KV7 Channel Expression and Function Within Rat Mesenteric Endothelial Cells

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).

FIGURE 2. Immunocytochemistry demonstrates KV7.1, KV7.4, and 7V.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. 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 Kv7.4 (A,B, respectively) and Kv7.5 (E,F, respectively). Confocal image stacks through the luminal EC to SM border (C,G) show diffuse/punctate Kv7.4/KV7.5 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,…

FIGURE 4. Sensitivity of Kv7 activator relaxations to 10 μmol-L–1 linopirdine. In vessels pre-incubated in DMSO solvent control, KV7.2-5 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 KV7 channel inhibitor linopirdine (10 μmol-L–1; gray; n = 5; A,B). In vessels pre-incubated in DMSO solvent control, KV7.1 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).

FIGURE 5. KV7.2-5 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).

FIGURE 6. KV7.2-5 channel activator-mediated vasorelaxation was attenuated by two structurally different KIR channel blockers, BaCl2 and ML133, but not In EC denuded vessels. KV7.2-5 channel activator S-1 and ML213 effects were significantly attenuated by pre-incubation with the KIR2 channel inhibitor BaCl2 (100 μmol-L–1; black; n = 5–7; A,C). KV7.2-5 channel activator S-1 and ML213 effects were significantly attenuated by pre-incubation with a selective KIr 2.1 channel inhibitor, ML133 (20 μmol-L–1; gray; n = 5–7; B,D). KV7.1 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, KV7.2-5 channel activator ML213 responses were not affected by pre-incubation with either KIR2 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).

FIGURE 7. KV7 channel activator mediated vasorelaxation was not affected by preincubation with IKCa/SKCa channel antagonists. Pre-incubation with both IKCa channel inhibitor TRAM34 (1 μmol-L–1) and SKCa 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).

FIGURE 8. CCh evoked vasorelaxation is mediated through both eNOS and IKCa/SKCa channels and is partially attenuated by inhibition of KV7 channels upstream of NO. Both eNOS inhibitor L-nitroarginine methyl ester (L-NAME; 100 μmol-L–1; gray) and IKCa/SKCa 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 KV7 channel inhibitor linopirdine (10 μmol-L–1; gray) significantly attenuated CCh mediated vasorelaxation (n = 6; B). The KV7.1 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) +…

FIGURE 9. Schematic of KV7.2-5 activator-mediated vasorelaxation. KV7.2-5 activator-mediated vasorelaxation occurs predominantly via KV7.4/7.5 activation within VSMCs. However, our findings suggest a novel contribution of endothelial-KV7.4/7.5 channels to KV7.2-5 activator mediated responses which augments relaxation. A process which is sensitive to both gap-junction and endothelial-KIR2.x inhibition (modified from Senadheera et al., 2012).