteries isolated from TREK1^−/− mice (n=4) and TREK1^+/+ mice (n=4). ACh, acetylcholine; BK, bradykinin; L-NAME, N^G-nitro-L-arginine methyl ester; SNP, sodium nitroprusside.

Figure 2

TREK1 protein in scalp microvessels and its role in in vivo vasodilation in response to pressure, SNP, acetylcholine and bradykinin. (A) TREK1 localization by immunohistochemistry and in situ methods in blood vessels from skull skin. Sections were stained with TREK1 antibody (green), CD31 (red), an endothelial cell marker, or both (yellow; Merged). Scale bar, 10 μm. The arrow-head shows that TREK1 channels are localized to endothelial cells in the microcirculation blood vessels. The high magnification of an in situ-labelled endothelial cell of a microvessel shows that TREK1 messenger RNA (arrowheads) is localized in the endothelial cell cytoplasm (label 3). The numbers identify an erythrocyte (1), the endothelial cell nucleus (2) and the endothelial cell cytoplasm (3). Scale bar, 5 μm. (B) In vivo changes in cutaneous blood flow in response to a progressive local pressure applied at 2.2 Pa/s (PIV) in TREK1^−/− mice (n=8) and TREK1^+/+ mice (n=8) (a.u., arbitrary units). Inset: representation of the mechanical device used to apply progressive pressure on the skin and simultaneously measure cutaneous blood flow with a laser Doppler probe at the same site. ^*P<0.05 and ^**P<0.01 versus TREK1^+/+ mice. (C) Maximal percentage of vasodilation in response to a local pressure of 0.13 kPa in TREK1^−/− mice (n=8) and TREK1^+/+ mice (n=8). ^*P<0.05 versus TREK1^+/+ mice. (D) Percentage of vasodilation following iontophoretic delivery of SNP in TREK1^−/− mice (n=8) and TREK1^+/+ mice (n=7). (E) Percentage of vasodilation after iontophoretic delivery of acetylcholine in TREK1^−/− mice (n=13) and TREK1^+/+ (n=11). ^*P<0.05 versus TREK1^+/+ mice. (F) Percentage of vasodilation after iontophoretic delivery of bradykinin in TREK1^−/− mice (n=7) and TREK1^+/+ (n=7). ^***P<0.001 versus TREK1^+/+ mice. ACh, acetylcholine; BK, bradykinin; LDF, laser Doppler flow; PIV, pressure-induced vasodilation; SNP, sodium nitroprusside.