Endothelin-1 impairs coronary arteriolar dilation: Role of p38 kinase-mediated superoxide production from NADPH oxidase

Abstract

Elevated levels of endothelin-1 (ET-1), a potent vasoactive peptide, are implicated as a risk factor for cardiovascular diseases by exerting vasoconstriction. The aim of this study was to address whether ET-1, at sub-vasomotor concentrations, elicits adverse effects on coronary microvascular function. Porcine coronary arterioles (50-100μm) were isolated, cannulated and pressurized without flow for in vitro study. Diameter changes were recorded using a videomicrometer. Arterioles developed basal tone (60±3μm) and dilated to the endothelium-dependent nitric oxide (NO)-mediated vasodilators serotonin (1nmol/L to 0.1μmol/L) and adenosine (1nmol/L to 10μmol/L). Treating the vessels with a clinically relevant sub-vasomotor concentration of ET-1 (10pmol/L, 60min) significantly attenuated arteriolar dilations to adenosine and serotonin but not to endothelium-independent vasodilator sodium nitroprusside. The arteriolar wall contains ETA receptors and the adverse effect of ET-1 was prevented by ETA receptor antagonist BQ123, the superoxide scavenger Tempol, the NADPH oxidase inhibitors apocynin and VAS2870, the NOX2-based NADPH oxidase inhibitor gp91 ds-tat, or the p38 kinase inhibitor SB203580. However, ETB receptor antagonist BQ788, H2O2 scavenger catalase, scrambled gp91 ds-tat, or inhibitors of xanthine oxidase (allopurinol), PKC (Gö 6983), Rho kinase (Y27632), and c-Jun N-terminal kinase (SP600125) did not protect the vessel. Immunohistochemical staining showed that ET-1 elicited Tempol-, apocynin- and SB203580-sensitive superoxide productions in the arteriolar wall. Our results indicate that exposure of coronary arterioles to a pathophysiological, sub-vasomotor concentration of ET-1 leads to vascular dysfunction by impairing endothelium-dependent NO-mediated dilation via p38 kinase-mediated production of superoxide from NADPH oxidase following ETA receptor activation.

Keywords: Arterioles; Endothelin-1; Endothelium; NADPH oxidase; Superoxide.

Fig. 1

Response of isolated coronary arterioles to ET-1, serotonin, adenosine, and SNP. The low concentrations of ET-1 (1 and 10 pmol/L) had no effect on vascular tone, but higher concentrations (> 10 pmol/L) caused significant vasoconstriction (A) (8 vessels; *P < 0.05 vs. 1 pmol/L ET-1, one-way ANOVA followed by the Bonferroni multiple range test). Dilations of coronary arterioles to serotonin (B) and adenosine (C) were examined before and after incubation with 1 or 10 pmol/L ET-1 for 60 minutes. ET-1 at 10 pmol/L, but not 1 pmol/L, significantly inhibited dilations to serotonin (5 vessels for each set of experiments) and adenosine (5 vessels for each set of experiments). D, A sub-vasomotor concentration of ET-1 (10 pmol/L) did not affect coronary arteriolar dilation to sodium nitroprusside (SNP; 5 vessels for each set of experiments). *P < 0.05 vs. Control, two-way ANOVA followed by the Bonferroni multiple range test.

Fig. 2

Role of NOS and ET receptors in the adverse action of ET-1. Administration of NOS inhibitor (L-NAME) did not affect the adverse action of ET-1 (10 pmol/L) on vasodilations to serotonin (A) and adenosine (B). However, blockade of ET_A receptors by BQ-123 (1 μmol/L) prevented ET-1-induced reduction of vasodilations in response to serotonin and adenosine. The ET_B receptor antagonist BQ-788 (0.1 μmol/L) had no impact on the adverse effect of ET-1. 5 vessels for each set of experiments. *P < 0.05 vs. Control, two-way ANOVA followed by the Bonferroni multiple range test. The ET_A receptors (green staining) were detected in the vascular wall (C; n = 3). The expression of eNOS protein (red staining) was used to localize the endothelium. Scale bar indicates 50 μm.

Fig. 3

Effect of superoxide, hydrogen peroxide and peroxynitrite scavengers on the adverse action of ET-1. The attenuated vasodilations to serotonin (A) and adenosine (B) by ET-1 were prevented by superoxide scavenger Tempol (1 mmol/L). The hydrogen peroxide scavenger catalase (1,000 units/mL) or peroxynitrite scavenger urate (100 μmol/L) had no impact on the adverse effect of ET-1. The preserved vasodilations to serotonin (C) and adenosine (D) by Tempol were abolished in the presence of L-NAME. 5 vessels for each set of experiments. *P < 0.05 vs. Control, two-way ANOVA followed by the Bonferroni multiple range test.

Fig. 4

Roles of xanthine oxidase and NOX in the adverse action of ET-1. The attenuated vasodilations to serotonin (A) and adenosine (B) by ET-1 were prevented by NOX inhibitors apocynin (100 μmol/L) and VAS2870 (10 μmol/L), but not by xanthine oxidase inhibitor allopurinol (100 μmol/L). Blockade of NOX2 with gp91 ds-tat peptides (100 μmol/L) but not scrambled gp91 ds-tat peptides (10 μmol/L) prevented ET-1-induced reduction of coronary arteriolar dilations to serotonin (C) and adenosine (D). 5 vessels for each set of experiments. *P < 0.05 vs. Control, two-way ANOVA followed by the Bonferroni multiple range test.

Fig. 5

Roles of PKC, ROCK, JNK and p38 MAPK in the adverse action of ET-1. Blockade of p38 MAPK by SB203580 (0.1 μmol/L) prevented ET-1-induced reduction of coronary arteriolar dilations to serotonin (A) and adenosine (B). Administration of inhibitors for PKC (Gö 6983, 1 μmol/L), JNK (SP600125, 5 μmol/L), or ROCK (Y27632, 0.1 μmol/L) had no impact on the adverse effect of ET-1. 5 vessels for each set of experiments. *P < 0.05 vs. Control, two-way ANOVA followed by the Bonferroni multiple range test.

Fig. 6

Dihydroethidium imaging of superoxide production in coronary arterioles. (A) The elevated superoxide level by ET-1 (10 pmol/L) was prevented by superoxide scavenger Tempol (1 mmol/L), NOX inhibitors apocynin (100 μmol/L) and VAS2870 (VAS; 10 μmol/L), and the p38 MAPK inhibitor SB203580 (SB; 0.1 μmol/L). (B) The corresponding quantitative analysis of DHE fluorescence signals is shown. 4 vessels for each set of experiments. Scale bar indicates 100 μm. *P < 0.05 vs. Control, Student’s t test.

Figure 7

The diagram shows the proposed signaling pathway exerting the adverse effect of ET-1 on endothelium-dependent, nitric oxide-mediated vasodilation. The bioavailability of nitric oxide, following activation of eNOS by G protein-coupled adenosine and serotonin receptors, is reduced by the elevated level of superoxide in the vascular wall. A sub-vasomotor level of ET-1 (10 pmol/L) activates NOX2 for superoxide production through ET_A receptor-dependent p38 MAPK activation and consequently compromises vasodilation to nitric oxide. Alleviating the overproduction of superoxide by agents that inhibit the activation of ET_A receptors, p38 MAPK and NOX2 or that reduce superoxide level directly protects the vascular insult from ET-1.