Potassium channel‐mediated NO‐induced vasodilation during maturation: Dominance of Kv7 channels

Figures

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  FIGURE 1. Demonstration of how the anticontractile effect of SNP was assessed. For each individual preparation treated with 50 mM potassium solution, the differences between the AUCs with and without SNP (difference shown in A as hatched area) are determined; attention, here mean data are shown for illustration purposes. The same procedure is used when preparations were treated with a mixture of potassium channel blockers with and without SNP (difference shown in B as hatched area). The individual AUCs are plotted in panel C. Panel D shows a scheme explaining the experimental protocol when vessels were treated with a potassium channel blocker mixture leaving just one potassium channel available to study its role in the anticontractile effect of SNP.

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  FIGURE 2. mRNA expression of isoforms of potassium channels, known to potentially mediate SNP‐induced vasodilation, in saphenous arteries from adult (A) and young (B) rats. Kcna1‐7, Kv1.1‐Kv1.7; Kcna10, Kv1.8; Kcnb1‐2, Kv2.1‐Kv2.2; Kcnq1‐5, Kv7.1‐Kv7.5; Kcnj2, Kir2.1; Kcnj12, Kir2.2; Kcnj4, Kir2.3; Kcnj14, Kir2.4; Kcnj8, Kir6.1; Kcnj11, Kir6.2; Kcnma1, KCa1.1. Relative expression is reported in relation to the expression of four reference (housekeeping) genes, B2M, Eif2b1, Gusb, and Ywhaz. . Data are presented as the median with 95% confidence interval, n=4.

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  FIGURE 3. Anticontractile effect of SNP under conditions of potassium channel blockade. Concentration–response relationships for methoxamine in the presence of 50 mM KCl alone or of KCl and SNP (0.1 μM) from arteries of adult (A) and young rats (D), respectively. *p < 0.05 (repeated measures ANOVA). Concentration–response relationships for methoxamine in the presence of a combination of potassium channels blockers (BaCl2 (Ba) 30 μM, DPO‐1 1 μM, stromatoxin (STX) 0.1 μM, iberiotoxin (IBTX) 0.1 μM, XE991 3 μM, glibenclamide (GLIB) 3 μM) without and with SNP (0.1 μM) from arteries of adult (B) and young rats (E), respectively. *p < 0.05 (repeated measures ANOVA). Anticontractile effect of SNP, presented as the difference between the area under the curves in the presence of KCl alone and together with SNP (circles) and in the presence of potassium channels blockers alone and together with SNP (triangles) from arteries of adult (C) (unpaired Student's t‐test) and young rats (F) (unpaired Student's t‐test).

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  FIGURE 4. Anticontractile effect of SNP under conditions of potassium channel blockade, except Kir2 or/and Kir6 channels. Concentration–response relationships for methoxamine in the presence of 50 mM KCl alone or of KCl and SNP (0.1 μM) from arteries for adult (A, D) and young rats (G), respectively. *p < 0.05 (repeated measures ANOVA). Concentration–response relationships for methoxamine in the presence of a combination of potassium channels blockers, except BaCl2 (B) or glibenclamide (E) without and with SNP (0.1 μM) from arteries of adult rats. *p < 0.05 (repeated measures ANOVA). Concentration–response relationships for methoxamine in the presence of a combination of potassium channels blockers, except BaCl2 and glibenclamide (H) without and with SNP (0.1 μM) from arteries of young rats. *p < 0.05 (repeated measures ANOVA). Anticontractile effect of SNP, presented as the difference between the area under the curves in the presence of KCl alone and together with SNP (circles) and in the presence of corresponding potassium channels blockers alone and together with SNP (triangles) from arteries of adult (C, F) (unpaired Student's t‐test) and young rats (I) (unpaired Student's t‐test).

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  FIGURE 5. Anticontractile effect of SNP under conditions of potassium channel blockade, except BKCa channels. Concentration–response relationships for methoxamine in the presence of 50 mM KCl alone or of KCl and SNP (0.1 μM) from arteries of adult (A) and young rats (D), respectively. *p < 0.05 (repeated measures ANOVA). Concentration–response relationships for methoxamine in the presence of a combination of potassium channels blockers, except iberiotoxin, without and with SNP (0.1 μM) from arteries of adult (B) and young rats (E), respectively. *p < 0.05 (repeated measures ANOVA). Anticontractile effect of SNP, presented as the difference between the area under the curves in the presence of KCl alone and together with SNP (circles) and in the presence of corresponding potassium channels blockers alone and together with SNP (triangles) from arteries of adult (C) (unpaired Student's t‐test) and young rats (F) (unpaired Student's t‐test), #p < 0.05. For adult rats, only the range of methoxamine concentrations between 1 and 10 μM was used; this is shown by fading out the range of methoxamine concentrations between 0.01 and 1 μM (see results section for the justification of this approach).

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  FIGURE 6. Anticontractile effect of SNP under conditions of potassium channel blockade, except Kv1 or/and Kv2 channels. Concentration–response relationships for methoxamine in the presence of 50 mM KCl alone or of KCl and SNP (0.1 μM) from arteries of adult (A, D) and young rats (G), respectively. *p < 0.05 (repeated measures ANOVA). Concentration–response relationships for methoxamine in the presence of a combination of potassium channels blockers, except DPO‐1 (B) or stromatoxin (E) without and with SNP (0.1 μM) from arteries of adult rats. *p < 0.05 (repeated measures ANOVA). Concentration–response relationships for methoxamine in the presence of a combination of potassium channels blockers, except DPO‐1 and stromatoxin (H) without and with SNP (0.1 μM) from arteries of young rats. *p < 0.05 (repeated measures ANOVA). Anticontractile effect of SNP, presented as the difference between the area under the curves in the presence of KCl alone and together with SNP (circles) and in the presence of corresponding potassium channels blockers alone and together with SNP (triangles) from arteries of adult (C, F) (unpaired Student's t‐test) and young rats (I) (unpaired Student's t‐test), #p < 0.05.…

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  FIGURE 7. Anticontractile effect of SNP under conditions of potassium channel blockade, except Kv7 channels. Concentration–response relationships for methoxamine in the presence of 50 mM KCl alone or of KCl and SNP (0.1 μM) from arteries of adult (A) and young rats (E), respectively. *p < 0.05 (repeated measures ANOVA). Concentration–response relationships for methoxamine in the presence of a combination of potassium channels blockers, except XE991, without and with SNP (0.1 μM) from arteries of adult (B) and young (F) rats. *p < 0.05 (repeated measures ANOVA). Anticontractile effect of SNP, presented as the difference between the area under the curves in the presence of KCl alone and together with SNP (circles) and in the presence of corresponding potassium channels blockers alone and together with SNP (triangles) from arteries of adult (C) and young rats (G). #p < 0.05 (unpaired Student's t‐test). Active force of adult (D) and young (H) arteries, induced by the combination of potassium channel blockers before and after administration of 3 μM of XE991 in the absence (‐SNP) and presence (+SNP) of SNP (0.1 μM, J). #p < 0.05 (unpaired Student's t‐test).

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  FIGURE 8. The effects of SNP on force and membrane potential in arterial smooth muscle of adult and young rats. The values of force (A, C) and membrane potential (B, D) were determined under six conditions: In 50 mM KCl alone (first bar from left in A–D) and together with SNP (0.1 μM) (second bar from left in A–D), in the absence of any substances (third bar from left in A–D), after incubation with a mixture of blockers (except XE991) without SNP (fourth bar from left in A–D), with SNP (fifth bar from left in A‐D) and after addition of 3 μM XE991 (sixth bar from left in A–D). @p < 0.05 (paired Student's t‐test). Changes in force (E) and membrane potential (F) in response to described stimuli for adult and young arteries. #p < 0.05 (unpaired Student's t‐test).

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  FIGURE 9. Scheme illustrating the key findings: Potassium channels, even multiple ones, contribute to SNP‐induced vasorelaxation in young and adult rats and these channels change from Kv1 and Kv7 channels to BKCa and Kv7 channels during postnatal development.