Gillian Edwards

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.

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.

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.

Role of gap junctions in the responses to EDHF in rat and guinea‐pig small arteries

In guinea‐pig internal carotid arteries with an intact endothelium, acetylcholine (10 μM) and levcromakalim (10 μM) each hyperpolarized the smooth muscle whereas a 5 mM elevation of extracellular K+ was without effect. Incubation of the carotid artery with the gap junction inhibitors carbenoxolone (100 μM) or gap 27 (500 μM) essentially abolished the hyperpolarization to acetylcholine but it was without effect on that to levcromakalim. Carbenoxolone had no effect on the acetylcholine‐induced endothelial cell hyperpolarization but inhibited the smooth muscle hyperpolarization induced by the endothelial cell K+ channel opener, 1‐ethyl‐2‐benzimidazolinone (600 μM). In rat hepatic and mesenteric arteries with endothelium, carbenoxolone (100 or 500 μM) depolarized the smooth muscle but did not modify hyperpolarizations induced by KCl or levcromakalim. In the mesenteric (but not the hepatic) artery, the acetylcholine‐induced hyperpolarization was inhibited by carbenoxolone. Phenylephrine (1 μM) depolarized the smooth muscle cells of intact hepatic and mesenteric arteries, an effect enhanced by carbenoxolone. Gap 27 did not have a depolarizing action. In the presence of phenylephrine, acetylcholine‐induced hyperpolarization of both hepatic and mesenteric artery myocytes was partially inhibited by each of the gap junction inhibitors. Collectively, the data suggest that gap junctions play some role in the EDHF (endothelium‐derived hyperpolarizing factor) response in rat hepatic and mesenteric arteries. However, in the guinea‐pig internal carotid artery, electrotonic propagation of endothelial cell hyperpolarizations via gap junctions may be the sole mechanism underlying the response previously attributed to EDHF.

Ion channel modulation by NS 1619, the putative BKCa channel opener, in vascular smooth muscle

1 The effects of NS 1619, the putative BKCa channel opener, were investigated on rat intact portal veins and on single smooth muscle cells enzymatically separated from the same tissue. 2 Under whole‐cell patch clamp conditions with K‐rich pipettes, exposure of single cells held at −10 mV to NS 1619 (10–33 μm) induced a noisy, outward current which reached a maximum (33 μm NS 1619; mean 35.8 ± 17 pA, n = 8) within about 6min. 3 On stepping to test potentials (range − 50 to + 50 mV) from a holding potential of − 10 mV, the NS 1619‐induced noisy current exhibited time‐dependent activation and marked outward rectification. 4 The stimulation of outward currents by NS 1619 at − 10 mV was independent of the presence of Ca2+ in the bath or pipette solutions but was antagonized by either charybdotoxin (250 nm) or penitrem A (100 nm) in the bath solution. 5 Stationary fluctuation analysis of the noisy current induced by NS 1619 at − 10 mV yielded a value of 70 ± 8 pS (n = 4) (under the quasi‐physiological conditions of the experiment) for the unitary conductance of the channel involved. 6 At − 10 mV, NS 1619 (10–33 μm) rapidly inhibited spontaneous transient outward currents. 7 With a holding potential of − 90 mV, NS 1619 (10–33 μm) produced a reduction of outward currents evoked by depolarizing steps to + 50 mV, an effect associated with marked inhibition of the delayed rectifier current, IK(V). 8 NS 1619 (3–100 μm) produced a concentration‐dependent inhibition of spontaneous activity in rat portal vein characterized by a reduction in the amplitude and duration of the tension waves. This inhibition was slightly potentiated in the presence of either charybdotoxin (250 nm) or penitrem A (1 μm). NS 1619 also totally inhibited contractions of rat aorta induced by KC1 (both 20 mm and 80 μm). 9 Under whole‐cell recording conditions and using Cs‐rich pipettes, Ca‐currents evoked in portal vein cells by stepping from a holding potential of − 90 mV to test potentials in the range − 30 to + 50 mV were totally inhibited in the presence of 33 μm NS 1619. 10 NS 1619 (33 μm) inhibited the induction of IK(ATP) by levcromakalim (10 μm). 11 It is concluded that NS 1619 activates the large conductance, Ca2+‐sensitive channel, BKCa and over the same concentration range it inhibits both Kv and L‐type Ca‐channels. The observed NS 1619‐induced mechanical inhibition in rat portal vein and aorta seems most likely to be due to the observed inhibition of Ca‐currents.

Evidence in Favor of a Calcium-Sensing Receptor in Arterial Endothelial Cells: Studies With Calindol and Calhex 231

Small increases in extracellular Ca2+ dilate isolated blood vessels. In the present study, the possibility that a vascular, extracellular Ca2+-sensing receptor (CaSR) could mediate these vasodilator actions was investigated. Novel ligands that interact with the CaSR were used in microelectrode recordings from rat isolated mesenteric and porcine coronary arteries. The major findings were that (1) raising extracellular Ca2+ or adding calindol, a CaSR agonist, produced concentration-dependent hyperpolarizations of vascular myocytes, actions attenuated by Calhex 231, a negative allosteric modulator of CaSR. (2) Calindol-induced hyperpolarizations were inhibited by the intermediate conductance, Ca2+-sensitive K+ (IKCa) channel inhibitors, TRAM-34, and TRAM-39. (3) The effects of calindol were not observed in the absence of endothelium. (4) CaSR mRNA and protein were present in rat mesenteric arteries and in porcine coronary artery endothelial cells. (5) CaSR and IKCa proteins were restricted to caveolin-poor membrane fractions. We conclude that activation of vascular endothelial CaSRs opens endothelial cell IKCa channels with subsequent myocyte hyperpolarization. The endothelial cell CaSR may have a physiological role in the control of arterial blood pressure.

Role of gap junctions and EETs in endothelium-dependent hyperpolarization of porcine coronary artery

The effects of endothelium‐derived hyperpolarizing factor (EDHF: elicited using substance P or bradykinin) were compared with those of 11,12‐EET in pig coronary artery. Smooth muscle cells were usually impaled with microelectrodes through the adventitial surface. Substance P (100 nM) and 11,12‐EET (11,12‐epoxyeicosatrienoic acid; 3 μM) hyperpolarized endothelial cells in intact arteries. These actions were unaffected by 100 nM iberiotoxin but were abolished by charybdotoxin plus apamin (each 100 nM). Substance P (100 nM) and bradykinin (30 nM) hyperpolarized intact artery smooth muscle; Substance P had no effect after endothelium removal. 11,12‐EET hyperpolarized de‐endothelialized vessels by 12.6±0.3 mV, an effect abolished by 100 nM iberiotoxin. 11,12‐EET hyperpolarized intact arteries by 18.6±0.8 mV, an action reduced by iberiotoxin, which was ineffective against substance P. Hyperpolarizations to 11,12‐EET and substance P were partially inhibited by 100 nM charybdotoxin and abolished by further addition of 100 nM apamin. 30 μM barium plus 500 nM ouabain depolarized intact artery smooth muscle but responses to substance P and bradykinin were unchanged. 500 μM gap 27 markedly reduced hyperpolarizations to substance P and bradykinin which were abolished in the additional presence of barium plus ouabain. Substance P‐induced hyperpolarizations of smooth muscle cells immediately below the internal elastic lamina were unaffected by gap 27, even in the presence of barium plus ouabain. In pig coronary artery, 11,12‐EET is not EDHF. Smooth muscle hyperpolarizations attributed to ‘EDHF’ are initiated by endothelial cell hyperpolarization involving charybdotoxin‐ (but not iberiotoxin) and apamin‐sensitive K+ channels. This may spread electrotonically via myoendothelial gap junctions but the involvement of an unknown endothelial factor cannot be excluded.