KV7 Channel Expression and Function Within Rat Mesenteric Endothelial Cells

Background and Purpose: Arterial diameter is dictated by the contractile state of the vascular smooth muscle cells (VSMCs), which is modulated by direct and indirect inputs from endothelial cells (ECs). Modulators of KCNQ-encoded k_V7 channels have considerable impact on arterial diameter and these channels are known to be expressed in VSMCs but not yet defined in ECs. However, expression of k_V7 channels in ECs would add an extra level of vascular control. This study aims to characterize the expression and function of K_V7 channels within rat mesenteric artery ECs.

Experimental Approach: In rat mesenteric artery, KCNQ transcript and K_V7 channel protein expression were determined via RT-qPCR, immunocytochemistry, immunohistochemistry and immunoelectron microscopy. Wire myography was used to determine vascular reactivity.

Key Results: KCNQ transcript was identified in isolated ECs and VSMCs. K_V7.1, K_V7.4 and K_V7.5 protein expression was determined in both isolated EC and VSMC and in whole vessels. Removal of ECs attenuated vasorelaxation to two structurally different K_V7.2-5 activators S-1 and ML213. K_IR2 blockers ML133, and BaCl₂ also attenuated S-1 or ML213-mediated vasorelaxation in an endothelium-dependent process. K_V7 inhibition attenuated receptor-dependent nitric oxide (NO)-mediated vasorelaxation to carbachol, but had no impact on relaxation to the NO donor, SNP.

Conclusion and Implications: In rat mesenteric artery ECs, K_V7.4 and K_V7.5 channels are expressed, functionally interact with endothelial K_IR2.x channels and contribute to endogenous eNOS-mediated relaxation. This study identifies K_V7 channels as novel functional channels within rat mesenteric ECs and suggests that these channels are involved in NO release from the endothelium of these vessels.

Introduction

KCNQ-encoded K_V7 channels are key regulators of arterial reactivity. Within the vasculature, of the five KCNQ subtypes, KCNQ4 is predominantly expressed, followed by KCNQ5 > KCNQ1; with little to no contribution from KCNQ2/3 (Ohya et al., 2003; Yeung et al., 2007; Ng et al., 2011). In human and rodent blood vessels K_V7 channels contribute to resting tone (Ohya et al., 2003; Yeung et al., 2007; Mackie et al., 2008; Ng et al., 2011), whereby their blockers such as linopirdine or XE991 produce contractions or enhance vasoconstrictor responses. In addition, a range of K_V7 channel activators including retigabine, S-1 and ML213 are effective relaxants of pre-contracted arterial tone. Furthermore, K_V7 channels also represent functional end targets for a myriad of endogenous vasoactive responses, wherein channel activity is impaired during PKC-mediated vasoconstriction (Brueggemann et al., 2006) and enhanced as a result of cGMP and cAMP dependent receptor-mediated vasodilation (e.g., Chadha et al., 2012; Khanamiri et al., 2013; Stott et al., 2014, 2015; Mani et al., 2016; Brueggemann et al., 2018; Mondéjar-Parreño et al., 2019). To date, vascular K_V7 channel studies have focused predominantly on vascular smooth muscle cells (VSMCs) or whole arteries, and as a result it is currently unclear whether endothelial cells (ECs) express K_V7 channels and if so, what their functional role may be.

Endothelial cells form the inner layer of blood vessels and constitute a paracrine signaling platform which regulates VSMC contractility, vascular resistance and ultimately blood flow through the release of nitric oxide (NO), prostacyclin, epoxyeicosatrienoic acid and others; including the generation and spread of endothelium-derived hyperpolarization (EDH) (McGuire et al., 2001). Myoendothelial (ME) projections within fenestrations (holes) of the internal elastic lamina (IEL) facilitate the presence gap junctions (MEGJs) at a proportion of such sites (∼50% in adult rat 1st–3rd order ‘large’ mesenteric arteries; MA; Sandow et al., 2009) which permits heterocellular electrochemical coupling via connexin proteins (Sandow et al., 2012). These sites enable the transfer of EC-derived signals via the flow of both small molecules < ∼1 kDa and current between the cells. In endothelium-dependent relaxation of rat MA, a fundamental role for small/intermediate conductance calcium-activated potassium (SK_Ca and IK_Ca, respectively; Sandow et al., 2006; Dora et al., 2008), transient receptor potential canonical type 3 (Senadheera et al., 2012), inwardly rectifying potassium channels (K_IR2) (Goto et al., 2004), as well as inositol-1,3,4 trisphosphate receptor/s (Fukao et al., 1997) has been demonstrated.

Identification of K_V7 expression in ECs would open up a new layer of vascular control by these channels and is a necessary requisite for understanding the role of these channels in vascular health and disease. This study aims to ascertain whether rat mesenteric ECs express K_V7 channels, and if so, what their functional implications may be. This study shows that K_V7 channels are expressed in rat mesenteric artery ECs and contribute to both K_V7 activator-mediated relaxation via a potential functional interaction with K_IR2 channels and endothelial NO synthase (eNOS)-dependent axis of carbachol (CCh)-mediated vasorelaxation.

Materials and Methods

Animal Models

Experiments were performed on MA from Male Wistar rats (Charles River, Margate, United Kingdom) ages 11–14 weeks (200–350 g) from the Biological Research Facility, St George’s, London, United Kingdom; and from the Animal Resources Center, Perth, Australia. Animals were housed in cages with free access to water and food (RM1; Dietex Inter-national, United Kingdom) ad libitum, with a 12-h light/dark cycle and constant temperature and humidity (21 ± 1°C; 50% ± 10% humidity) in accordance with the Animal (Scientific Procedures) Act 1986, the guidelines of the National Health and Medical Research Council of Australia and the UNSW Animal Ethics and Experimentation Committee (AEEC #18/86B). Animals were kept in LSB Aspen woodchip bedding. Animals were culled by either cervical dislocation with secondary confirmation via cessation of the circulation by femoral artery severance or were anesthetized with sodium pentathol (intraperitoneal, 100 mg/kg) in accordance with Schedule 1 of the ASPA 1986.

Either whole mesenteric plexus or 2nd/3rd/4th order MA were used with vessel order identified from the second bifurcation of the superior MA. Arteries were dissected, cleaned of fat and adherent tissue and stored on ice within physiological salt solution (PSS) of the following composition (mmol-L^–1); 119 NaCl, 4.5 KCl, 1.17 MgSO₄.7H₂0, 1.18 NaH₂PO₄, 25 NaHCO₃, 5 glucose, 1.25 CaCl₂.

Reverse Transcription Quantitative Polymerase Chain Reaction

Second and third order MA segments were enzymatically digested to obtain either freshly isolated ECs (as previous, Greenberg et al., 2016) or VSMCs. Briefly, vessels were washed in Hanks’ Balanced Salt Solution (HBSS; ThermoFisher Scientific, GIBCO, 14170-088) containing 50 μmol-L^–1 CaCl₂ for 5 min at 37°C, the media was then replaced in HBSS containing 50 μmol-L^–1 CaCl₂ with 1 mg/mL collagenase IA (Sigma Aldrich, C9891, United Kingdom) for 15 min at 37°C. Vessels were washed in HBSS containing 50 μmol-L^–1 CaCl₂ for 10 min at 37°C. The supernatant was removed and the vessels suspended in fresh HBSS containing 75 μmol-L^–1 CaCl₂. For RT-qPCR, ECs were dissociated using a wide-bore smooth-tipped pipette and identified under the microscope (x10) as sheets of cells independent from the vessel which were harvested and stored separately from the residual VSMCs.

mRNA from both isolated ECs and VSMCs was extracted using Monarch Total RNA Miniprep Kit (New England BioLabs, Ipswich, MA, United States) and reverse transcribed via LunaScript RT SuperMix Kit (New England BioLabs, Ipswich, MA, United States). Quantitative analysis of relative gene expression was assessed via CFX-96 Real-Time PCR Detection System (BioRad, Hertfordshire, United Kingdom). Samples were run in duplicate to account for variation. Samples were run in BrightWhite qPCR plate (Primer Design, Camberley, United Kingdom), with each well containing 20 μL of reaction solution containing: 10 μL of PrecisionPLUS qPCR Master Mix (Primer Design, Camberley, United Kingdom), 300 nmol-L^–1 of gene specific target primer (Thermofisher scientific, Waltham, MA, United States) and 10 ng of cDNA sample made up to 20 μL total volume with nuclease free water. Run protocol: (1) activation step (15 min:95°C), (2) denaturation step (15 s: 94°C), (3) annealing step (30 s: 55°C), and (4) extension step (30 s: 70°C). Steps 2- 4 were repeated x 40. Quantification cycle (Cq) was determined via Bio-Rad CFX96 Manager 3.0. Cq values were normalised to housekeeper genes expressed as a 2^–ΔCq when compared to appropriate reference genes including 14-3-3 Zeta (YWHAZ) and glyceraldehyde-3-phosphate dehydrogenase (GAPD). Cell isolation for VSMCs and ECs was validated by either positive expression of VSMC specific marker α-actin 2 (Acta2) or EC specific marker-platelet endothelial cell adhesion molecule-1 (Pecam-1) respectively. See Table 1 for a list of the primers used in the following (Jepps et al., 2011; Askew Page et al., 2019; ThermoFisher Scientific).

TABLE 1

Table 1. RT-qPCR primer sequences.

Immunocytochemistry

Freshly dispersed ECs (as above), together with residual VSMCs were left for 1hr before use. Cells were then fixed in 4% paraformaldehyde (Sigma-Aldrich, United Kingdom) in PBS for 20 min at RT as previously described (Barrese et al., 2018a). Cells were treated with 0.1 mol-L^–1 glycine for 5 min and incubated for 1 h with blocking solution (PBS-0.1% Triton X-100-10% bovine serum albumin) at RT. Following incubation overnight at 4°C with primary antibodies (Table 2) diluted in blocking solution (anti-PECAM-1 for ECs, anti-α-actin for VSMCs and anti-K_V7.1, K_V7.4, and K_V7.5 channel for ECs/VSMCs), cells were then washed for 20 min with PBS, incubated for 1 h at RT with the secondary conjugated antibodies diluted in blocking solution. Excess secondary antibody was removed by washing with PBS and cells mounted using media containing 4′,6-diamidino-2-phenylindole (DAPI) for nuclear counterstaining. Using triple staining, ECs and VSMC were differentiated via the following: ECs were positive for anti PECAM-1 and negative for anti-α-actin; while VSMC was positive for anti-α-actin and negative for anti-PECAM-1 (data not shown). Cells were analyzed using a Zeiss LSM 510 Meta argon/krypton laser scanning confocal microscope (Image Resource Facility, St George’s University, London).

TABLE 2

Table 2. Immunocyto/histochemistry reagents and use (Jepps et al., 2009; Chadha et al., 2012).

Cell Culture

Chinese Hamster Ovary (CHO) cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mmol-L^–1 L-glutamine, and 1% penicillin/streptomycin (Sigma Aldrich, Dorset, United Kingdom) and maintained at 37°C with 5% CO₂ in an incubator. Cells were plated in a 24-well plate, incubated for 24hr then transfected with either K_V7.1, K_V7.4 or K_V7.5 plasmids using Lipofecamine 2000 (Thermofisher, Paisley, United Kingdom) as described previously (Barrese et al., 2018a). After 24 h, cells were fixed and stained as described above.

Immunohistochemistry

Animals were anesthetized with sodium pentathol (intraperitoneal, 100 mg/kg) and perfusion fixed (Sandow et al., 2004) in 2% paraformaldehyde in 0.1 mol-L^–1 PBS. Third to 4th order MA segments were dissected, opened laterally and pinned as a sheet to a Sylgard dish. Segments were washed in PBS (3 × 5 min), incubated in blocking buffer (PBS with 1% BSA and 0.2% Triton) at room temperature (RT) for 2 h and then overnight with primary antibody (Table 2) in blocking buffer at 4°C, washed again (3 × 5 min with gentle agitation), and incubated in secondary antibody (Table 2; matched to the respective primary) in PBS with 0.1% Triton in PBS for 2 h at RT. Tissue was mounted on slides in anti-fade media containing propidium iodide (PI) or DAPI (Table 2) and imaged with uniform confocal settings. Incubation of tissue with secondary only was used as a ‘zero’ setting for confocal imaging. Controls involved substitution of primary with isotype control, with concentration (where provided by manufacturer) matched, or 10-fold higher than the respective antibody of interest (Table 2). Working Ab dilutions were prepared in accordance with previous work (Jepps et al., 2009; Chadha et al., 2012). Confocal image stacks were collected at 0.2 μm intervals. The optimal rinsing protocol was determined by incubating in secondary only; and rinsing after successive 5 min incubations until fluorescence was reduced to background. Note that if this was not done secondary alone was specifically and highly localized to IEL hole sites; as potential false positives at such sites; suggesting that such sites have an affinity for IgG-secondary label alone.

Electron Microscopy

Animals were anesthetized as above and perfusion fixed in 0.2% glutaraldehyde and 2% paraformaldehyde in 0.1 mol-L^–1 PBS (pH 7.4). MA segments (∼2 mm in length) were washed (3 × 5 min) and processed in a Leica EMPACT 2 high-pressure freezer using 0.7% low melting agarose as a cryoprotectant. Samples were then freeze-substituted in a Leica AFS2 into 0.2% uranyl acetate in 95% acetone (from −85 to −50°C) and infiltrated with Lowicryl (at −50°C), before UV polymerization (2 days each at −50 and 20°C; Zechariah et al., 2020). Conventional transmission electron microscopy (TEM) was conducted using standard procedures (Sandow et al., 2002, 2004).

Individual serial transverse sections (∼100 nm) were mounted on Formvar-coated slot grids and processed for antigen localization as for confocal immunohistochemistry (per above and Table 2). The secondary used was 5 or 10 nmol-L^–1 colloidal gold-conjugated antibody (1:40; 2 h) in 0.01% Tween-20. Sections were imaged at x10-40,000 on a JEOL transmission electron microscope at 16 MP (Emsis, Morada G3). Background gold label density was determined from randomly selected (4x) 1 × 1 μm regions per sample of lumen and IEL, compared to the same sized regions of interest in EC profiles.

Wire Myography

Second order MA segments (∼2 mm in length) were mounted on 40 μm diameter tungsten wire in a tension myograph chamber (Danish Myo Technology, Arhus, Denmark) containing 5 mL of PSS (composition, as above) oxygenated with 95% O₂ and 5% CO₂ at 37°C. Vessels then underwent a passive force normalization process to achieve an internal luminal circumference at a transmural pressure of 100 mmHg (13.3 kPa) to standardize pre-experimental conditions (Mulvany and Halpern, 1976). Force generated was first amplified by a PowerLab (ADInstruments, Oxford, United Kingdom), and recorded by LabChart software (ADInstruments, Oxford, United Kingdom). Vessels were then challenged with 60 mmol-L^–1 [K⁺] to determine viability, and then constricted with 10 μmol-L^–1 methoxamine (MO), an α-1 adrenoreceptor agonist, EC integrity was then determined via addition of 10 μmol-L^–1 carbachol (CCh), a synthetic acetylcholine analog. Vessels displaying ≥ 90% vasorelaxation in response to CCh were considered EC positive (EC+). Vessels were denuded of ECs by gently passing a human hair through the lumen. Vessels expressing ≤ 10% vasorelaxation in response to CCh were considered EC negative (EC−). During functional investigations, all vessels were pre-constricted with the thromboxane A2 receptor agonist U46619 (300 nmol-L^–1) to elicit an EC₈₀ contraction. Concentration-dependent relaxant responses to S-1 (0.1–10 μmol-L^–1), ML213 (0.1–10 μmol-L^–1), ML277 (0.03–10 μmol-L⁻¹), CCh (0.3–10 μmol-L^–1) and S-nitroprusside (SNP; 0.01–3 μmol-L^–1) were determined in the presence and absence of ECs, linopirdine (10 μmol-L^–1), HMR-1556 (10 μmol-L^–1), carbenoxolone (100 μmol-L^–1), ML133 (20 μmol-L^–1), barium chloride (BaCl₂; 100 μmol-L^–1), L-nitroarginine methyl ester (L-NAME; 100 μmol-L^–1), TRAM34 (1 μmol-L^–1), Apamin (10 nmol-L^–1), 4-aminopyridine (4-AP; 1 mmol-L^–1), and tetraethylamonium (TEA; 1 mmol-L^–1).

Data and Statistical Analysis

All functional figures express mean data from at least 5 animals ± standard error of the mean (SEM). Experiments comparing groups of unequal numbers are present due to technical failure or expiry of tissue during isometric tension recording. For functional experiments involving cumulative concentrations, a transformed data set was generated using; X = Log(X), to reduce representative skew. A four parametric linear regression analysis was then performed using the following equation; [Log(Agonist) vs. response – variable slope (four parameters bottom/hillslope/top/EC₅₀)] using GraphPad Prism (Version 8.2.0) to fit a concentration effect curve (CEC) to the figure. For data comparing multiple groups, a two way-ANOVA followed by a post hoc Bonferonni test in order to account for type 1 errors in multiple comparisons was performed for comparison of mean values. For data comparing two groups, an unpaired parametric T-test was was performed. Significance values are represented as follows; P < 0.05 (^∗). n = (x), number of animals used. N = (x), number of segments used. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018).

Results

Identification of K_V7 Channels Within MA ECs

Initial investigation sought to identify Kcnq/K_V7 transcript and protein within MA ECs. Transcript levels for Kcnq1-5 and EC and VSMC markers were determined in cell lysates from isolated MA VSMC and EC (per Methods). Both ECs and VSMCs expressed Kcnq4 > Kcnq5 > Kcnq1 with no expression of Kcnq2/3 (Figure 1A) similar to previous studies (Ohya et al., 2003; Yeung et al., 2007; Ng et al., 2011). However, a comparative reduction in the relative abundance of Kcnq1 was observed in MA ECs when compared to VSMCs (Figure 1A). Cell isolation efficiency is demonstrated by a reduction in EC marker Pecam within VSMCs cell lysates when compared to ECs (P ≤ 0.05) and a reduction in VSMCs marker Acta2 in EC cell lysates when compared to VSMCs (P = 0.065, Figures 1B,C).

FIGURE 1

Figure 1. Expression profile of Kcnq genes within isolated rat mesenteric vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). RT-qPCR analysis of relative expression of Kcnq1-5 in isolated VSMCs and ECs (A). Cell specific markers Acta2 and Pecam, for VSMCs and ECs respectively, were measured to determine sample purity (B,C). Values expressed as mean ± SEM of (2^–ΔCq) of ΔCq values generated from appropriate housekeeper genes (n = 3). Statistical significance is defined as *P < 0.05 (B,C).

K_V7.1, K_V7.4, and K_V7.5 were detected in isolated ECs by immunodetection (Figures 2A,C,E). K_V7.4 had a punctate distribution in isolated ECs (Figure 2C) whereas K_V7.5 label appeared to be predominantly cytoplasmic with some diffuse label around the nucleus (Figure 2E). Similar to previous reports (Zhong et al., 2010; Oliveras et al., 2014; Mills et al., 2015; Morales-Cano et al., 2015; Barrese et al., 2018a). K_V7.1, K_V7.4, and 7.5 were also identified in isolated MA VSMCs (Figures 2B,D,F). K_v7.1 staining in ECs was negligible compared to VMSCs (Figures 2A,B). ECs and VSMCs were identified by positive expression of either PECAM (Figures 2Ai,Ci,Ei) or α-actin (Figures 2Bi,Di,Fi) respectively. Antibody specificity was determined via positive staining in CHO cells transfected with purported target and negative staining in non-transfected control cells (Supplementary Figure 1). Importantly, as ion channel expression can alter in isolated cells K_V7.4 and K_V7.5 were also detected in both ECs and VSMCs in en face whole-mount arteries (Figures 3A,B,E,F). Notably, both K_V7.4 and 7.5 were expressed at a proportion of IEL hole sites at an apparently higher level than the associated EC membrane label (Figures 3C,D,G,H). We also detected K_V7.4 and K_V7.5 in EC by immuno-gold electron microscopy (Supplementary Figure 2). These studies identify K_V7.4 and K_V7.5 in ECs as well as smooth muscle cells.

FIGURE 2

Figure 2. Immunocytochemistry demonstrates K_V7.1, K_V7.4, and 7_V.5 localization in rat mesenteric artery isolated endothelial (EC; A,C,E) and smooth muscle cells (SMC; B,D,F). Punctate labelling for Kv7.1, Kv7.4, and 7.5 was present in isolated EC and SMCs (A,C,E and B,D,F). PECAM and smooth muscle a-actin were used as markers for isolated endothelial and smooth muscle cells, respectively (Ai,Ci,Ei and Bi,Di,Fi respectively); e.g. comparative-same cells, double arrows, A–Fi). 4′,6-diamidino-2-phenylindole (DAPI; e.g. inset, C) were used as nuclear markers to verify cell patency. n = 4–6, representative of different source tissue (A–Fi). Double arrows from insets indicate same panel as main, but to different marker.

FIGURE 3

Figure 3. Immunohistochemistry demonstrates Kv7.4 and 7.5 localization in rat mesenteric artery endothelium (EC) and smooth muscle cells (SMCs) with a focus on internal elastic lamina (IEL) hole sites in whole mount tissue. Whole mounts of confocal stacks through the EC and SMC of K_v7.4 (A,B, respectively) and Kv7.5 (E,F, respectively). Confocal image stacks through the luminal EC to SM border (C,G) show diffuse/punctate K_v7.4/K_V7.5 across the cell membrane with some punctate higher-level fluorescence at localized sites (e.g. full arrows; C). When single images were taken at the EC-IEL-SM interface (D,H), punctate higher-level fluorescence densities were present (e.g. D,H arrows; H, higher magnification inset) at a proportion of IEL hole sites (e.g., D,H arrowheads). Propidium iodide (PI; e.g., upper left inset, A,B) were used as nuclear markers to verify cell and layer patency. Incubation in secondary only separately for each of the 3 fluorophores (Table 2; e.g. lower left inset, B; inset, F) were used to set ‘zero’ confocal settings. Incubation in isotype IgG control only (e.g., E, inset) showed fluorescence equivalent to secondary alone (e.g., B, lower inset; F, inset). n = 4, representative of different source. Longitudinal vessel axis, left to right. Double arrows indicate same site, but at different focal planes (C,D,G,H), and location of inset (D). Bar, 25 μm.

Removal of ECs Modulates K_V7.2-5 Activator Efficacy

A comprehensive pharmacological analysis was undertaken to determine if K_V7 channels have a functional role in MA ECs via a reductive approach. Initially, the effects of K_V7 channel modulators were examined in endothelium intact or denuded MAs. The structurally dissimilar K_V7.2–7.5 activators S-1 and ML213 interact with the same pharmacophore centered around a tryptophan in the S5 domain (Schenzer, 2005; Bentzen et al., 2006; Brueggemann et al., 2014; Jepps et al., 2014). ML277 is a potent activator of K_V7.1 (Yu et al., 2013) with a 100-fold increase in selectivity for K_V7.1 compared to K_V7.2-5 (Yu et al., 2013). Consistent with previous findings (Chadha et al., 2012; Jepps et al., 2014), S-1- and ML213-mediated vasorelaxation was ablated by pre-incubation with 10 μmol-L^–1 of the pan-K_V7 channel inhibitor linopirdine (Schnee and Brown, 1998; Figures 4A,B,D). Relaxation produced by 10–300 nmol-L^–1 ML277 were also prevented by pre-incubation with linopirdine (Figure 4C). However, relaxation produced by concentrations > 1 μmol-L^–1 ML277 was not attenuated by linopirdine and are therefore not mediated by K_V7.1 activation.

FIGURE 4

Figure 4. Sensitivity of Kv7 activator relaxations to 10 μmol-L^–1 linopirdine. In vessels pre-incubated in DMSO solvent control, K_V7.2-5 channel activators S-1 and ML213 relax pre-constricted arterial tone (300 nmol-L^–1 U46619; black) and are ablated by pre-incubation with pan K_V7 channel inhibitor linopirdine (10 μmol-L^–1; gray; n = 5; A,B). In vessels pre-incubated in DMSO solvent control, K_V7.1 channel activator ML277 relaxes pre-constricted arterial tone (300 mol-L^–1 U46619; black) and is ablated by linopirdine (10 μmol-L^–1; gray) to a threshold of 300 nmol-L^–1 ML277 (n = 5; C). A representative trace of S-1-mediated vasorelaxation ± 10 μmol-L^–1 linopirdine (D). All values are expressed as mean + SEM. A two-way statistical ANOVA with a post hoc Bonferroni test was used to generate significance values. Statistical significance is defined as *P < 0.05 (A–C).

Endothelial removal for the following experiments was confirmed by ablation of vasorelaxation in response to 10 μmol-L^–1 CCh (Figure 5A). Endothelium denudation by mechanical abrasion has no impact on the peak contraction produced by 300 nmol-L^–1 U46619 (Figure 5B), but significantly attenuated the potency of S-1 mediated vasorelaxation increasing EC₅₀ from 2 ± 0.2 μmol-L^–1 to 3 ± 0.7 μmol-L^–1 (Figures 5C,D). S-1 mediated relaxation was also impaired via the non-selective gap junction inhibitor carbenoxolone (Tare et al., 2002) (water E_max = 6.11 ± 1.82% vs. carbenoxolone E_max = 18.7 ± 3.25%; Figure 5E). In addition, the potency of ML213 was also impaired by endothelial removal (EC(+) EC₅₀ = 1 ± 0.2 μmol-L^–1 vs. EC(−) EC₅₀ = 3 ± 0.2 μmol-L^–1; Figure 5F) in a fashion analogous to S-1. However, the linopirdine-sensitive relaxation produced by ML277 was not affected by endothelial removal (Figure 5G).

FIGURE 5

Figure 5. K_V7.2-5 channel activator mediated vasorelaxation was significantly attenuated by EC removal. Removal of ECs ablated vasorelaxation in response to 10 μmol-L^–1 CCh following pre-constriction with 10 μmol-L^–1 MO (A). Removal of ECs (gray) has no effect on vasoconstriction to U46619 (300 nmol-L^–1) when normalized to vasoconstriction to KCl (60 mmol-L^–1; n = 5–7; n = 15-21; B). Representative trace of EC S-1-mediated vasorelaxation in EC(–) denuded vessels (gray) vs. EC(+) vessels (black; C). S-1-mediated vasorelaxation was significantly attenuated by EC removal and 100 μmolL^–1 carbenoxolone (gray; n = 5–7; D,E). ML213 but not ML277-mediated vasorelaxation was significantly attenuated by EC removal (gray; n = 5–7; D,E). Removal of ECs (gray) has no effect on ML277 within its range of linopirdine sensitivity (n = 5; F). All values are expressed as mean + SEM (A–G). A two-way statistical ANOVA with a post hoc Bonferroni test was used to generate significance values. Statistical significance is defined as *P < 0.05 (A–G).

EC K_IR Channels Modulate K_V7.2-5 Activator Sensitivity

Having identified that the presence of the endothelium modulates responses to K_V7 activators, experiments were performed to identify the underlying mechanism/s involved. Murine endothelial KCNJ2-encoded K_IR2.1 channels have been identified as ‘signal boosters’ that enhance EC-derived relaxation (Sonkusare et al., 2016). Comparatively, the current literature regarding rat mesenteric K_IR2 channels is limited. In brief, rat MA endothelium expresses K_IR2.1 (Dora et al., 2008), inwardly rectifying Ba²⁺ sensitive channels are restricted to the endothelial layer (Crane et al., 2003a) and K_IR channels contribute to acetylcholine-mediated responses (Goto et al., 2004). We propose that like mice, rat mesenteric ECs express functional K_IR2 channels that propagate EC signals in a similar process. Therefore, we performed a series of studies investigating the effect of two characterized K_IR2 blockers, BaCl₂ (Hagiwara et al., 1978) and ML133 (Wang et al., 2011) on K_V7 activator-mediated vasorelaxation.

In arteries with a functional endothelium, K_IR2 blockers, BaCl₂ (100 μmol-L^–1) and ML133 (20 μmol-L^–1), significantly impaired relaxations produced by S-1 (Figure 6A, EC₅₀ = DMSO, 1.89 ± 0.2 μmol-L^–1/BaCl₂, 2.3 ± 0.31 μmol-L^–1; Figure 6B, EC₅₀ = DMSO, 0.52 ± 0.12 μmol-L^–1/ML133, 3.1 ± 1.5 μmol-L^–1) and ML213 (Figures 6C,D, EC₅₀ = DMSO, 0.9 ± 0.3 μmol/L^–1/BaCl₂, 2.2 ± 0.5 μmol/L^–1/ML133, 2.5 ± 0.25 μmol-L^–1) when compared to DMSO solvent control (Figures 6A–D). No attenuation of the response to ML277 was observed when pre-incubated with either blocker (Figures 6E,F), consistent with EC removal. Furthermore, in arteries where the endothelium had been removed neither ML133 nor BaCl₂ had any effect on ML213 mediated relaxations (Figures 6G,H).

FIGURE 6

Figure 6. K_V7.2-5 channel activator-mediated vasorelaxation was attenuated by two structurally different K_IR channel blockers, BaCl₂ and ML133, but not In EC denuded vessels. K_V7.2-5 channel activator S-1 and ML213 effects were significantly attenuated by pre-incubation with the K_IR2 channel inhibitor BaCl₂ (100 μmol-L^–1; black; n = 5–7; A,C). K_V7.2-5 channel activator S-1 and ML213 effects were significantly attenuated by pre-incubation with a selective K_Ir 2.1 channel inhibitor, ML133 (20 μmol-L^–1; gray; n = 5–7; B,D). K_V7.1 channel activator ML277-mediated vasorelaxation was not affected by pre-incubation with either BaCl₂ (100 μmol-L^–1) or ML133 (20 μmol-L^–1; gray; n = 6–7; E,F). In EC denuded vessels, K_V7.2-5 channel activator ML213 responses were not affected by pre-incubation with either K_IR2 channel inhibitor BaCl₂ (100 μmol-L^–1; gray; n = 5; G) or ML133 (20 μmol-L^–1; gray; n = 5–6; H). All values are expressed as mean + SEM (A–H). A two-way statistical ANOVA with a post hoc Bonferroni test was used to generate significance values. Statistical significance is defined as *P < 0.05 (A–H).

IK_Ca/SK_Ca Inhibitors Had No Impact on K_V7 Activator Mediated Relaxation

Endothelial IK_Ca and SK_Ca channels contribute to relaxation responses in rat MA (Crane et al., 2003b). Thus, it is feasible that K_V7 channels interact with other key endothelial potassium channels; particularly those expressed within microdomains (Sandow et al., 2009). However, consistent with previous reports (Jepps et al., 2016), pre-incubation with a combination of IK_Ca inhibitor TRAM-34 (1 μmol-L^–1; Wulff et al., 2000) and SK_Ca inhibitor apamin (100 nmol-L^–1; Spoerri et al., 1975) had no effect on K_V7 activator mediated vasorelaxation (Figures 7A–C). These data suggest that the endothelium-dependent increase in potency to the K_V7 activators involves endothelial K_IR, but not IK_Ca or SK_Ca channels.

FIGURE 7

Figure 7. K_V7 channel activator mediated vasorelaxation was not affected by preincubation with IK_Ca/SK_Ca channel antagonists. Pre-incubation with both IK_Ca channel inhibitor TRAM34 (1 μmol-L^–1) and SK_Ca channel inhibitor Apamin (100 nmol-L^–1) had no effect on S-1, ML213 nor ML277 mediated vasorelaxation (n = 5–7; gray, A–C). All values are expressed are expressed as mean values ± SEM. A two-way statistical ANOVA with a post hoc Bonferroni test was used to generate significance values; no significant values were observed (A–C).

K_V7 Channels Contribute to CCh Evoked Vasorelaxation

The expression of functional K_V7 channels within ECs begs the question - do they contribute to EC-derived responses? Acetylcholine produces endothelium-dependent relaxation through NO-, EDH- and prostanoid-dependent mechanisms in rat MA (Parsons et al., 1994; Shimokawa et al., 1996; Peredo et al., 1997).

A distinct rightward shift in the sensitivity to vasorelaxation in response to CCh, a synthetic acetylcholine analog, was produced by the eNOS inhibitor L-NAME (100 μmol-L^–1) when compared to DMSO control (EC₅₀ DMSO = 0.59 ± 0.1 μmol-L^–1; L-NAME = 0.94 ± 0.1 μmol-L^–1; Figure 8A). A combination IK_Ca and SK_Ca inhibitors, TRAM-34 (1 μmol-L^–1) and apamin (100 nmol-L^–1) respectively, suppress EDH in rat MA, and produced greater attenuation (EC₅₀ TRAM34/apamin = 1.5 ± 0.7 μmol-L^–1; Figure 8A) when compared to L-NAME. Pre-incubating vessels with the pan K_V7 channel inhibitor linopirdine (10 μmol-L^–1) significantly attenuated CCh-mediated relaxation when compared to DMSO control (EC₅₀ DMSO = 0.2 ± 0.08 μmol-L^–1; linopirdine = 0.7 ± 0.3 μmol-L^–1; Figure 8B). In contrast, pre-incubating vessels with either the K_V7.1 specific inhibitor HMR-1556 (10 μmol-L^–1) or a combination of non-specific K_V channel inhibitors TEA (1 mmol-L^–1; Choi et al., 1991) and 4-AP (1 mmol-L^–1; Kurata and Fedida, 2006) had no significant effect on CCh-evoked vasorelaxation (Figures 8C,D).

FIGURE 8

Figure 8. CCh evoked vasorelaxation is mediated through both eNOS and IK_Ca/SK_Ca channels and is partially attenuated by inhibition of K_V7 channels upstream of NO. Both eNOS inhibitor L-nitroarginine methyl ester (L-NAME; 100 μmol-L^–1; gray) and IK_Ca/SK_Ca channel inhibitors TRAM-34 (1 μmol-L^–1)/apamin (100 nmol-L^–1; red, dashed line) significantly attenuated CCh mediated vasorelaxation (n = 5–6; A). The pan K_V7 channel inhibitor linopirdine (10 μmol-L^–1; gray) significantly attenuated CCh mediated vasorelaxation (n = 6; B). The K_V7.1 channel inhibitor HMR-1556 (10 μmol-L^–1; gray) did not modulate CCh-mediated vasorelaxation (n = 6; C). A combination of non-specific K⁺ channel inhibitors 4-aminopyridine (4-AP; 1 mmol-L^–1) and tetraethylammonium (TEA; 1 mmol-L^–1; gray) did not modulate CCh-mediated vasorelaxation (n = 5; D). Additive attenuation to CCh-evoked relaxation in the presence of TRAM-34 (1 μmol-L^–1)/apamin (100 nmol-L^–1) + linopirdine (10 μmol-L^–1; gray, dashed line) was observed when compared to vessels pre-incubated in only TRAM34/apamin (gray; n = 5–6; E). No additive attenuation to CCh evoked vasorelaxation was observed between vessels pre-incubated in L-NAME (100 μmol-L^–1) + linopirdine (10 μmol-L^–1; gray, dashed line) when compared with L-NAME (100 μmol-L^–1; gray; n = 5; F). SNP-mediated vasorelaxation was no effected by the pan-K_V7 channel inhibitor linopirdine (10 μmol-L^–1; gray) when compared to DMSO solvent control (black; G). K_V7.2-5 channel activator S-1 was significantly attenuated by pre-incubation with eNOS inhibitor L-NAME (100 μmol-L^–1; gray; n = 5; H). All values are expressed as mean + SEM. A two-way statistical ANOVA with a post hoc Bonferroni test was used to generate significance values (*, drug v DMSO solvent control; #=Group B vs. Group C) Statistical significance is defined as ^#/*P < 0.05 (A–H).

Additionally, CCh-evoked relaxations were significantly attenuated in vessels pre-incubated in TRAM34/apamin (1 μmol-L^–1/100 nmol-L^–1) and linopirdine (10 μmol-L^–1) compared to vessels only pre-incubated in TRAM34/apamin alone (EC₅₀ DMSO = 0.24 ± 0.05 μmol-L^–1; TRAM34/Apamin = 0.27 ± 0.03 μmol-L^–1; TRAM34/Apamin + linopirdine = 0.61 ± 0.2 μmol-L^–1; Figure 8E). In contrast, linopirdine failed to attenuate CCh relaxation in arteries pre-incubated with L-NAME (100 μmol-L^–1; Figure 8F), thus suggesting K_V7 contribution to eNOS sensitive proportion of CCh-mediated relaxation.

Furthermore, the present data demonstrates that pre-incubation with linopirdine (10 μmol-L^–1) has no effect on vasorelaxation produced by the NO-donor SNP (Figure 8G). However, in contrast with previous reports (Jepps et al., 2016), pre-incubation with L-NAME (100 μmol-L^–1) significantly attenuated K_V7.2-5 activator mediated vasorelaxation (Figure 8H).

Discussion

The present study identified Kcnq1, Kcnq4, and Kcnq5 transcripts in EC marker expressing cells and the consequent K_V7.1, K_V7.4, and K_V7.5 protein in isolated and whole mount rat MA ECs/VSMCs. Functionally, the present study demonstrates that the relaxation produced by two structurally different K_V7.2-5 activators, but not a K_V7.1 activator, were modulated by the presence of the endothelium and gap junction inhibition. Said relaxation was also sensitive to K_IR2 inhibition, which was dependent on the presence of ECs, suggestive of a novel functional interaction between K_V7 and endothelial K_IR2.x channels. Furthermore, the present data suggest that K_V7.4/K_V7.5 channels contribute to the NO-mediated axis of CCh-evoked endothelium-dependent relaxation downstream of eNOS. Thus, K_V7 channels are expressed in ECs, when pharmacologically upregulated, are functionally coupled to other EC potassium channels in rat MA and contribute to endothelium-derived responses.

K_V7 Channel Expression and Function Within ECs

Kv7 channel modulators have considerable impact on arterial tone. In VSMCs, active K_V7 channels hyperpolarize the membrane potential, decreasing voltage-dependent calcium channel (VDCC) open probability and extracellular calcium influx resulting in relaxation. Within rodent models, the pharmacopeia of K_V7 channel modulators has revealed K_V7.4 and 7.5 channels are; (1) key determinants of resting vascular tone via regulation of resting membrane potential; (2) upregulated during cGMP and cAMP/EPAC/PKA-mediated vasodilation; (3) suppressed via PKC-mediated vasoconstriction. Comparatively, no functional role for K_V7.1 has been identified in arteries (see Barrese et al., 2018b; Byron and Brueggemann, 2018 for review). A caveat of these observations is a lack of differentiation between VSMCs and ECs. However, recently K_V7 channels were identified in pig coronary artery ECs (Chen et al., 2016), the novel findings presented here expand on these findings and demonstrate K_V7 transcript and channel expression, functional activity and contribution to EC-derived vasodilatory signaling cascades in rat MA endothelium.

In most arteries, the endothelium and smooth muscle are electrochemically linked via MEGJs formed from connexin proteins within heterocellular communicating microdomains present within holes in the IEL (see Sandow et al., 2012 for review). Via these connections, current injection in to ECs passes into VSMCs (Sandow et al., 2002) supporting the presence of such coupling. The present data suggests a potential role for electrochemical heterocellular communication during K_V7.2-5 activator-mediated vasorelaxation (Figure 9). This conjecture is supported by apparently higher level expression of K_V7.4/K_V7.5 in a proportion of IEL holes than that at the EC membrane as well as a significant attenuation of relaxation of pre-constricted arterial tone by two structurally different K_V7.2-5 activators in the absence of ECs and presence of a (putative) gap junction inhibitor. In contrast, EC removal had no impact on K_V7.1 activator-mediated vasorelaxation, which was observed in conjunction with reduced expression of Kcnq1 transcript and K_V7.1 labeling within ECs compared to VSMCs. Collectively suggesting that only K_V7.4/K_V7.5 channels of the K_V7 sub-family are functionally expressed within MA ECs. An extrapolation of these results indicates a novel role for K_V7.4/K_V7.5 channels in the regulation of EC membrane potential within myoendothelial projections. It is plausible, that localized EC K_V7 channels negate the influx of depolarization from VSMCs into the endothelium, providing tight regulation of EC V_m during VSMC contraction. However, further studies would be required to validate this hypothesis.

FIGURE 9

Figure 9. Schematic of K_V7.2-5 activator-mediated vasorelaxation. K_V7.2-5 activator-mediated vasorelaxation occurs predominantly via K_V7.4/7.5 activation within VSMCs. However, our findings suggest a novel contribution of endothelial-K_V7.4/7.5 channels to K_V7.2-5 activator mediated responses which augments relaxation. A process which is sensitive to both gap-junction and endothelial-K_IR2.x inhibition (modified from Senadheera et al., 2012).

A Novel Functional Interaction With Endothelial K_IR Channels

K_IR2.x channels have been characterized in a variety of rat vascular beds including cerebral and coronary arteries (Smith et al., 2008), where their selective inhibition by Ba²⁺ revealed K_IR channel amplification of a K⁺ channel activator conductance (Smith et al., 2008). However, there is a degree of conflict regarding the role of K_IR2.x channels within rat MAs. As above, Ba²⁺ sensitive currents and K_IR2.x expression has been demonstrated in rat MA ECs (Crane et al., 2003a) which are purported to contribute to K_Ca mediated-hyperpolarization during ACh-derived EC-dependent responses (Goto et al., 2004). In contrast, Smith et al. (2008) demonstrated that within 3rd order MAs, ACh-mediated K⁺ conductance-dependent relaxation was insensitive to Ba²⁺, indicating that K_IR2.x channels do not augment K⁺ conductance in these vessels during receptor-mediated vasodilation.

In the present study, significant attenuation of both S-1 and ML213 K_V7.2-5 activator-mediated vasorelaxation was found following pre-incubation with two structurally different K_IR2.x blockers ML133 (Wu et al., 2010) and Ba²⁺ (Hagiwara et al., 1978) when compared to a solvent control. ML133 has been identified via high-throughput and mutagenesis investigation as a novel inhibitor of K_IR2.1 channels via D172 and I176 residues within the M2 region of K_IR2.1 (Wang et al., 2011) with an IC₅₀ of 1.8 μmol-L^–1 at pH 7.4, with little to no effect against other K_IR2.x family members (Wu et al., 2010; Wang et al., 2011). Presently, ML133 is the most selective inhibitor of the K_IR2 family. In analogous fashion to our endothelium denudation experiments, no effect was seen on the K_V7.1 activator ML277-mediated vasorelaxation; implying a specific interplay with K_V7.4 and 7.5 channels. Furthermore, K_IR2 blockers had no effect on K_V7.2–7.5 activator-mediated relaxation in EC-denuded arteries, supporting the notion that functional K_IR2 channels (in the rat MA bed) are restricted to the endothelium (Crane et al., 2003b). The present study suggests that pharmacological activation of endothelial K_V7.4/7.5 channels also activates an endothelial K_IR2 channels, increasing K⁺ conductance, which in turn accounts for a proportion of EC augmentation of K_V7.2-5 activator-mediated vasorelaxation (Figure 9). This may be due to a localized increase in potassium ion concentration in the proximity of the K_IR2 channels or an alternative modulation. However, based on the findings described by Goto et al. (2004) and Smith et al. (2008), the collective data suggest that this phenomenon is dependent on the branch order of MA. Furthermore, it remains unclear if this occurs during receptor mediated signaling, or if it is only present during pharmacological activation of endothelial K_V7 channels.

A primary concern for identification of novel functional interactions between ion channels using pharmacological tools is potential off-target effects. However, K_V7 activator-mediated relaxation in vessels pre-incubated in 10 μmol-L^–1 linopirdine was abolished. If S-1 or ML213 were activating non-K_V channels such as K_IR, a degree of relaxation would still be observed in the presence of linopirdine. The present findings therefore suggest that both S-1 and ML213 work exclusively via K_V7 channels and that attenuation of their response within rat MA occurs via a novel functional coupling of K_V7.4/7.5 and K_IR2 within ECs.

The Contribution of K_V7 Channels to CCh Evoked Relaxations Within Rat MA

Our findings demonstrate that both NO- and EDH-dependent signaling contributes to CCh-mediated vasodilation, though the main contributor to endothelial-dependent vasodilation in 2nd order MA appears to be EDH, consistent with previous studies (Shimokawa et al., 1996). In light of the significant impact of K_V7 inhibition on CCh-mediated vasorelaxation during the suppression of EDH, the present study suggests that K_V7 channels contribute to the eNOS sensitive axis of CCh-evoked relaxation within rat MA.

However, in a similar manner to rat renal artery (Stott et al., 2015), rat MA K_V7 channels do not represent downstream targets of NO signaling as K_V7 inhibition does not impair NO-donor SNP-mediated relaxation. However, eNOS inhibition does impair relaxation to a K_V7 activator, potentially implying K_V7 channel involvement in the production or release of NO in response to CCh. Interestingly, this appears to be a vascular bed specific phenomenon, as L-NAME has no effect on K_V7 activator-mediated relaxation within rat penile artery (Jepps et al., 2016).

Limitations

Ideally, direct functional evidence for a functional role of K_v7 channels in ECs would be provided by either single cell electrophysiology of sharp microelectrode impalement in whole arteries. However, this was not possible within the constraints of this study. Instead, we inferred a functional role for EC K_v7 channels using a reductive approach, i.e., comparison of functional responses without or with EC ablation. These studies implicate K_v7 channels as functional component of the EC physiology that merit consideration in future studies.

Conclusion

In conclusion, the present data reveal that mesenteric ECs express K_V7.4/K_V7.5 channels and their presence boosts K_V7 activator-mediated relaxation. Furthermore, the data indicated a novel functional interaction with endothelial K_IR2 channels and support the proposition that endothelial K_V7 channels contribute to endogenous endothelium-derived responses. These findings highlight the complex nature of the vascular response to K_V7 channel upregulation and emphasize the importance of these channels to vascular signaling. The present data are consistent with K_V7 channels representing a novel therapeutic target in endothelial dysfunction.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

Ethics Statement

The animal study was reviewed and approved by UNSW Animal Ethics and Experimentation Committee (AEEC #18/86B). For investigations performed at St George’s University, London, investigators strictly adhered to the Animal (Scientific Procedures) Act 1986.

Author Contributions

SB, SS, GM-P, and JS performed the research. IG and JS designed the research study. SS contributed essential reagents or tools. SB, SS, and GM-P analyzed the data. SB and IG wrote the manuscript. All the authors contributed to the article and approved the submitted version.

Funding

SB was funded by the British Heart Foundation (Grant #FS/18/41/33762) awarded to IG.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors would like to thank Miss Aditi Gunjal for her excellent assistance in wire myography.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys.2020.598779/full#supplementary-material

Abbreviations

4-AP, 4-aminopyridine; Acta2, α-smooth muscle actin-2; CCh, carbachol; cGMP, cyclic guanosine monophosphate; Cq, quantification cycle; CYC1, cytochrome C1; EC, endothelial cell; EDH, endothelium derived hyperpolarization; GAPD, glyceraldehyde-3-phosphate dehydrogenase; eNOS, endothelial nitric oxide synthase; IK_Ca, intermediate conductance calcium-activated potassium channel; IEL, internal elastic lamina; K_IR, inwardly rectifying potassium channel; K_V, voltage-gated potassium channel; L-NAME, L-nitroarginine methyl ester; MA, mesenteric artery; MO, methoxamine; Myh11, myosin heavy chain 11; NO, nitric oxide; PECAM-1, platelet endothelial cell adhesion molecule-1; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; SEM, standard error of the mean; SK_Ca, small conductance calcium-activated potassium channel; SNP, S-nitroprusside; TEA, tetraethylammonium; VSMC, vascular smooth muscle cell; vWBF, von-Willebrand factor.

References

Askew Page, H. R., Dalsgaard, T., Baldwin, S. N., Jepps, T. A., Povstyan, O., Olesen, S. P., et al. (2019). TMEM16A is implicated in the regulation of coronary flow and is altered in hypertension. Br. J. Pharmacol. 176, 1635–1648. doi: 10.1111/bph.14598

PubMed Abstract | CrossRef Full Text | Google Scholar

Barrese, V., Stott, J. B., Figueiredo, H. B., Aubdool, A. A., Hobbs, A. J., Jepps, T. A., et al. (2018b). Angiotensin II Promotes K_V7.4 channels degradation through reduced interaction with HSP90. Hypertension 71, 1091–1100. doi: 10.1161/HYPERTENSIONAHA.118.11116

PubMed Abstract | CrossRef Full Text | Google Scholar

Barrese, V., Stott, J. B., and Greenwood, I. A. (2018a). KCNQ-encoded potassium channels as therapeutic targets. Ann. Rev. Pharmacol. Toxicol. 58, 625–648. doi: 10.1146/annurev-pharmtox-010617-052912

PubMed Abstract | CrossRef Full Text | Google Scholar

Bentzen, B. H., Schmitt, N., Calloe, K., Dalby Brown, W., Grunnet, M., and Olesen, S. P. (2006). The acrylamide (S)-1 differentially affects Kv7 (KCNQ) potassium channels. Neuropharmacology 51, 1068–1077. doi: 10.1016/j.neuropharm.2006.07.001

PubMed Abstract | CrossRef Full Text | Google Scholar

Brueggemann, L. I., Cribbs, L. L., Schwartz, J., Wang, M., Kouta, A., and Byron, K. L. (2018). Mechanisms of PKA-dependent potentiation of Kv7.5 channel activity in human airway smooth muscle cells. Int. J. Mol. Sci. 12:2223. doi: 10.3390/ijms19082223

PubMed Abstract | CrossRef Full Text | Google Scholar

Brueggemann, L. I., Haick, J. M., Cribbs, L. L., and Byron, K. L. (2014). Differential activation of vascular smooth muscle Kv7.4, Kv7.5, and Kv7.4/7.5 channels by ML213 and ICA-069673. Mol. Pharmacol. 86, 330–341. doi: 10.1124/mol.114.093799

PubMed Abstract | CrossRef Full Text | Google Scholar

Brueggemann, L. I., Moran, C. J., Barakat, J. A., Yeh, J. Z., Cribbs, L. L., and Byron, K. L. (2006). Vasopressin stimulates action potential firing by protein kinase C-dependent inhibition of KCNQ5 in A7r5 rat aortic smooth muscle cells. AJP Heart Circ. Physiol. 292, H1352–H1363. doi: 10.1152/ajpheart.00065.2006

PubMed Abstract | CrossRef Full Text | Google Scholar

Byron, K. L., and Brueggemann, L. I. (2018). Kv7 potassium channels as signal transduction intermediates in the control of microvascular tone. Microcirculation 25:e12419. doi: 10.1111/micc.12419

PubMed Abstract | CrossRef Full Text | Google Scholar

Chadha, P. S., Zunke, F., Zhu, H. L., Davis, A. J., Jepps, T. A., Olesen, S. P., et al. (2012). Reduced KCNQ4-encoded voltage-dependent potassium channel activity underlies impaired β-adrenoceptor-mediated relaxation of renal arteries in hypertension. Hypertension 59, 877–884. doi: 10.1161/HYPERTENSIONAHA.111.187427

PubMed Abstract | CrossRef Full Text | Google Scholar

Chen, X., Li, W., Hiett, S. C., and Obukhov, A. G. (2016). Novel Roles for Kv7 channels in shaping histamine-induced contractions and bradykinin-dependent relaxations in Pig Coronary Arteries. PLoS One 11:e1048569. doi: 10.1371/journal.pone.0148569

PubMed Abstract | CrossRef Full Text | Google Scholar

Choi, K. L., Aldricht, R. W., Yellen, G., and Hughes, H. (1991). Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated Ki channels. Proc. Natl. Acad. Sci. U. S. A. 88, 5092–5095. doi: 10.1073/pnas.88.12.5092

PubMed Abstract | CrossRef Full Text | Google Scholar

Crane, G. J., Gallagher, N., Dora, K. A., and Garland, C. J. (2003a). Small-and intermediate-conductance calcium-activated K+ channels provide different facets of endothelium-dependent hyperpolarization in rat mesenteric artery. J. Physiol. 553(Pt 1), 183–189. doi: 10.1113/jphysiol.2003.051896

PubMed Abstract | CrossRef Full Text | Google Scholar

Crane, G. J., Walker, S. D., Dora, K. A., and Garland, C. J. (2003b). Evidence for a differential cellular distribution of inward rectifier K channels in the rat isolated mesenteric artery. J. Vasc. Res. 40, 159–168. doi: 10.1159/000070713

PubMed Abstract | CrossRef Full Text | Google Scholar

Curtis, M. J., Alexander, S., Cirino, G., Docherty, J. R., George, C. H., Giembycz, M. A., et al. (2018). Experimental design and analysis and their reporting II: updated and simplified guidance for authors and peer reviewers. Br. J. Pharmacol. 175, 987–993. doi: 10.1111/bph.14153

PubMed Abstract | CrossRef Full Text | Google Scholar

Dora, K. A., Gallagher, N. T., McNeish, A., and Garland, C. J. (2008). Modulation of endothelial cell KCa3.1 channels during endothelium-derived hyperpolarizing factor signaling in mesenteric resistance arteries. Circ. Res. 102, 1247–1255. doi: 10.1161/CIRCRESAHA.108.172379

PubMed Abstract | CrossRef Full Text | Google Scholar

Fukao, M., Hattori, Y., Kanno, M., Sakuma, I., and Kitabatake, A. (1997). Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery. Br. J. Pharmacol. 120, 1328–1334. doi: 10.1038/sj.bjp.0701027

PubMed Abstract | CrossRef Full Text | Google Scholar

Goto, K., Rummery, N. M., Grayson, T. H., and Hill, C. E. (2004). Attenuation of conducted vasodilatation in rat mesenteric arteries during hypertension: role of inwardly rectifying potassium channels. J. Physiol. 561(Pt 1), 215–231. doi: 10.1113/jphysiol.2004.070458

PubMed Abstract | CrossRef Full Text | Google Scholar

Grayson, T. H., Chadha, P. S., Bertrand, P. P., Chen, H., Morris, M. J., Senadheera, S., et al. (2013). Increased caveolae density and caveolin-1 expression accompany impaired NO-mediated vasorelaxation in diet-induced obesity. Histochem. Cell Biol. 139, 309–321. doi: 10.1007/s00418-012-1032-2

PubMed Abstract | CrossRef Full Text | Google Scholar

Greenberg, H. Z. E., Shi, J., Jahan, K. S., Martinucci, M. C., Gilbert, S. J., Ho, W. S., et al. (2016). Stimulation of calcium-sensing receptors induces endothelium-dependent vasorelaxations via nitric oxide production and activation of IKCa channels. Vasc. Pharmacol. 80, 75–84. doi: 10.1016/j.vph.2016.01.001

PubMed Abstract | CrossRef Full Text | Google Scholar

Hagiwara, S., Miyazaki, S., Moody, W., and Patlak, J. (1978). Blocking effects of barium and hydrogen ions on the potassium current during anomalous rectification in the starfish egg. J. Physiol. 279, 167–185. doi: 10.1113/jphysiol.1978.sp012338

PubMed Abstract | CrossRef Full Text | Google Scholar

Howitt, L., Hilton Grayson, T., Morris, M. J., Sandow, S. L., and Murphy, T. V. (2012). Dietary obesity increases NO and inhibits BKCa-mediated, endothelium-dependent dilation in rat cremaster muscle artery: association with caveolins and caveolae. Am. J. Physiol. Heart Circ. Physiol. 302, H2464–H2476. doi: 10.1152/ajpheart.00965.2011

PubMed Abstract | CrossRef Full Text | Google Scholar

Jepps, T. A., Bentzen, B. H., Stott, J. B., Povstyan, O. V., Sivaloganathan, K., Dalby-Brown, W., et al. (2014). Vasorelaxant effects of novel Kv7.4 channel enhancers ML213 and NS15370. Br. J. Pharmacol. 171, 4413–4424. doi: 10.1111/bph.12805

PubMed Abstract | CrossRef Full Text | Google Scholar

Jepps, T. A., Chadha, P. S., Davis, A. J., Harhun, M. I., Cockerill, G. W., Olesen, S. P., et al. (2011). Downregulation of Kv7.4 channel activity in primary and secondary hypertension. Circulation 124, 602–611. doi: 10.1161/CIRCULATIONAHA.111.032136

PubMed Abstract | CrossRef Full Text | Google Scholar

Jepps, T. A., Greenwood, I. A., Moffatt, J. D., Sanders, K. M., and Ohya, S. (2009). Molecular and functional characterization of K_v7 K ⁺ channel in murine gastrointestinal smooth muscles. Am. J. Physiol. Gastrointest. Liver Physiol. 297, G107–G115. doi: 10.1152/ajpgi.00057.2009

PubMed Abstract | CrossRef Full Text | Google Scholar

Jepps, T. A., Olesen, S. P., Greenwood, I. A., and Dalsgaard, T. (2016). Molecular and functional characterization of Kv7 channels in penile arteries and corpus cavernosum of healthy and metabolic syndrome rats. Br. J. Pharmacol. 173, 1478–1490. doi: 10.1111/bph.13444

PubMed Abstract | CrossRef Full Text | Google Scholar

Khanamiri, S., Soltysinska, E., Jepps, T. A., Bentzen, B. H., Chadha, P. S., Schmitt, N., et al. (2013). Contribution of K_v7 channels to basal coronary flow and active response to ischemia. Hypertension 62, 1090–1097. doi: 10.1161/HYPERTENSIONAHA.113.01244

PubMed Abstract | CrossRef Full Text | Google Scholar

Kurata, H. T., and Fedida, D. (2006). A structural interpretation of voltage-gated potassium channel inactivation. Prog. Biophys. Mol. Biol. 92, 185–208. doi: 10.1016/j.pbiomolbio.2005.10.001

PubMed Abstract | CrossRef Full Text | Google Scholar

Mackie, A. R., Brueggemann, L. I., Henderson, K. K., Shiels, A. J., Cribbs, L. L., Scrogin, K. E., et al. (2008). Vascular KCNQ potassium channels as novel targets for the control of mesenteric artery constriction by vasopressin, based on studies in single cells, pressurized arteries, and in vivo measurements of mesenteric vascular resistance. J. Pharmacol. Exp. Ther. 325, 475–483. doi: 10.1124/jpet.107.135764

PubMed Abstract | CrossRef Full Text | Google Scholar

Mani, B. K., Robakowski, C., Brueggemann, L. I., Cribbs, L. L., Tripathi, A., Majetschak, M., et al. (2016). Kv7.5 potassium channel subunits are the primary targets for PKA-dependent enhancement of vascular smooth muscle Kv7 Currents. Mol. Pharmacol. 89, 323–334. doi: 10.1124/mol.115.101758

PubMed Abstract | CrossRef Full Text | Google Scholar

McGuire, J. J., Ding, H., and Triggle, C. R. (2001). Endothelium-derived relaxing factors: a focus on endothelium-derived hyperpolarizing factor(s). Can. J. Physiol. Pharmacol. 79, 443–470. doi: 10.1139/y01-025

CrossRef Full Text | Google Scholar

Mills, T. A., Greenwood, S. L., Devlin, G., Shweikh, Y., Robinson, M., Cowley, E., et al. (2015). Activation of K_V7 channels stimulates vasodilatation of human placental chorionic plate arteries. Placenta 36, 638–644. doi: 10.1016/j.placenta.2015.03.007

PubMed Abstract | CrossRef Full Text | Google Scholar

Mondéjar-Parreño, G., Moral-Sanz, J., Barreira, B., De la Cruz, A., Gonzalez, T., Callejo, M., et al. (2019). Activation of Kv7 channels as a novel mechanism for NO/cGMP-induced pulmonary vasodilation. Br. J. Pharmacol. 176, 2131–2145. doi: 10.1111/bph.14662

PubMed Abstract | CrossRef Full Text | Google Scholar

Morales-Cano, D., Moreno, L., Barreira, B., Pandolfi, R., Chamorro, V., Jimenez, R., et al. (2015). Kv7 channels critically determine coronary artery reactivity: left-right differences and down-regulation by hyperglycaemia. Cardiovasc. Res. 106, 98–108. doi: 10.1093/cvr/cvv020

PubMed Abstract | CrossRef Full Text | Google Scholar

Mulvany, M. J., and Halpern, W. (1976). Mechanical properties of vascular smooth muscle cells in situ. Nature 260, 617–619. doi: 10.1038/260617a0

PubMed Abstract | CrossRef Full Text | Google Scholar

Ng, F. L., Davis, A. J., Jepps, T. A., Harhun, M. I., Yeung, S. Y., Wan, A., et al. (2011). Expression and function of the K + channel KCNQ genes in human arteries. Br. J. Pharmacol. 162, 42–53. doi: 10.1111/j.1476-5381.2010.01027.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Ohya, S., Sergeant, G. P., Greenwood, I. A., and Horowitz, B. (2003). Molecular variants of KCNQ channels expressed in murine portal vein myocytes: a role in delayed rectifier current. Circ. Res. 92, 1016–1023. doi: 10.1161/01.RES.0000070880.20955.F4

CrossRef Full Text | Google Scholar

Oliveras, A., Roura-Ferrer, M., Solé, L., De La Cruz, A., Prieto, A., Etxebarria, A., et al. (2014). Functional assembly of Kv7.1/Kv7.5 channels with emerging properties on vascular muscle physiology. Arterioscler. Thromb. Vasc. Biol. 23, 1522–1530. doi: 10.1161/ATVBAHA.114.303801

PubMed Abstract | CrossRef Full Text | Google Scholar

Parsons, S. J. W., Hill, A., Waldron, G. J., Plane, T., and Garland, C. J. (1994). The relative importance of nitric oxide and nitric oxide−independent mechanisms in acetylcholine−evoked dilatation of the rat mesenteric bed. Br. J. Pharmacol. 113, 1275–1280. doi: 10.1111/j.1476-5381.1994.tb17136.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Peredo, H. A., Feleder, E. C., and Adler-Graschinsky, E. (1997). Differential effects of acetylcholine and bradykinin on prostanoid release from the rat mesenteric bed: role of endothelium and of nitric oxide. Prostaglandins Leukot. Essent. Fatty Acids 56, 253–258. doi: 10.1016/S0952-3278(97)90567-6

CrossRef Full Text | Google Scholar

Sandow, S. L., Goto, K., Rummery, N. M., and Hill, C. E. (2004). Developmental changes in myoendothelial gap junction mediated vasodilator activity in the rat saphenous artery. J. Physiol. 556(Pt 3), 875–886. doi: 10.1113/jphysiol.2003.058669

PubMed Abstract | CrossRef Full Text | Google Scholar

Sandow, S. L., Haddock, R. E., Hill, C. E., Chadha, P. S., Kerr, P. M., Welsh, D. G., et al. (2009). What’s where and why at a vascular myoendothelial microdomain signalling complex. Clin. Exp. Pharmacol. Physiol. 36, 67–76. doi: 10.1111/j.1440-1681.2008.05076.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Sandow, S. L., Neylon, C. B., Chen, M. X., and Garland, C. J. (2006). Spatial separation of endothelial small- and intermediate-conductance calcium-activated potassium channels (KCa) and connexins: possible relationship to vasodilator function? J. Anat. 209, 689–698. doi: 10.1111/j.1469-7580.2006.00647.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Sandow, S. L., Senadheera, S., Bertrand, P. P., Murphy, T. V., and Tare, M. (2012). Myoendothelial contacts, gap junctions, and microdomains: anatomical links to function? Microcirculation 19, 403–415. doi: 10.1111/j.1549-8719.2011.00146.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Sandow, S. L., Tare, M., Coleman, H. A., Hill, C. E., and Parkington, H. C. (2002). Involvement of myoendothelial gap junctions in the actions of endothelium-derived hyperpolarizing factor. Circ. Res. 90, 1108–1113. doi: 10.1161/01.RES.0000019756.88731.83

CrossRef Full Text | Google Scholar

Schenzer, A. (2005). Molecular Determinants of KCNQ (Kv7) K+ channel sensitivity to the anticonvulsant retigabine. J. Neurosci. 25, 5051–5060. doi: 10.1523/JNEUROSCI.0128-05.2005

PubMed Abstract | CrossRef Full Text | Google Scholar

Schnee, M. E., and Brown, B. S. (1998). Selectivity of linopirdine (DuP 996), a neurotransmitter release enhancer, in blocking voltage-dependent and calcium-activated potassium currents in hippocampal neurons. J. Pharmacol. Exp. Ther. 286, 709–717.

Google Scholar

Senadheera, S., Kim, Y., Grayson, T. H., Toemoe, S., Kochukov, M. Y., Abramowitz, J., et al. (2012). Transient receptor potential canonical type 3 channels facilitate endothelium-derived hyperpolarization-mediated resistance artery vasodilator activity. Cardiovasc. Res. 95, 439–447. doi: 10.1093/cvr/cvs208

PubMed Abstract | CrossRef Full Text | Google Scholar

Shimokawa, H., Yasutake, H., Fujii, K., Owada, M. K., Nakaike, R., Fukumoto, Y., et al. (1996). The importance of the hyperpolarizing mechanism increases as the vessel size decreases in endothelium-dependent relaxations in rat mesenteric circulation. J. Cardiovasc. Pharmacol. 28, 703–711. doi: 10.1097/00005344-199611000-00014

PubMed Abstract | CrossRef Full Text | Google Scholar

Smith, P. D., Brett, S. E., Luykenaar, K. D., Sandow, S. L., Marrelli, S. P., Vigmond, E. J., et al. (2008). KIR channels function as electrical amplifiers in rat vascular smooth muscle. J. Physiol. 586, 1147–1160. doi: 10.1113/jphysiol.2007.145474

PubMed Abstract | CrossRef Full Text | Google Scholar

Sonkusare, S. K., Dalsgaard, T., Bonev, A. D., and Nelson, M. T. (2016). Inward rectifier potassium (Kir2.1) channels as end-stage boosters of endothelium-dependent vasodilators. J. Physiol. 594, 3271–3285. doi: 10.1113/JP271652

PubMed Abstract | CrossRef Full Text | Google Scholar

Spoerri, E., Jentsch, J., and Glees, P. (1975). Apamin from bee venom. Effects of the neurotoxin on subcellular particles of neural cultures. FEBS Lett. 52, 143–147. doi: 10.1016/0014-5793(75)80006-8

CrossRef Full Text | Google Scholar

Stott, J. B., Barrese, V., Jepps, T. A., Leighton, E. V., and Greenwood, I. A. (2015). Contribution of Kv7 channels to natriuretic peptide mediated vasodilation in normal and hypertensive rats. Hypertension 65, 676–682. doi: 10.1161/HYPERTENSIONAHA.114.04373

PubMed Abstract | CrossRef Full Text | Google Scholar

Stott, J. B., Jepps, T. A., and Greenwood, I. A. (2014). K_V7 potassium channels: a new therapeutic target in smooth muscle disorders. Drug Discov. Today 19, 413–424. doi: 10.1016/j.drudis.2013.12.003

PubMed Abstract | CrossRef Full Text | Google Scholar

Tare, M., Coleman, H. A., and Parkington, H. C. (2002). Glycyrrhetinic derivatives inhibit hyperpolarization in endothelial cells of guinea pig and rat arteries. Am. J. Physiol. Heart Circ. Physiol. 282, H335–H341. doi: 10.1152/ajpheart.2002.282.1.H335

PubMed Abstract | CrossRef Full Text | Google Scholar

Wang, H. R., Wu, M., Yu, H., Long, S., Stevens, A., Engers, D. W., et al. (2011). Selective inhibition of the Kir2 family of inward rectifier potassium channels by a small molecule probe: The discovery, SAR, and pharmacological characterization of ML133. ACS Chem. Biol. 6, 845–856. doi: 10.1021/cb200146a

PubMed Abstract | CrossRef Full Text | Google Scholar

Wu, M., Wang, H., Yu, H., Makhina, E., Xu, J., Dawson, E. S., et al. (2010). “A potent and selective small molecule Kir2.1 inhibitor,” in Probe Reports from the NIH Molecular Libraries Program, (Bethesda, MD: National Center for Biotechnology Information).

Google Scholar

Wulff, H., Miller, M. J., Hänsel, W., Grissmer, S., Cahalan, M. D., and Chandy, K. G. (2000). Design of a potent and selective inhibitor of the intermediate- conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant. Proc. Natl. Acad. Sci. U. S. A. 97, 8151–8156. doi: 10.1073/pnas.97.14.8151

PubMed Abstract | CrossRef Full Text | Google Scholar

Yeung, S. Y. M., Pucovský, V., Moffatt, J. D., Saldanha, L., Schwake, M., Ohya, S., et al. (2007). Molecular expression and pharmacological identification of a role for Kv7 channels in murine vascular reactivity. Br. J. Pharmacol. 151, 758–770. doi: 10.1038/sj.bjp.0707284

PubMed Abstract | CrossRef Full Text | Google Scholar

Yu, H., Lin, Z., Xu, K., Huang, X., Long, S., Wu, M., et al. (2013). “Identification of a novel, small molecule activator of KCNQ1 channels,” in Probe Reports from the NIH Molecular Libraries Program, (Bethesda, MD: National Center for Biotechnology Information).

Google Scholar

Zechariah, A., Tran, C. H. T., Hald, B. O., Sandow, S. L., Sancho, M., Kim, M. S. M., et al. (2020). Intercellular conduction optimizes arterial network function and conserves blood flow homeostasis during cerebrovascular challenges. Arterioscler. Thromb. Vasc. Biol. 40, 733–750. doi: 10.1161/ATVBAHA.119.313391

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhong, X. Z., Harhun, M. I., Olesen, S. P., Ohya, S., Moffatt, J. D., Cole, W. C., et al. (2010). Participation of KCNQ (Kv7) potassium channels in myogenic control of cerebral arterial diameter. J. Physiol. 588(Pt 17), 3277–3293. doi: 10.1113/jphysiol.2010.192823

PubMed Abstract | CrossRef Full Text | Google Scholar