Openers of small conductance calcium-activated potassium channels selectively enhance NO-mediated bradykinin vasodilatation in porcine retinal arterioles

Abstract

Background and purpose: Small (SK(Ca) or K(Ca)2) and intermediate (IK(Ca) or K(Ca)3.1) conductance calcium-activated potassium channels are involved in regulation of vascular tone and blood pressure. The present study investigated whether NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime) and CyPPA (cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine), which are selective openers of SK(Ca) and IK(Ca) channels and of SK(Ca)2 and SK(Ca)3 channels, respectively, enhance endothelium-dependent vasodilatation in porcine retinal arterioles.

Experimental approach: In porcine retinal arterioles, SK(Ca)3 and IK(Ca) protein localization was examined by immunolabelling. Endothelial cell calcium was measured by fluorescence imaging. For functional studies, arterioles with internal diameters of 116 +/- 2 microm (n = 276) were mounted in microvascular myographs for isometric tension recordings.

Key results: SK(Ca)3 and IK(Ca) protein was localized in the endothelium. Bradykinin, but not NS309 or CyPPA increased endothelial cell calcium. Pre-incubation with NS309 or CyPPA enhanced bradykinin relaxation without changing endothelial cell calcium. This enhanced relaxation was abolished by blocking SK(Ca) channels with apamin. In the presence of NS309 or CyPPA, mainly inhibition of NO synthase with asymmetric dimethylarginine, but also inhibition of cyclooxygenase with indomethacin, reduced bradykinin relaxation. Bradykinin relaxation was completely abolished by NO synthase and cyclooxygenase inhibition together with a NO scavenger, oxyhaemoglobin.

Conclusions and implications: In porcine retinal arterioles, bradykinin increases endothelial cell calcium leading to activation of SK(Ca) and IK(Ca) channels. Without altering endothelial cell calcium, NS309 and CyPPA open SK(Ca) channels that enhance NO-mediated bradykinin relaxations. These results imply that opening SK(Ca) channels improves endothelium-dependent relaxation and makes this channel a potential target for treatments aimed at restoring retinal blood flow.

Figure 1

H&E staining (A, E) and localization of SK_Ca3 protein (C, D) and IK_Ca protein (G, H) by immunoreaction (green fluorescence, arrows) in retinal arterioles. B and F are negative controls without primary antibody. D and H are enlargements of C and G. The scale bars in A-C, E-F are 50 µm, and in D and H 10 µm.

Figure 2

NS309 relaxation (A) or CyPPA relaxation (B) in the absence or presence of apamin (0.5 µM), in segments without endothelium, or contracted with 125 mM KPSS. Results are mean ± SEM (n = 7). Two-way anova. *P < 0.05 from control.

Figure 3

CyPPA relaxation in the presence of asymmetric dimethylarginine (300 µM, ADMA), ADMA + oxyhaemoglobin (25 µM, OxyHb), indomethacin (3 µM, Indo), Indo + ADMA and Indo + ADMA + OxyHb in porcine retinal arterioles contracted with U46619 (0.1 µM). Results are mean ± SEM (n = 7). Two-way anova. *P < 0.05 from control.

Figure 4

Effect of the bradykinin-receptor B₂ antagonist, HOE-140 (0.1, 1.0, 10 and 100 nM), on bradykinin relaxation (A), HOE-140 (10 nM) on NS309 relaxation (B) and HOE-140 (10 nM) on CyPPA relaxation (C) in porcine retinal arterioles contracted with U46619 (0.1 µM). Results aremean ± SEM (n = 6–8). Two-way anova. *P < 0.05 from control.

Figure 5

Original traces showing the response to increasing concentrations of bradykinin (A), bradykinin in the presence of sodium nitroprusside (SNP, 1 µM) (B), bradykinin in the presence of NS309 (1 µM) (C) and bradykinin in the presence of CyPPA (6 µM) (D), in porcine retinal arterioles contracted with U46619 (0.1 µM).

Figure 6

Effect of sodium nitroprusside (SNP, 1 µM), NS309 (1 µM) and CyPPA (6 µM) on bradykinin relaxation (A). Effect of NS309 (1 µM) and CyPPA (6 µM) on SNP relaxation (B). Effect of NS309 (1 µM) on bradykinin relaxation in the absence and presence of apamin (0.5 µM), and/or charybdotoxin (ChTx, 0.1 µM) (C). Effect of CyPPA (6 µM) on bradykinin relaxation in the absence and presence of apamin (0.5 µM) (D). Results are mean ± SEM (n = 6–8). Two-way anova. *P < 0.05 from control.

Figure 7

Relative change in intracellular calcium (Δ[Ca²⁺]_i) for bradykinin (0.1 nM, 1 nM, 3 nM and 10 nM), NS309 (1 µM), NS309 (1 µM) + bradykinin (BK, 3 nM), CyPPA (6 µM) and CyPPA (6 µM) + BK (3 nM) in endothelial cells from porcine retinal arterioles. Results are mean ± SEM (n = 4–6). Wilcoxon matched pair test, *P < 0.05 from zero [Ca²⁺]_i.

Figure 8

Bradykinin relaxation in the presence of NS309 (1 µM) (A) or CyPPA (6 µM) (B) and asymmetric dimethylarginine (300 µM, ADMA), ADMA + oxyhaemoglobin (25 µM, OxyHb), indomethacin (3 µM, indo), indo + ADMA and indo + ADMA + OxyHb in porcine retinal arterioles contracted with U46619 (0.1 µM). Results are mean ± SEM (n = 6–8). Two-way anova. *P < 0.05 from their respective controls.

Figure 9

Bradykinin relaxation in the absence or presence of indomethacin (3 µM, Indo) + asymmetric dimethylarginine (ADMA; 300 µM) + oxyhaemoglobin (OxyHb; 25 µM) in porcine retinal arterioles contracted with U46619 (0.1 µM) or endothelin-1 (0.03 µM) (C). Results are mean ± SEM (n = 6–8). Two-way anova. *P < 0.05 from their respective controls.

Figure 10

Relaxation to NO solution (A) and H₂S (B) in the absence and presence of oxyhaemoglobin (25 µM, OxyHb) in porcine retinal arterioles contracted with U46619 (0.1 µM). Results are mean ± SEM (n = 6–8). Two-way anova. *P < 0.05 from control.