Abstract-The endothelium-derived hyperpolarizing factor (EDHF) contributes critically to the regulation of vascular tone.Its dependency on direct signaling through myoendothelial gap junctions composed of connexins (Cx) is controversially discussed. We studied the impact of Cx40 in EDHF-type dilations in vivo and in vitro (wire and pressure myography) in small arteries (A. gracilis) using different Cx40-deficient mouse models. Acetylcholine induced prominent EDHF-type dilations (inhibition of NO synthase and cyclooxygenase) of Ϸ90% (maximum effect) in wild-type and Cx40-deficient vessels (Cx40) in vitro under isobaric conditions. In contrast, under isometric conditions, EDHF-type relaxations were nearly abrogated in Cx40 Ϫ/Ϫ (9Ϯ3%) but only slightly reduced in wild-type vessels (45Ϯ4%; PϽ0.05). Vessels expressing Cx45 instead of Cx40 exhibited similarly reduced relaxations (13Ϯ1%), demonstrating that Cx45 cannot replace Cx40 functionally. The necessity of Cx40 in EDHF-type dilations under isometric conditions was verified by the attenuation in vessels being specifically deficient for Cx40 in endothelial cells (Cx40 fl :TIE2-Cre: 17Ϯ3%; Cx40-floxed controls: 67Ϯ6%; PϽ0.05). Nevertheless, EDHF-type dilations were Cx40 independent when studied isobarically. The EDHF-type dilation in vivo resembled the isobaric situation, being virtually Cx40 independent and similar powerful. Distinct EDHF mechanisms can be distinguished by their Cx40 dependency. A powerful EDHF is present in vivo and in vitro under isobaric conditions but is lacking in wire myography (isometric conditions). Herein, a less potent EDHF depends on Cx40 and may represent signaling through myoendothelial gap junctions. We suggest that distinct EDHFs (even in the same artery) explain partially the controversy on the role of myoendothelial gap junctions in EDHF signaling. (Hypertension. 2011;57:802-808.) • Online Data Supplement Key Words: myoendothelial coupling Ⅲ gap junctions Ⅲ connexins Ⅲ microcirculation Ⅲ endothelium-derived hyperpolarizing factor T he endothelium is the key player in arteries to control the contractile state of the adjacent smooth muscle and thereby vascular diameter. This function is achieved by the release of NO, prostaglandins, and the endothelium-derived hyperpolarizing factor (EDHF). Of these autocaids, NO predominates in large conducting arteries, whereas the importance of EDHF increases as the size of the arteries decreases. 1-5 NO and prostaglandins are well characterized with respect to chemistry and signaling cascades, but the nature of EDHF is controversially discussed. 6 -8 By definition, EDHF-type dilations are associated with smooth muscle hyperpolarization, which is followed by its relaxation, most likely through decreasing the open probability of voltagedependent L-type Ca 2ϩ -channels. Because EDHF-type dilations require an endothelial hyperpolarization through activation of Ca 2ϩ -activated K ϩ -channels (K Ca ) 9 -11 as an initial step the smooth muscle hyperpolarization may be caused by a direct charge transfer ...

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