Endothelial Ca²⁺-activated K⁺ channels in normal and impaired EDHF–dilator responses – relevance to cardiovascular pathologies and drug discovery

Abstract

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 Ca²⁺-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.

British Journal of Pharmacology (2009) 157, 509–526; doi:10.1111/j.1476-5381.2009.00132.x; published online 19 March 2009

This article is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009

Introduction

Robert Furchgott's seminal work showing that the vascular endothelium releases a short-living factor capable of producing arterial dilation revolutionized our understanding of the regulation of vascular tone and blood pressure. This endothelium-derived relaxing factor turned out to be the gas nitric oxide (NO) (Furchgott and Zawadzki, 1980; Palmer et al., 1987). Today we know two more relaxing factors, the cyclooxygenase product prostacyclin (PGI₂) (Moncada et al., 1976) and the latest identified endothelium-derived hyperpolarizing factor (EDHF) (Busse et al., 2002; Feletou and Vanhoutte, 2006). The contribution of NO and PGI₂ to endothelium-dependent vasodilation and to local and systemic blood pressure control is well established. Although the contribution of the EDHF is less well characterized, a considerable body of experimental evidence suggests that EDHF could be equally important as NO in controlling vascular tone and blood pressure in vivo (Shimokawa et al., 1996; Feletou and Vanhoutte, 2006; Köhler and Hoyer, 2007).

Endothelium-derived hyperpolarizing factor as the third endothelium-derived vasodilating system was first projected in the early 1980s (De Mey et al., 1982; Taylor and Weston, 1988), and the naming resulted from the observation that this non-NO- and non-PGI₂-mediated component of endothelium-dependent relaxation was accompanied by smooth muscle hyperpolarization. The hyperpolarization closes the smooth muscle voltage-gated Ca²⁺ channels, leading to a drop in [Ca²⁺]_i (intracellular calcium concentration) and ultimately relaxation (see scheme in Figure 1). The molecular identity and signalling pathways of EDHF are still a matter of vigorous discussion. Indeed, EDHF–dilator responses have been ascribed to a variety of diffusible and structurally very diverse EDHFs such as cytochrome P450-generated epoxyeicosatrienoic acids (EETs) (metabolites of arachidonic acid) (Fisslthaler et al., 1999; Campbell and Falck, 2007), lipoxygenase products (Faraci et al., 2001), NO itself (Bolotina et al., 1994), hydrogen peroxide [H₂O₂ (Ellis and Triggle, 2003; Shimokawa and Morikawa, 2005)], cAMP (Popp et al., 2002) and possibly C-type natriuretic peptide (Wei et al., 1994). The arguments in favour of or against their action as EDHFs were excellently reviewed recently (Feletou and Vanhoutte, 2006; 2007). Moreover, the respective contribution of these EDHF candidates to endothelium-dependent dilator responses seems to vary considerably among the different vascular beds and species, and it is likely that there are several distinct EDHFs that may act separately or interact synergistically.

Figure 1.

Putative EDHF-signalling pathways related to endothelial and smooth muscle ion channel opening. AA, arachidonic acid; ACh, acetylcholine, [Ca²⁺]_i, intracellular calcium concentration; CYP, cytochrome P450 epoxygenase; EC, endothelial cell; EDHF, endothelium-derived hyperpolarizing factor; EETs, epoxyeicosatrienoic acids; ER, endoplasmic reticulum; GPCR, G protein-coupled receptor; KCa1.1, large-conductance Ca²⁺-activated K⁺ channel; KCa2.3, small-conductance Ca²⁺-activated K⁺ channel subtype 3; KCa3.1, intermediate-conductance Ca²⁺-activated K⁺ channel; K_ir, inwardly rectifying K⁺ channel; meGJ, myo-endothelial gap-junction; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; VDCC, voltage-dependent Ca²⁺ channel; VSMC, vascular smooth muscle cell.

In addition to the concept of EDHF as a diffusible factor, there is also good evidence for EDHF-type dilation to be a rather ‘biophysical’ phenomenon (Figure 1), based on direct electrical coupling of endothelium and smooth muscle via myo-endothelial gap-junctions (Yamamoto et al., 1998; Hutcheson et al., 1999; Emerson and Segal, 2000; Sandow et al., 2002) (for review see Griffith, 2004; Beny et al., 2006; de Wit et al., 2006). Hence, compounds disrupting intercellular electrical coupling were found to abrogate EDHF signalling in a variety of vascular beds and species (Yamamoto et al., 1998; Hutcheson et al., 1999; Ungvari et al., 2002; Ujiie et al., 2003).

In this scenario of electrical communication between the two cell types, activation of the Ca²⁺-activated K⁺ channels (K_Ca), which are located in the endothelium (Edwards et al., 1998; Köhler et al., 2000; 2001; Bychkov et al., 2002; Eichler et al., 2003; Taylor et al., 2003), seems to play a major role in the initiation of hyperpolarization (Waldron and Garland, 1994; Zygmunt and Hogestatt, 1996; Si et al., 2006a). This ‘myo-endothelial coupling hypothesis’ is a very attractive explanation for the EDHF phenomenon, as direct electrical coupling is an ‘economic’ and instant mechanism, which does not require the synthesis and release of a diffusible factor. However, myo-endothelial electrical coupling does not seem to apply to EDHF–dilator response in all vascular beds, for example, in the cremaster muscle microcirculation, in which myo-endothelial coupling is weak but EDHF–dilator responses are rather strong (Siegl et al., 2005).

An additional and equally intriguing possibility of how activation of endothelial K_Ca may elicit smooth muscle hyperpolarization and relaxation is that K⁺ release through the channels causes a small increase in K⁺ in the intercellular space between endothelium and smooth muscle and thus activates smooth muscle inwardly rectifying K⁺ channels and/or Na⁺/K⁺ ATPases (Na⁺/K⁺ pump), which finally evoke membrane hyperpolarization (Edwards et al., 1998; Zaritsky et al., 2000) (Figure 1).

At present, both myo-endothelial electrical coupling and K⁺ as EDHF appear comparably attractive explanations for EDHF responses in different preparations, but definitely not in all. Perhaps, these two EDHF pathways act separately, in parallel or even synergistically. In any case, both pathways require activation of endothelial K_Ca channel as the starting point.

In this review, we wish to focus on the endothelial K_Ca, intermediate-conductance K_Ca (KCa3.1) (also known as, a.k.a. IK_Ca, IK1) and small-conductance K_Ca type 3 (KCa2.3) (a.k.a. SK_Ca, SK3), their pharmacology, subcellular compartmentation, as well as their contribution to the EDHF–dilator response in vivo. In addition, we spotlight on alterations in arterial blood pressure in KCa3.1- and KCa2.3-transgenic animals and on defects of channel expression and EDHF–dilator responses in cardiovascular pathologies. Finally, we wish to convey that pharmacological manipulation of the KCa3.1/KCa2.3-dilator system could represent a novel treatment option for cardiovascular pathologies such as hypertension, diabetes and atherosclerosis.

Molecular and electrophysiological characteristics of Ca²⁺-activated K⁺ channels

The complete gene family of K_Ca is subdivided into two well-defined groups, which are phylogenetically only distantly related. It consists of eight members and belongs to the group of K⁺ channels with four pore-forming subunits (Wei et al., 2005). Each subunit consists of six/seven membrane spanning α-helical segments (S1–S6/7) and a pore-forming loop (between S5 and S6) (Figure 2) harbouring the selectivity filter with the signature motif GYG for a K⁺-selective channel.

Figure 2.

Molecular and pharmacological characteristics of KCa3.1, KCa2.1–3 and KCa1.1 channels. (A) Membrane topology of KCa3.1/KCa2.1–3 and KCa1.1 channels. Left: schematic illustration of a single KCa3.1/KCa2.1–3 subunit with six transmembrane domains (1–6) and a pore loop between S5 and S6. Ca²⁺ sensitivity is conferred by constitutively bound calmodulin (CaM) to the intracellular c-terminus. Right: illustration of a single α-subunit of KCa1.1 with seven transmembrane domains (0–6) and an associated β-subunit with two transmembrane domains. The extremely long intracellular c-terminus contains additional hydrophobic segments (7–10) and the so-called Ca²⁺ bowl, conferring Ca²⁺ sensitivity to gather with the hydrophobic segments 7 and 8. (B) Pharmacology of KCa3.1, KCa2.1–3 and KCa1.1 channels. Left: blockers and openers of KCa3.1 and KCa2.1–3 channels. Note that the openers have a higher affinity to KCa3.1 over KCa2.1–3 channels. Right: blockers and openers of the KCa1.1 channel. (C) KCa3.1 model (side view) based on the crystal structure of the bacterial KcsA channel (Doyle et al., 1998). Only two of the four subunits are shown to have a better view on the selectivity filter with the glycine (Gly)/tyrosine (Tyr)/glycine signature motive (in green) for a K⁺-selective and the cavity of the channel. The hydrophobic residues of threonine (Thr²⁵⁰) and valine (Val²⁷⁵) are lining the water filled of the cavity and are required for TRAM-34 and arachidonic acid (AA) binding just below the selectivity filter. 1-EBIO, 1-ethyl-2-benzimidazolinone; BMS-204352, [3S]-[+]-[5-chloro-2-methoxyphenyl]-1,3-dihydro-3-fluoro-6-[trifluoromethyl]-2H-indol-2-one; clotrimazole, 1-[(2-chlorophenyl)diphenylmethyl]-1H-imidazole; DC-EBIO, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazole-2-one; DHS-1, dehydrosoyasaponin-1; KCa1.1, large-conductance Ca²⁺-activated K⁺ channel; KCa2.3, small-conductance Ca²⁺-activated K⁺ channel subtype 3; KCa3.1, intermediate-conductance Ca²⁺-activated K⁺ channel; NS11021, 1-(3,5-bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea; NS1608, (N-(3-trifluoromethyl)phenyl) N′-(2-hydroxy-5-chlorophenyl) urea; NS1619, 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one; NS309, 3-oxime-6,7-dichloro-1H-indole-2,3-dione; SKA-20, anthra[2,1-d]thiazol-2-amine; SKA-31, naphtho[1,2-d]thiazol-2-amine; TRAM-34, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; UCL1684, 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-metheno-7H-dibenzo[b,m][1,5,12,16]tetraazacyclotricosine-5,13-diium.

Small/intermediate-conductance Ca²⁺-activated K⁺ channels

Group one comprises three subtypes of channels with a small unitary conductance of 5–10 pS, KCa2.1, KCa2.2 and KCa2.3 (according to the IUHAR (Wei et al., 2005) and GRAC (Alexander et al., 2008) nomenclature; a.k.a. SK1, SK2 and SK3). The fourth member in this group, KCa3.1 (a.k.a. SK4, IK1) (Ishii et al., 1997a) has a higher unitary conductance of 20–40 pS and thus has a unique position in this group (Figure 2A). All four subtypes (KCa2.1–3 and KCa3.1) have in common that Ca²⁺ sensitivity is conferred by calmodulin that is constitutively bound to the intracellularly located carboxyl-terminus of each subunit (Figure 2B, left) (Xia et al., 1998; Fanger et al., 1999; Schumacher et al., 2001). Thus calmodulin acts as a type of channel activating ‘β-subunit’. Moreover, an important feature of these channels is that their activity (gating) is voltage-independent as they do not possess a voltage sensor domain. This means that KCa2.1–3/KCa3.1 channels do not inactivate at negative membrane potentials and can therefore evoke a robust hyperpolarization response (towards the K⁺ equilibrium potential of −89 mV).

Large-conductance Ca²⁺-activated K⁺ channels

The second group in this gene family consists of the large-conductance K_Ca channel (KCa1.1; a.k.a. Maxi K, BK_Ca) (Atkinson et al., 1991) with a unitary conductance of 200–300 pS and the phylogenetically related K⁺ channels, KCa4.1, KCa4.2 and KCa5.1 (Wei et al., 2005). Unlike KCa2.1–3 and the KCa3.1 channels, Ca²⁺ sensitivity of KCa1.1 is conferred by direct high affinity binding of Ca²⁺ to the intracellular carboxyl-terminus of the pore-forming α-subunits of the channel complex (Figure 2A, right) (Schreiber and Salkoff, 1997). In further contrast to KCa2.1–3 and KCa3.1 channels, KCa1.1 contains a voltage sensor domain (positively charged arginines in S4) and is thus voltage-dependent (Diaz et al., 1998). At membrane potentials more negative than −40 to −50 mV the channel rapidly inactivates. Additionally, KCa1.1 channel activity is further regulated by associated β-subunits (KCNMB1–4) (Jiang et al., 1999) (Figure 2A).

K_Ca channel pharmacology

K_Ca channel modulators comprise metal ions, small organic compounds and venom-derived peptides [for in-depth review see (Wulff and Zhorov, 2008)]. These different classes of chemicals modulate the channels by binding to either the external or the internal face of the ion-conducting pore. In some cases, their modulating effects are mediated via binding to other domains, for example, the voltage sensors, or to associated subunits. Among the different ion modulators, venom-derived toxins usually block their targets with high affinity, that is, with IC₅₀ in the nanomolar or even femtomolar range and are therefore considered as the most selective and potent ion channel inhibitors. The disadvantage of venom-derived toxins is that they are expensive, especially if used for vessel studies or in vivo requiring higher amounts. Some small organic molecules also exhibit high affinity to their targets, but at higher concentrations, they usually exert non-specific actions. The advantage of this class of substances is that it offers both blockers and openers. Moreover, organic molecules are less cost-intensive and more suitable for in vivo applications. Metal ions (such as Ba²⁺ and Cs⁺) are the least potent class of channel modulators as they block channels often in the millimolar range and usually lack specificity for the different K⁺ channels. An advantage of these metal ions is that they do not interfere with Na⁺ and Ca²⁺ channels, even at high concentrations.

Concerning KCa2.1–3, KCa3.1 and KCa1.1 channels, several venom-derived peptides and small organic molecules (for overview see Figure 2B) are widely used to pharmacologically discriminate their contributions, especially to vascular functions.

Venom-derived toxins

Apamin (a venom-peptide from Apis mellifera, honey bee) blocks with high affinity (IC₅₀ 1–10 pmol·L⁻¹) all three types of KCa2.1–3 (Köhler et al., 1996; Ishii et al., 1997b) but has no appreciable blocking effects on KCa3.1 and KCa1.1 channels. KCa3.1 channels are inhibited by charybdotoxin (from the scorpion Leiurus quinquestriatus (Giangiacomo et al., 1993; Kaczorowski et al., 1996; Ishii et al., 1997a; Joiner et al., 1997) and by maurotoxin (from the scorpion Scorpio maurus) at nanomolar concentrations (IC₅₀s ∼5 and ∼1 nmol·L⁻¹ respectively) (Visan et al., 2004). Of these two toxins, charybdotoxin is less specific as it also blocks some voltage-gated K⁺ channels [delayed rectifier K⁺ channels, K_V1.2 (IC₅₀∼14 nmol·L⁻¹), K_V1.3 (IC₅₀∼2 nmol·L⁻¹) (Rauer et al., 2000) and K_V1.6 (IC₅₀∼2 nmol·L⁻¹)] and the KCa1.1 (IC₅₀∼3 nmol·L⁻¹) with high affinity. Unlike KCa3.1 and KCa2.1–3 channels, KCa1.1 is selectively inhibited by iberiotoxin (from the scorpion Buthus tamulus) with an IC₅₀ of ∼2 nmol·L⁻¹ (Kaczorowski et al., 1996).

Small organic molecules

A well-characterized and selective small molecule blocker for KCa2.1–3 is UCL1684 (6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-metheno-7H-dibenzo[b,m][1,5,12,16]tetraazacyclotricosine-5,13-diium) (Rosa et al., 1998) with a reported IC₅₀ in the lower nanomolar range (∼3 nmol·L⁻¹). The antifungal clotrimazole (Ishii et al., 1997a) is a fairly selective blocker of KCa3.1, inhibiting the channel with an IC₅₀ of ∼70 nmol·L⁻¹. The disadvantage of clotrimazole is that it promiscuously inhibits cytochrome P450 enzymes at submicromolar concentrations because of its imidazole moiety. A more potent and selective blocker of KCa3.1 is the non-cytochrome P450 blocking clotrimazole-derivative TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole) (IC₅₀∼20 nmol·L⁻¹) in which the imidazole ring is replaced by a pyrazole ring (Wulff et al., 2000). As shown in Figure 2C (left), TRAM-34 and clotrimazole were reported to have the same binding site in the internal cavity of the channel just below the selectivity filter (Wulff et al., 2001). Interestingly, TRAM-34 and clotrimazole seem to use the same binding site as arachidonic acid (Figure 2C, right), which also blocks KCa3.1 (Hamilton et al., 2003). Arachidonic acid thus may serve as an endogenous modulator of channel activity.

Small molecule blockers of KCa1.1 are paxilline (IC₅₀∼2 nmol·L⁻¹) and the rather non-selective tetraethylammonium (TEA, IC₅₀∼1 mmol·L⁻¹) (Sanchez and McManus, 1996).

K_Ca openers

KCa2.1–3 and KCa3.1 channels are activated by 1-EBIO (1-ethyl-2-benzimidazolinone) (Devor et al., 1996) and the 10-fold more potent DC-EBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazole-2-one) (Singh et al., 2001). 1-EBIO and DC-EBIO have a 10–30-fold higher selectivity for the KCa3.1 channel (EC₅₀s ∼30 and 1 µmol·L⁻¹ respectively) over KCa2.1–3 channels (EC₅₀s ∼300 and ∼30 µmol·L⁻¹ respectively). NS309 (3-oxime-6,7-dichloro-1H-indole-2,3-dione) (Strobaek et al., 2004) was reported to be even more potent than DC-EBIO. A disadvantage of DC-EBIO and NS309 is that both compounds block L-type Ca²⁺ channels with IC₅₀s of 70 and 10 µmol·L⁻¹, respectively, (Morimura et al., 2006) and NS309 at micromolar concentrations also inhibits cardiac hERG channels (Strobaek et al., 2004) thus raising concerns about its in vivo usage. The recently developed riluzole derivatives SKA-20 (anthra[2,1-d]thiazol-2-amine) and SKA-31 (naphtho[1,2-d]thiazol-2-amine) (Sankaranarayanan et al., 2008) are also potent and selective KCa3.1 openers with EC₅₀s of ∼100 and ∼200 nmol·L⁻¹, respectively, and with a 5–10-fold higher selectivity over KCa2.1–3 channels. A rather non-selective opener of KCa3.1 and KCa2.1–3 channels is the mother compound, the neuroprotective agent riluzole (EC₅₀∼2 and ∼20 µmol·L⁻¹, for KCa3.1 and KCa2.1–3 respectively) (Cao et al., 2002). The NeuroSearch A/S compounds, NS1608 [(N-(3-trifluoromethyl)phenyl) N′-(2-hydroxy-5-chlorophenyl) urea] and NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one), with EC₅₀s of ∼3 and ∼30 µmol·L⁻¹, respectively, are widely used and fairly selective openers of the KCa1.1 channel (Strobaek et al., 1996). These compounds activate KCa1.1 channels directly via the α-subunit, but without changing Ca²⁺ sensitivity. A disadvantage of NS1619 is that it also blocks certain voltage-gated calcium channels and the KCa3.1 channel (Cai et al., 1998). The recently developed NS11021 [1-(3,5-bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea] (EC₅₀∼400 nmol·L⁻¹) (Bentzen et al., 2007) and BMS-204352 ([3S]-[+]-[5-chloro-2-methoxyphenyl]-1,3-dihydro-3-fluoro-6-[trifluoromethyl]-2H-indol-2-one) (EC₅₀∼350 nmol·L⁻¹) (Gribkoff et al., 2001) seem to be the most potent and selective openers at present, and BMS-204352 was found to have beneficial effects in rodent stroke models (Starrett et al., 2000) and entered clinical trials, which were however discontinued due to lack of efficacy (Jensen, 2002). Another KCa1.1 channel opener, DHS-1 (dehydrosoyasaponin-1) potentiates KCa1.1 activity via the regulatory β-subunit (McManus et al., 1993) and is a bioactive compound from a medicinal herb used in Ghana for the treatment of asthma.

In aggregate, there is a variety of valuable pharmacological tools available to study the contribution of a specific K_Ca channel to the mechanism of endothelium-dependent dilation in isolated vessels and to blood pressure control in vivo. However, in keeping with the uncertain or weak selectivity of some of the blocker and opener compounds, caution is indicated in interpreting results when using higher dosage in vitro and in vivo.

Expression of Ca²⁺-activated K⁺ channels in the endothelium

Among different K_Ca channel subtypes, the KCa2.3 channel (Köhler et al., 1996) has been convincingly shown to be the predominantly expressed isoform of the KCa2 subgroup in vascular endothelia from different species and vascular beds (Feletou and Vanhoutte, 2006; Köhler and Hoyer, 2007). For instance, the presence of the KCa2.3 protein has been demonstrated by combined electrophysiological and mRNA expression studies in rat carotid arteries (Köhler et al., 2001), by combined electrophysiological and immunohistochemical analysis in murine mesenteric artery (Taylor et al., 2003), and by a combination of electrophysiological studies, mRNA expression analysis and immunohistochemistry in porcine coronary artery (Burnham et al., 2002), by Western blotting in rat mesenteric artery and by immunohistochemistry in porcine coronary artery (Absi et al., 2007). Other groups detected KCa2.3 protein expression by immunohistochemical means in rat mesenteric artery (Sandow et al., 2006; Dora et al., 2008). Some studies also have shown the expression of the KCa2.2 gene in human saphenous vein endothelium (Sultan et al., 2004) and in porcine coronary endothelium (Burnham et al., 2002), whereas the KCa2.1 expression has not been detected.

The other K_Ca subtype predominantly expressed in endothelia is the KCa3.1. This channel was found in human, porcine, bovine and rodent endothelium by using immunoblotting, immunohistochemical, mRNA expression approaches and electrophysiology (Köhler et al., 2000; 2001; Burnham et al., 2002; Weston et al., 2005; Sandow et al., 2006; Si et al., 2006a; Dora et al., 2008; Ledoux et al., 2008a,b). Interestingly, KCa3.1 mRNA expression is up-regulated by shear stress exposure of umbilical vein endothelial cells (HUVEC) and by pro-angiogenic factors (vascular endothelial growth factor and basic fibroblast growth factor) requiring activation of the ras/raf/MEK/ERK MAP kinase signal transduction cascade. This suggests that, besides its role in the mechanism of endothelium-dependent vasodilation and EDHF signalling (see also below), KCa3.1 also contributes to vascular adaptation to altered haemodynamics as well as to endothelial mitogenesis and angiogenesis (Brakemeier et al., 2003a; Grgic et al., 2005). The latter role is further supported by the inhibitory effects of the selective KCa3.1 blocker TRAM-34 on vascularization of matrigel plugs and by the higher expression levels of the channel in mesenteric endothelium of colon cancer patients.

Apart from KCa2.3 and KCa3.1, some endothelia also express KCa1.1. For instance, KCa1.1 expression was detected electrophysiologically and molecular biologically (mRNA expression) in human endothelia (HUVEC) (Köhler et al., 1998; 2000) and in two human endothelial cell lines, EAhy.926 (umbilical) (Papassotiriou et al., 2000) and microvascular HMEC-1 (Grgic et al., 2005) and in porcine endocardium (Hoyer et al., 1994) and renal endothelium in situ (Brakemeier et al., 2003b). Bovine endothelia do not express KCa1.1 (Gauthier et al., 2002). The significance for this apparent heterogeneity among different species is presently unclear. Interestingly, KCa1.1 expression was detected in low amounts in endothelium of mesenteric artery from colon adenocarcinoma patients while channel expression was not detectable in diverticulitis patients, suggesting that similar to KCa3.1 up-regulation, induction of KCa1.1 expression in endothelium may play a role in phenotypic modulation of endothelial cells and neo-angiogenesis due to malignancies (Köhler et al., 2000). While expression of KCa1.1 channels in endothelia is debatable and thus requires further investigation, we wish to emphasize here that KCa1.1 channels confer the voltage-dependent K_Ca conductance in differentiated vascular smooth muscle (Brayden et al., 1991; Nelson and Quayle, 1995). Importantly, a phenotypic switch from KCa1.1 to KCa3.1 occurs in proliferating smooth cells (Neylon et al., 1999; Si et al., 2006b) and in cardiovascular disease states characterized by abnormal smooth muscle cell proliferation (Köhler et al., 2003; Cheong et al., 2005; Tharp et al., 2008).

Endothelial KCa2.3 and KCa3.1, as crucial effectors in the EDHF–dilator response

Endothelial hyperpolarization responses mediated by K_Ca channels occur in response to the agonist-induced Ca²⁺ mobilization (Colden-Stanfield et al., 1987; Marchenko and Sage, 1994) [for review see (Nilius and Droogmans, 2001)] as well as to Ca²⁺ mobilization under shear stress stimulation (Hoyer et al., 1998) and also during membrane stretch (Köhler et al., 1999). That these hyperpolarization responses are mediated by activation of KCa2.3 and KCa3.1 channels is pharmacologically evidenced by a number of studies on animal vessels, using a combination of apamin and charybdotoxin (Edwards et al., 1998; Stankevicius et al., 2006), or a combination of apamin and a small molecule blocker of KCa3.1 (clotrimazole/TRAM-34) (Köhler et al., 2001; Eichler et al., 2003; Absi et al., 2007; Leuranguer et al., 2008). Interestingly, inhibition of KCa3.1 alone was sufficient to abrogate endothelial hyperpolarizations in human mesenteric (Köhler et al., 2000) and rat cerebral arteries (Marrelli et al., 2003), suggesting a major role of KCa3.1 in endothelial hyperpolarization in some vascular beds. In contrast, iberiotoxin as the more selective KCa1.1 blocker had no effect on endothelial hyperpolarization in human mesenteric artery (Köhler et al., 2000) and in various rodent vascular beds (Feletou and Vanhoutte, 2006), further demonstrating a predominant role of KCa3.1 and KCa2.3 channels and a minor or no role of the KCa1.1 channel in endothelial hyperpolarization responses.

Yet, pharmacological tools to study the contribution of a specific channel to hyperpolarization responses in isolated vessels or in vivo are problematic as selectivity of the toxins or small molecular blockers, as mentioned before, is not unambiguously given. For instance, charybdotoxin blocks endothelial KCa3.1 and smooth muscle KCa1.1 but also voltage-gated K⁺ channels (delayed rectifier K⁺ channels) of which some contribute to the resting membrane potential and repolarization events in smooth muscle and perhaps also in endothelium [for review see (Jackson, 2005)]. At concentrations >1 µmol·L⁻¹, the KCa3.1 blocker clotrimazole additionally blocks Ca²⁺-influx channels of the transient receptor gene family (Meseguer et al., 2008) and cytochrome P450 enzymes, which have been proposed to contribute to endothelial Ca²⁺-signalling pathways (Vriens et al., 2005). Thus and also in keeping with the tight intercellular electrical coupling, blocking effects could be due to complex disturbance of ionic signalling. Therefore, a more definitive proof for the involvement of endothelial KCa2.3 and KCa3.1 channels in hyperpolarization and vascular function may arise from gene-knockout models. In a recent study we showed that genetically encoded deletion of the KCa3.1 gene reduced endothelial K_Ca currents (Figure 3A) and severely dampened the hyperpolarization response to acetylcholine in carotid endothelium of KCa3.1-deficient mice (Si et al., 2006a). Likewise, suppression or over-expression of endothelial KCa2.3 in KCa2.3^T/T mice (Taylor et al., 2003), in which KCa2.3 expression can be manipulated by dietary doxycycline (Dox) (Bond et al., 2000), had a strong impact on total K_Ca currents and acetylcholine-induced hyperpolarization in mesenteric endothelium. In the Dox-treated animals, endothelial KCa2.3 currents and KCa2.3-mediated hyperpolarization to acetylcholine were almost abolished while in the untreated animals KCa2.3 currents were 10-fold higher than in wild-type animals and the hyperpolarization response was restored. Mice deficient of both KCa3.1 and KCa2.3 channel (KCa3.1^−/−/KCa2.3^T/T+Dox) virtually lack K_Ca currents (Figure 3A) and hyperpolarization to acetylcholine (Köhler et al., 2008). Thus, the pharmacological proofs together with the more recent evidences from gene-knockout animals demonstrate that KCa3.1 and KCa2.3 channels are the major determinants in mediating endothelial hyperpolarization to Ca²⁺-mobilizing stimuli.

Figure 3.

Impact of KCa3.1/KCa2.3 deficiency on K_Ca currents in freshly isolated carotid artery endothelial cells (CAEC). (A) Diminished currents in CAEC from KCa3.1^−/− mice (KCa3.1^−/−) and almost complete suppression of K_Ca currents in doxycycline (Dox)-administered KCa3.1^−/−/KCa2.3^T/T mice; wt indicates wild-type control (wt). (B) Deficiency of KCa3.1/KCa2.3 impairs acetylcholine (ACh)-induced vasodilation in murine carotid artery. Representative traces show EDHF-type vasodilation (in the presence of L-NNA and indomethacin) in response to increasing concentrations of ACh (−9 to −5 = logM [ACh]) and to the nitric oxide donor sodium nitroprusside (SNP, 10 µmol·L⁻¹) in wt carotid artery (left) and in carotid artery from Dox-administered KCa3.1^−/−/KCa2.3^T/T (+Dox) mice (right). Carotid arteries were pre-constricted with phenylephrine (PE, 1 µmol·L⁻¹). EDHF, endothelium-derived hyperpolarizing factor; KCa2.3, small-conductance Ca²⁺-activated K⁺ channel subtype 3; KCa3.1, intermediate-conductance Ca²⁺-activated K⁺ channel.

The idea that KCa2.3 and KCa3.1 channels are involved in the EDHF-type dilator response is based on the seminal observation that the combination of apamin plus charybdotoxin, but not of apamin plus the KCa1.1 blocker iberiotoxin, abolished the EDHF-mediated dilations in isolated hepatic and mesenteric arteries of the rat (Waldron and Garland, 1994; Zygmunt and Hogestatt, 1996). Meanwhile a large number of studies have further substantiated this role of endothelial KCa2.3 and KCa3.1 in the EDHF–dilator response by pharmacological means using combinations of apamin and charybdotoxin or apamin and a small molecule blocker of KCa3.1, for example, TRAM-34 (Edwards et al., 1998; Köhler et al., 2001; Eichler et al., 2003; Stankevicius et al., 2006; Absi et al., 2007; Leuranguer et al., 2008) (for review see Feletou and Vanhoutte, 2006). However, only a few studies have investigated the role of these channels in human vessels. For instance, KCa3.1 has been proposed to contribute to EDHF–dilator responses in isolated human gastroepiploic arteries and distal microvessels (Urakami-Harasawa et al., 1997). In omental and myometrial arteries from healthy women undergoing caesarean section, combined inhibition of KCa3.1 and KCa2.3 by a combination of charybdotoxin and apamin or TRAM-34 and apamin attenuated EDHF–dilator response in vessels pre-constricted with the thromboxane-mimetic U46619 (Gillham et al., 2007). Also in human interlobar arteries, EDHF-mediated responses involve activation of endothelial KCa3.1 and KCa2.3 channels (Bussemaker et al., 2003a). In human small-sized skeletal muscle arterioles from patients undergoing cardiopulmonary bypass surgery, the KCa2.1–3 and KCa3.1 opener NS309 caused endothelium-dependent vasodilation, demonstrating an involvement of endothelial KCa3.1 and KCa2.3 channels in the regulation of arteriolar tone in humans (Liu et al., 2008).

Besides ample evidence for pivotal roles of KCa2.3 and KCa3.1 channels in EDHF–dilator responses in isolated vessels, the exact contribution of KCa2.3 and KCa3.1 channels to EDHF–dilator responses in vivo as well as, the contribution of the KCa2.3/KCa3.1-EDHF–dilator system to blood pressure control, remains unclear to date. However, there is still enough evidence from new K_Ca-transgenic animal models showing that endothelial KCa2.3 and KCa3.1 channels contribute to endothelium-dependent dilation and EDHF–dilator responses in vivo. For instance, continuous KCa2.3 over-expression in KCa2.3^T/T mice enlarged arterial diameter of the mesenteric vasculature (Taylor et al., 2003). Interestingly, KCa2.3 over-expression also increased the degree of vessel branching, suggesting that over-expression of endothelial KCa2.3 impacts overall vessel morphology and growth (Taylor et al., 2003). The larger vessel diameter was accompanied by a continuous hyperpolarization of the mesenteric endothelium leading to a reduction in myogenic and phenylephrine-induced tone. This suggests that endothelial KCa2.3 exert a tonic dilating influence on vascular tone (Taylor et al., 2003). Deficiency of KCa2.3 reversed these effects on vascular tone and increased systemic blood pressure in these animals. EDHF signalling upon acetylcholine stimulation was only moderately reduced in mice lacking KCa2.3 (Köhler et al., 2008).

Regarding the other endothelial K_Ca channel, the genetic disruption of KCa3.1 in mice (KCa3.1^−/−) (Si et al., 2006a) resulted in a severe impairment of EDHF–dilator responses in resistance-sized arterioles of the cremaster muscle in vivo as well as in isolated carotid arteries suggesting that the KCa3.1 channel is of critical importance for the EDHF–dilator response. Similar to mice deficient for endothelial KCa2.3, mean arterial blood pressure in KCa3.1-deficient mice was elevated by ∼7 mmHg as measured by 72 h telemetry. Combined deficiency of both KCa3.1 and KCa2.3 channels in KCa3.1^−/−/KCa2.3^T/T+Dox mice almost completely abolished EDHF–dilator responses (Figure 3B) and further elevated mean arterial blood pressure (Köhler et al., 2008).

Thus, these findings from KCa3.1- and/or KCa2.3-deficient mice indicate that endothelial KCa3.1 and KCa2.3 are indeed fundamental components of the EDHF-signalling pathway and thus of blood pressure control in vivo.

Differential subcellular localization of KCa2.3 and KCa3.1 within the endothelium: a clue to subtype-specific functions in endothelium-dependent dilation

It appears rather surprising that a single endothelial cell expresses two readily similar types of K_Ca channels, both being regulated by calmodulin and equally capable of producing hyperpolarization. Although the total amount of each channel per endothelial cell seems to differ to some extent (∼2–3 times more KCa2.3 than KCa3.1 channels) (Si et al., 2006a; Ledoux et al., 2008a), the effective current generated by each channel is similar due to the higher unitary conductance of the KCa3.1 channel. This raises the question whether this dual expression of both channels is just a kind of evolutionary redundancy or whether they serve distinct functions. In this regard, suppression of either channel by pharmacological means or by genetic disruption has each been shown to dampen the endothelial hyperpolarization to acetylcholine (Eichler et al., 2003; Si et al., 2006a). This clearly suggests that they serve different functions beyond causing merely hyperpolarization. One possibility is that one of the channels is needed to amplify the response generated by the other. This could be a useful mechanism for fine tuning hyperpolarization responses and thus EDHF–dilator responses at variable contractile and depolarization states of the smooth muscle, possibly depending on sympathetic stimulation. This hypothesis is supported by a study showing that KCa3.1 channels contribute to acetylcholine-induced smooth muscle hyperpolarization in rat mesenteric artery only during phenylephrine-induced depolarization (Crane et al., 2003). Moreover, repetitive stimulation with the potent vasoconstrictor U46619, a thromboxane mimetic, has been reported to cause a rundown of the KCa2.3-mediated component of hyperpolarization and relaxation without affecting the KCa3.1-mediated component (Crane and Garland, 2004). These findings thus suggest that KCa3.1 channels may play a role in counteracting strong vasoconstriction and vasospasm.

Another possibility arguing in favour of distinct functions of KCa3.1 and KCa2.3 channels is their differential compartmentation in the endothelium. For instance, by biochemical approaches using membrane fractionation protocols, KCa2.3 channels were found in membrane fractions rich in caveolin-1 (the principle structural and functional component of caveolae (Frank et al., 2003; Gratton et al., 2004), suggesting their localization in caveolae (Weston et al., 2005; Absi et al., 2007). Interestingly, endothelial nitric oxide synthase as well as a broad variety of membrane protein such as G protein-coupled receptors, for example, the bradykinin receptors and Ca²⁺-influx channels of the TRP gene family (Remillard and Yuan, 2006; Saliez et al., 2008) are located in the same caveolin-1-rich and cholesterol-rich membrane fraction, as schematically illustrated in Figure 4A[for review see (Gratton et al., 2004)]. This suggests that KCa2.3 might participate in caveolar functions such as endothelial mechanosensation (Yu et al., 2006), transport and permeability, as well as NO formation by generating the electrochemical driving force for Ca²⁺ entry. Such a functional link between KCa2.3 channel and NO synthesis has been reported in cerebral arteries (McNeish et al., 2006). Interestingly, a complete absence of EDHF-mediated vasodilation was reported recently in isolated mesenteric arteries from caveolin-1-deficient mice (Saliez et al., 2008), demonstrating an important role of caveolae also in the EDHF–dilator response.

Figure 4.

Hypothetical compartmentation of KCa3.1 and KCa2.3 channels in the endothelium. (A) Hypothetical co-localization of the small-conductance Ca²⁺-activated K_Ca channel subtype 3 (KCa2.3) together with the endothelial nitric oxide synthase (eNOS) and G protein-coupled receptor (GPCR) in caveolae [based on biochemical evidence (Weston et al., 2005)]. Note that the intermediate-conductance K_Ca channel (KCa3.1) is located in the less buoyant membrane fractions together with the calcium-sensing receptor (CaR). Other abbreviations: Cav-1, caveolin-1 (hairpin-like and structure-giving protein of caveolae); DAG, diacylglycerol, PLC, phospholipase C. Note that the intermediate-conductance K_Ca (KCa3.1) is located in the less buoyant membrane fractions. (B) KCa3.1 localization in endothelial projections facing smooth muscle cells [based on immunohistochemical evidence (Sandow et al., 2006; Dora et al., 2008; Ledoux et al., 2008b)]. Possible activation of KCa3.1 by so-called Ca²⁺-pulsars in response to agonist stimulation (based on high-resolution calcium-imaging experiments in arterial preparations (Ledoux et al., 2008b). EC, endothelial cell; K_ir, inwardly rectifying K⁺ channel; meGJ, myo-endothelial gap-junction; VSMC, vascular smooth muscle cell.

In contrast, KCa3.1 channels are not present in the caevolin-1-rich membrane fractions but in the less buoyant non-caveolar fractions (Weston et al., 2005), thus suggesting their localization in a different membrane compartment. Interestingly, KCa3.1 co-localizes with the Ca²⁺-sensing receptor (CaR) in non-caveolar membrane fractions, and pharmacological activation of CaR causes smooth muscle hyperpolarization and an EDHF–dilator response by using KCa3.1 channels (Absi et al., 2007).

Other studies characterizing the membrane localization of endothelial KCa3.1 and KCa2.3 by immunohistochemical approaches (Sandow et al., 2006; Dora et al., 2008; Ledoux et al., 2008b) showed that KCa2.3 immunoreactivity is predominantly seen at inter-endothelial junctions whereas KCa3.1 immunoreactivity was limited to endothelial projections passing through the internal elastic lamina and coming into a direct contact with smooth muscle cells. These are also the sites where myo-endothelial gap-junctions are formed and thus electrical coupling of both tissues arises. Interestingly at such endothelial projections, acetylcholine induces IP₃-mediated and fluctuating Ca²⁺-release events [recently termed ‘Ca²⁺-pulsars’ (Ledoux et al., 2008b)] in close proximity to KCa3.1 channels. This suggest that these ‘Ca²⁺-pulsars’ trigger KCa3.1 activation and in that way EDHF–dilator responses, via either spread of hyperpolarization through myo-endothelial gap-junction or K⁺ efflux, spatially very close to the smooth muscle (Scheme in Figure 4B).

In aggregate, these recent findings support the concept that KCa3.1 and KCa2.3 channels do not have simply overlapping functions, but rather contribute to mechanistically distinct EDHF-signalling pathways, perhaps because of their different subcellular compartmentation.

KCa1.1 and EDHF–dilator responses?

The role of KCa1.1 channels in some endothelia is still unclear; however, in the smooth muscle KCa1.1 channels are undoubtedly important in controlling the membrane potential and thus the contractile state (Brayden et al., 1991; Nelson and Quayle, 1995). Importantly in EDHF–dilator responses, KCa1.1 channels are the presumed target of diffusible EDHFs (e.g. EETs, H₂O₂ and NO). Therefore, it is worth to include this channel in this review, which although primarily deals with endothelial KCa2.3 and KCa3.1 channels.

Regarding EETs and EDHF signalling, the KCa1.1 channel was shown to be the target of EETs, which enhance the ‘open probability’ of this channel and thereby elicit smooth muscle hyperpolarization (Li and Campbell, 1997; Larsen et al., 2006). This enhancement of KCa1.1 channel open probability is either due to a direct interaction of for example 14,15-EETs (Wu et al., 2000) and dihydroxyeicosatrienoic acids (Lu et al., 2001) with the channel or requires a more complex signal transduction pathway involving a still undefined EET receptor, phosphorylation/dephosphorylation events (Dimitropoulou et al., 2007; Imig et al., 2008), or the interplay with co-localized Ca²⁺-permeable TRPV4 channels (Earley et al., 2005) that have been shown to be modulated by EETs (Vriens et al., 2005).

With respect to the EDHF candidate molecules, H₂O₂ and NO, a similar enhancement of KCa1.1 open probability is caused either by a direct effect on the channel protein itself (Bolotina et al., 1994; Hayabuchi et al., 1998) or by stimulating the generation of cGMP or possibly cAMP, which are well-known positive modulators of KCa1.1 channels [for review see (Schubert and Nelson, 2001)]. Although, H₂O₂ has also been shown to block endothelial KCa1.1 α-subunits at the single-channel level (isolated membrane patch) (Soto et al., 2002; Brakemeier et al., 2003b), perhaps by modifying disulfide bridges.

In keeping with the availability of KCa1.1-null mice, it is somewhat surprising that there are no published studies investigating the impact of KCa1.1 deficiency on H₂O₂- or EET-dependent and/or EDHF–dilator pathways. Nonetheless, the overall importance of smooth muscle KCa1.1 channels is highlighted by the higher myogenic tone and hypertension of α-subunit- as well as β1-subunit-deficient mice (Brenner et al., 2000; Plüger et al., 2000; Sausbier et al., 2005; Werner et al., 2005). Moreover, α-subunit-deficient mice show erectile dysfunction (Werner et al., 2005) and primary hyperaldosteronism (Sausbier et al., 2005) as the putative reason for hypertension.

Role of EDHF in systemic blood pressure control and in cardiovascular disease states

The contribution of EDHF to the overall endothelium-mediated vasodilation in the arterial system increases when vessel diameter decreases (Shimokawa et al., 1996; Urakami-Harasawa et al., 1997). In contrast, the contribution of the NO system decreases with the decrease of vessel diameter. Accordingly, EDHF contributes only to a small extent, if at all, to endothelium-dependent dilations in the aorta. In medium-sized conduit arteries such mesenteric arteries with a diameter <500 µm, EDHF accounts for at least half of the complete dilator response, while EDHF is the main dilator system in resistance-sized arterioles with a diameter <100 µm (Shimokawa et al., 1996; Koeppen et al., 2004). Causality for these calibre-dependent differences is unclear. However, it is possible that EDHF's capability to produce a vasodilating effect depends on structural determinants, for example, media size and myo-endothelial electrical coupling, or functional aspects such as degree of basal myogenic tone, metabolic state and/or sympathetic nerve activity. In any case, the strong impact of EDHF on tone of resistance-sized arteries suggests that the EDHF system plays a significant role in systemic blood pressure control. Concerning the KCa3.1/KCa2.3-EDHF–dilator system, this has been demonstrated by elevated blood pressures in KCa3.1/KCa2.3-deficient mice as mentioned previously. However, such a role may also apply to diffusible EDHFs, such as EETs, as concluded from alterations in arterial blood pressure in the soluble epoxide hydrolase-deficient mice (Sinal et al., 2000). Of note, besides the impact of such defects in EDHF on blood pressure, EDHF has also the capacity to counteract hypertension caused by deficiency of the other two dilator system NO and PGI₂ (Brandes et al., 2000; Scotland et al., 2005; Taddei et al., 2006a), which underpins that EDHF can also serve as an important compensatory dilator system in cardiovascular diseases.

Most cardiovascular pathologies such as hypertension, uraemia, hypercholesterolemia, diabetes and restenosis disease and also, aging are associated with endothelial dysfunction (for extensive review see Endemann and Schiffrin, 2004; Spieker et al., 2006; Sudano et al., 2006; Yildiz, 2007), and there is growing evidence that this endothelial dysfunction is not only related to diminished NO production or NO availability (for review also see Forstermann and Munzel, 2006; Taddei et al., 2006b), but in some disease states also to defects in the EDHF system(s) [for review see (Feletou and Vanhoutte, 2004)]. This raises the possibility that impairment of particularly the KCa3.1/KCa2.3-EDHF–dilator system may contribute to the development or to the progression of endothelial dysfunction and thus cardiovascular disease.

Regarding hypertension, defects in the EDHF system have been reported, although not consistently in all animal models. For instance, in experimental genetic hypertension, an impairment of EDHF–dilator response has been reported in aged spontaneously hypertensive rats (SHR) (Bussemaker et al., 2003b; Kansui et al., 2004) and in SHR-stroke prone (SHR-SP) with more manifest hypertension (Sunano et al., 1999). In both strains endothelium-dependent hyperpolarization was diminished, and EDHF–dilator responses were reduced in renal arteries of aged SHR (Bussemaker et al., 2003b) and in mesenteric arteries of SHR-SP (Sunano et al., 1999). In contrast, NO-mediated relaxation was only slightly reduced, and production of endothelium-derived contracting factors, cyclooxygenase-generated prostanoids, was found to be enhanced in SHR. In the rat 2-kidneys/1-clip (2K1C) model of renovascular hypertension (Goldblatt hypertension), aortic smooth muscle hyperpolarization and EDHF–dilator responses were also found to be disturbed (Callera et al., 2000). However, (mREN-2)-27-transgenic hypertensive rats do not show a disturbed EDHF–dilator response, and overall endothelium-dependent vasodilation appears to be intact (Randall and March, 1998). Likewise, in angiotensin-II-infused hypertensive rats, EDHF–dilator responses seem to be normal (Hilgers and Webb, 2007). Similarly, in the murine 2K1C model and in angiotensin-II-infused mice, EDHF–dilator responses are also unaffected in carotid arteries (I. Grgic, B.P. Kaistha, J. Hoyer and R. Köhler, unpubl. obs.) and in mesenteric resistance vessels (Wang et al., 2006). Intriguingly, angiotensin-II hypertension can impair the EDHF system involving EETs as diffusible EDHF(s) by inducing expression of soluble expoxide hydrolase, the EET-degrading enzyme (Ai et al., 2007). Therefore inhibitors of soluble expoxide hydrolase are now being considered as novel antihypertensive drugs (Chiamvimonvat et al., 2007).

Interestingly, in pregnancies complicated by pre-eclampsia, the absence of EDHF–dilator responses in the mother's myometrial arteries has been proposed to contribute to endothelial dysfunction in this disease state (Kenny et al., 2002).

Impairments of the EDHF system have also been reported in other models characterized by endothelial dysfunction with or without hypertension. In albuminuria-prone hypertensive Munich Wistar Fromter rats, an impairment of the EDHF–dilator response is present in coronary but not in mesenteric arteries suggesting that hypertension and/or renal failure can effect the EDHF system in a vascular bed-specific manner (Gschwend et al., 2002). Moreover in uraemic hypertensive and non-hypertensive rats, the EDHF system was reported to be impaired in a similar fashion (Köhler et al., 2005; Vettoretti et al., 2006) suggesting that renal failure, in particular, impairs this dilator system.

In other cardiovascular pathologies such as hypercholesterolemia and atherosclerosis EDHF–dilator responses are preserved or even enhanced, perhaps to counterbalance the defects of NO activity and NO-mediated dilations present in these disease states. For instance, normal EDHF–dilator responses have been reported in hypercholesterolemic and apolipoprotein-E-deficient (ApoE^−/−) mice (Brandes et al., 1997; Ding et al., 2005; Morikawa et al., 2005; Wolfle and de Wit, 2005) and dyslipidemic ApoB-deficient mice (Krummen et al., 2005). Interestingly, EDHF–dilator responses were found to be disturbed in gastroepiploic arteries from hypercholesterolemic as well as aged patients (Urakami-Harasawa et al., 1997), suggesting that hypercholesterolemia and aging are cardiovascular risk factors affecting the EDHF–dilator system in humans.

Defects in the EDHF system have been reported in experimental diabetes in rats as well as in human diabetes (De Vriese et al., 2000; Fitzgerald et al., 2005). Diabetes seems to impair the EDHF system, particularly in small-sized arteries, where it plays a major role, as outlined above. For instance, impaired EDHF–dilator responses have been reported in human penile resistance arteries from diabetic man (Angulo et al., 2003) and in small mesenteric arteries of type I and type II diabetic rats (Makino et al., 2000; Wigg et al., 2001; Burnham et al., 2006; Weston et al., 2008), in coronary arteries of type II diabetic rats (Miller et al., 1999) as well as type I diabetic mice, and more severely in type I diabetic ApoE^−/− mice (Ding et al., 2005; Morikawa et al., 2005).

Although the suggested molecular and cellular bases for the reported defects in EDHF–dilator responses may vary and are not yet fully characterized, in some cases; however, impairments of EDHF–dilator responses are clearly shown to involve defects in the function or expression of KCa3.1/KCa2.3 channels (Table 1 provides an overview of these findings). For instance, a diminished expression of endothelial KCa3.1 and KCa2.3 channels underlies the impaired EDHF–dilator response in carotid arteries of uraemic 5/6 nephrectomized rats (Köhler et al., 2005). A similarly reduced expression of KCa2.3 and KCa3.1 in regenerated endothelium parallels the impaired EDHF–dilator responses in carotid arteries after balloon catheter injury (Köhler et al., 2001). In contrast, EDHF–dilator responses seem to be normal in small mesenteric arteries of angiotensin-II hypertensive rats, albeit with a reduced mRNA expression and protein levels of KCa2.3 without any change in KCa3.1 (Hilgers and Webb, 2007). In diabetic Zucker Diabetic Fatty rats, acetylcholine-induced smooth muscle hyperpolarization is impaired due to a loss of KCa2.3 functions without any reduction in mRNA expression (Burnham et al., 2006). Interestingly, KCa3.1 functions in these diabetic rats are conserved despite a modest decrease in mRNA expression. However, inappropriate channel activation seems to occur due to diminished expression of the CaR as reported recently (Weston et al., 2008). According to some studies on human patients, KCa3.1/KCa2.3 function seems to be compromised in a number of cardiovascular pathologies. For instance, the disturbed dilator responses in skeletal muscle arterioles and coronary microvasculature following cardiopulmonary bypass surgery were associated with diminished endothelial KCa2.3 and KCa3.1 functions (Feng et al., 2008; Liu et al., 2008). Defective EDHF–dilator responses were also observed in omental arteries from patients with essential hypertension, possibly related to a reduction in KCa2.3 expression (Li et al., 2007). Moreover, aging and hypercholesterolemia were shown to significantly impair EDHF-mediated relaxations in isolated human gastroepiploic arteries and distal microvessels, which appear to involve a disturbed function of KCa3.1 channels (Urakami-Harasawa et al., 1997).

Table 1.

EDHF in cardiovascular disease

Animal model/subjects	Phenotype/cardiovascular disease/hypertension	Origin/cause	Overall endothelial function	EDHF-activity	Vessel	Possible reason for altered EDHF-activity/other mechanism	References
Rat models
SHR	Hypertension/‘aging’	Genetic (unknown)	↓	↓	Renal A./mesenteric A.	↓ EETs ↓ Cx37/40 mRNA expression	Bussemaker et al. (2003b)
SHR-SP	Severe hypertension	Genetic (unknown)	↓	↓	Mesenteric A.	?	Sunano et al. (1999)
Transgenic hypertensive rat	Severe hypertension	Transgenic mREN-2)-27 over-expression	→	↑	Mesenteric A.	↓ NO	Randall and March (1998)
Zucker Diabetic Fatty rats	Diabetes type II	Insulin resistance	↓	↓	Small mesenteric arteries	↓ KCa3.1 functions/↓ CaR mRNA expression	Burnham et al. (2006); Weston et al. (2008)
Chronic renal failure in rats	Uraemia/hypertension	5/6 nephrectomy	↓	↓	Carotid A./small mesenteric A.	↓ KCa3.1/KCa2.3 mRNA expression/function, ↓ hyperpolarization	Köhler et al. (2005); Vettoretti et al. (2006)
Balloon catheter injury in rats	Restenosis	Endothelial ablation/regeneration	↓	↓	Carotid A.	↓ KCa3.1/KCa2.3 mRNA expression	Köhler et al. (2001)
Knockout mice
Diabetic apolipoprotein-E-knockout mice (ApoE−/−)	Diabetes/atherosclerosis	Streptozotocin/ApoE gene deletion	↓/→	↓	Small mesenteric arteries/aorta	↓ KCa2.3/KCa2.2/Cx37 mRNA expression ↑ eNOS mRNA expression	Ding et al. (2005); Morikawa et al. (2005)
eNOS-knockout mice (eNOS−/−)	Hypertension	eNOS gene deletion	↓/→	↑	Resistance arteries/aorta	↑ Cyclooxygenase-1 activity, KCa→	(Huang et al. (1995); Scotland et al. (2005)
KCa2.3-transgenic mice (KCa2.3T/T)	Parturition defects, increased vessel diameter	Constitutive over-expression or suppression with dietary doxycycline	↑?	↑?	Mesenteric A.	↑ KCa2.3 mRNA expression, ↑ resting membrane potential in endothelial cell, diminished basal tone	Taylor et al. (2003)
KCa2.3 over-expression	Hypertension		↓	↓	Mesenteric A.	↓ KCa2.3 mRNA expression	Taylor et al. (2003)
KCa2.3 suppression	Hypertension	KCa3.1 gene deletion	↓	↓↓	Carotid A./ microcirculation	Endothelial dysfunction/diminished endothelial and smooth muscle hyperpolarization, normal conducted vasodilations	(Si et al. (2006a)
KCa3.1-knockout mice (KCa3.1−/−)	Arteriosclerosis/hypertension/‘aging’	Genetic/multifactorial	↓	↓	Large gastroepiploic A.	↓ KCa function?	Urakami-Harasawa et al. (1997)
Human subjects	Arteriosclerosis/coronary bypass surgery	Genetic/multifactorial		↓	Skeletal arterioles coronary microvessels	↓ KCa3.1/KCa2.3 function	Feng et al. (2008); Liu et al. (2008)

Note that this is a list of selected studies and therefore should be regarded as incomplete.

↓, dysfunctional; ↑, up-regulated.

CaR, Ca²⁺-sensing receptor; EDHF, endothelium-derived hyperpolarizing factor; EETs, epoxyeicosatrienoic acids; eNOS, endothelial nitric oxide synthase; SHR, spontaneously hypertensive rats; SHR-SP, SHR-stroke prone; KCa2.3, small-conductance Ca²⁺-activated K⁺ channel subtype 3; KCa3.1, intermediate-conductance Ca²⁺-activated K⁺ channel.

In aggregate, these studies suggest that the intact KCa2.3/KCa3.1-EDHF system plays a substantial role in blood pressure regulation and that the impairment of this system contributes substantially to endothelial dysfunction in a number of cardiovascular pathologies.

Openers of KCa2.3 and KCa3.1 channels as a new therapeutic option for the treatment of hypertension?

In light of the pivotal role of KCa2.3 and KCa3.1 channels in endothelial function and particularly in EDHF–dilator responses, it is intriguing to speculate that compounds opening KCa2.3 and KCa3.1 channels could exert blood pressure lowering actions and thus be useful to lower blood pressure in hypertensive disease states or in other cardiovascular pathologies characterized by endothelial dysfunction. Indeed, the novel riluzole-derivative SKA-31 (Sankaranarayanan et al., 2008) opens preferentially KCa3.1 channels in murine endothelial cells and potentiates at nanomolar concentrations acetylcholine-induced EDHF–dilator responses in carotid arteries from mice. Moreover, intraperitoneal injection of SKA-31 has been shown to lower blood pressure by 6 mmHg in normotensive mice and by 12 mmHg in angiotensin-II-induced hypertension. These effects are not seen in KCa3.1^−/− mice thus demonstrating selectivity of SKA-31 for KCa3.1 channels in vivo (Sankaranarayanan et al., 2008). Likewise, the NeuroSearch A/S compound NS309 has been shown to produce endothelium-dependent hyperpolarizations in guinea pig carotid arteries by opening of KCa2.3 and KCa3.1 channels (Leuranguer et al., 2008). Of note, NS309 has been shown to improve endothelium-dependent dilation by also enhancing NO formation in mesenteric artery and in porcine retinal arterioles (Dalsgaard et al., 2008; Stankevicius et al., 2008). Thus, these promising results from recent studies indeed suggest that KCa2.3 and KCa3.1 channels may represent new drug targets for the treatment of hypertension. Henceforth, further studies are required to test their therapeutic efficacy and safety in the different forms of hypertension and types of endothelial dysfunction, that is, in cardiovascular disease states in which NO formation or NO availability is compromised and/or the KCa3.1/KCa2.3-EDHF–dilator system is reduced.

Concluding remarks

Based on recent findings derived from genetic models, there is substantial evidence that the KCa3.1/KCa2.3-EDHF–dilator system is a fully emancipated endothelial dilator system, next to the classical NO and PGI₂ systems and other presumed EDHF systems. The recent data furthermore suggest that KCa3.1 and KCa2.3 channels exert distinct as well as overlapping functions, by either contributing predominantly to EDHF–dilator responses (KCa3.1), or influencing EDHF and NO signalling (KCa2.3). These distinct roles can be explained by their differential subcellular compartmentation. The blood pressure alterations in KCa3.1/KCa2.3-transgenic animals strongly indicate that both channels are of individual importance for appropriate blood pressure control. Moreover, the current data corroborate the idea that the KCa3.1/KCa2.3-EDHF system acts independently and is non-compensable by other endothelium-dependent vasodilator systems, NO and PGI₂. Importantly, the KCa3.1/KCa2.3-EDHF–dilator system appears to be impaired in cardiovascular disease states such as hypertension, diabetes, and perhaps in atherosclerosis, and this defect may thus contribute to the overall endothelial dysfunction present in these cardiovascular pathologies. Finally, the recent advances in the field of selective KCa3.1/KCa2.3 openers suggest that KCa3.1 and KCa2.3 channel may represent novel attractive targets for especially the development of alternative antihypertensive therapies. Intriguingly, KCa3.1 and KCa2.3 opener NS309 (Strobaek et al., 2004) and SKA-31, opening preferentially KCa3.1 with high affinity (Sankaranarayanan et al., 2008), may emerge as promising candidates or lead compounds to improve EDHF responses and thus endothelial function in various cardiovascular pathologies.

Glossary

Abbreviations:

EDHF

endothelium-derived hyperpolarizing factor

KCa2.3

small-conductance Ca²⁺-activated K⁺ channel type 3

KCa3.1

intermediate-conductance Ca²⁺-activated K⁺ channel

VSMC

vascular smooth muscle cell

Conflict of interest

None.