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The arterial endothelium critically contributes to blood pressure control by releasing vasodilating autacoids such as nitric oxide, prostacyclin and a third factor or pathway termed 'endothelium-derived hyperpolarizing factor' (EDHF). The nature of EDHF and EDHF-signalling pathways is not fully understood yet. However, endothelial hyperpolarization mediated by the Ca(2+)-activated K(+) channels (K(Ca)) has been suggested to play a critical role in initializing EDHF-dilator responses in conduit and resistance-sized arteries of many species including humans. Endothelial K(Ca) currents are mediated by the two K(Ca) subtypes, intermediate-conductance K(Ca) (KCa3.1) (also known as, a.k.a. IK(Ca)) and small-conductance K(Ca) type 3 (KCa2.3) (a.k.a. SK(Ca)). In this review, we summarize current knowledge about endothelial KCa3.1 and KCa2.3 channels, their molecular and pharmacological properties and their specific roles in endothelial function and, particularly, in the EDHF-dilator response. In addition we focus on recent experimental evidences derived from KCa3.1- and/or KCa2.3-deficient mice that exhibit severe defects in EDHF signalling and elevated blood pressures, thus highlighting the importance of the KCa3.1/KCa2.3-EDHF-dilator system for blood pressure control. Moreover, we outline differential and overlapping roles of KCa3.1 and KCa2.3 for EDHF signalling as well as for nitric oxide synthesis and discuss recent evidence for a heterogeneous (sub) cellular distribution of KCa3.1 (at endothelial projections towards the smooth muscle) and KCa2.3 (at inter-endothelial borders and caveolae), which may explain their distinct roles for endothelial function. Finally, we summarize the interrelations of altered KCa3.1/KCa2.3 and EDHF system impairments with cardiovascular disease states such as hypertension, diabetes, dyslipidemia and atherosclerosis and discuss the therapeutic potential of KCa3.1/KCa2.3 openers as novel types of blood pressure-lowering drugs.

Aims: Potassium channels are essential elements of endothelial function. Recently, evidence emerged that the TWIK (tandem of P domains in a weak inwardly rectifying K+ channel)-related K+ channel (TREK-1) of the two-pore domain potassium channel gene family (K2P) may be involved in the regulation of vascular tone. However, the functional and molecular characterization of vascular TREK-1 is incomplete. In this study, we therefore analysed the functional expression of TREK-1 in the endothelium. Moreover, we hypothesized that changes in channel expression may contribute to altered endothelial vasodilator response under conditions of elevated blood pressure., Methods and Results: Gene expression and function of endothelial TREK-1 were analysed by single-cell RT-PCR, the patch-clamp technique and pressure myography in murine carotid arteries (CA). K+ outward currents displaying the characteristics of TREK-1 were observed following various TREK-1-activating stimuli such as membrane stretch, intracellular acidosis, polyunsaturated fatty acids, isoflurane (ISOFL), riluzole, and acetylcholine (ACh). In K(Ca)3.1(-/-) mice exhibiting elevated blood pressure, endothelial TREK-1 currents and TREK-1 mRNA expression were enhanced as compared with normotensive control mice. TREK-1-mediated vasodilator responses to alpha-linolenic acid, ISOFL, or ACh were increased. A similar up-regulation of endothelial TREK-1 was observed in spontaneously hypertensive rats., Conclusion: We have found that TREK-1 is an endothelial K+ channel capable of producing hyperpolarization and vasodilation. A correlation between hypertension and up-regulation of TREK-1 was observed in two different animal models of elevated blood pressure. Thus, TREK-1 may play a protective role in the cardiovascular system by providing a novel type of endothelial hyperpolarization-mediated vasodilator response.

Background: In blood vessels, the endothelium is a crucial signal transduction interface in control of vascular tone and blood pressure to ensure energy and oxygen supply according to the organs' needs. In response to vasoactive factors and to shear stress elicited by blood flow, the endothelium secretes vasodilating or vasocontracting autacoids, which adjust the contractile state of the smooth muscle. In endothelial sensing of shear stress, the osmo- and mechanosensitive Ca(2+)-permeable TRPV4 channel has been proposed to be candidate mechanosensor. Using TRPV4(-/-) mice, we now investigated whether the absence of endothelial TRPV4 alters shear-stress-induced arterial vasodilation., Methodology/principal Findings: In TRPV4(-/-) mice, loss of the TRPV4 protein was confirmed by Western blot, immunohistochemistry and by in situ-patch-clamp techniques in carotid artery endothelial cells (CAEC). Endothelium-dependent vasodilation was determined by pressure myography in carotid arteries (CA) from TRPV4(-/-) mice and wild-type littermates (WT). In WT CAEC, TRPV4 currents could be elicited by TRPV4 activators 4alpha-phorbol-12,13-didecanoate (4alphaPDD), arachidonic acid (AA), and by hypotonic cell swelling (HTS). In striking contrast, in TRPV4(-/-) mice, 4alphaPDD did not produce currents and currents elicited by AA and HTS were significantly reduced. 4alphaPDD caused a robust and endothelium-dependent vasodilation in WT mice, again conspicuously absent in TRPV4(-/-) mice. Shear stress-induced vasodilation could readily be evoked in WT, but was completely eliminated in TRPV4(-/-) mice. In addition, flow/reperfusion-induced vasodilation was significantly reduced in TRPV4(-/-) vs. WT mice. Vasodilation in response to acetylcholine, vasoconstriction in response to phenylephrine, and passive mechanical compliance did not differ between genotypes, greatly underscoring the specificity of the above trpv4-dependent phenotype for physiologically relevant shear stress., Conclusions/significance: Genetically encoded loss-of-function of trpv4 results in a loss of shear stress-induced vasodilation, a response pattern critically dependent on endothelial TRPV4 expression. Thus, Ca(2+)-influx through endothelial TRPV4 channels is a molecular mechanism contributing significantly to endothelial mechanotransduction.

Objective: Ca2+-influx through transient receptor potential (TRP) channels was proposed to be important in endothelial function, although the precise role of specific TRP channels is unknown. Here, we investigated the role of the putatively mechanosensitive TRPV4 channel in the mechanisms of endothelium-dependent vasodilatation., Methods and Results: Expression and function of TRPV4 was investigated in rat carotid artery endothelial cells (RCAECs) by using in situ patch-clamp techniques, single-cell RT-PCR, Ca2+ measurements, and pressure myography in carotid artery (CA) and Arteria gracilis. In RCAECs in situ, TRPV4 currents were activated by the selective TRPV4 opener 4alpha-phorbol-12,13-didecanoate (4alphaPDD), arachidonic acid, moderate warmth, and mechanically by hypotonic cell swelling. Single-cell RT-PCR in endothelial cells demonstrated mRNA expression of TRPV4. In FURA-2 Ca2+ measurements, 4alphaPDD increased [Ca2+]i by &140 nmol/L above basal levels. In pressure myograph experiments in CAs and A gracilis, 4alphaPDD caused robust endothelium-dependent and strictly endothelium-dependent vasodilatations by &80% (K(D) 0.3 microL), which were suppressed by the TRPV4 blocker ruthenium red (RuR). Shear stress-induced vasodilatation was similarly blocked by RuR and also by the phospholipase A2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF3). 4alphaPDD produced endothelium-derived hyperpolarizing factor (EDHF)-type responses in A gracilis but not in rat carotid artery. Shear stress did not produce EDHF-type vasodilatation in either vessel type., Conclusions: Ca2+ entry through endothelial TRPV4 channels triggers NO- and EDHF-dependent vasodilatation. Moreover, TRPV4 appears to be mechanistically important in endothelial mechanosensing of shear stress.

Endothelial cation channel are important regulators of vascular tone by modulating intracellular Ca(2+)-signaling and thus adequate synthesis of vasodilating factors. The overall importance of these ion channels suggests that they may represent novel pharmacotherapeutic targets in addition to the well-known voltage-gated calcium channels in vascular smooth muscle. In this short overview we summarize the current knowledge about endothelial ion channels and their roles for endothelium-dependent vasodilatation. Furthermore, we perspectively discuss the usefulness of specific openers of endothelial Ca(2+)-activated K(+)-channels and TRPV-channels as novel antihypertensive drugs.

Background: Chronic renal failure (CRF) is associated with increased cardiovascular morbidity, abnormal arterial tone, and endothelial dysfunction. Ca(2+)-activated K(+)-channels (K(Ca)) are important regulators of endothelial function by controlling endothelial hyperpolarization and thus endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilations. Here we tested the hypothesis whether an altered function of endothelial K(Ca) and diminished EDHF-mediated vasodilation contribute to the endothelial dysfunction in the rat remnant kidney model of chronic renal failure., Methods: Functional expression of endothelial K(Ca) currents and endothelium-dependent vasodilations in rat carotid arteries were assessed by using patch-clamp techniques, single-cell reverse transcription-polymerase chain reaction (RT-PCR), and a pressure myograph 8 weeks after either subtotal 5/6 nephrectomy in normotensive or hypertensive, or sham-operated rats., Results: Acetylcholine (ACh)-induced EDHF-mediated vasodilations were present in sham-operated rats, but almost absent in both normotensive 5/6 nephrectomy rats and hypertensive 5/6 nephrectomy rats. In experiments without blocking nitric oxide/prostacyclin synthesis, endothelium-dependent vasodilation to ACh was significantly reduced in both normotensive 5/6 nephrectomy rats and hypertensive 5/6 nephrectomy rats. In sham-operated rats, 1-ethyl-2-benzimidazolinone (1-EBIO), a selective opener of endothelial small and intermediate K(Ca), induced a substantial EDHF-mediated vasodilation, which was greatly reduced in hypertensive 5/6 nephrectomy rats and in normotensive 5/6 nephrectomy rats. In patch-clamp experiments, mean K(Ca) currents were significantly reduced in endothelial cells from hypertensive 5/6 nephrectomy rats and normotensive 5/6 nephrectomy rats when compared to sham-operated rats. Concordantly, single-cell reverse-transcription-polymerase chain reaction (RT-PCR) analysis revealed a greatly reduced frequency of endothelial cells expressing the K(Ca) genes, SKCa3 and IKCa1 in 5/6 nephrectomy rats compared to sham-operated rats., Conclusion: Experimental CRF leads to a loss of EDHF-type vasodilation which was caused at least in part by an impaired functional expression of endothelial hyperpolarizing K(Ca). The loss of EDHF-type vasodilation may contribute to endothelial dysfunction and abnormal arterial tone in CRF.

Objective: Ca2+-activated K+ (K(Ca)) channels have been proposed to promote mitogenesis in several cell types. Here, we tested whether the intermediate-conductance K(Ca) channel (IKCa1) and the large-conductance K(Ca) channel (BK(Ca)) contribute to endothelial cell (EC) proliferation and angiogenesis., Material and Results: Function and expression of IKCa1 and BK(Ca)/Slo were investigated by patch-clamp analysis and real-time RT-PCR in human umbilical vein ECs (HUVECs) and in dermal human microvascular ECs 1 (HMEC-1). HMEC-1 expressed IKCa1 and BK(Ca)/Slo, whereas HUVECs expressed IKCa1. A 48-hour exposure to basic fibroblast growth factor (bFGF) augmented IKCa1 current amplitudes and induced a 3-fold increase in IKCa1 mRNA expression in HUVECs and HMEC-1. Vascular endothelial growth factor (VEGF) was also effective in upregulating IKCa1. BK(Ca)/Slo expression and current amplitudes in HMEC-1 were not altered by bFGF. bFGF- and VEGF-induced EC proliferation was suppressed by charybdotoxin, clotrimazole, or the selective IKCa1 blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), whereas inhibition of BK(Ca)/Slo by iberiotoxin was ineffective. In the Matrigel plug assay in mice, administration of TRAM-34 for 2 weeks significantly suppressed angiogenesis by approximately 85%., Conclusions: bFGF and VEGF upregulate expression of IKCa1 in human ECs. This upregulation of IKCa1 seems to be required for mitogen-induced EC proliferation and angiogenesis in vivo. Selective IKCa1 blocker might be of therapeutic value to prevent tumor angiogenesis.

Objective: Wall shear stress associated with blood flow is a major stimuli for generation of endothelial vasodilating and antithrombotic factors and it also regulates endothelial gene expression. Activation of endothelial intermediate-conductance Ca(2+)-activated K(+) channels (IK(Ca)) is important for the control of endothelial function by inducing cell hyperpolarization and thus generation of the endothelium-derived hyperpolarizing factor. In the present study we tested whether the IK(Ca) encoding IKCa1 gene is regulated by laminar shear stress (LSS)., Methods: Human umbilical vein endothelial cells (HUVEC) were subjected to LSS with a magnitude of 0.5-15 dyn/cm(2) and time intervals of 2-24 h in a flow cone apparatus. Expression of the IKCa1 gene and IK(Ca)-functions were determined by using real time RT-PCR and patch-clamp techniques., Results: A short 2-4 h-or long 24 h-exposure to a LSS with a low (venous) magnitude of 0.5 dyn/cm(2) had no effect on IKCa1 expression levels. An exposure for 2 and 4 h to LSS with an intermediate magnitude of 5 dyn/cm(2) was also ineffective, whereas an exposure for 24 h induced a significant threefold up-regulation of IKCa1 expression levels. An exposure to LSS with a higher (arterial) magnitude of 15 dyn/cm(2), resulted in an eightfold up-regulation of IKCa1 expression levels after a 4 h-exposure and a fourfold increase of IKCa1 expression levels at 24 h. The increased IKCa1 expression levels following exposure to high levels of LSS resulted in enhanced IK(Ca) whole-cell currents and in an increased hyperpolarization of the endothelium in response to ATP and the IK(Ca) opener 1-EBIO. Inhibition of the mitogen-activated protein kinase/extracellular-signal-regulated kinase (ERK) kinase 1/2 (MEK/ERK) pathway by PD98059 prevented the LSS-induced up-regulation of IKCa1 expression levels and IK(Ca) whole-cell currents indicating that augmentation of IKCa1 expression levels is mediated by the LSS-induced activation of the MEK/ERK pathway., Conclusion: Long term exposure to LSS up-regulates expression and function of endothelial IK(Ca). This increase might represent a new important mechanism in endothelial adaptation to altered hemodynamics.

1. Activation of Ca(2+)-activated K(+)-channels (K(Ca)) has been suggested to play a key role in endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation. However, due to the low selectivity of commonly used K(Ca)-channel blockers it is still elusive which endothelial K(Ca)-subtypes mediate hyperpolarization and thus initiate EDHF-mediated vasodilation. 2. Using the non-cytochrome P450 blocking clotrimazole-derivatives, 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) and 2-(2-chlorophenyl)-2,2-diphenylacetonitrile (TRAM-39) as highly selective IK1-inhibitors, we investigated the role of the intermediate-conductance K(Ca) (rIK1) in endothelial hyperpolarization and EDHF-mediated vasodilation. 3. Expression and function of rIK1 and small-conductance K(Ca) (rSK3) were demonstrated in situ in single endothelial cells of rat carotid arteries (CA). rIK1-currents were blocked by TRAM-34 or TRAM-39, while rSK3 was blocked by apamin. In current-clamp experiments, endothelial hyperpolarization in response to acetylcholine was abolished by the combination of apamin and TRAM-34. 4. In phenylephrine-preconstricted CA, acetylcholine-induced NO and prostacyclin-independent vasodilation was almost completely blocked by ChTX, CLT, TRAM-34, or TRAM-39 in combination with the SK3-blocker apamin. Apamin, TRAM-34, and CLT alone or sulphaphenzole, a blocker of the cytochrome P450 isoform 2C9, were ineffective in blocking the EDHF-response. 5. In experiments without blocking NO and prostacyclin synthesis, the combined blockade of SK3 and IK1 reduced endothelium-dependent vasodilation. 6. In conclusion, the use of selective IK1-inhibitors together with the SK3-blocker apamin revealed that activation of both K(Ca), rIK1 and rSK3 is crucial in mediating endothelial hyperpolarization and generation of the EDHF-signal while the cytochrome P450 pathway seems to play a minor or no role in rat CA.