Pirfenidone Is a Vasodilator: Involvement of KV7 Channels in the Effect on Endothelium-Dependent Vasodilatation in Type-2 Diabetic Mice

Figures

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  FIGURE 1. Direct vasodilator effect of pirfenidone in rat pulmonary arteries involve the endothelium and potassium channels. (A) In U44619-contracted rat pulmonary arteries, pirfenidone (n = 10) induced concentration-dependent relaxations. Mannitol (n = 5) failed to relax the preparations, while tempol (n = 5) only induced relaxations at very high concentrations. (B) Inhibition of the degradation of deoxyribose by •OH by the hydroxyl scavenger D-mannitol by up to 60% inhibition, while there is no effect of pirfenidone on the degradation of deoxyribose by •OH (averages of three independent experiments). (C) In U46619 (3 × 10−8 M) contracted arteries, pirfenidone (n = 8) induced relaxations, which were reduced in the presence of an inhibitor of nitric oxide synthase, L-NOARG, and of cyclooxygenase (3 × 10−6 M) (n = 8). In arteries without endothelium (n = 6), pirfenidone relaxation was markedly reduced. Vehicle did not affect vascular tone (n = 6). (D) In preparations contracted with 30 mM KPSS, prefenidone relaxations were markedly reduced compared to effect in U46619-contracted preparations. The results are means ± s. e.mean, where n indicates the number of animals examined. *p < 0.05 vs.…

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  FIGURE 2. Pirfenidone induces endothelium-dependent nitric oxide-mediated relaxations in mouse coronary arteries. (A) Original trace showing contraction to the thromboxane analogue, U46619 and relaxations to increasing concentrations of the antifibrotic drug, pirfenidone. Horizontal bar shows time and vertical bar increase in tension (ΔT, 2 Nm−1). (B) Average increases in tension (ΔT) in control conditions, after incubation with an inhibitor of nitric oxide synthase L-NOARG (10−3 M), addition of U46619 (10−7 M), and L-NOARG plus U46619. (C) Original trace showing pirfenidone relaxations are abolished in coronary artery contracted with U46619 in the presence of L-NOARG. (D) Average relaxations induced by pirfenidone in the absence (n = 10) and the presence of L-NOARG (n = 7), and in the presence of L-NOARG plus indomethacin (3 × 10−6 M, n = 6), an inhibitor of cyclooxygenase. The results are means ± s.e. mean, where n indicates the number of animals examined. *p < 0.05 versus pirfenidone control curves, two-way ANOVA.

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  FIGURE 3. Involvement of potassium channels in pirfenidone relaxation of mouse coronary arteries. (A) Average relaxations induced by increasing concentrations of pirfenidone in preparations contracted by, respectively, U46619 (n = 10) and high extracellular potassium (30 mM, n = 7). Concentration-response curves were performed for pirfenidone in U46619-contracted arteries in the absence the presence of (B) a blocker of calcium-activated K channels, tetraethylammonium (1 mM TEA, n = 6), (C) TRAM-34 (10−6 M, n = 5), a blocker of intermediate-conductance calcium-activated potassium channels, (D) TRAM-34 and UCL1684 (10−6 M, n = 5) a blocker of small-conductance calcium-activated potassium channels, (E) a blocker of voltage-gated potassium channels of the KV7 subtype, XE0991 (10−5 M, n = 6), and (F) a selective blocker of large-conductance calcium-activated K channels, iberiotoxin (10−8 M, n = 6). The results are means ± s. e.mean, where n indicates the number of animals examined. *p < 0.05 vs. pirfenidone control curves, two-way ANOVA.

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  FIGURE 4. Effect of iberiotoxin on pirfenidone-induced current in vascular smooth muscle. (A) Original traces of whole-cell patch clamp showing current induced by voltage ramps before (control) and after the addition of pirfenidone (10−4 M). (B) Original traces showing the current increases in the presence of iberiotoxin without (control) and with pirfenidone. (C) Average current-voltage (I-V) relationships of isolated pulmonary vascular smooth muscle cells before and after short incubation with pirfenidone (10−4 M) in control conditions (n = 7) and (D) in the presence of iberiotoxin (10−7 M) (n = 7), and posterior addition of XE991 (10−5 M, n = 3). I-V relationship were determined using 600 ms voltage ramps (−100 to 100 mV; holding voltage, Vh = −65 mV). The results are means ± s. e.mean, where n indicates the number of animals examined. *p < 0.05 vs. current in the corresponding control conditions, two-way ANOVA. #p < 0.05 vs. currents in the presence of iberiotoxin after the addition of pirfenidone.

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  FIGURE 5. Pirfenidone enhances acetylcholine relaxation in aorta from male diabetic mice. Average relaxations induced by the endothelium-dependent vasodilator, acetylcholine in the absence and the presence of pirfenidone in phenylephrine-contracted aorta segments arteries from (A) heterozygous control (db/db+) (n = 6) and (B) diabetic (db/db) mice (n = 6). Average relaxations induced by the nitric oxide donor, sodium nitroprusside (SNP) in the absence and the presence of pirfenidone in phenylephrine-contracted aorta segments arteries from (C) db/db+ (n = 8) and (D) db/db mice (n = 6). The results are means ± s. e.mean, where n indicates the number of animals examined. *p < 0.05 vs. respective control curves, two-way ANOVA.

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  FIGURE 6. Effect of a KV7 channel opener on acetylcholine relaxation in aorta from male diabetic animals. Average relaxations induced by (A, B) acetylcholine (ACh) and (C, D) sodium nitroprusside (SNP) in the absence and the presence of the KV7 channel opener flupirtine (10−6 M) or flupirtine plus pirfenidone (3 × 10−5 M) in (A, C) heterozygous db/db + mice (n = 6), and in (B, D) diabetic db/db mice (n = 6). The results are means ± s. e.mean, where n indicates the number of animals examined. *p < 0.05 vs. respective control curves, two-way ANOVA.

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  FIGURE 7. The KV7 channel blocker XE991 inhibits the potentiating effect of pirfenidone on acetylcholine relaxation in aorta from diabetic animals. Average relaxations induced by acetylcholine in the absence and absence of KV7 channel blocker, XE0991 (10−5 M), or XE0991 combined with pirfenidone in (A) db/db+ mice (n = 6) and (B) diabetic db/db mice (n = 5). The results are means ± s. e.mean, where n indicates the number of animals examined. *p < 0.05 vs. respective control curves, two-way ANOVA.

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  FIGURE 8. Effect of iberiotoxin, a blocker on large-conductance calcium-activated potassium channels (BKCa) on acetylcholine and sodium nitroprusside relaxations in aorta from diabetic (db/db) mice. Average relaxations induced by (A) acetylcholine in control conditions (n = 6) and in the presence of iberiotoxin with (n = 5) and without pirfenidone (n = 6) and by (B) sodium nitroprusside (SNP) in control conditions (n = 6) and in the presence of iberiotoxin with (n = 5) and without pirfenodine (n = 6). *p < 0.05 vs. respective control curves; # p < 0.05 vs. concentration-response curve in the presence of iberiotoxin, two-way ANOVA.

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  FIGURE 9. Immunoblotting for subunits of the large-conductance calcium-activated potassium channels (BKCaα and BKCaβ) and for voltage-gated KV7.4 and KV7.5 channels in the aorta of male diabetic db/db mice. The Figures show inserts of original immunoblottings and average values expressed as a ratio to the loading control pan-actin in aorta from heterozygote db/db + control mice (n = 8) and diabetic db/db (n = 8) mice. (A) BKCaα (n = 6 from each group), (B) BKCaβ1 (n = 8 from each group), (C) KV7.4 (n = 7 from each group), and (D) KV7.5 (n = 7 from each group). The results are means ± s. e. mean from. There were no significant differences, Student’s t-test.

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  FIGURE 10. Effect of pirfenidone alone and in combination with acetylcholine on heart rate and mean arterial blood pressure in anesthetized db/db+ and diabetic (db/db) mice. Acetylcholine (ACh) infusion decreased mean arterial blood pressure and heart rate, while pirfenidone (PFD) by itself failed to change heart rate and blood pressure in anesthetized (A, B) db/db+ and (C, D) db/db mice. The response to acetylcholine was unaltered in the presence of the pirfenidone. The results are means ± s. e. mean, where n indicates the number of animals examined. *p < 0.05 compared to baseline, Wilcoxon test.

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  FIGURE 11. Schematic presentation of the mechanism involved in the vasodilator effects of pirfenidone. (A) Mechanisms involved in pirfenidone-induced vasodilatation in mouse coronary arteries involve pirfenidone through targeting upstream pathway (v) leading to opening of BKCa and KV7 channels increase the relaxations induced by basally-released nitric oxide (NO). (B) In the aorta from diabetic mice, acetylcholine relaxations are potentiated by pirfenidone supporting opening of KV7.4 channels. There is no effect of pirfenidone in the aorta from normoglycaemic animals.