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 AT₁ 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.

1. Introduction

Endothelins (ET-1, ET-2, and ET-3) are autocrine/paracrine signalling molecules and growth factors produced and released by vascular endothelium, cardiomyocytes, and other cells. Up-regulation of ET-1, the major cardiac myocyte isoform, is observed in cardiovascular disorders including congestive heart failure, myocardial ischaemia, atrial fibrillation, hypertension, and sepsis-induced dysfunction.¹ This peptide modulates cardiac contractile and electrical activity, and it is a potent mitogenic, fibrotic, and inflammatory factor that contributes to structural remodelling and triggers pro-survival signalling. ET-1 acts primarily via G protein-coupled ET_A receptors, which are much more abundant on cardiomyocytes than ET_B receptors.

The effects of ET-1 on cardiac electrical activity are complex. In atria, for example, ET-1 abbreviates action potential duration and modulates L-type Ca²⁺, ACh-activated K⁺, and inwardly rectifying K⁺ currents.^2,3 Moreover, endothelins regulate the volume-sensitive Cl⁻ current, I_Cl,swell. After first eliciting I_Cl,swell by osmotic swelling or hydrostatic inflation of atrial cells, Du and Sorota⁴ reported that ET-1 and ET-2 further augment the current, but the underlying signalling mechanisms are unknown.

Osmotic swelling and stretch of β_1D integrins stimulate I_Cl,swell in ventricular myocytes via angiotensin II (AngII) AT₁ receptors, epidermal growth factor receptor (EGFR) kinase, and phosphoinositide-3-kinase (PI-3K) signalling that leads to production of reactive oxygen species (ROS) by NADPH oxidase (NOX).^5–8 Hydrogen peroxide (H₂O₂) appears to be the active species; I_Cl,swell activation is reversed by catalase, and exogenous H₂O₂ elicits I_Cl,swell. ROS generated by NOX and exogenous H₂O₂ also stimulate I_Cl,swell in HeLa and haematoma cells,^9,10 cultured primary astrocytes,¹¹ and microglia.¹²

The signalling that triggers I_Cl,swell overlaps pathways implicated in the cardiac response to endothelin. Endothelins are downstream effectors for several actions of AngII,^13–15 are released by mechanical stretch,^14,15 and activate EGFR kinase¹⁶ and PI-3K.¹⁷ ROS production is also necessary for at least some of the endothelin-dependent responses in heart.^18,19

We tested the hypothesis that ET-1 activates I_Cl,swell in atrial myocytes⁴ by the same pathway triggered by osmotic swelling and AngII in ventricular myocytes. We found that ET-1 elicited I_Cl,swell via ET_A receptors under isosmotic conditions and that ET_A receptors were downstream effectors for osmotic swelling- and AngII-induced I_Cl,swell. In contrast, ET-1–ET_A receptors were upstream to EGFR kinase and PI-3K. Furthermore, ET-1-induced current and ROS production were fully inhibited by blocking either NOX or mitochondrial electron transport, whereas interventions that more directly augmented mitochondrial ROS production were insensitive to NOX inhibitors. Confirming results in atria, ET-1 evoked DCPIB-sensitive I_Cl,swell via ET_A receptors and both NOX and mitochondrial ROS in ventricular myocytes. These data suggest that ET-1 regulates I_Cl,swell as part of a complex signalling cascade that ultimately triggers mitochondrial ROS production.

2. Methods

This study conforms to Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23, 1996) and was approved by Virginia Commonwealth University Institutional Animal Care and Use Committee (AM10290). Expanded Methods are available in Supplementary material online.

Atrial and ventricular myocytes were isolated from New Zealand white rabbits (2.8–3.4 kg) using collagenase (Cls 4) and pronase (Type XIV).^5,6 Recordings were made in the whole-cell patch-clamp mode. Successive 500 ms steps were made from −60 mV to potentials between −100 and +60 mV in +10 mV increments, and current–voltage (I–V) relationships were plotted from quasi-steady-state currents.

ROS was detected by flow cytometry (EPICS XL; Beckman Coulter) of mouse atrial HL-1 cells loaded with C-H₂DCFDA-AM (2.5 or 5 µmol/L), which primarily detects H₂O₂. This permitted analysis of cell populations and accurate cell-by-cell quantification of ROS production.

Solutions were designed to isolate Cl⁻ currents. Isosmotic bath solution (300 mOsm/kg; 1T; T, times-isosmotic) contained (in mmol/L): 90 N-methyl-d-glucamine-Cl, 3 MgCl₂, 10 HEPES, 10 glucose, 5 CsCl, 0.5 CdCl₂, 70 mannitol (pH 7.4). Hypoosmotic (0.7T) and hyperosmotic (1.5T) bath solutions were made by omitting or adding mannitol. Standard pipette solution contained: 110 Cs-aspartate, 20 TEA-Cl, 5 Mg-ATP, 0.1 Tris-GTP, 0.15 CaCl₂, 8 Cs₂-EGTA, 10 HEPES (pH 7.1), and CsCl partially replaced equimolar Cs–aspartate in symmetrical Cl⁻ pipette solution.

Data are reported as mean ± SEM; n denotes the number of cells for electrophysiology or experiments for flow cytometry. One-way repeated-measures ANOVA were performed and pairwise comparisons made by the Holm–Sidak method. Fluorescence histograms were analysed using the Kruskal–Wallis one-way ANOVA on ranks and compared with control by Dunn's method.

3. Results

3.1. ET-1 activates I_Cl,swell via ET_A receptors

Figure 1A–C shows that ET-1 rapidly elicited an outwardly rectifying Cl⁻ current in atrial myocytes in isosmotic solutions that reversed near the Cl⁻ equilibrium potential, –43 mV. The ET-1-induced current was abolished by adding DCPIB, a highly selective I_Cl,swell inhibitor. Overall, ET-1 increased Cl⁻ current at +60 mV from 0.54 ± 0.04 to 1.80 ± 0.10 pA/pF (n = 61, P< 0.01), and DCPIB blocked 99 ± 4% (n = 5, P< 0.01) of the increase. The half-time for activation was 5.1 ± 0.5 min (n = 9), and current was maintained for at least 45 min (n = 3). Furthermore, 95 ± 6% (n = 4, P< 0.01) of ET-1-induced current was inhibited by osmotic shrinkage in 1.5T bath solution containing ET-1 (Figure 1D–F). Complete suppression of the outwardly rectifying Cl⁻ current with DCPIB and osmotic shrinkage indicates that ET-1 activated I_Cl,swell in atrial myocytes under isosmotic conditions and that prior stimulation by osmotic or hydrostatic swelling⁴ was not required. ET-1 also evoked an outwardly rectifying DCPIB-sensitive current in symmetrical Cl⁻ media in atrial HL-1 myocytes,²⁰ a characteristic diagnostic for I_Cl,swell.

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.

Stimulation of I_Cl,swell involved ET_A rather than ET_B receptors (see Supplementary material online, Figure S1). Exposure to BQ123 (10 µmol/L, 5–10 min), a selective ET_A inhibitor, suppressed ET-1-induced currents by 90 ± 3% (n = 4, P < 0.01), whereas the selective ET_B blocker BQ788 (100 nmol/L, 15–20 min) was ineffective (n = 4, NS).

Supplementary material online, Figure S2, shows that ET-1 also regulated I_Cl,swell in ventricular myocytes. The ET-1-induced current was 3.3 ± 0.4 pA/pF at +60 mV (n = 16, P< 0.01) and was fully suppressed by DCPIB (93 ± 6%; n = 4, P< 0.01). Moreover, the response to ET-1 was fully blocked by adding BQ123 (97 ± 2%; n = 4 P< 0.01), implicating ET_A receptors in ventricular as well as atrial myocytes.

3.2. Role of ET-1 and ET_A receptors in signalling

Stimulation of I_Cl,swell by β_1D integrin stretch, osmotic swelling, or exogenous AngII in ventricular myocytes requires the sequential activation of AngII AT₁ receptors, EGFR kinase, and PI-3K.^5,7,8 Because AngII can act via ET-1 and ET_A receptors^13,19 and ET-1 activates EGFR kinase in cardiac myocytes,¹⁶ we tested the hypothesis that ET_A receptors are upstream from EGFR kinase and PI-3K and downstream from AT₁ receptors and osmotic swelling in the signalling pathway for I_Cl,swell. Figure 2A illustrates the effect of blocking downstream signalling in atrial myocytes. As predicted, selective inhibition of EGFR kinase with AG1478 suppressed ET-1-induced I_Cl,swell by 88 ± 3% (n = 5, P< 0.01), and exogenous EGF (10 nmol/L, 10 min) activated I_Cl,swell (n = 9, P< 0.01). Moreover, inhibiting PI-3K with LY294002 or wortmannin abrogated ET-1-induced current, blocking 102 ± 7% (n = 4, P< 0.02) and 92 ± 3% (n = 4, P< 0.01), respectively (Figure 2B).

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.

The role of ET_A receptors in responding to upstream signalling is depicted in Figure 2C–E. If ET_A receptors are downstream from AT₁ receptors, BQ123 should block AngII-induced I_Cl,swell, whereas the AT₁ receptor blocker losartan should be ineffective in suppressing ET-1-induced I_Cl,swell. Consistent with this idea, AngII increased current at +60 mV from 0.40 ± 0.02 to 1.38 ± 0.24 pA/pF, and BQ123 blocked 92 ± 2% (n = 5, P< 0.01) of AngII-induced current in the presence of AngII. In contrast, the AT₁ receptor blocker losartan did not alter ET-1-induced I_Cl,swell (n = 4, NS).

The proposed scheme also predicts that blockade of ET_A receptors should suppress the activation of I_Cl,swell by osmotic swelling (Figure 2E). Osmotic swelling increased current at +60 mV from 0.67 ± 0.16 to 1.80 ± 0.42 pA/pF, and adding BQ123 to 0.7T solution blocked 91 ± 4% of swelling-induced current (n = 4, P< 0.01). In the presence of BQ123, both the swelling-induced current in 0.7T and AngII-induced current in 1T were indistinguishable from 1T control currents.

3.3. Reactive oxygen species

I_Cl,swell activation by stretch, osmotic swelling, AngII, and EGF requires ROS, and exogenous H₂O₂ elicits I_Cl,swell at a downstream site.^5,7,8 To test whether ET-1 also utilizes ROS as a downstream effector in atrial myocytes, we used ebselen, a membrane-permeant glutathione peroxidase mimetic that scavenges H₂O₂. Figure 3 shows that the stimulation of I_Cl,swell by ET-1 was fully reversed by adding ebselen. Ebselen blocked 113 ± 10% (n = 4, P< 0.01) of ET-1-induced current. As expected if ebselen scavenges H₂O₂, exogenous H₂O₂ rapidly activated I_Cl,swell (n = 4, P< 0.01), as previous shown in the ventricle.^5,7,8

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.

What is the source of ROS? One possibility is NOX. NOX subunit assembly and ROS production are stimulated by AngII, PI-3K, and ET-1,^21,22 and block of NOX suppresses swelling- and stretch-induced I_Cl,swell in ventricular myocytes^5,7,8 and other cells.^9–12 To assess whether ET-1 depends on NOX to evoke I_Cl,swell, we used apocynin and the fusion peptide gp91ds-tat, two chemically distinct inhibitors of NOX assembly. Figure 4A–C demonstrates that adding apocynin suppressed 86 ± 6% (n = 5, P< 0.01) of ET-1-induced current in atrial myocytes, and current after block was not significantly different than control. Similarly, adding gp91ds-tat suppressed 101 ± 5% (n = 4, P< 0.01) of ET-1-induced I_Cl,swell (Figure 4C).

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.

Although these data implicate NOX as a required source of ROS in ET-1 signalling, NOX may not be a sufficient source. An important alternative is mitochondria, and the complex III Q cycle is a major site for extra-mitochondrial ROS release. Rotenone, a selective complex I inhibitor, suppresses electron flow to complex III and inhibits ROS generation by intact cardiac mitochondria. As shown in Figure 4D–F, adding rotenone to ET-1 blocked 85 ± 4% (n = 4, P< 0.01) of ET-1-induced current in atrial myocytes and restored current to its control value. I_Cl,swell evoked by ET-1 in ventricular myocytes also was fully abrogated by both gp91ds-tat (102 ± 0.8%, n = 4, P < 0.01) and rotenone (105 ± 0.8%, n = 4, P < 0.01), as shown in Supplementary material online, Figure S3. Finding inhibitors of NOX and mitochondrial ROS production both completely blocked I_Cl,swell suggests that these ROS sources are likely to act in series rather than by independent pathways.

To verify that mitochondrial ROS can activate I_Cl,swell, we used electron transport chain inhibitor antimycin A and the mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP) agonist diazoxide; both stimulate mitochondrial ROS production at complex III. As expected, if mitochondrial ROS participate, both antimycin A and diazoxide elicited atrial I_Cl,swell (Figure 5A and B). Antimycin A increased Cl⁻ current at +60 mV from 0.54 ± 0.11 to 1.40 ± 0.14 pA/pF (n = 11, P< 0.01) and diazoxide from 0.60 ± 0.13 to 1.78 ± 0.47 pA/pF (n = 5, P< 0.02). Next, we tested whether production of ROS by NOX was downstream from mitochondrial ROS in the pathway regulating I_Cl,swell. Contrary to this idea, I_Cl,swell evoked by antimycin A and diazoxide was insensitive to two NOX blockers, apocynin (n = 5, NS) and the membrane-permeant fusion peptide gp91ds-tat (Figure 5A: n = 6, NS; Figure 5B: n = 5, NS). Taken together, Figures 4 and 5 suggest that mitochondria must be downstream from NOX in the I_Cl,swell signalling cascade.

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.

Osmotic shrinkage fully blocks I_Cl,swell elicited by exogenous ET-1 (Figure 1), EGF, and upstream signalling effectors, but shrinkage fails to reverse I_Cl,swell activation by exogenous H₂O₂, which is the furthest downstream regulator identified to date.⁸ Figure 5C–E demonstrates that mitochondrial ROS production elicited by antimycin A also was insensitive to osmotic shrinkage in 1.5T bath solution (n = 4, NS). These data imply that mitochondrial ROS production is a distal step in the cascade and is downstream to the site of action of osmotic shrinkage.

3.4. Measurement of ROS production

ET-1-induced ROS production was directly assessed in atrial HL-1 myocytes by flow cytometry after loading cells with C-H₂DCFDA-AM, a fluorophore that senses H₂O₂. ET-1 itself was not fluorescent in the absence of the probe (Figure 6A and D). Basal ROS production (Ctrl) was detected in C-H₂DCFDA-AM-loaded cells, and exposure to ET-1 significantly augmented fluorescence; exogenous H₂O₂ served as a positive control (Figure 6B and D). Importantly, pre-treatment with either rotenone or gp91ds-tat fully prevented ET-1-induced ROS production (Figure 6C and D), consistent with the idea that NOX and mitochondria must act in series. Flow cytometry also confirmed that antimycin A-induced mitochondrial ROS production was independent of NOX. Antimycin A increased ROS production 3.6-fold (n = 3, P< 0.05). After pre-treatment with gp91ds-tat, antimycin A still elevated ROS production 3.5-fold (n = 3, P< 0.05), whereas pre-treatment with rotenone decreased antimycin-induced ROS production to 1.2-times background (n = 3, NS). Thus, mitochondrial ROS production must be downstream from NOX, as also suggested by the electrophysiological data.

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.

4. Discussion

Endothelins were initially reported to modulate I_Cl,swell after the current was first activated by osmotic swelling or hydrostatic inflation of atrial myocytes.⁴ We found that I_Cl,swell was also elicited by ET-1 via ET_A receptors under isosmotic conditions in both atrial and ventricular myocytes, identified its position the signalling cascade, and demonstrated the involvement of ROS production by NOX and mitochondria. A diagram summarizing the I_Cl,swell cascade is presented in Figure 7. Although components of this pathway were known to regulate I_Cl,swell in cardiomyocytes, the role of ET-1 and obligatory mitochondrial ROS production was not previously recognized.

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.

Consistent with the proposed cascade, exogenous AngII^5,8 and EGF^7,8 elicited I_Cl,swell, and inhibitors of AT₁ receptors, EGFR kinase, and PI-3K suppressed the current evoked by osmotic swelling^6,8 or integrin stretch.^5,7 ET_A receptors must be downstream from osmotic swelling and AT₁ receptors but upstream from EGFR kinase. Swelling- and AngII-induced I_Cl,swell was fully blocked by the ET_A inhibitor BQ123. Moreover, ET-1-induced I_Cl,swell was blocked by suppressing EGFR kinase with AG1478 or PI-3K with LY294002 or wortmannin but was unaffected by the AT₁ blocker losartan. Because integrin stretch activates I_Cl,swell via AT₁ receptors, EGFR kinase, and PI-3K, it is likely but not proven that ET_A receptors also participate in this pathway. A similar scheme for regulating I_Cl,swell may operate in other tissues. For example, EGF activates I_Cl,swell in HTC cells,¹⁰ and blocking PI-3K inhibits I_Cl,swell in pulmonary artery smooth muscle.²³ Nevertheless, the proposed scheme may be an over simplification. We did not establish whether the response to swelling is due to an autocrine/paracrine release of endothelin and/or AngII or another mechanism. AT₁ receptors appear to be activated by stretch in the absence of AngII binding,²⁴ but an analogous mechanism has not been explored for ET_A receptors. Alternatively, formation of AT₁–ET_A heterodimers might modulate crosstalk, as found for heterodimers between several G protein-coupled receptors.²⁵ Src-dependent transactivation of ET_A by AT₁ reported in mesenteric arteries²⁶ seems unlikely because blocking Src stimulates rather than inhibits osmotic swelling-induced I_Cl,swell in cardiac myocytes.^5,27 Owing to the rapidity of the response, it also seems unlikely that osmotic swelling and AngII act by altering ET-1 synthesis. Finally, contributions from other branches of the signalling cascade triggered by ET_A receptors and from feedback by ROS that modulates signalling cannot be excluded.

4.1. Reactive oxygen species

Activation of I_Cl,swell by swelling, stretch, AngII, and EGF has been attributed to ROS production by NOX in cardiac myocytes^5,7,8 and other cells.^9–12 H₂O₂ is thought to be the active ROS because I_Cl,swell activation is reversed by catalase and exogenous H₂O₂ elicits the current. ET-1-induced I_Cl,swell studied here must also depend on ROS production because it was suppressed by ebselen, a glutathione peroxidase mimetic that scavenges H₂O₂. Although NOX activity was required for ET-1 to elicit both I_Cl,swell and ROS production, we found that it was not a sufficient stimulus. Blocking either NOX with apocynin or gp91ds-tat or mitochondrial ROS production with rotenone fully suppressed both ET-1-induced I_Cl,swell and ROS production. This suggests that the NOX and mitochondria must have acted in series. If instead these ROS generators lead to independent paths, blocking only one would be expected to give partial inhibition. It seems likely that NOX was an upstream trigger for mitochondrial ROS production by complex III. Activation of I_Cl,swell was mimicked by augmenting mitochondrial complex III ROS production with antimycin A, an inhibitor of mitochondrial electron transport distal to the Q_o site in complex III, or diazoxide, a mitoK_ATP channel opener. Furthermore, both the resulting current and ROS-induced fluorescence were insensitive apocynin and gp91ds-tat. Therefore, mitochondria must be downstream from NOX in the cascade activating I_Cl,swell.

Superoxide (O₂^−•) produced by NOX rapidly undergoes dismutation to H₂O₂, a longer-lived, membrane-permeant species. ROS may stimulate mitochondrial ROS production by several mechanisms. First, mitochondria can undergo ROS-induced ROS release, a process by which an initial release of ROS triggers oscillations of mitochondrial potential and ROS release that propagates from mitochondrion to mitochondrion.^28,29 Although NOX has not been evaluated as a trigger for oscillatory ROS-induced ROS release, subsarcolemmal mitochondria are optimally positioned to respond to ROS generated by sarcolemma NOX. Secondly, membrane-permeant H₂O₂ acting on PKCε causes mitoK_ATP channels to open³⁰ and thereby stimulates mitochondrial ROS production at complex III.³¹ A third possibility is that a signalosome, rather than free diffusion, delivers O₂^−• and H₂O₂ to mitochondria.³⁰ Ligand binding to G protein-coupled receptors causes receptor internalization as part of multimolecular signalling complexes assembled in caveolae. Garlid et al.³⁰ proposed such a mechanism to explain the coupling of multiple G protein-coupled receptor agonists to mitoK_ATP channels and complex III ROS production that underlies cardiac preconditioning. Although NOX was not included in the signalosome proposed by these authors, NOX is incorporated in agonist-induced early signalling endosomes in vascular smooth muscle cells and is an important source of O₂^−• and H₂O₂ in this setting.³²

Both the NOX2 and NOX4 isoforms are expressed in cardiac myocytes.³³ NOX4 is localized to endoplasmic reticulum in endothelial cells and to mitochondria in renal mesangial cells and cortex. It is likely, however, that only NOX2 is involved here. Downstream signalling by AngII depends on NOX2 but not on NOX4 in the heart.^21,33 Moreover, both apocynin and gp91ds-tat act by preventing subunit assembly that is required for ROS production by NOX2. In contrast, NOX4 is generally considered constitutively active and is insensitive to subunit assembly blockers.³³ Apocynin does not alter O₂^−• production by cardiac mitochondria³⁴ but appears to inhibit P450 monooxygenase in endothelial cells.³⁵ The fusion peptide gp91ds-tat³⁶ is thought to be highly selective because it contains the 9-mer-binding site from gp91^phox (NOX2) that associates with p47^phox or its homologues (e.g. NOXO1, assembles with NOX1).

Rapid induction of cardiac NOX-dependent ROS production by ET_A receptor activation initial was reported in cultured neonatal rat myocytes.³⁷ Recently, mechanical stretch, AngII/AT₁ receptors,³⁸ and ET-1/ET_A receptors³⁹ also were shown to rapidly augment ROS production in cat ventricular myocytes by NOX- and mitochondria-dependent processes. These authors did not identify the interaction between the sources of ROS or the signalling cascade. Signalling regulating ROS production is highly tissue- and species-dependent, however. Whereas AngII-induced ROS produced in rat vascular smooth muscle peaks in 10 min, it takes 6 h when ET_A receptors are stimulated with ET-1.⁴⁰ In human vascular smooth muscle, AngII-induced activation of NOX is suppressed by apocynin, but blocking NOX has no effect on ET-1-induced ROS.⁴¹ Rather, ET-1-triggered ROS production was suppressed by inhibiting mitochondrial complex II with TIFT, complex IV with CCCP, but not blocking complex I with rotenone.

4.2. Implications

Thinking of I_Cl,swell as a current regulated by NOX and mitochondrial ROS rather than a current regulated by cell volume or stretch may help clarify its role. ROS production is augmented in a variety of physiological and pathophysiological settings, and up-regulation of ET-1 occurs in congestive heart failure, myocardial ischaemia, atrial fibrillation, hypertension, and sepsis-induced dysfunction.¹ We suspect that the same signalling cascade described here is likely to regulate I_Cl,swell in human cardiac disease, but to date only the involvement of EGFR kinase has been studied in atrial myocytes from patients.⁶ If I_Cl,swell is activated in human disease, it may influence outcomes by modulating action potential duration, cardiac cell volume, cell growth, and apoptosis. Moreover, in addition to I_Cl,swell, NOX and mitochondrial ROS production triggered by this signalling cascade are very likely to influence other ion channels and transporters in the heart.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Funding

This work was supported by grants to C.M.B. (American Heart Association grant 0855044E) and to Massey Cancer Center Flow Cytometry and Imaging Shared Resource Facility (National Institutes of Health grant P30CA16059).