Endothelium-dependent Smooth Muscle Hyperpolarization Research Articles

Interactions between thromboxane A2, thromboxane/prostaglandin (TP) receptors, and endothelium-derived hyperpolarization

Endothelium-dependent smooth muscle hyperpolarization (EDH) increasingly predominates over endothelium-derived nitric oxide (NO) as a participant in vasodilation as vessel size decreases. Its underlying nature is highly variable between vessel types, species, disease states, and exact experimental conditions, and is variably mediated by one or more transferable endothelium-derived hyperpolarizing factors and/or the electrotonic spread of endothelial hyperpolarization into the media via gap junctions. Although generally regarded (and studied) as a mechanism that is independent of NO and prostanoids, evidence has emerged that the endothelium-derived contracting factor and prostanoid thromboxane A2 can modulate several signalling components central to EDH, and therefore potentially curtail vasodilation through mechanisms that are distinct from those putatively involved in direct smooth muscle contraction. Notably, vascular production of thromboxane A2 is elevated in a number of cardiovascular disease states that promote endothelial dysfunction. This review will therefore discuss the mechanisms through which thromboxane A2 interacts with and modulates EDH, and will also consider the implications of such cross-talk in vasodilator control in health and disease.

Endothelial control of vasodilation: integration of myoendothelial microdomain signalling and modulation by epoxyeicosatrienoic acids

Endothelium-derived epoxyeicosatrienoic acids (EETs) are fatty acid epoxides that play an important role in the control of vascular tone in selected coronary, renal, carotid, cerebral and skeletal muscle arteries. Vasodilation due to endothelium-dependent smooth muscle hyperpolarization (EDH) has been suggested to involve EETs as a transferable endothelium-derived hyperpolarizing factor. However, this activity may also be due to EETs interacting with the components of other primary EDH-mediated vasodilator mechanisms. Indeed, the transfer of hyperpolarization initiated in the endothelium to the adjacent smooth muscle via gap junction connexins occurs separately or synergistically with the release of K(+) ions at discrete myoendothelial microdomain signalling sites. The net effects of such activity are smooth muscle hyperpolarization, closure of voltage-dependent Ca(2+) channels, phospholipase C deactivation and vasodilation. The spatially localized and key components of the microdomain signalling complex are the inositol 1,4,5-trisphosphate receptor-mediated endoplasmic reticulum Ca(2+) store, Ca(2+)-activated K(+) (KCa), transient receptor potential (TRP) and inward-rectifying K(+) channels, gap junctions and the smooth muscle Na(+)/K(+)-ATPase. Of these, TRP channels and connexins are key endothelial effector targets modulated by EETs. In an integrated manner, endogenous EETs enhance extracellular Ca(2+) influx (thereby amplifying and prolonging KCa-mediated endothelial hyperpolarization) and also facilitate the conduction of this hyperpolarization to spatially remote vessel regions. The contribution of EETs and the receptor and channel subtypes involved in EDH-related microdomain signalling, as a candidate for a universal EDH-mediated vasodilator mechanism, vary with vascular bed, species, development and disease and thus represent potentially selective targets for modulating specific artery function.

Endothelial H 2 O 2

About a decade ago everyone who read, at least occasionally, the science part of the local newspapers or followed popular science broadcasts “knew” that reactive oxygen species (ROS) are dangerous. They were thought to be bad, really bad, and many looking for a healthier lifestyle took action against these nasty small molecules accused of harming lipids and proteins by taking antioxidants like vitamin E. Food industry teamed up with pharmaceutical companies and brought up functional food enriched or supplemented with trustful antioxidants in the fight against ROS, which seemed to be produced under pathological conditions with just one mission: to damage the body. Things have changed during recent years, and there is emerging evidence that ROS are not only continuously produced even in healthy individuals but also exert important physiological functions. While we are still in-between a period that provides exciting new findings on the physiology of ROS, one can already summarize our present knowledge rather accurately by citing Paracelsus’ adage of “ Dosis (sola) facit venenum ” ( tertio defensio , 1538). See accompanying article on page 1774 The present work of Edwards et al1 is one of these exciting new reports that open our eyes to recognize the good side of the previously dammed ROS. This article deals with the effect of hydrogen peroxide (H2O2) on endothelium-derived hyperpolarization and is quite remarkable in several ways: First, the article is focused on EDHF (formerly termed endothelium-derived hyperpolarizing factor), a nitric oxide (NO) and prostacyclin-independent phenomenon of endothelium-dependent relaxation, of which the molecular mechanism and nature are still somewhat mysterious. Thus, this endothelium-dependent smooth muscle hyperpolarization has been attributed to either the formation and release of diffusible factors from the endothelium2 such as potassium ions (K+),3 cytochrome P450-derived arachidonic acid metabolites (epoxyeicosatrienoic acids), …

Closing the Gap at Hot Spots

See related article, pages 1026–1035 Coordination of cellular behavior is indispensable for vascular function. Because of the structural design of the vascular tree only an orchestrated diameter change along the vessel length increases conductance significantly and ensures optimal tissue perfusion. Mechanisms that suffice these needs include locally generated signals at upstream sites which reflect downstream requirements (flow-induced dilation)1 and longitudinal signal transmission through communication channels residing in the vessel wall itself which enable the synchronisation of cellular behavior. This latter is achieved by homocellular gap junctions which are composed of connexins that connect adjacent cells by intercellular low-resistance channels establishing a functional syncitium.2 Electrical communication through these channels is experimentally studied by locally initiated vasomotor responses which conduct along the vessel wall and promote synchronized dilations or constrictions of arteriolar segments reflecting the coordination of cellular behavior.3 Longitudinal communication through gap junctions is also required for the synchronisation of intracellular Ca2+ ([Ca2+]i) changes in smooth muscle and subsequent vasomotion, ie, rhythmic oscillations of vascular diameter. Although the physiological significance of vasomotion is still to be clearly defined it depends critically on Ca2+ release from intracellular stores which occurs in an oscillating fashion and the synchronization of [Ca2+]i transients through gap junctional communication.4,5 In addition, endothelial and smooth muscle cells are coupled heterocellularly (myoendothelial junctions) allowing the direct spread of current from the endothelium to the smooth muscle6 or other signaling molecules.7 The importance of gap junctional communication in vascular function contrasts with the poor knowledge of its regulation in vessels which is complicated by the existence of functional and structural different types of gap junctions. Moreover, longterm regulation because of degradation or upregulation of connexins has to be separated from shortterm effects which reflect an …

Analysis of effects of connexin-mimetic peptides in rat mesenteric small arteries

Synthetic peptides homologous to the extracellular loops of the major vascular connexins represent a novel class of gap junction blockers that have been used to assess the role of direct cellular communication in arteries and veins. However, the specificity of action of such peptides on the coupling between smooth muscle cells (SMCs) has not yet been fully characterized. Isolated third-order rat mesenteric arteries were therefore studied with respect to isometric tension (myography), intracellular Ca2+ concentration ([Ca2+]i) (Ca2+ -sensitive dyes), membrane potential, and input resistance (sharp intracellular glass electrodes). Confocal imaging was used for visualization of [Ca2+]i events in individual SMCs in the arterial wall and membrane currents (patch clamp) measured in individual SMCs isolated from the same arteries. A triple peptide combination (37,43Gap 27 + 40Gap 27 + 43Gap 26) increased intercellular resistance (measured as input resistance) in intact arterial segments without affecting the membrane conductance of individual cells and also interrupted electrical coupling between pairs of rat aortic A7r5 myocytes. In intact arterial segments, the peptides desynchronized [Ca2+]i transients in individual SMCs and abolished vasomotion without suppressing Ca2+ transients in individual cells. They also depolarized SMCs, increased [Ca2+]i, and attenuated acetylcholine-induced, endothelium-dependent smooth muscle hyperpolarization. Experiments with endothelium-denuded arteries suggested that the depolarization produced by the peptides under basal conditions was in part secondary to electrical uncoupling of the endothelium from SMCs with loss of a tonic hyperpolarizing effect of the endothelium. Taken together, the results indicate that connexin-mimetic peptides block electrical signaling in rat mesenteric small arteries without exerting major nonjunctional effects.

The obligatory link: role of gap junctional communication in endothelium-dependent smooth muscle hyperpolarization

Although an endothelium-derived hyperpolarizing factor (EDHF) has often been hypothesized to underpin vascular relaxations that are independent of nitric oxide (NO) and prostanoids, bioassay techniques have failed to confirm the existence of a freely transferable EDHF in a consistent fashion. Indeed, observations that inhibitors of direct cell–cell coupling such as connexin–mimetic peptides (e.g. Gap 26 and 27) and glycyrrhetinic acid derivatives attenuate “EDHF-type” smooth muscle hyperpolarizations and relaxations suggest that an electrotonic spread of endothelial hyperpolarization via myoendothelial and homocellular smooth muscle gap junctions plays an obligatory role in such responses. The endothelial hyperpolarization that initiates relaxation results from the opening of K Ca channels and is sustained by capacitative Ca 2+ entry triggered by the depletion of intracellular Ca 2+ stores in the endoplasmic reticulum. EDHF-type relaxations are also associated with a prostanoid-independent synthesis of cAMP that increases the conductance of gap junction channels and enhances the transmission of endothelial hyperpolarization through the vascular wall in a permissive fashion. This review will discuss the roles of these interacting signalling pathways in the mediation of the EDHF phenomenon.

Endothelium-independent, ouabain-sensitive relaxation of bovine coronary arteries by EETs.

Endothelium-derived hyperpolarizing factor (EDHF) is released in response to agonists such as ACh and bradykinin and regulates vascular smooth muscle tone. Several studies have indicated that ouabain blocks agonist-induced, endothelium-dependent hyperpolarization of smooth muscle. We have demonstrated that epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of arachidonic acid, function as EDHFs. To further test the hypothesis that EETs represent EDHFs, we have examined the effects of ouabain on the electrical and mechanical effects of 14,15- and 11,12-EET in bovine coronary arteries. These arteries are relaxed in a concentration-dependent manner to 14,15- and 11,12-EET (EC(50) = 6 x 10(-7) M), bradykinin (EC(50) = 1 x 10(-9) M), sodium nitroprusside (SNP; EC(50) = 2 x 10(-7) M), and bimakalim (BMK; EC(50) = 1 x 10(-7) M). 11,12-EET-induced relaxations were identical in vessels with and without an endothelium. Potassium chloride (1-15 x 10(-3) M) inhibited [(3)H]ouabain binding to smooth muscle cells but failed to relax the arteries. Ouabain (10(-5) to 10(-4) M) increased basal tone and inhibited the relaxations to bradykinin, 11,12-EET, and 14,15-EET, but not to SNP or BMK. Barium (3 x 10(-5) M) did not alter EET-induced relaxations and ouabain plus barium was similar to ouabain alone. Resting membrane potential (E(m)) of isolated smooth muscle cells was -50.2 +/- 0.5 mV. Ouabain (3 x 10(-5) and 1 x 10(-4) M) decreased E(m) (-48.4 +/- 0.2 mV), whereas 11,12-EET (10(-7) M) increased E(m) (-59.2 +/- 2.2 mV). Ouabain inhibited the 11,12-EET-induced increase in E(m). In cell-attached patch clamp studies, 11,12-EET significantly increased the open-state probability (NP(o)) of a calcium-activated potassium channel compared with control cells (0.26 +/- 0.06 vs. 0.02 +/- 0.01). Ouabain did not change NP(o) but blocked the 14,15-EET-induced increase in NP(o). These results indicate that: 1) EETs relax coronary arteries in an endothelium-independent manner, 2) unlike EETs, potassium chloride does not relax the coronary artery, and 3) ouabain inhibits bradykinin- and EET-induced relaxations as has been reported for EDHF. These findings provide further evidence that EETs are EDHFs.

Role of endothelial cell hyperpolarization in EDHF-mediated responses in the guinea-pig carotid artery.

1. Experiments were performed to identify the potassium channels involved in the acetylcholine-induced endothelium-dependent hyperpolarization of the guinea-pig internal carotid artery. Smooth muscle and endothelial cell membrane potentials were recorded in isolated arteries with intracellular microelectrodes. Potassium currents were recorded in freshly-dissociated smooth muscle cells using patch clamp techniques. 2. In single myocytes, iberiotoxin (0.1 microM)-, charybdotoxin (0.1 microM)-, apamin (0.5 microM)- and 4-aminopyridine (5 mM)-sensitive potassium currents were identified indicating the presence of large- and small-conductance calcium-sensitive potassium channels (BK(Ca) and SK(Ca)) as well as voltage-dependent potassium channels (K(V)). Charybdotoxin and iberiotoxin inhibited the same population of BK(Ca) but a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected. 4-aminopyridine (0. 1 - 25 mM) induced a concentration-dependent inhibition of K(V) without affecting the iberiotoxin- or the apamin-sensitive currents. 3. In isolated arteries, both the endothelium-dependent hyperpolarization of smooth muscle and the hyperpolarization of endothelial cells induced by acetylcholine or by substance P were inhibited by 5 mM 4-aminopyridine. 4. These results indicate that in the vascular smooth muscle cells of the guinea-pig carotid artery, a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected, comforting the hypothesis that the combination of these two toxins should act on the endothelial cells. Furthermore, the inhibition by 4-aminopyridine of both smooth muscle and endothelial hyperpolarizations, suggests that in order to observe an endothelium-dependent hyperpolarization of the vascular smooth muscle cells, the activation of endothelial potassium channels is likely to be required.

Further investigation of endothelium-derived hyperpolarizing factor (EDHF) in rat hepatic artery: studies using 1-EBIO and ouabain.

1. The characteristics of endothelium-dependent hyperpolarization in rat hepatic artery have been further investigated in the presence of inhibitors of cyclo-oxygenase and nitric oxide synthase. 2. Using sharp micro-electrodes, the smooth muscle hyperpolarization induced by acetylcholine, KCl or 1-ethyl-2-benzimidazolinone (1-EBIO) in intact hepatic arteries was abolished by 30 micronM barium plus 500 nM ouabain. 3. In vessels without endothelium, the smooth muscle hyperpolarization induced by KCl was not reduced by 30 micronM barium alone. However, in the presence of barium the effects of KCl were partially inhibited by 100 nM ouabain and essentially abolished by 500 nM ouabain. 4. Using sharp micro-electrodes, the hyperpolarization of both the smooth muscle and the endothelium induced by 1-EBIO or by acetylcholine was unaffected by 100 nM iberiotoxin. However, in the presence of 100 nM charybdotoxin, the effects of 1-EBIO were abolished whereas those of acetylcholine were only partially reduced. The hyperpolarization induced by levcromakalim was unaffected by either charybdotoxin or iberiotoxin. 5 Under whole-cell patch-clamp recording conditions, 1-EBIO induced a voltage-insensitive, charybdotoxin-sensitive K+ current in cultured endothelial cells but was without effect on K+ currents in smooth muscle cells isolated from hepatic arteries. 6 It is concluded that the endothelium-dependent hyperpolarization of smooth muscle induced by either acetylcholine or by 1-EBIO in rat hepatic artery is initially associated with the opening of endothelial calcium-sensitive K+-channels insensitive to iberiotoxin. The resulting accumulation of K+ in the myoendothelial space activates an isoform of Na+/K+-ATPase which is sensitive to low concentrations of ouabain.

Role of Heterocellular Gap Junctional Communication in Endothelium-Dependent Smooth Muscle Hyperpolarization: Inhibition by a Connexin-Mimetic Peptide

A synthetic connexin-mimetic peptide (Gap 27 peptide) was used to evaluate the contribution of gap junctional communication to smooth muscle responses mediated by the endothelium-dependent agonist acetylcholine (ACh) in rabbit mesenteric arteries. Hyperpolarizations and relaxations to 0.1 and 1 μM ACh observed in the presence of nitric oxide synthase and cyclooxygenase inhibition were markedly attenuated by the peptide at a concentration of 300 μM, whereas the hyperpolarizing response to levcromakalim, aKATPchannel opener, was unaffected. The peptide also attenuated intercellular transfer of Lucifer yellow in confluent cultures of COS-7 cells, thus confirming its ability to modulate the permeability of gap junctions. The findings demonstrate that heterocellular gap junctional communication contributes to NO- and prostanoid-independent mechanisms of vasorelaxation that are widely attributed to an endothelium-derived hyperpolarizing factor.

Role of potassium channels in endothelium-dependent relaxation resistant to nitroarginine in the rat hepatic artery.

1. In the presence of indomethacin (IM, 10 microM) and N omega-nitro-L- arginine (L-NOARG, 0.3 mM), acetylcholine (ACh) induces an endothelium-dependent smooth muscle hyperpolarization and relaxation in the rat isolated hepatic artery. The potassium (K) channel inhibitors, tetrabutylammonium (TBA, 1 mM) and to a lesser extent 4-aminopyridine (4-AP, 1 mM) inhibited the L-NOARG/IM-resistant relaxation induced by ACh, whereas apamin (0.1-0.3 microM), charybdotoxin (0.1-0.3 microM), iberiotoxin (0.1 microM) and dendrotoxin (0.1 microM) each had no effect. TBA also inhibited the relaxation induced by the receptor-independent endothelial cell activator, A23187. 2. When combined, apamin (0.1 microM) + charybdotoxin (0.1 microM), but not apamin (0.1 microM) + iberiotoxin (0.1 microM) or a triple combination of 4-AP (1 mM) + apamin (0.1 microM) + iberiotoxin (0.1 microM), inhibited the L-NOARG/IM-resistant relaxation induced by ACh. At a concentration of 0.3 microM, apamin + charybdotoxin completely inhibited the relaxation. This toxin combination also abolished the L-NOARG/ IM-resistant relaxation induced by A23187. 3. In the absence of L-NOARG, TBA (1 mM) inhibited the ACh-induced relaxation, whereas charybdotoxin (0.3 microM) + apamin (0.3 microM) had no effect, indicating that the toxin combination did not interfere with the L-arginine/NO pathway. 4. The gap junction inhibitors halothane (2 mM) and 1-heptanol (2 mM), or replacement of NaCl with sodium propionate did not affect the L-NOARG/IM-resistant relaxation induced by ACh. 5. Inhibition of Na+/K(+)-ATPase by ouabain (1 mM) had no effect on the L-NOARG/IM-resistant relaxation induced by ACh. Exposure to a K(+)-free Krebs solution, however, reduced the maximal relaxation by 13% without affecting the sensitivity to ACh. 6. The results suggest that the L-NOARG/IM-resistant relaxation induced by ACh in the rat hepatic artery is mediated by activation of K-channels sensitive to TBA and a combination of apamin + charybdotoxin. Chloride channels, Na+/K(+)-ATPase and gap junctions are probably not involved in the response. It is proposed that endothelial cell activation induces secretion of an endothelium-derived hyperpolarizing factor(s) (EDHF), distinct from NO and cyclo-oxygenase products, which activates more than one type of K-channel on the smooth muscle cells. Alternatively, a single type of K-channel, to which both apamin and charybdotoxin must bind for inhibition to occur, may be the target for EDHF.

Endothelium-dependent relaxation and hyperpolarization in aorta from control and renal hypertensive rats.

Endothelium-dependent relaxations are depressed in hypertension. In this study we investigated the possible involvement of endothelium-dependent smooth muscle hyperpolarization in this phenomenon. In isolated aortic segments from control rats, acetylcholine (10(-8)-10(-5) M) elicits relaxations after precontraction with norepinephrine (10(-7) M), and acetylcholine or carbachol (10(-5) M) induce smooth muscle hyperpolarization (10.6 +/- 0.9 mV). Both effects disappear after removal of the endothelium and are depressed by tetraethylammonium (3 x 10(-3) M), a rather nonspecific blocker of K+ channels, but not by glibenclamide (10(-5) M), a potent blocker of the ATP-regulated K+ channels, which has a marked effect on the relaxation induced by BRL 38227. The relaxation effect of acetylcholine is impaired in norepinephrine-contracted preparations from hypertensive rats but is not further depressed by tetraethylammonium. In aorta from hypertensive rats, hyperpolarization induced by carbachol was significantly reduced to a mean of only 21.8% of the values obtained in preparations from normotensive rats. From the relaxation-hyperpolarization relation obtained with BRL 38227 (opening K+ channels), it is derived that the endothelium-dependent hyperpolarization (approximately 10 mV) contributes for at least 20-30% of the maximal relaxation effect of acetylcholine on rat aorta. It is concluded that the diminished endothelium-dependent hyperpolarization may contribute to the depression of the endothelium-dependent relaxation in hypertension.

Thimerosal hyperpolarizes arterial smooth muscles in an endothelium-dependent manner

Thimerosal activates the production of endothelium-derived relaxing factor (EDRF). I examined whether thimerosal also causes the release of an endothelium-dependent hyperpolarizing factor (EDHF). Thimerosal caused an endothelium-dependent hyperpolarization of smooth muscle. This effect is unlikely to be caused by the property of thimerosal to inhibit the acyl-coenzyme A: lysolecithin acyltransferase (LAT) since neither arachidonic acid nor lysolecithin hyperpolarized the muscles. I conclude that thimerosal is not a pure activator of EDRF (nitric oxide) production, but that it also releases a distinct EDHF.