Endothelin signalling regulates volume-sensitive Cl- current via NADPH oxidase and mitochondrial reactive oxygen species

Abstract

Aims: We assessed regulation of volume-sensitive Cl(-) current (I(Cl,swell)) by endothelin-1 (ET-1) and characterized the signalling pathway responsible for its activation in rabbit atrial and ventricular myocytes.

Methods and results: ET-1 elicited I(Cl,swell) under isosmotic conditions. Outwardly rectified Cl(-) current was blocked by the I(Cl,swell)-selective inhibitor DCPIB or osmotic shrinkage and involved ET(A) but not ET(B) receptors. ET-1-induced current was abolished by inhibiting epidermal growth factor receptor (EGFR) kinase or phosphoinositide-3-kinase (PI-3K), indicating that these kinases were downstream. Regarding upstream events, activation of I(Cl,swell) by osmotic swelling or angiotensin II (AngII) was suppressed by ET(A) blockade, whereas AngII AT(1) receptor blockade failed to alter ET-1-induced current. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) stimulate I(Cl,swell). As expected, blockade of NOX suppressed ET-1-induced I(Cl,swell), but blockade of mitochondrial ROS production with rotenone also suppressed I(Cl,swell). I(Cl,swell) was activated by augmenting complex III ROS production with antimycin A or diazoxide; in this case, I(Cl,swell) was insensitive to NOX inhibitors, indicating that mitochondria were downstream from NOX. ROS generation in HL-1 cardiomyocytes measured by flow cytometry confirmed the electrophysiological findings. ET-1-induced ROS production was inhibited by blocking either NOX or mitochondrial complex I, whereas complex III-induced ROS production was insensitive to NOX blockade.

Conclusion: ET-1-ET(A) signalling activated I(Cl,swell) via EGFR kinase, PI-3K, and NOX ROS production, which triggered mitochondrial ROS production. ET(A) receptors were downstream effectors when I(Cl,swell) was elicited by osmotic swelling or AngII. These data suggest that ET-1-induced ROS-dependent I(Cl,swell) is likely to participate in multiple physiological and pathophysiological processes.

Figure 1

ET-1 activated I_Cl,swell in atrial myocytes under isosmotic (1T) conditions. (A and D) Families of currents before (Ctrl) and after exposure to ET-1 (10 nmol/L, 10 min) and after addition of DCPIB (10 µmol/L, 5 − 10 min) or osmotic shrinkage in 1.5T solution (5 − 10 min) in the presence of ET-1. (B and E) Current–voltage (I–V) relationships for (A) and (D), respectively. (Inset) Activation kinetics at +60 mV; t_1/2, 5.1 ± 0.5 min (n = 9). (C and F) Currents at +60 mV in Ctrl, after ET-1, and after exposed to DCPIB (n = 5) or 1.5T (n = 4) in the presence of ET-1. **P < 0.01.

Figure 2

Atrial ET-1–ET_A receptors were upstream from EGFR kinase and PI-3K and downstream from AngII AT₁ receptors and osmotic swelling. (A) ET-1-induced I_Cl,swell (10 nmol/L, 10 min) was inhibited by the EGFR kinase blocker AG1478 (AG; 10 µmol/L, 10–15 min; n = 5) in the presence of ET-1, and EGF (10 nmol/L, 10 min; n = 9) elicited I_Cl,swell. (B) PI-3K blockers LY294002 (LY; 20 µmol/L, 10–15 min; n = 4) or wortmannin (Wort; 500 nmol/L, 10–15 min; n = 4) also inhibited ET-1-induced I_Cl,swell. (C) AngII-induced I_Cl,swell (5 nmol/L, 10 min; n = 5) was suppressed by BQ123. (D) AT₁ blocker losartan (Los; 5 µmol/L, 15 min, n = 4) did not affect ET-1-induced I_Cl,swell. (E) Osmotic swelling-induced I_Cl,swell (0.7T, 5 min; n = 4) was reversed by BQ123. *P < 0.02; **P < 0.01.

Figure 3

I_Cl,swell activation required ROS. (A) Currents and (B) I–V relationships before and after eliciting I_Cl,swell with ET-1 (10 nmol/L, 10 min) and after adding ebselen (15 µmol/L, 15 min), a glutathione peroxidase mimetic that dismutates H₂O₂. (C) Ebselen inhibited ET-1-induced current at +60 mV (n = 4), whereas H₂O₂ (100 µmol/L, 5 min) activated I_Cl,swell. **P < 0.01.

Figure 4

Inhibiting either NOX ROS production with apocynin or gp91ds-tat or mitochondrial ROS production with rotenone fully blocked atrial I_Cl,swell. (A and D) Currents and (B and E) I–V relationships before and after exposure to ET-1 (10 nmol/L, 10 min) and after adding NOX inhibitor apocynin (Apo; 500 µmol/L, 10–15 min), or the mitochondrial complex I electron transport inhibitor rotenone (Rot; 10 µmol/L, 25–45 min) in the presence of ET-1. (C and F) Apocynin (n = 5), gp91ds-tat (gp; 500 nmol/L, 15 min; n = 4), and rotenone (n = 4) inhibited ET-1-induced I_Cl,swell. **P < 0.01.

Figure 5

Mitochondrial ROS elicited I_Cl,swell and acted downstream from NOX and osmotic shrinkage. (A) Antimycin A (Anti; 10 µmol/L, 10 min) and (B) diazoxide (Diaz; 50 µmol/L, 10 min) augment ROS production by complex III, and both elicited I_Cl,swell. Mitochondrial ROS-induced I_Cl,swell was insensitive to two NOX blockers, apocynin (Apo; 500 µmol/L, 10 − 15 min; n = 5) and gp91ds-tat [500 nmol/L, 15 − 20 min; (A) n = 6; (B) n = 5]. (C–E) Currents and I–V relationship activated by antimycin and response to osmotic shrinkage (1.5T, 15 − 25 min) in the presence of antimycin. Osmotic shrinkage failed to suppress antimycin-induced I_Cl,swell (n = 4). **P < 0.01.

Figure 6

ET-1-induced ROS production was inhibited by blocking either NOX or mitochondrial electron transport. Fluorescence (FL) histograms without (A) or with (B and C) C-H₂-DCFDA-AM loading. (A) ET-1 was not fluorescent. (B) Basal ROS production (Ctrl) was increased by ET-1 (10 nmol/L, 10 min) or H₂O₂ (100 µmol/L, 10 min). (C) ET-1-induced ROS production was suppressed by pre-treatment with rotenone (Rot; 10 µmol/L) or gp91ds-tat (gp; 500 nmol/L). (D) Fold-increase in fluorescence from Ctrl (FL/FL₀) for ET-1 (n = 9), ET-1 + Rot (n = 5), ET-1 + gp91ds-tat (n = 4), and H₂O₂ (n = 9). *P < 0.05, ANOVA on ranks. (E) Sigmoidal fit to kinetics of ET-1-induced ROS production (n = 5); t_1/2, 9.5 ± 1.3 min, maximum FL/FL₀, 6.1 ± 0.6. **P< 0.01 vs. Ctrl.

Figure 7

Proposed I_Cl,swell signalling cascade. ET-1 elicited I_Cl,swell via ET_A receptors. ET-1/ET_A signalling was downstream from osmotic swelling and AngII AT₁ receptors and was upstream from EGFR kinase, PI-3K, and ROS production. O₂^−• and/or H₂O₂ generated by NOX, most likely NOX2, stimulated mitochondrial (Mito) ROS production by complex III, and antimycin A and diazoxide augmented Complex III ROS production independent of NOX. The resulting H₂O₂ elicited I_Cl,swell, perhaps through intermediates. Ebselen scavenges H₂O₂ from NOX and/or mitochondria. Osmotic shrinkage inhibited the cascade upstream to complex III and, based on prior results in the ventricle, downstream to EGFR kinase. Interventions that stimulate (green) or suppress (red) I_Cl,swell. Activation (→), inhibition (⊥), and excluded pathways (×) are indicated.