Endothelial Hyperpolarization Research Articles

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.

N‐arachidonoyl glycine suppresses Na+/Ca2+ exchanger‐mediated Ca2+ entry into endothelial cells and activates BKCa channels independently of GPCRs

Background and PurposeN-arachidonoyl glycine (NAGly) is a lipoamino acid with vasorelaxant properties. We aimed to explore the mechanisms of NAGly's action on unstimulated and agonist-stimulated endothelial cells.Experimental ApproachThe effects of NAGly on endothelial electrical signalling were studied in combination with vascular reactivity.Key ResultsIn EA.hy926 cells, the sustained hyperpolarization to histamine was inhibited by the non-selective Na+/Ca2+ exchanger (NCX) inhibitor bepridil and by an inhibitor of reversed mode NCX, KB-R7943. In cells dialysed with Cs+-based Na+-containing solution, the outwardly rectifying current with typical characteristics of NCX was augmented following histamine exposure, further increased upon external Na+ withdrawal and inhibited by bepridil. NAGly (0.3–30 μM) suppressed NCX currents in a URB597- and guanosine 5′-O-(2-thiodiphosphate) (GDPβS)-insensitive manner, [Ca2+]i elevation evoked by Na+ removal and the hyperpolarization to histamine. In rat aorta, NAGly opposed the endothelial hyperpolarization and relaxation response to ACh. In unstimulated EA.hy926 cells, NAGly potentiated the whole-cell current attributable to large-conductance Ca2+-activated K+ (BKCa) channels in a GDPβS-insensitive, paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches, NAGly stimulated single BKCa channel activity.Conclusion and ImplicationsOur data showed that NCX is a Ca2+ entry pathway in endothelial cells and that NAGly is a potent G-protein-independent modulator of endothelial electrical signalling and has a dual effect on endothelial electrical responses. In agonist pre-stimulated cells, NAGly opposes hyperpolarization and relaxation via inhibition of NCX-mediated Ca2+ entry, while in unstimulated cells, it promotes hyperpolarization via receptor-independent activation of BKCa channels.

Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

BackgroundArachidonic acid (AA) and/or its enzymatic metabolites are important lipid mediators contributing to endothelium‐derived hyperpolarizing factor (EDHF)–mediated dilation in multiple vascular beds, including human coronary arterioles (HCAs). However, the mechanisms of action of these lipid mediators in endothelial cells (ECs) remain incompletely defined. In this study, we investigated the role of the transient receptor potential vanilloid 4 (TRPV4) channel in AA‐induced endothelial Ca2+ response and dilation of HCAs.Methods and ResultsAA induced concentration‐dependent dilation in isolated HCAs. The dilation was largely abolished by the TRPV4 antagonist RN‐1734 and by inhibition of endothelial Ca2+‐activated K+ channels. In native and TRPV4‐overexpressing human coronary artery ECs (HCAECs), AA increased intracellular Ca2+ concentration ([Ca2+]i), which was mediated by TRPV4‐dependent Ca2+ entry. The AA‐induced [Ca2+]i increase was inhibited by cytochrome P450 (CYP) inhibitors. Surprisingly, the CYP metabolites of AA, epoxyeicosatrienoic acids (EETs), were much less potent activators of TRPV4, and CYP inhibitors did not affect EET production in HCAECs. Apart from its effect on [Ca2+]i, AA induced endothelial hyperpolarization, and this effect was required for Ca2+ entry through TRPV4. AA‐induced and TRPV4‐mediated Ca2+ entry was also inhibited by the protein kinase A inhibitor PKI. TRPV4 exhibited a basal level of phosphorylation, which was inhibited by PKI. Patch‐clamp studies indicated that AA activated TRPV4 single‐channel currents in cell‐attached and inside‐out patches of HCAECs.ConclusionsAA dilates HCAs through a novel mechanism involving endothelial TRPV4 channel‐dependent Ca2+ entry that requires endothelial hyperpolarization, PKA‐mediated basal phosphorylation of TRPV4, and direct activation of TRPV4 channels by AA.

Inwardly rectifying potassium channels 2.x contribute to endothelial hyperpolarization and vasorelaxation to P2Y receptor stimulation in murine cerebral artery

Inwardly rectifying potassium channels (Kir) 2.x are expressed in cerebral endothelial cells (EC) but little is known about their role in vascular tone control. We tested the hypothesis that endothelial Kir amplify membrane hyperpolarization and cerebral vasodilation to ATP and UTP.MethodsExpression of Kir 2.1 and 2.2 was demonstrated in freshly isolated EC from rat middle cerebral artery (MCA) by PCR. Vasorelaxation (pressure myography) and endothelial hyperpolarization (di‐8‐ANEPPS) were measured in pressurized MCA following intraluminal delivery of UTP. Membrane current and voltage changes were recorded from mouse cerebral EC using perforated‐patch voltage clamp and current clamp techniques.ResultsUnstimulated EC showed Kir‐like (sensitive to Ba2+ and Kir 2.x blocker ML 133) and transient outward KCa‐like currents. Stimulation with ATP activated large outward currents that were sensitive to SKCa and IKCa blockers (UCL1684 and TRAM34). The ensuing membrane hyperpolarization was significantly attenuated in cells pretreated with Ba2+ or ML 133. In pressurized MCA, intraluminal application of Ba2+ significantly decreased hyperpolarization and relaxation responses to UTP.ConclusionEndothelial Kir2.x channels play a significant role in cerebral vasorelaxation by maintaining endothelial hyperpolarization in response to P2Y receptor stimulation. Supported by R01HL088435 to SPM

G Protein–Coupled Estrogen Receptor Agonist Improves Cerebral Microvascular Function After Hypoxia/Reoxygenation Injury in Male and Female Rats

Reduced risk and severity of stroke in adult females are thought to depend on normal levels of endogenous estrogen, which is a known neuro- and vasoprotective agent in experimental cerebral ischemia. Recently, a novel G protein-coupled estrogen receptor (GPER, formerly GPR30) has been identified and may mediate the vasomotor and -protective effects of estrogen. However, the signaling mechanisms associated with GPER in the cerebral microcirculation remain unclear. We investigated the mechanism of GPER-mediated vasoreactivity and also its vasoprotective effect after hypoxia/reoxygenation (H/RO) injury. Rat cerebral penetrating arterioles from both sexes were isolated, cannulated, and pressurized. Vessel diameters were recorded by computer-aided videomicroscopy. To investigate vasomotor mechanism of the GPER agonist (G-1), several inhibitors with or without endothelial impairment were tested. Ischemia/reperfusion injury was simulated using H/RO. Vasomotor responses to adenosine triphophate after H/RO were measured with or without G-1 and compared with controls. G-1 produced a vasodilatory response, which was partially dependent on endothelium-derived nitric oxide (NO) but not arachidonic acid cascades and endothelial hyperpolarization factor. Attenuation of G-1-vasodilation by the NO synthase inhibitor and endothelium-impairment were greater in vessels from female than male animals. G-1 treatment after H/RO injury fully restored arteriolar dilation to adenosine triphophate compared with controls. GPER agonist elicited dilation, which was partially caused by endothelial NO pathway and induced by direct relaxation of smooth muscle cells. Further, GPER agonist restored vessel function of arterioles after H/RO injury and may play an important role in the ability of estrogen to protect the cerebrovasculature against ischemia/reperfusion injury.

What is the Efficiency of ATP Signaling from Erythrocytes to Regulate Distribution of O2 Supply Within the Microvasculature?

Erythrocytes appear to be ideal sensors for regulating microvascular O(2) supply as they release the potent vasodilator ATP in an O(2) saturation-dependent manner. Whether erythrocytes play a significant role in regulating O(2) supply in the complex environment of diffusional O(2) exchange among capillaries, arterioles, and venules, depends on the efficiency with which erythrocytes signal the vascular endothelium. If one assumes that the distribution of purinergic receptors is uniform throughout the microvasculature, then the most efficient site for signaling should occur in capillaries, where the erythrocyte membrane is in close proximity to the endothelium. ATP released from erythrocytes would diffuse a short distance to P(2y) receptors inducing an increase in blood flow, possibly the result of endothelial hyperpolarization. We hypothesize that this hyperpolarization varies across the capillary bed depending upon erythrocyte supply rate and the flux of O(2) from these erythrocytes to support O(2) metabolism. This would suggest that the capillary bed would be the most effective site for erythrocytes to communicate tissue oxygen needs. Electrically coupled endothelial cells conduct the integrated signal upstream where arterioles adjust vascular resistance, thus enabling ATP released from erythrocytes to regulate the magnitude and distribution of O(2) supply to individual capillary networks.

Reduced hyperpolarization of endothelial cells following high dietary Na+: effects of enalapril and tempol

1. High dietary Na(+) is associated with impaired vascular endothelial function. However, the underlying mechanisms are not completely understood. In the present study, we investigated whether the endothelial hyperpolarization response to acetylcholine (ACh) exhibited any abnormalities in Wistar rats fed a high-salt diet (HSD) for 1 month and, if so, whether chronic treatment with the angiotensin-converting enzyme inhibitor enalapril or the anti-oxidant tempol could normalize the response. Membrane potential was recorded using the perforated patch-clamp technique on the endothelium of rat aorta. 2. Acetylcholine (2 μmol/L) produced a hyperpolarization sensitive to TRAM-34, a blocker of intermediate-conductance Ca(2+) -sensitive K(+) channels (IK(Ca)), but not to apamin, a blocker of small-conductance Ca(2+)-sensitive K(+) channels (SK(Ca)). NS309 (3 μmol/L), an activator of SK(Ca) and IK(Ca) channels, produced a hyperpolarization of similar magnitude as ACh. 3. In the HSD group, the ACh-evoked hyperpolarization was significantly attenuated compared with that in the control group, which was fed normal chow rather than an HSD. Similarly, the hyperpolarization produced by NS309 was weaker in tissues from HSD-fed rats. 4. Combination of HSD with chronic enalapril treatment (20 mg/kg per day for 1 month) normalized endothelial hyperpolarizing responses to ACh. Chronic tempol treatment (1 mmol/L in tap water for 1 month) prevented the reduced hyperpolarization to ACh. 5. The results of the present study indicate that excess in dietary Na(+) results in a failure of endothelial cells to generate normal IK(Ca) channel-mediated hyperpolarizing responses. Our observations implicate oxidative stress mediated by increased angiotensin II signalling as a mechanism underlying altered endothelial hyperpolarization during dietary salt loading.

Endothelial calcium-activated potassium channels as therapeutic targets to enhance availability of nitric oxide

The vascular endothelium plays a critical role in vascular health by controlling arterial diameter, regulating local cell growth, and protecting blood vessels from the deleterious consequences of platelet aggregation and activation of inflammatory responses. Circulating chemical mediators and physical forces act directly on the endothelium to release diffusible relaxing factors, such as nitric oxide (NO), and to elicit hyperpolarization of the endothelial cell membrane potential, which can spread to the surrounding smooth muscle cells via gap junctions. Endothelial hyperpolarization, mediated by activation of calcium-activated potassium (K(Ca)) channels, has generally been regarded as a distinct pathway for smooth muscle relaxation. However, recent evidence supports a role for endothelial K(Ca) channels in production of endothelium-derived NO, and indicates that pharmacological activation of these channels can enhance NO-mediated responses. In this review we summarize the current data on the functional role of endothelial K(Ca) channels in regulating NO-mediated changes in arterial diameter and NO production, and explore the tempting possibility that these channels may represent a novel avenue for therapeutic intervention in conditions associated with reduced NO availability such as hypertension, hypercholesterolemia, smoking, and diabetes mellitus.

Recruitment of the Vascular Endothelium into Neurovascular Coupling

In neurovascular coupling (NVC), neuronal activity in the brain leads to elevated local blood flow to satisfy the increased metabolic demands of the active tissue. The vascular endothelium has a major role in the modulation of blood flow, and here we explore the hypothesis that the endothelium has an important role in NVC.Brain slices were prepared from male C57 Bl/6 mice. Intraparenchymal arterioles were pre‐constricted with U46619 (125 nM). Neurons were depolarized with electrical field stimulation (EFS) to induce NVC and arteriolar dilation. In some experiments, the endothelium was loaded with di‐8‐ANEPPS (20 μM).EFS‐evoked arteriolar dilation was accompanied by endothelial hyperpolarization, as indicated by a 1.4% increase in the fluorescence ratio of di‐8‐ANEPPS (n=7). Blockade of I prostanoid receptors with 1 μM R01138452 had no effect on NVC (n=4). Blockade of neuronal nitric oxide synthase (NOS) with 7‐NI (100 μM) reduced the magnitude of vasodilation by 63% (n=7) with no effect on vessel tone (n=5). Blockade of both neuronal and endothelial NOS with L‐NNA (100 μM) caused a tonic constriction (17%, n=5) and reduced vasodilation by 89% (n=7). In the absence of U46619, L‐NNA constricted arterioles by 28% and reduced vasodilation by 90% (n=5).These data are consistent with the concept that the vascular endothelium is actively engaged during neurovascular coupling. Supported by NIH award P01 HL095489.

Pharmacological activation of KCa3.1/KCa2.3 channels produces endothelial hyperpolarization and lowers blood pressure in conscious dogs

In rodents, the endothelial KCa channels, KCa3.1 and KCa2.3, have been shown to play a crucial role in initiating endothelium-derived hyperpolarizing factor (EDHF) vasodilator responses. However, it is not known to what extent these channels are involved in blood pressure regulation in large mammals, which would also allow us to address safety issues. We therefore characterized canine endothelial KCa3.1 and KCa2.3 functions and evaluated the effect of the KCa3.1/KCa2.3 activator SKA-31 on blood pressure and heart rate in dogs. Canine endothelial KCa3.1/KCa2.3 functions were studied by patch-clamp electrophysiology and wire myography in mesenteric arteries. Systemic cardiovascular actions of acute SKA-31 administration were monitored in conscious, unstressed beagle dogs. Mesenteric endothelial cells expressed functional KCa3.1 and KCa2.3 channels that were strongly activated by SKA-31. SKA-31 hyperpolarized the endothelial membrane and doubled endothelial hyperpolarization-dependent vasodilator responses in mesenteric arteries. SKA-31 (2 mg·kg(-1), i.v.) rapidly decreased the MAP by 28 ± 6 mmHg; this response was transient (8 ± 1 s), and the initial drop was followed by a fast and pronounced increase in HR (+109 ± 7 beats min(-1)) reflecting baroreceptor activation. SKA-31 significantly augmented similar transient depressor responses elicited by ACh (20 ng·kg(-1)) and doubled the magnitude of the response over time. Activation of endothelial KCa3.1 and KCa2.3 lowers arterial blood pressure in dogs by an immediate electrical vasodilator mechanism. The results support the concept that pharmacological activation of these channels may represent a potential unique endothelium-specific antihypertensive therapy.

Endothelial-Derived Hyperpolarization Factor (EDHF) Contributes to PlGF-Induced Dilation of Mesenteric Resistance Arteries from Pregnant Rats

The aim of this study was to investigate the cellular mechanism involved in the potent vasodilatory action of PlGF on mesenteric resistance arteries from pregnant rats. PlGF (3 nM) induced a vasodilation of 64 ± 3.8% that was completely abolished by endothelial denudation. Significant dilation (28 ± 4.0%) remained, however, in the presence of nitric oxide synthase and cyclooxygenase inhibition, and was associated with significant reductions in vascular smooth muscle cell calcium. Absence of dilation in potassium-depolarizing solution (30 mM) confirmed its dependence on endothelial-derived hyperpolarization factor. Subsequent studies established that vasodilation was abolished by pharmacologic inhibition of SK_Ca (apamin) and BK_Ca (iberiotoxin) but not IK_Ca (tram-34) potassium channels. In summary, PlGF acts through the release of a combination of endothelium-derived relaxation factors. Based on the results of potassium channel blockade, we suggest that it induces endothelial hyperpolarization via SK_Ca channel activation; this, in turn, leads to the release of a diffusible mediator that activates vascular smooth muscle BK_Ca channels, hyperpolarization and vasodilation. This is the first study to identify the mechanism for PlGF/VEGFR-1 resistance artery dilation in the pregnant state, whose attenuation likely contributes to the systemic hypertension characteristic of pre- eclampsia.

The Endothelium, Part II: EDHF-Mediated Responses "The Classical Pathway"

ABSTRACT The endothelium controls vascular tone by releasing various vasoactive substances. Additionally, another pathway associated with the hyperpolarization of both endothelial and vascular smooth muscle cells contributes also to endothelium-dependent relaxations (EDHF-mediated responses). These responses involve an increase in the intracellular Ca₂⁺ concentration of the endothelial cells followed by the opening of Ca₂⁺-activated K⁺ channels of small and intermediate conductances (SKCa and IKCa). These channels show a distinct subcellular distribution, suggesting that their activation could be elicited by distinct stimuli. Following KCa activation, the endothelial hyperpolarization can be conducted to the underlying smooth muscle cells by electrical coupling through myo-endothelial gap junctions. In addition, the potassium efflux can lead to the accumulation of potassium ions in the intercellular space and the subsequent activation of smooth muscle Kir2.1 and/or Na⁺/K⁺-ATPase. The hyperpolarization of ...

Endothelium-Derived Hyperpolarizing Factor and Vascular Function

Endothelial function refers to a multitude of physiological processes that maintain healthy homeostasis of the vascular wall. Exposure of the endothelium to cardiac risk factors results in endothelial dysfunction and is associated with an alteration in the balance of vasoactive substances produced by endothelial cells. These include a reduction in nitric oxide (NO), an increase in generation of potential vasoconstrictor substances and a potential compensatory increase in other mediators of vasodilation. The latter has been surmised from data demonstrating persistent endothelium-dependent vasodilatation despite complete inhibition of NO and prostaglandins. This remaining non-NO, non-prostaglandin mediated endothelium-dependent vasodilator response has been attributed to endothelium-derived hyperpolarizing factor/s (EDHF). Endothelial hyperpolarization is likely due to several factors that appear to be site and species specific. Experimental studies suggest that the contribution of the EDHFs increase as the vessel size decreases, with a predominance of EDHF activity in the resistance vessels, and a compensatory up-regulation of hyperpolarization in states characterized by reduced NO availability. Since endothelial dysfunction is a precursor for atherosclerosis development and its magnitude is a reflection of future risk, then the mechanisms underlying endothelial dysfunction need to be fully understood, so that adequate therapeutic interventions can be designed.

The Arterial Pulse: Vascular Biology, Vascular Function Testing, and Therapies

In this special issue on The Arterial Pulse: Vascular Biology, Vascular Function, and Therapies, we have compiled five reviews, a clinical trial and a translational pilot study for the readership of Cardiology Research and Practice. Techniques and approaches for modern vascular function testing have evolved over time from invasive methods restricted to smaller studies in the research laboratory to more standardized noninvasive methods, which are suitable for use in large prospective cohort studies and clinical trials. E. A. Ellins and J. P. J. Halcox describe currently available methods for the assessment of endothelial function and their potential application in cardiovascular research and clinical practice. In the second contribution E. Patvardhan and co-authors present a clinical trial investigating the relationship of the augmentation index (AIx) obtained from pressure waveforms via arterial applanation tonometry and cardiovascular risk factors and coronary artery disease (CAD). The authors conclude that AIx may be a useful measure of assessing overall risk for coronary heart disease. J. Steppan and colleagues review the effects of age-associated increase in vascular stiffness on systolic blood pressure, pulse pressure, AIx, and cardiac workload in the third paper. In this paper they describe evidence for the use of pulse wave velocity testing to measure vascular stiffness as an index of vascular health and as a predictor of adverse cardiovascular outcomes. In the fourth paper, M. Weber and colleagues present data from a pilot study about the potential diagnostic role of microRNAs (miRNAs) in blood samples of patients with angiographically documented CAD and healthy controls. The authors show that a distinct miRNA expression profile discriminates patients with CAD from healthy controls, which in turn is altered by vasoactive medications such as angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. In the fifth paper, C. R. Martens and D. G. Edwards review the current literature pertaining to the potential mechanisms of peripheral vascular dysfunction in chronic kidney disease and propose possible targets for treatment. Vascular endothelial dysfunction is associated with a reduction in nitric oxide (NO) bioavailability, an increase in vasoconstrictors, including superoxide anions and endothelin-1 in parallel with a potential compensatory increase in other mediators of vasodilatation. This non-NO, non-prostaglandin-mediated endothelium-dependent vasodilatation has been partly attributed to endothelium-derived hyperpolarizing factors (EDHFs), the topic of the comprehensive review by M. A. Ozkor and A. A. Quyyumi who describe the role of endothelial hyperpolarization in human circulatory physiology. Finally, N. Ghasemzadeh and A. M. Zafari provide a historical review about the evolution of our knowledge of the arterial pulse from ancient times to the modern era. The authors describe the revolutionary scientific concepts and the technological innovations that provided the foundation of our current understanding of the arterial pulse as a wave that can be accurately measured with various noninvasive techniques in standardized fashion suitable for clinical patient-oriented research as well as clinical practice. A. Maziar Zafari Arshed A. Quyyumi Julian P. J. Halcox Sanjay Rajagopalan

Gas attack or gently, the gases!

The article contains the current understanding of gas communication in smooth muscle cells, the basic mechanisms of action of gaseous transmitters, analyzes the different views on the nature of the endothelial relaxing and endothelial hyperpolarizing factors. We discuss the controversial issues of the mechanisms of endothelium-dependent relaxation of vascular smooth muscle.

Endothelium-Dependent Hyperpolarization: Out of the Dish and into the Brain

Certain agonists are known to dilate cerebral arteries and/or arterioles by hyperpolarizing the endothelium through activation of endothelial intermediate-conductance calcium-activated K channels (IKCa or KCa3.1) alone or in combination with small-conductance calcium-activated K channels (SKCa or KCa2.X, most likely KCa2.3) (Faraci et al, 2004; Marrelli et al, 2003; McNeish et al, 2006; You et al, 1999). A major component of this dilatory mechanism involves spread of the endothelial hyperpolarization to the vascular smooth muscle (VSM) through myo-endothelial gap junctions (Bryan et al, 2005). This hyperpolarization relaxes VSM by reducing cytoplasmic Ca2+ through closing voltage-operated calcium channels that reside in the plasmalemma of VSM but not endothelium. Although traditionally known as endothelium-derived hyperpolarizing factor, a more appropriate term for this mechanism of dilation is endothelium-dependent hyperpolarization (EDH), as no ‘factor', per se, is transferred between cells (Bryan et al, 2005). Utilizing techniques that covered a spectrum from single-cell K+ currents to isolated pressurized parenchymal arterioles (PAs) to laser Doppler perfusion, a measure of cerebral blood flow (CBF), Hannah et al (this issue) now demonstrate that this EDH mechanism does not require receptor stimulation but is active in the basal state and is responsible for helping to maintain a resting CBF. Although it has been known that IKCa and SKCa are expressed by endothelium, this is the first time that K+ currents from these channels have been measured in freshly isolated endothelial cells from cerebral vessels and the first for any arteriole. Given the difficulty in obtaining single-cell currents in freshly isolated endothelium in general, the fact that these were from arterioles is impressive. More importantly, Hannah et al demonstrated that IKCa and SKCa contribute to the resting tone of PA and contribute significantly to resting CBF. Combined blockade of IKCa and SKCa decreased CBF by ∼15%, a response similar to that produced by the inhibition of NO. Whereas previous studies have demonstrated EDH as an important mechanism in isolated vessels, Hannah et al now show that it is an important mechanism in vivo during resting conditions. Several observations point to the fact that EDH is a major regulator of CBF: (1) EDH is more important than NO as a dilator mechanism in isolated PA upon activation of P2Y2 receptors (You et al, 1999); (2) EDH contributes to resting CBF and the resting tone of arterioles (Hannah et al, this issue) (Cipolla and Bullinger, 2008; Cipolla et al, 2009); (3) EDH is as important as NO in controlling resting CBF (Hannah et al, this issue); and (4) EDH dilations persist or are even upregulated in cerebral vessels, following a number of pathological states, when NO bioavailability is diminished (Cipolla and Bullinger, 2008; Cipolla et al, 2009; Golding et al, 2001; Marrelli et al, 1999; Prisby et al, 2006). Thus, EDH must be considered as a significant mechanism for the regulation of CBF and as a potential target for increasing CBF in pathological states where flow has been compromised. Certainly, more in vivo studies, similar to the one highlighted by this editorial, are needed to fully understand the role of EDH in the cerebral circulation.

The endothelium at the brink of calamity in storage disease: more than just overloaded with junk?

This editorial refers to ‘Globotriaosylceramide leads to KCa3.1 channel dysfunction: a new insight into endothelial dysfunction in Fabry disease’ by S. Park et al ., pp. 290–299, this issue. The endothelium crucially controls the contractile status of vascular smooth muscle and thereby vascular diameter. This is achieved by the release of dilating endothelial autacoids such as nitric oxide (NO), prostaglandins, and a third factor that elicits relaxation through smooth muscle hyperpolarization and is therefore known as ‘endothelium-derived hyperpolarizing factor’ (EDHF). Although the importance of EDHF is well accepted, its chemical nature is still elusive, and a wide range of agents, such as K+ ions, eicosanoids, H2O2, H2S, or C-type natriuretic peptide, have been suggested to serve as EDHF. Despite this diversity, it is firmly established that endothelial hyperpolarization is critical to initiate EDHF-type dilation. It is either achieved by releasing one of the above-mentioned molecules or by a direct current transfer from endothelium to smooth muscle via the gap junctions that interconnect these cells.1 The initial endothelial hyperpolarization results from opening of Ca2+-activated K+-channels (KCa), which have been identified as KCa3.1 and/or KCa2.3 (also named IKCa and SKCa, respectively) by pharmacological blockers as well as by genetic approaches. Both endothelial KCa channels are activated by Ca2+ and are voltage independent, which is an important biophysical feature that prevents their inactivation by the ensuing hyperpolarization, thus …

Amplification of EDHF‐type vasodilatations in TRPC1‐deficient mice

TRPC1 channels are expressed in the vasculature and are putative candidates for intracellular Ca(2+) handling. However, little is known about their role in endothelium-dependent vasodilatations including endothelium-derived hyperpolarizing factor (EDHF) vasodilatations, which require activation of Ca(2+) -activated K(+) channels (K(Ca)). To provide molecular information on the role of TRPC1 for K(Ca) function and the EDHF signalling complex, we examined endothelium-dependent and independent vasodilatations, K(Ca) currents and smooth muscle contractility in TRPC1-deficient mice (TRPC1-/-). Vascular responses were studied using pressure/wire myography and intravital microscopy. We performed electrophysiological measurements, and confocal Ca(2+) imaging for studying K(Ca) channel functions and Ca(2+) sparks. TRPC1 deficiency in carotid arteries produced a twofold augmentation of TRAM-34- and UCL1684-sensitive EDHF-type vasodilatations and of endothelial hyperpolarization to acetylcholine. NO-mediated vasodilatations were unchanged. TRPC1-/- exhibited enhanced EDHF-type vasodilatations in resistance-sized arterioles in vivo associated with reduced spontaneous tone. Endothelial IK(Ca) /SK(Ca)-type K(Ca) currents, smooth muscle cell Ca(2+) sparks and associated BK(Ca)-mediated spontaneous transient outward currents were unchanged in TRPC1-/-. Smooth muscle contractility induced by receptor-operated Ca(2+) influx or Ca(2+) release and endothelium-independent vasodilatations were unaltered in TRPC1-/-. TRPC1-/- exhibited lower systolic blood pressure as determined by tail-cuff blood pressure measurements. Our data demonstrate that TRPC1 acts as a negative regulator of endothelial K(Ca) channel-dependent EDHF-type vasodilatations and thereby contributes to blood pressure regulation. Thus, we propose a specific role of TRPC1 in the EDHF-K(Ca) signalling complex and suggest that pharmacological inhibition of TRPC1, by enhancing EDHF vasodilatations, may be a novel strategy for lowering blood pressure.

Crucial importance of the endothelial K + channel SK3 and connexin40 in arteriolar dilations during skeletal muscle contraction

Skeletal muscle activity requires substantial increases in blood flow, and the underlying vasodilation involves endothelial activity, but the contribution of the endothelium-dependent hyperpolarizing factor (EDHF) is only poorly defined. In EDHF signaling, endothelial hyperpolarization mediated by the Ca(2+)-activated K(+) channels SK3 and IK1 is a key step and also initiates gap junction-dependent conducted dilations. We assessed the role of SK3, IK1, and connexin40 (Cx40) in muscular contraction-induced dilations in the microcirculation in vivo. Hitherto, arterioles were observed in the electrically stimulated cremaster skeletal muscle of anesthetized mice lacking SK3, IK1, or Cx40 using intravital microscopy. Genetic deficiency of SK3, but not of IK1, strongly attenuated dilations to muscular contraction. Similarly, pharmacologic blockade of SK3 by the specific blocker UCL1684 impaired such dilations in wild-type and IK1-deficient mice. In contrast, IK1 was required for acetylcholine-induced dilations. Genetic deficiency of Cx40 also attenuated dilations induced by muscular contraction but not by acetylcholine. These data support the concept that endothelial hyperpolarization through activation of SK3 contributes to exercise hyperemia and the hyperpolarization ascends the vascular tree through gap junctions formed by Cx40 to orchestrate dilation. The differential impact of SK3- and IK1-deficiency on dilations to distinct stimuli suggests stimulus-dependent activation of these endothelial channels.

Connexins and gap junctions in the EDHF phenomenon and conducted vasomotor responses

It is becoming increasingly evident that electrical signaling via gap junctions plays a central role in the physiological control of vascular tone via two related mechanisms (1) the endothelium-derived hyperpolarizing factor (EDHF) phenomenon, in which radial transmission of hyperpolarization from the endothelium to subjacent smooth muscle promotes relaxation, and (2) responses that propagate longitudinally, in which electrical signaling within the intimal and medial layers of the arteriolar wall orchestrates mechanical behavior over biologically large distances. In the EDHF phenomenon, the transmitted endothelial hyperpolarization is initiated by the activation of Ca(2+)-activated K(+) channels channels by InsP(3)-induced Ca(2+) release from the endoplasmic reticulum and/or store-operated Ca(2+) entry triggered by the depletion of such stores. Pharmacological inhibitors of direct cell-cell coupling may thus attenuate EDHF-type smooth muscle hyperpolarizations and relaxations, confirming the participation of electrotonic signaling via myoendothelial and homocellular smooth muscle gap junctions. In contrast to isolated vessels, surprisingly little experimental evidence argues in favor of myoendothelial coupling acting as the EDHF mechanism in arterioles in vivo. However, it now seems established that the endothelium plays the leading role in the spatial propagation of arteriolar responses and that these involve poorly understood regenerative mechanisms. The present review will focus on the complex interactions between the diverse cellular signaling mechanisms that contribute to these phenomena.