Involvement of H2O2 in superoxide-dismutase-induced enhancement of endothelium-dependent relaxation in rabbit mesenteric resistance artery

Abstract

1 The mechanism underlying the enhancement by superoxide dismutase (SOD) of endothelium-dependent relaxation was investigated in rabbit mesenteric resistance arteries. 2 SOD (200 U ml(-1)) increased the production of H(2)O(2) in smooth muscle cells (as indicated by the use of an H(2)O(2)-sensitive fluorescent dye). 3 Neither SOD nor catalase (400 U ml(-1)) modified either the resting membrane potential or the hyperpolarization induced by acetylcholine (ACh, 1 micro M) in smooth muscle cells. 4 In arteries constricted with noradrenaline, the endothelium-dependent relaxation induced by ACh (0.01-1 micro M) was enhanced by SOD (200 U ml(-1)) (P<0.01). This action of SOD was inhibited by L-N(G)-nitroarginine (nitric oxide (NO)-synthase inhibitor) but not by either charybdotoxin+apamin (Ca(2+)-activated-K(+)-channel blockers) or diclofenac (cyclooxygenase inhibitor). 5 Neither ascorbate (50 micro M) nor tiron (0.3 mM), superoxide scavengers, had any effect on the ACh-induced relaxation, but each attenuated the enhancing effect of SOD on the ACh-induced relaxation. Similarly, catalase (400 U ml(-1)) inhibited the effect of SOD without changing the ACh-induced relaxation. 6 In endothelium-denuded strips constricted with noradrenaline, SOD enhanced the relaxation induced by the NO donor 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7) (P<0.05). Ascorbate and catalase each attenuated this effect of SOD. 7 H(2)O(2) (1 micro M) enhanced the relaxation on the noradrenaline contraction induced by NOC-7 and that induced by 8-bromo-cGMP, a membrane-permeable analogue of guanosine 3',5' cyclic monophosphate (cGMP). 8 SOD had no effect on cGMP production, whether measured in endothelium-intact strips following an application of ACh (0.1 micro M) or in endothelium-denuded strips following an application of NOC-7 (0.1 micro M). 9 It is suggested that in rabbit mesenteric resistance arteries, SOD increases the ACh-induced, endothelium-dependent relaxation by enhancing the action of NO in the smooth muscle via its H(2)O(2)-producing action (rather than via a superoxide-scavenging action).

Figure 1

Effects of charybdotoxin (CTx)+apamin, diclofenac and SOD on ACh-induced hyperpolarization in smooth muscle cells of the rabbit mesenteric resistance artery. (a) Effect of ACh on membrane potential in the absence (‘control') and presence of CTx (0.1 μM)+apamin (0.1 μM). ACh produced an initial, followed by a second-phase hyperpolarization. ACh (1 μM) was applied for 2 min as indicated by the bar. (b) Effect of diclofenac (3 μM) on ACh-induced hyperpolarization. (c) Effect of SOD (200 U ml⁻¹) on ACh-induced response. Broken lines indicate original resting membrane potential level in each cell (in a given panel, recordings were made from one and the same cell). Traces in (a)–(c) were obtained from different strips. The data were representative of five to six experiments and the results were reproducible.

Figure 2

Effect of SOD on ACh-induced relaxation in endothelium-intact strips. (a) Effect of SOD (200 U ml⁻¹) on the ACh-induced relaxation on the contraction induced by noradrenaline (10 μM). ‘Control': in the absence of SOD, ‘SOD': in the presence of SOD. Agents were applied as indicated by the bars. (b) Summary of the effect of SOD on ACh-induced relaxation. Data are shown as means and s.e.m. **P<0.01 vs control (two-way repeated-measures ANOVA and Scheffé's F test).

Figure 3

Effects of ascorbate, tiron and catalase on ACh-induced relaxation in the absence or presence of SOD (200 U ml⁻¹) in endothelium-intact strips. (a) Effect of ascorbate (50 μM) on ACh-induced relaxation. ‘Control': in the absence of agents; ‘Ascorbate': in the presence of ascorbate; ‘Ascorbate+SOD': the action of SOD in the presence of ascorbate. (b) Effect of tiron (0.3 mM) on ACh-induced relaxation. ‘Control': in the absence of agents; ‘Tiron': in the presence of tiron; ‘Tiron+SOD': the action of SOD in the presence of tiron. (c) Effect of catalase (400 U ml⁻¹) on ACh-induced relaxation. ‘Control': in the absence of agents; ‘Catalase': in the presence of catalase; ‘Catalase+SOD': the action of SOD in the presence of catalase. Data are shown as means and s.e.m.

Figure 4

Effects of L-N^G-nitroarginine (L-NOARG), CTx+apamin and diclofenac on the SOD (200 U ml⁻¹)-induced enhancement of the ACh-induced relaxation in endothelium-intact strips. Effect of SOD on the ACh relaxation on the ongoing contraction induced by (a) 0.5 μM noradrenaline in L-NOARG (0.1 mM)-treated strips, (b) 10 μM noradrenaline in strips treated with diclofenac (3 μM), and (c) 10 μM noradrenaline in the presence of CTx (0.1 μM)+apamin (0.1 μM) in strips treated with diclofenac (3 μM). Data are shown as means and s.e.m. *P<0.05, **P<0.01 vs corresponding response in the absence of SOD (two-way repeated-measures ANOVA and Scheffé's F test). †† P<0.01 vs ‘Diclofenac'.

Figure 5

Effects of SOD, ascorbate and catalase on the NOC-7-induced relaxation on the ongoing noradrenaline (10 μM)-contraction in endothelium-denuded strips. (a) Effect of SOD (200 U ml⁻¹) on NOC-7-induced relaxation. ‘Control': in the absence of agent; ‘SOD': the action of SOD. (b) Effect of ascorbate (50 μM) on NOC-7-induced relaxation in the absence and presence of SOD (200 U ml⁻¹). ‘Control': in the absence of agents; ‘Ascorbate': in the presence of ascorbate; ‘Ascorbate+SOD': the action of SOD in the presence of ascorbate; ‘SOD': the action of SOD after a 60-min removal of ascorbate. (c) Effect of catalase (400 U ml⁻¹) on NOC-7-induced relaxation in the absence and presence of SOD (200 U ml⁻¹). ‘Control': in the absence of agents; ‘Catalase': in the presence of catalase; ‘Catalase+SOD': the action of SOD in the presence of catalase. Data are shown as means and s.e.m. **P<0.01 vs control (two-way repeated-measures ANOVA and Scheffé's F test). † P<0.05, †† P<0.01 vs ‘Ascorbate+SOD'.

Figure 6

Effects of MnCl₂ and H₂O₂ (in the absence or presence of diclofenac) on the relaxation induced by NOC-7 during the contraction induced by 10 μM noradrenaline in endothelium-denuded strips. (a) Effect of MnCl₂ (30 μM) on the NOC-7-induced relaxation. ‘Control': in the absence of MnCl₂; ‘MnCl₂': in the presence of MnCl₂. (b) Effect of H₂O₂ (1 μM) on the NOC-7-induced relaxation. ‘Control': in the absence of H₂O₂; ‘H₂O₂': in the presence of H₂O₂. (c) Effect of H₂O₂ (1 μM) on the NOC-7-induced relaxation in the absence and presence of diclofenac. ‘Control': in the absence of diclofenac and H₂O₂; ‘Diclofenac': in the presence of diclofenac alone. ‘Diclofenac+H₂O₂': in the presence of both diclofenac and H₂O₂. Data are shown as means and s.e.m. *P<0.05, **P<0.01 vs control (two-way repeated-measures ANOVA and Scheffé's F test).

Figure 7

Effect of H₂O₂ on relaxation induced by 8-bromo-cGMP in endothelium-denuded strips. Effect of H₂O₂ (1 μM) on the 8-bromo-cGMP-induced relaxation obtained during a contraction induced by 10 μM noradrenaline. ‘Control': in the absence of H₂O₂; ‘H₂O₂': in the presence of H₂O₂. Data are shown as means and s.e.m. *P<0.05, **P<0.01 vs control (two-way repeated-measures ANOVA and Scheffé's F test).

Figure 8

Effects of MnCl₂, SOD and H₂O₂ on the fluorescence obtained with the H₂O₂-sensitive dye CM-H₂DCF in smooth muscle cells of endothelium-denuded strips. (A) fluorescent images: a, image obtained just before application of SOD; b, at 15 min after application of SOD (200 U ml⁻¹); c, at 15 min after application of H₂O₂ (10 μM). Similar results were obtained in three other preparations. (B) Summary of the effects of MnCl₂ (30 μM), SOD (200 U ml⁻¹) and H₂O₂ (10 μM) on the fluorescence. To obtain the fluorescence ratio, the fluorescence value was normalized with respect to that recorded just before the application of agent. Data are shown as means and s.e.m. *P<0.05, **P<0.01 vs untreated (Student's unpaired t-test).

Figure 9

Effects of SOD on the production of cGMP. Effects of SOD (200 U ml⁻¹) on cGMP production (a) in the absence or presence of ACh (0.1 μM) in endothelium-intact (E(+)) strips and (b) in the absence or presence of NOC-7 (0.1 μM) in endothelium-denuded (E(−)) strips. Data are shown as means and s.e.m. *P<0.05 vs basal (Student's unpaired t-test). N.S. not significantly different.