In arteries, muscarinic agonists such as acetylcholine release an unidentified, endothelium-derived hyperpolarizing factor (EDHF) which is neither prostacyclin nor nitric oxide. Here we show that EDHF-induced hyperpolarization of smooth muscle and relaxation of small resistance arteries are inhibited by ouabain plus Ba2+; ouabain is a blocker of Na+/K+ ATPase and Ba2+ blocks inwardly rectifying K+ channels. Small increases in the amount of extracellular K+ mimic these effects of EDHF in a ouabain- and Ba2+-sensitive, but endothelium-independent, manner. Acetylcholine hyperpolarizes endothelial cells and increases the K+ concentration in the myoendothelial space; these effects are abolished by charbdotoxin plus apamin. Hyperpolarization of smooth muscle by EDHF is also abolished by this toxin combination, but these toxins do not affect the hyperpolarizaiton of smooth muscle by added K+. These data show that EDHF is K+ that effluxes through charybdotoxin- and apamin-sensitive K+ channels on endothelial cells. The resulting increase in myoendothelial K+ concentration hyperpolarizes and relaxes adjacent smooth-muscle cells by activating Ba2+-sensitive K+ channels and Na+/K+ ATPase. These results show that fluctuations in K+ levels originating within the blood vessel itself are important in regulating mammalian blood pressure and flow.

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1 The effects of haemoglobin and methylene blue on the acetylcholine (ACh)-induced electrical and mechanical responses of smooth muscle cells were investigated in rat aorta and rat main pulmonary artery.2 When the endothelium was intact, ACh induced a transient hyperpolarization and sustained relaxation of tissues precontracted with noradrenaline. Both hyperpolarization and relaxation were absent in preparations without endothelium. 3 Haemoglobin and methylene blue inhibited the ACh-induced relaxation, but not the transient hyperpolarization. 4 In aorta with an intact endothelium, ACh produced an increase in both the rate of 86Rb efflux and tissue cyclic GMP levels. The changes in ion flux were unaffected by either haemoglobin or methylene blue in concentrations which almost abolished the increase in cyclic GMP concentrations. 5 In arteries with an intact endothelium, indomethacin had no effect on the ACh-induced electrical and mechanical responses or on the increase in 86Rb effiux and tissue cyclic GMP levels. 6 It is concluded that in the rat aorta and rat main pulmonary artery, ACh releases two different substances, an endothelium-derived relaxing factor (EDRF) and a hyperpolarizing factor (EDHF), from the endothelial cells. Neither substance appears to be derived from a pathway dependent on cyclo-oxygenase. EDHF seems to play a minor role in the relaxation of noradrenaline-induced contractions.

The term endothelium-derived hyperpolarising factor (EDHF) was introduced in 1987 to describe the hypothetical factor responsible for myocyte hyperpolarisations not associated with nitric oxide (EDRF) or prostacyclin. Two broad categories of EDHF response exist. The classical EDHF pathway is blocked by apamin plus TRAM-34 but not by apamin plus iberiotoxin and is associated with endothelial cell hyperpolarisation. This follows an increase in intracellular [Ca(2+)] and the opening of endothelial SK(Ca) and IK(Ca) channels preferentially located in caveolae and in endothelial cell projections through the internal elastic lamina, respectively. In some vessels, endothelial hyperpolarisations are transmitted to myocytes through myoendothelial gap junctions without involving any EDHF. In others, the K(+) that effluxes through SK(Ca) activates myocytic and endothelial Ba(2+)-sensitive K(IR) channels leading to myocyte hyperpolarisation. K(+) effluxing through IK(Ca) activates ouabain-sensitive Na(+)/K(+)-ATPases generating further myocyte hyperpolarisation. For the classical pathway, the hyperpolarising "factor" involved is the K(+) that effluxes through endothelial K(Ca) channels. During vessel contraction, K(+) efflux through activated myocyte BK(Ca) channels generates intravascular K(+) clouds. These compromise activation of Na(+)/K(+)-ATPases and K(IR) channels by endothelium-derived K(+) and increase the importance of gap junctional electrical coupling in myocyte hyperpolarisations. The second category of EDHF pathway does not require endothelial hyperpolarisation. It involves the endothelial release of factors that include NO, HNO, H(2)O(2) and vasoactive peptides as well as prostacyclin and epoxyeicosatrienoic acids. These hyperpolarise myocytes by opening various populations of myocyte potassium channels, but predominantly BK(Ca) and/or K(ATP), which are sensitive to blockade by iberiotoxin or glibenclamide, respectively.