Arthur H. Weston

K + is an endothelium-derived hyperpolarizing factor in rat arteries

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.

EDHF: bringing the concepts together.

Endothelial cells synthesize and release vasoactive mediators in response to various neurohumoural substances (e.g. bradykinin or acetylcholine) and physical stimuli (e.g. cyclic stretch or fluid shear stress). The best-characterized endothelium-derived relaxing factors are nitric oxide and prostacyclin. However, an additional relaxant pathway associated with smooth muscle hyperpolarization also exists. This hyperpolarization was originally attributed to the release of an endothelium-derived hyperpolarizing factor (EDHF) that diffuses to and activates smooth muscle K(+) channels. More recent evidence suggests that endothelial cell receptor activation by these neurohumoural substances opens endothelial cell K(+) channels. Several mechanisms have been proposed to link this pivotal step to the subsequent smooth muscle hyperpolarization. The main concepts are considered in detail in this review.

Experimental design and analysis and their reporting: new guidance for publication in BJP

This Editorial is part of a series. To view the other Editorials in this series, visit: http://onlinelibrary.wiley.com/doi/10.1111/bph.12956/abstract; http://onlinelibrary.wiley.com/doi/10.1111/bph.12954/abstract; http://onlinelibrary.wiley.com/doi/10.1111/bph.12955/abstract and http://onlinelibrary.wiley.com/doi/10.1111/bph.13112/abstract

Acetylcholine releases endothelium-derived hyperpolarizing factor and EDRF from rat blood vessels.

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 efflux 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.

Comparison of the effects of brl 34915 and verapamil on electrical and mechanical activity in rat portal vein

1 The effects of the novel anti‐hypertensive agent BRL 34915, (±) 6‐cyano‐3,4‐dihydro‐2,2‐dimethyl‐trans‐4‐(2‐oxo‐1‐pyrrolidyl)‐2H‐benzo[b]pyran‐3‐ol, have been compared with those of verapamil on rat isolated portal vein. 2 BRL 34915 produced a concentration‐dependent reduction in mechanical responses to noradrenaline but had relatively little inhibitory effect on K+‐induced contractions. Verapamil reduced the magnitude of both noradrenaline and K+‐induced mechanical responses. 3 BRL 34915 delayed the appearance of the reduced noradrenaline contractions, a property not shared by verapamil. 4 BRL 34915 abolished spontaneous electrical and mechanical discharges and hyperpolarized the portal vein cells close to their calculated potassium equilibrium potential. Verapamil inhibited spontaneous electrical and mechanical discharges, effects associated with a small depolarization. 5 BRL 34915 produced a significant increase in the 86Rb efflux rate coefficient whilst verapamil was without effect on this parameter. 6 The inhibitory effects of BRL 34915 were rapid in onset and readily reversible by washing, whilst those of verapamil were slower in onset and only slowly reversible. 7 It is concluded that the inhibitory effects of BRL 34915 in rat portal vein are produced by the opening of potassium channels in the smooth muscle cells. This inhibits spike activity and in sufficient concentration holds the membrane potential at or close to the potassium equilibrium potential, thereby reducing the effects of excitatory agents.

Endothelium-derived hyperpolarising factors and associated pathways: a synopsis

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 [Ca2+] and the opening of endothelial SKCa and IKCa 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 SKCa activates myocytic and endothelial Ba2+-sensitive KIR channels leading to myocyte hyperpolarisation. K+ effluxing through IKCa 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 KCa channels. During vessel contraction, K+ efflux through activated myocyte BKCa channels generates intravascular K+ clouds. These compromise activation of Na+/K+-ATPases and KIR 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, H2O2 and vasoactive peptides as well as prostacyclin and epoxyeicosatrienoic acids. These hyperpolarise myocytes by opening various populations of myocyte potassium channels, but predominantly BKCa and/or KATP, which are sensitive to blockade by iberiotoxin or glibenclamide, respectively.

The effects of BRL 34915 and nicorandil on electrical and mechanical activity and on 86Rb efflux in rat blood vessels

1 The effects of the antihypertensive agent BRL 34915 on a variety of responses of the aorta and portal vein of the rat have been compared with those of nicorandil. 2 On portal vein, BRL 34915 (0.01–50 × 10−6 M) and nicorandil (0.1–500 × 10−6 M) abolished spontaneous mechanical activity and reduced mechanical responses to noradrenaline (0.1–100 × 10−6 M) and K+ (5–20 × 10−3 M) but had little inhibitory effect on responses to K+ (40–80 × 10−3 M). The onset of the reduced responses to noradrenaline was delayed by both agents. 3 On portal vein, BRL 34915 (0.1–50 × 10−6 M) and nicorandil (0.5–500 × 10−6 M) abolished spontaneous electrical and mechanical activity, hyperpolarized the smooth muscle cells to a value close to their calculated potassium equilibrium potential and increased the 86Rb efflux rate coefficient. 4 On aorta, BRL34915 (0.2‐0.8 × 10−6 M) and nicorandil (8–32 × 10−6 M) reduced mechanical responses to noradrenaline (0.001‐1 × 10−6 M) and K+ (5–20 × 10−3 M) but had little inhibitory effect on responses to K+ (40–80 × 10−3 M). 5 On aorta, BRL 34915 (0.2‐0.8 × 10−6 M) increased the 86Rb efflux rate coefficient whereas nicorandil (8–32 × 10−6 M) was without effect. 6 It is concluded that the inhibitory actions of BRL 34915 on both aorta and portal vein result from the opening of membrane potassium channels. The resulting membrane shunt inhibits the effects of excitatory agents. The inhibitory effects of nicorandil result from a combination of the opening of potassium channels together with an additional, undefined action.

Structure-activity relationships of K+ channel openers

Seven groups of synthetic agent, distinguished by a combination of their chemical and pharmacological characteristics exert some or all of their effects by opening plasmalemmal K+ channels primarily in smooth muscle. Progress over the past two years now allows broad structure-activity relationships to be formulated within many of the individual groups of agent. Gillian Edwards and Arthur Weston review the historical basis of these discoveries and comment on the significance of new developments. They focus on the search for tissue and channel selectivity, two factors likely to be important for the successful clinical deployment of these substances as antihypertensive and bronchodilator agents.

Characterization of an apamin-sensitive small-conductance Ca2+-activated K+ channel in porcine coronary artery endothelium: relevance to EDHF

The apamin‐sensitive small‐conductance Ca2+‐activated K+ channel (SKCa) was characterized in porcine coronary arteries. In intact arteries, 100 nM substance P and 600 μM 1‐ethyl‐2‐benzimidazolinone (1‐EBIO) produced endothelial cell hyperpolarizations (27.8±0.8 mV and 24.1±1.0 mV, respectively). Charybdotoxin (100 nM) abolished the 1‐EBIO response but substance P continued to induce a hyperpolarization (25.8±0.3 mV). In freshly‐isolated endothelial cells, outside‐out patch recordings revealed a unitary K+ conductance of 6.8±0.04 pS. The open‐probability was increased by Ca2+ and reduced by apamin (100 nM). Substance P activated an outward current under whole‐cell perforated‐patch conditions and a component of this current (38%) was inhibited by apamin. A second conductance of 2.7±0.03 pS inhibited by d‐tubocurarine was observed infrequently. Messenger RNA encoding the SK2 and SK3, but not the SK1, subunits of SKCa was detected by RT – PCR in samples of endothelium. Western blotting indicated that SK3 protein was abundant in samples of endothelium compared to whole arteries. SK2 protein was present in whole artery nuclear fractions. Immunofluorescent labelling confirmed that SK3 was highly expressed at the plasmalemma of endothelial cells and was not expressed in smooth muscle. SK2 was restricted to the peri‐nuclear regions of both endothelial and smooth muscle cells. In conclusion, the porcine coronary artery endothelium expresses an apamin‐sensitive SKCa containing the SK3 subunit. These channels are likely to confer all or part of the apamin‐sensitive component of the endothelium‐derived hyperpolarizing factor (EDHF) response.

Characterization of a charybdotoxin-sensitive intermediate conductance Ca2+-activated K+ channel in porcine coronary endothelium: relevance to EDHF

This study characterizes the K+ channel(s) underlying charybdotoxin‐sensitive hyperpolarization of porcine coronary artery endothelium. Two forms of current‐voltage (I/V) relationship were evident in whole‐cell patch‐clamp recordings of freshly‐isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, charybdotoxin also inhibited a current but did not alter the form. Single‐channel, outside‐out patch recordings revealed a 17.1±0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca2+ demonstrated that the open probability was increased by Ca2+. This channel was blocked by charybdotoxin but not by iberiotoxin or apamin. Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1±0.7 mV) were reduced by apamin (100 nM; 17.0±1.8 mV) whereas those to 1‐ethyl‐2‐benzimidazolinone (1‐EBIO, 600 μM, 21.0±0.3 mV) were unaffected (21.7±0.8 mV). Substance P, bradykinin (100 nM) and 1‐EBIO evoked charybdotoxin‐sensitive, iberiotoxin‐insensitive whole‐cell perforated‐patch currents. A porcine homologue of the intermediate‐conductance Ca2+‐activated K+ channel (IK1) was identified in endothelial cells. In conclusion, porcine coronary artery endothelial cells express an intermediate‐conductance Ca2+‐activated K+ channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the charybdotoxin‐sensitive component of the EDHF response.