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CARDIOVASCULAR

Endothelin-1 Regulates Cardiac L-Type Calcium Channels via NAD(P)H Oxidase-Derived Superoxide

Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota (F.Y., S.T.O., C.S.); the Laboratory of Molecular & Cellular Physiology, School of Life Sciences, Northeast Normal University, Changchun, China (Q.Ze.); and Frontier Medical Sciences Institute, Jilin University, Changchun, China (Q.Zh.)

Received for publication April 22, 2008
Accepted June 4, 2008.

	Abstract

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It has been shown that reactive oxygen species (ROS) are involved in the intracellular signaling response to G-protein coupled receptor stimuli in vascular smooth muscle cells and in neurons. In the present study, we tested the hypothesis that NAD(P)H oxidase-derived ROS are involved endothelin-1 (ET-1)-induced L-type calcium channel activation in isolated cardiac myocytes. ET-1 (10 nM) induced a 2-fold increase in L-type calcium channel open-state probability (NPo). This effect of ET-1 was abolished by the ET_A receptor antagonist cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) [BQ-123 (1 µM)] but was not altered in the presence of an ET_B receptor antagonist N-cis-2,6-dimethylpiperidinocarbonyl-b-tBu-Ala-D-Trp(1-methoxycarbonyl)-D-Nle-OH [BQ-788 (1 µM)]. Pretreatment of cells with the ROS scavenger tempol (100 µM), polyethylene glycol-superoxide dismutase (SOD, 25 U/ml), or the NAD(P)H-oxidase inhibitor gp91ds-tat ([H]RKKRRQRRR-CSTRIRRQL[NH₃]) (5 µM) significantly attenuated ET-1-induced increases in calcium channel NPo. Tempol, SOD, and gp91ds-tat alone had no effect on basal calcium channel activity. In addition, ET-1 significantly increased NAD(P)H oxidase activity and elevated intracellular superoxide levels in cultured cardiac myocytes. The superoxide generator, xanthine-xanthine oxidase (10 mM, 20 mU/ml), also increased calcium channel NPo in cardiac myocytes, mimicking the effect of ET-1. These observations provide the first evidence that ET-1 induces the activation of L-type Ca²⁺ channels via stimulation of NAD(P)H-derived superoxide production in cardiac myocytes.

In cardiac myocytes, the L-type Ca²⁺ current (I_CaL) is the main depolarizing current contributing to the plateau-shape of the action potential and regulation of Ca²⁺ release from the sarcoplasmic reticulum (Cleemann and Morad, 1991). This channel is regulated by several second messengers, which explains why it mediates the action of numerous hormones and neuromediators, including ET-1. However, the effect of ET-1 on I_CaL still remains unclear. For example, some studies of ET-1 have shown clear increases in I_CaL (Lauer et al., 1992; Boixel et al., 2001), whereas others have shown a decrease in I_CaL (Cheng et al., 1995; Ono et al., 1995) or no effect (Tohse et al., 1990). These contradictory data may be due to the wide differences in experimental conditions, including different species, tissues, and preparations. In addition, the method employed to measure I_CaL could be the most important reason. Most of the initial studies were performed using the ruptured whole-cell patch clamp technique. In contrast, cell-attached single channel recording allows characterization of I_CaL without the alteration of intracellular components that occurs with whole-cell patch clamp technique. Therefore, it is essential to examine the effect of ET-1 on I_CaL and the underlying mechanism in intact cardiac myocytes.

Reactive oxygen species (ROS) are well recognized as important mediators of cardiovascular pathology, including hypertrophy and heart failure (Dhalla et al., 2000; Byrne et al., 2003b). Both mitochondria and NAD(P)H oxidase are capable of participating directly in the reduction of oxygen and producing superoxide. In vascular tissue the NAD(P)H oxidase is recognized as the predominant source of superoxide (Griendling et al., 2000). Recent studies are providing evidence for a role for NAD(P)H oxidase-derived ROS in cardiac pathology. Mice lacking the pg91phox subunit do not develop angiotensin II-induced cardiac hypertrophy (Byrne et al., 2003a). With the exception of the importance of ROS in cardiac pathophysiology, it is still not clear whether NAD(P)H oxidase-derived ROS play a role in the rapid response to ET-1 in cardiac myocytes. Identifying the mechanisms responsible for the action of ET-1 could help identify a new therapeutic target for ET-1-associated cardiac disorders.

In this study, we examined whether ET-1 increases intracellular ROS production via increased NAD(P)H-oxidase activity and whether the elevated ROS subsequently modulates the function of L-type calcium channels in cardiac myocytes.

	Materials and Methods

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Animals and Drugs. Adult male Sprague-Dawley rats (200–250 g) were obtained from Charles River Farms (Wilmington, MA). Rats were housed individually and kept on a 12/12-h light/dark cycle in a climate-controlled room. Rat chow and water were provided ad libitum. All animal protocols were approved by the Institutional Animal Care and Use Committee of Northeast Normal University and by the North Dakota State University Institutional Animal Care and Use Committee.

Dihydroethidium (DHE) was purchased from Molecular Probes (Carlsbad, CA). Dulbecco's modification of Eagle's medium was obtained from GIBCO (Carlsbad, CA). Bay K 8644 was purchased from Calbiochem (San Diego, CA). The selective NAD(P)H oxidase inhibitor gp91ds-tat ([H]RKKRRQRRR-CSTRIRRQL[NH₃]) and its control, scrambled gp91ds-tat ([H]RKKRRQRRR-CLRITRQSR[NH₃]), were synthesized by Tufts University Core Facility (Medford, MA). BQ-123, BQ-788, ET-1, ATP, GTP, HEPES, and others were purchased from Sigma-Aldrich (St. Louis, MO).

Ventricular Myocytes Isolation. Cardiac myocytes were enzymatically isolated from hearts obtained from rats anesthetized with sodium pentobarbitone (30 mg/kg i.p. injection). Each heart was rapidly excised and cooled in ice-cold Tyrode's solution containing 135 mM NaCl, 5.4 mM KCl, 0.33 mM NaH₂PO₄, 1.0 mM MgCl₂, 5.5 mM glucose, 1.8 mM CaCl₂, and 10 mM HEPES, pH 7.4 (NaOH). After cannulation of the aorta, retrograde perfusion was started with Tyrode's solution via a recirculating system for 2 min. The heart was then perfused with Ca²⁺-free normal Tyrode's solution for 5 min, followed by perfusion with Ca²⁺-free normal Tyrode's solution containing collagenase (0.15 mg/ml) for 50 min. The ventricle was excised, cut into small pieces, and incubated at 37°C for 10 min in a storage solution containing 70 mM KOH, 50 mM glutamic acid, 40 mM KCl, 20 mM KH₂PO₄, 20 mM taurine, 3 mM MgCl₂, 10 mM glucose, 10 mM HEPES, and 0.5 mM EGTA, pH 7.4 (KOH). The separated ventricular myocytes were stored at 4°C in a refrigerator until they were used for electrophysiological recording.

Single Calcium Channel Recording. Single L-type calcium channel activity was measured by the patch-clamp technique in cell-attached mode. Patch pipettes were pulled with a three-step puller (Zeitz, Augsburg, Germany) and fire-polished to a final resistance of 2 to 5 M. Cells were bathed in a bath solution containing 140 mM potassium aspartate, 10 mM EGTA, and 10 mM HEPES, pH 7.4 (KOH). The patch pipettes were filled with an internal pipette solution containing 110 mM BaCl₂, and 10 mM HEPES, pH 7.4 [Ba(OH)₂]. The potential of the electrode was adjusted to zero between the pipette solution and the bath solution immediately before seal formation. After a giga-seal between the pipette and myocyte had formed, the activity of L-type calcium channels was induced by stepping from a holding potential of -60 to +10 mV for 256-ms duration at a rate of 0.5 Hz. Single Ca²⁺ channel activity was recorded by L/M-PC patch-clamp amplifier (List, Darmstadt, Germany). Analysis of the unitary currents was performed with pClamp 8.0 software (Axon Instruments, Downingtown, PA), and the results were expressed as open-state probability (NPo).

Neonatal Rat Cardiomyocyte Culture. Primary cultured cardiomyocytes were prepared from neonatal rats according to previously published procedures with minor modulation (Gruh et al., 2006). In brief, ventricles from 1-day-old Sprague-Dawley rats were digested at 37°C in Hanks' solution containing collagenase (class II; Worthington Biochemical, Lakewood, NJ). Isolated cells were suspended in culture medium composed of 8% fetal bovine serum, Dulbecco's modification of Eagle's medium, and 0.2% penicillin-streptomycin solution. The cell suspension was placed in 35-mm diameter tissue culture dishes at a cell density of 4 x 10⁶/dish and incubated at 37°C in a humidified atmosphere with 95% O₂ + 5% CO₂.

Measurement of Intracellular ROS Level. ROS generation was determined using the oxidant-sensitive fluorogenic probe DHE (excitation wavelength, 488 nm; emission wavelength, 610 nm) essentially as described elsewhere (Hool et al., 2005). In brief, primary cultured cardiomyocytes were loaded with 100 nM DHE for 30 min at 37°C. The cells were then incubated with PBS for 5 min, followed by 10 nM ET-1 for 5 min. In another set of cardiomyocytes, cells were pretreated with BQ-123 (1 µM) or vehicle control for 10 min before ET-1 treatment. Ethidium fluorescence within cardiomyocytes was detected by a fluorescent microscope (Nikon, Melville, NY), and its intensity in individual cells was analyzed using Quantity One Software (Bio-Rad, Hercules, CA). Each treatment condition was run in triplicate within experiments, and each set of experiments was performed using three separate culture dishes.

Measurement of NAD(P)H Oxidase Activity. The lucigenin-derived chemiluminescence method was used to measure ET-1-induced NAD(P)H oxidase activity in primary cultured cardiomyocytes. Cardiomyocytes were treated with ET-1 (10 nM) or PBS for 5 min and washed in ice-cold PBS, and cells were scraped and then sonicated for 1 s. Ten minutes before recording luminescence, NAD(P)H (100 µM) and lucigenin (5 µM) were added and light emission was recorded during the next 10 s by a Wallac 1450 Micro-Beta JET Luminometer (PerkinElmer Life and Analytical Sciences, Waltham, MA). Protein concentrations were determined using a Bio-Rad protein assay kit with bovine serum albumin standards. Data were presented as counts per minute per milligram of protein.

Data Analysis. All data are expressed as mean ± S.E. Comparisons between experimental groups were performed using analysis of variance followed by a Newman-Keuls test. Differences were considered significant at P < 0.05.

	Results

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ET-1 Stimulates L-type Calcium Channel. ET-1 has positive inotropic and chronotropic actions in a variety of cardiac preparations (Kramer et al., 1991). To investigate the role of I_CaL in these responses to ET-1, we used cell-attached patch-clamp single channel recording to assess the effect of ET-1 on I_CaL in isolated rat cardiac myocytes without disruption of intracellular environment. Application of ET-1 (10 nM) evoked a significant increase in the calcium channel NPo (Fig. 1, A and B). The channel open time was increased from 2.95 ± 0.33 to 4.74 ± 0.42 ms (n = 11, P < 0.05), and the channel close time was decreased from 88.40 ± 14.73 to 35.31 ± 12.41 ms (n = 11, P < 0.05) by application of ET-1 (10 nM). However, the perfusion of cardiomyocytes with ET-1 (10 nM) did not significantly alter the unitary current amplitude, as shown in Fig. 1A (1.42 ± 0.11 and 1.40 ± 0.18 pA in cells before and after treatment with ET-1, respectively, n = 11, P > 0.05). The increases in NPo of I_CaL evoked by ET-1 were partially reversed after 10-min washout with fresh bath solution (from 0.021 ± 0.002 to 0.017 ± 0.002, n = 4, P < 0.05; the baseline NPo was 0.014 ± 0.03). In another series of experiments, the addition of a specific I_CaL activator Bay K 8644 (100 nM) significantly increased NPo of I_CaL without changing the unitary current amplitude, mimicking the effect of ET-1 on this channel in cardiac myocytes (Fig. 1, C and D). The Bay K 8644-induced increases in NPo of I_CaL were completely reversed after 10-min washout with fresh bath solution (from 0.026 ± 0.03 to 0.017 ± 0.01, n = 4, P < 0.05; the baseline NPo was 0.015 ± 0.02)

View larger version (19K): [in this window] [in a new window]   Fig. 1. Effects of ET-1 and Bay K 8644 on ICaL recorded in cell-attached patches of isolated cardiomyocytes. A, representative tracings showing ICaL activity recorded in a cardiomyocyte before and after application of ET-1 (10 nM). The bars show the baseline (ICaL closing state). The patch membrane was depolarized to 10 mV from the holding potential of -60 mV for 256 ms. B, bar graphs are mean ± S.E. of ICaL open probability in cardiomyocytes (n = 11) before and after application of ET-1 (10 nM). *, P < 0.01 compared with control. C, representative tracings showing ICaL activity recorded in a cardiomyocyte under control condition and after superfusion with Bay K 8644 (Bay K, 100 nM). The bars indicate the channel closing state. D, bar graphs are mean ± S.E. of calcium channel open probability in cardiomyocytes (n = 12) before and after application of Bay K 8644 (Bay K, 100 nM). *, P < 0.005 compared with control.

It has been shown that both ET_A and ET_B receptors are expressed in cardiomyocytes (Wainwright et al., 2005). Thus, we examined the endothelin-receptor subtype(s) involved in the L-type calcium current response to ET-1 by pretreatment of the cells with specific receptor antagonists. Data are shown in Fig. 2A, indicating that the ET_A receptor antagonist BQ-123 (1 µM) completely blocked the ET-1-induced increase in calcium channel NPo. By contrast, the ET_B receptor antagonist BQ-788 (1 µM) did not alter the calcium channel responsiveness to ET-1 under the same treatment conditions. In these experiments, neither BQ-123 (1 µM) nor BQ-788 (1 µM) alone significantly changed the basal calcium channel activity.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Role of ETA receptor, ETB receptor, and superoxide in ET-1-induced increases in ICaL in isolated cardiomyocytes. A, bar graphs showing the effect of an ETA receptor antagonist, BQ-123 (BQ123, 1 µM), and an ETB receptor antagonist, BQ-788 (BQ788, 1 µM), on ET-1-induced increases in ICaL in cell-attached patches. Data are mean ± S.E. channel open probability in cardiomyocytes (n = 7 and 8) treated under the following conditions: Control, ET-1 (10 nM), BQ-123 or BQ-788, and BQ-123 + ET-1 or BQ-788 + ET-1. *, P < 0.05 compared with the respective control. B, bar graphs showing the effect of a superoxide scavenger, tempol (TP, 100 µM), and a NAD(P)H oxidase inhibitor, gp91ds-tat (GP, 5 µM), on ET-1-induced increases in ICaL in cell-attached patches. Data are mean ± S.E. of channel open probability in cardiomyocytes (n = 9 and 7) treated under the following conditions: control, ET-1 (10 nM), TP or GP, and TP + ET-1 or GP + ET-1. *, P < 0.05 compared with control.

ET-1 Increases ROS Production in Cardiomyocytes. The fluorogenic probe, DHE, was used to assess the effect of ET-1 on ROS production in primary cultured cardiomyocytes. Under control conditions, ethidium fluorescence was low in PBS-treated cardiomyocytes (Fig. 3B); however, treatment of these same cells with 10 nM ET-1 resulted in a significant increase in the density of ethidium fluorescence within cardiomyocytes (Fig. 3, C and G). The ET-1-induced increase in fluorescence was completely inhibited by the ET_A endothelin receptor antagonist BQ-123 (1 µM; Fig. 3, F and G). BQ-123 (1 µM) alone did not alter the basal ethidium fluorescence (Fig. 3, E and G). In contrast, the ET_B endothelin receptor antagonist BQ-788 (1 µM) had no significant effect on this action of ET-1 (Fig. 3G). In addition, the ET-1-induced increase in ethidium fluorescence was attenuated by gp91ds-tat (5 µM) (845 ± 61 and 926 ± 77 ethidium fluorescence intensity of cells treated with gp91ds-tat and gp91ds-tat + ET-1. respectively, n = 13, P > 0.05). However, the scrambled control gp91ds-tat did not alter the stimulatory action of ET-1 on ROS production (751 ± 79 and 1522 ± 98 ethidium fluorescence intensity of cells treated with scrambled gp91ds-tat and scrambled gp91ds-tat + ET-1, respectively, n = 11, P < 0.01). These results indicate that, via activation of ET_A receptors, ET-1 increases superoxide production, which is blocked by inhibition of NAD(P)H oxidase.

View larger version (64K): [in this window] [in a new window]   Fig. 3. Effects of ET-1 on superoxide production within cardiomyocytes. Superoxide levels were detected using the fluorogenic probe DHE in primary cultured cardiomyocytes as detailed under Materials and Methods.A through C, cardiomyocytes that were treated under the following conditions: cardiomyocytes in normal optical phase (A); fluorescence micrograph of cardiomyocytes loaded with DHE and treated with PBS (B); and fluorescence micrograph of the same cardiomyocytes as shown in B (C), following treatment with ET-1 (10 nM). D through F, cardiomyocytes that were treated under the following conditions: cardiomyocytes in normal optical phase (D); fluorescence micrograph of cardiomyocytes loaded with DHE after treatment with BQ-123 (BQ123, 1 µM) for 5 min (E); and fluorescence micrograph of the same cardiomyocytes as shown in E (F), following treatment with ET-1 (10 nM). Bar = 20 µm. G, bar graphs summarizing ethidium fluorescence intensity before and after treatment with ET-1 in the presence of vehicle control (PBS), BQ-123 (BQ123, 1 µM), or BQ-778 (BQ788, 1 µM). In each treatment condition, 20 to 23 cardiomyocytes were used for quantification of fluorescence. They were derived from three experiments and at least three dishes in each experiment. Data are mean ± S.E. *, significant difference from the respective control (P < 0.01).

ET-1 Stimulates NAD(P)H Oxidase in Cardiomyocytes. Treatment of primary cultured cardiomyocytes with ET-1 (10 nM) resulted in a 2-fold increase in NAD(P)H oxidase activity (Fig. 4A). This effect of ET-1 was completely blocked by preincubation of cardiomyocytes with the ET_A endothelin receptor antagonist BQ-123 (1 µM). However, the ET_B endothelin receptor antagonist, BQ-788 (1 µM), has no effect on ET-1-induced increases in NAD(P)H oxidase activity (Fig. 4A). In addition, preincubation of cardiomyocytes with gp91ds-tat (5 µM), but not with scrambled gp91ds-tat (5 µM), significantly attenuated the ET-1-induced activation of NAD(P)H oxidase (Fig. 4B).

View larger version (14K): [in this window] [in a new window]   Fig. 4. Effects of ET-1 and gp91ds-tat on NAD(P) H oxidase activity in cardiomyocytes. A, effect of ET-1 and receptor involvement. Primary cultured cardiomyocytes were pretreated under the following conditions: vehicle control, BQ-123 (BQ123 1 µM), or BQ-788 (BQ788 1 µM) for 10 min. This was followed by incubation with 10 nM ET-1 for an additional 5 min. Cells were collected, and NAD(P)H activity was measured and expressed as mean light emission (counts per milligram of protein per minute). Data are mean ± S.E. (n = 7). *, significantly different from respective control treatment (P < 0.05). B, effect of gp91ds-tat. Primary cultured cardiomyocytes were pretreated under the following conditions: vehicle control, gp91ds-tat (GP, 5 µM), or scrambled gp91ds-tat control (ScrGP, 5 µM) for 10 min before treatment with 10 nM ET-1 for 5 min as described in A. Data are mean ± S.E. (n = 9). *, significantly different from respective control (P < 0.05).

View larger version (12K): [in this window] [in a new window]   Fig. 5. Identification of ROS involved in ET-1-induced increases in ICaL activity. A, role of superoxide. ICaL were recorded in cell-attached patches of isolated cardiomyocytes under the following conditions: control, superfusion of ET-1 (10 nM), superfusion of PEG-SOD (SOD, 25 U/ml), and PEG-SOD plus ET-1 or control, superfusion of xanthine-xanthine oxidase (X-XO: X, 10 mM; XO, 20 mU/ml). Data are mean ± S.E. of calcium channel open probability in cardiomyocytes (n = 9–10). *, P < 0.05 compared with respective control. B, role of hydrogen peroxide. ICaL were recorded in cell-attached patches of cardiomyocytes under the following treatment conditions: control, superfusion of ET-1 (10 nM), superfusion with PEG-catalase (CAT, 250 U/ml), and PEG-Catalase plus ET-1 or control, superfusion of H2O2 (1 µM). Data are mean ± S.E. of calcium channel open probability in cardiomyocytes (n = 11 and 9). *, P < 0.05 compared with respective control.

	Discussion

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ET-1 has a positive inotropic effect in many isolated cardiac preparations (Kramer et al., 1991). It is well known that an increase in the intracellular calcium transient contributes to this positive inotropic effect and that the basis for the increased Ca²⁺ transient is stimulation of I_CaL. The results of the present study are consistent with this hypothesis, inasmuch as ET-1 caused an increase in the open-state probability of I_CaL in rat cardiac myocytes. However, previous investigations examining the effect of ET-1 on I_CaL have produced conflicting results, with decreases (Lauer et al., 1992; Boixel et al., 2001), increases (Cheng et al., 1995; Ono et al., 1995), and no effect (Tohse et al., 1990) on basal I_CaL reported. These discrepancies may be due to differences in experimental conditions, particularly differences in the electrophysiologic recording methods used (e.g., rupture of the cell membrane). One important reason for the discrepant findings regarding the effects of ET-1 on I_CaL in cardiac myocytes may be related to the expression of ET receptors. Cardiac myocytes express both ET_A-and ET_B endothelin receptors (Moe et al., 2003; Wainwright et al., 2005). In general, the ET_A receptor subtype is more abundant (90%) and is considered more important for the cardiac stimulatory effects of ET-1 on I_CaL as demonstrated in the present study. Although ET_B receptors may not contribute to the action of ET-1 on I_CaL, as shown in the present study in rat cardiac myocytes, they may contribute to inhibitory effects of ET-1 (Kedzierski and Yanagisawa, 2001). Thus, the ratio between ET_A and ET_B receptors in cardiac myocytes from different animal species could play a role in the observed action of ET-1 on I_CaL. Another important reason for these discrepant findings may be related to the interaction with other regulatory factors and intracellular second messengers, such as cyclic AMP, β-adrenoceptor agonists, protein kinase A, and protein kinase C (PKC). It has been shown that the effect of ET-1 on cardiac contractility and L-type calcium current varies depending on the presence of norepinephrine, β-adrenoceptor agonists, or cAMP analogs (Watanabe and Endoh, 1999; Chu et al., 2003). For example, in canine ventricular myocardium, ET-1 induces either a positive inotropic effect or a negative inotropic effect, which is determined by the presence of a low concentration or high concentration of norepinephrine, respectively. Moreover, in rabbit cardiomyocytes, ET-1 has a biphasic effect on calcium current, causing a transient decrease followed by a long-lasting increase in current (Watanabe and Endoh, 1999). The ET-1-induced inhibition of L-type calcium current was enhanced in the presence of an activated cAMP-dependent pathway. This interaction between ET-1 and norepinephrine in the regulation of cardiomyocyte contractility and calcium channel current could play an important role in the pathophysiology of heart failure, which is associated with increased sympathetic nerve activity and elevated plasma norepinephrine levels. Nevertheless, studying the effect of ET-1 on calcium channel current under different neurohormonal conditions will help improve our understanding of the cellular mechanisms underlying the pathogenesis of cardiac diseases, such as heart failure and cardiac remodeling.

The present observations demonstrate that ET-1 increases intracellular superoxide levels by stimulation of NAD(P)H oxidase and that increased superoxide production is involved in the stimulatory effect of ET-1 on I_CaL in cardiac myocytes. Although superoxide may be rapidly converted to hydrogen peroxide, it is unlikely that the stimulatory effect of ET-1 on I_CaL is mediated by hydrogen peroxide because the hydrogen peroxide scavenger, catalase, had no effect on the response to ET-1 and authentic hydrogen peroxide itself had no effect on I_CaL. These results are supported by data from others in vascular smooth muscle cells showing that ET-1 increases superoxide production via ET_A-receptor/NAD(P)H oxidase pathways (Callera et al., 2003; Li et al., 2003). Given that extracellular calcium influx plays essential roles in ET-1-induced vascular contraction and cell proliferation, it is not surprising that the ET-1-ROS signaling pathway is involved in the action of other cardiovascular regulatory factors, such as angiotensin II-induced positive inotropic effects, leptin-induced cardiac hypertrophy, and atrial natriuretic peptide-mediated antihypertrophic effects (Xu et al., 2004; Cingolani et al., 2006; Laskowski et al., 2006). However, the nature of the intracellular signaling mechanisms by which ROS increases I_CaL in cardiac myocytes remains to be clarified. One possibility may be a direct redox-dependent mechanism, because the pore-forming _1C-subunit of the cardiac L-type Ca²⁺ channel contains more than 10 cysteine residues (Mikami et al., 1989) that can potentially undergo redox modification. Indeed, oxidization of Src homology groups causes stimulation of I_CaL, whereas GSH and dithiothreitol, which reduce disulfide bonds, inhibit this current in ferret ventricular myocytes (Campbell et al., 1996).

Another major question that arises from our studies concerns the mechanisms that are involved in the ET-1-stimulated NAD(P)H oxidase activation in cardiac myocytes. At present, little is known with regard to such mechanisms in cardiac myocytes. However, several clues are emerging from other research areas that may provide future direction for addressing this question. Activation of PKC is involved in ET-1-induced increases in cardiac I_CaL via an unknown mechanism (He et al., 2000), and PKC-dependent NAD(P)H oxidase activation is the main source of intracellular ROS production in response to angiotensin II in vascular smooth muscle cells (Touyz and Schiffrin, 2001; Yasunari et al., 2002; Ungvari et al., 2003) and in neurons (Wang et al., 2006). Therefore, future studies examining whether PKC-mediated phosphorylation of NAD(P)H oxidase contributes to the ET-1-induced ROS elevation in cardiac myocytes should provide important information regarding intracellular signaling.

In summary, ET-1 increases I_CaL activation via stimulation of ET_A receptors in cardiac myocytes. The ET-1-induced increase in I_CaL is mediated by NAD(P)H oxidase-derived superoxide production. Given that activation of L-type calcium channels is involved in ET-1-related cardiac pathophysiological conditions (Chu et al., 2003; Hirotani et al., 2004; Angerio, 2005; Sugden and Clerk, 2006), ROS-dependent activation of I_CaL may contribute to ET-1-induced intracellular calcium mobilization, cardiac excitation-contraction, myocyte proliferation, and consequently disorders, such as heart failure and cardiac hypertrophy.

	Footnotes

 
This work was supported by the American Heart Association. The project described was also supported by Grant 2P20RR015566 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH).

Q.Ze. and Q.Zh. contributed equally to this work

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.140301.

ABBREVIATIONS: ET-1, endothelin-1; NAD(P)H, nicotinamide adenine dinucleotide phosphate; ROS, reactive oxygen species; NPo, channel open-state probability; DHE, dihydroethidium; I_CaL, L-type calcium channel current; SOD, superoxide dismutase; PEG, polyethylene glycol; X-XO, xanthine-xanthine oxidase; PKC, protein kinase C; BQ-123, cyclo(D-Trp-D-Asp-Pro-D-Val-Leu); BQ-788, N-cis-2,6-dimethylpiperidinocarbonyl-b-tBu-Ala-D-Trp(1-methoxycarbonyl)-D-Nle-OH; gp91ds-tat, [H]RKKRRQRRR-CSTRIRRQL[NH₃]; PBS, phosphate-buffered saline; Bay K 8644, 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2'-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester.

Address correspondence to: Dr. Chengwen Sun, Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105. E-mail: chengwen.sun{at}ndsu.edu

	References

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Angerio AD (2005) The role of endothelin in heart failure. Crit Care Nurs Q 28: 355-359.[Medline]
Boixel C, Dinanian S, Lang-Lazdunski L, Mercadier JJ, and Hatem SN (2001) Characterization of effects of endothelin-1 on the L-type Ca²⁺ current in human atrial myocytes. Am J Physiol Heart Circ Physiol 281: H764-H773.[Abstract/Free Full Text]
Byrne JA, Grieve DJ, Bendall JK, Li JM, Gove C, Lambeth JD, Cave AC, and Shah AM (2003a) Contrasting roles of NAD(P)H oxidase isoforms in pressure-overload versus angiotensin II-induced cardiac hypertrophy. Circ Res 93: 802-805.[Abstract/Free Full Text]
Byrne JA, Grieve DJ, Cave AC, and Shah AM (2003b) Oxidative stress and heart failure. Arch Mal Coeur Vaiss 96: 214-221.[Medline]
Callera GE, Touyz RM, Teixeira SA, Muscara MN, Carvalho MH, Fortes ZB, Nigro D, Schiffrin EL, and Tostes RC (2003) ET_A receptor blockade decreases vascular superoxide generation in DOCA-salt hypertension. Hypertension 42: 811-817.[Abstract/Free Full Text]
Campbell DL, Stamler JS, and Strauss HC (1996) Redox modulation of L-type calcium channels in ferret ventricular myocytes. Dual mechanism regulation by nitric oxide and S-nitrosothiols. J Gen Physiol 108: 277-293.[Abstract/Free Full Text]
Cheng TH, Chang CY, Wei J, and Lin CI (1995) Effects of endothelin 1 on calcium and sodium currents in isolated human cardiac myocytes. Can J Physiol Pharmacol 73: 1774-1783.[Medline]
Chu L, Takahashi R, Norota I, Miyamoto T, Takeishi Y, Ishii K, Kubota I, and Endoh M (2003) Signal transduction and Ca²⁺ signaling in contractile regulation induced by crosstalk between endothelin-1 and norepinephrine in dog ventricular myocardium. Circ Res 92: 1024-1032.[Abstract/Free Full Text]
Cingolani HE, Villa-Abrille MC, Cornelli M, Nolly A, Ennism IL, Garciarena C, Suburo AM, Torbidoni V, Correa MV, Camilionde Hurtado MC, et al. (2006) The positive inotropic effect of angiotensin II: role of endothelin-1 and reactive oxygen species. Hypertension 47: 727-734.[Abstract/Free Full Text]
Cleemann L and Morad M (1991) Role of Ca²⁺ channel in cardiac excitation-contraction coupling in the rat: evidence from Ca²⁺ transients and contraction. J Physiol 432: 283-312.[Abstract/Free Full Text]
Dhalla NS, Temsah RM, and Netticadan T (2000) Role of oxidative stress in cardiovascular diseases. J Hypertens 18: 655-673.[CrossRef][Medline]
Griendling KK, Sorescu D, and Ushio-Fukai M (2000) NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res 86: 494-501.[Abstract/Free Full Text]
Gruh I, Beilner J, Blomer U, Schmiedl A, Schmidt-Richter I, Kruse ML, Haverich A, and Martin U (2006) No evidence of transdifferentiation of human endothelial progenitor cells into cardiomyocytes after coculture with neonatal rat cardiomyocytes. Circulation 113: 1326-1334.[Abstract/Free Full Text]
He JQ, Pi Y, Walker JW, and Kamp TJ (2000) Endothelin-1 and photoreleased diacylglycerol increase L-type Ca²⁺ current by activation of protein kinase C in rat ventricular myocytes. J Physiol 524: 807-820.[Abstract/Free Full Text]
Hirotani S, Higuchi Y, Nishida K, Nakayama H, Yamaguchi O, Hikoso S, Takeda T, Kashiwase K, Watanabe T, Asahi M, et al. (2004) Ca²⁺-sensitive tyrosine kinase Pyk2/CAK beta-dependent signaling is essential for G-protein-coupled receptor agonist-induced hypertrophy. J Mol Cell Cardiol 36: 799-807.[CrossRef][Medline]
Hool LC, Di Maria CA, Viola HM, and Arthur PG (2005) Role of NAD(P)H oxidase in the regulation of cardiac L-type Ca²⁺ channel function during acute hypoxia. Cardiovasc Res 67: 624-635.[Abstract/Free Full Text]
Ito H, Hirata Y, Adachi S, Tanaka M, Tsujino M, Koike A, Nogami A, Murumo F, and Hiroe M (1993) Endothelin-1 is an autocrine/paracrine factor in the mechanism of angiotensin II-induced hypertrophy in cultured rat cardiomyocytes. J Clin Invest 92: 398-403.[Medline]
Ito H, Hirata Y, Hiroe M, Tsujino M, Adachi S, Takamoto T, Nitta M, Taniguchi K, and Marumo F (1991) Endothelin-1 induces hypertrophy with enhanced expression of muscle-specific genes in cultured neonatal rat cardiomyocytes. Circ Res 69: 209-215.[Abstract/Free Full Text]
Kedzierski RM and Yanagisawa M (2001) Endothelin system: the double-edged sword in health and disease. Annu Rev Pharmacol Toxicol 41: 851-876.[CrossRef][Medline]
Krämer BK, Smith TW, and Kelly RA (1991) Endothelin and increased contractility in adult rat ventricular myocytes. Role of intracellular alkalosis induced by activation of the protein kinase C-dependent Na⁺-H⁺ exchanger. Circ Res 68: 269-279.[Abstract/Free Full Text]
Laskowski A, Woodman OL, Cao AH, Drummond GR, Marshall T, Kaye DM, and Ritchie RH (2006) Antioxidant actions contribute to the antihypertrophic effects of atrial natriuretic peptide in neonatal rat cardiomyocytes. Cardiovasc Res 72: 112-123.[Abstract/Free Full Text]
Lauer MR, Gunn MD, and Clusin WT (1992) Endothelin activates voltage-dependent Ca²⁺ current by a G protein-dependent mechanism in rabbit cardiac myocytes. J Physiol 448: 729-747.[Abstract/Free Full Text]
Li L, Fink GD, Watts SW, Northcott CA, Galligan JJ, Pagano PJ, and Chen AF (2003) Endothelin-1 increases vascular superoxide via endothelin(A)-NAD(P)H oxidase pathway in low-renin hypertension. Circulation 107: 1053-1058.[Abstract/Free Full Text]
Mikami AK, Imoto T, Tanabe T, Niidome T, Mori Y, and Takeshima H (1989) Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel, Nature 340: 230-233.[CrossRef][Medline]
Moe GW, Rouleau JL, Nguyen QT, Cernacek P, and Stewart DJ (2003) Role of endothelins in congestive heart failure. Can J Physiol Pharmacol 81: 588-597.[CrossRef][Medline]
Ono K, Eto K, Sakamoto A, Masaki T, Shibata K, Sada T, Hashimoto K, and Tsujimoto G (1995) Negative chronotropic effect of endothelin 1 mediated through ET_A receptors in guinea pig atria. Circ Res 76: 284-292.[Abstract/Free Full Text]
Sugden PH (2003) An overview of endothelin signaling in the cardiac myocyte. J Mol Cell Cardiol 35: 871-886.[CrossRef][Medline]
Sugden PH and Clerk A (2006) Oxidative stress and growth-regulating intracellular signaling pathways in cardiac myocytes. Antioxid Redox Signal 8: 2111-2124.[CrossRef][Medline]
Suzuki YJ, Nagase H, Nie K, and Park AM (2005) Redox control of growth factor signaling: recent advances in cardiovascular medicine. Antioxid Redox Signal 7: 829-834.[CrossRef][Medline]
Tohse N, Hattori Y, Nakaya H, Endou M, and Kanno M (1990) Inability of endothelin to increase Ca²⁺ current in guinea-pig heart cells. Br J Pharmacol 99: 437-438.[Medline]
Touyz RM and Schiffrin EL (2001) Increased generation of superoxide by angiotensin II in smooth muscle cells from resistance arteries of hypertensive patients: role of phospholipase D-dependent NAD(P)H oxidase-sensitive pathways. J Hypertens 19: 1245-1254.[CrossRef][Medline]
Ungvari Z, Csiszar A, Huang A, Kaminski PM, Wolin MS, and Koller A (2003) High pressure induces superoxide production in isolated arteries via protein kinase C-dependent activation of NAD(P)H oxidase. Circulation 108: 1253-1258.[Abstract/Free Full Text]
Wainwright CL, McCabe C, and Kane KA (2005) Endothelin and the ischaemic heart. Curr Vasc Pharmacol 3: 333-341.[CrossRef][Medline]
Wang G, Anrather J, Glass MJ Tarsitano MJ, Zhou P, Frys KA, Pickel VM, and Iadecola C (2006) Nox2, Ca²⁺ and protein kinase C play a role in angiotensin II-induced free radical production in nucleus tractus solitarius. Hypertension 48: 482-489.[Abstract/Free Full Text]
Watanabe T and Endoh M (1999) Characerization of the endotheline-1-induced regulation of L-type Ca²⁺ current in rabbit ventricular myocytes. Naunyn Schmiedebergs Arch Pharmacol 360: 654-664.[CrossRef][Medline]
Xu FP, Chen MS, Wang YZ, Yi Q, Lin SB, Chen AF, and Luo JD (2004) Leptin induces hypertrophy via endothelin-1-reactive oxygen species pathway in cultured neonatal rat cardiomyocytes. Circulation 110: 1269-1275.[Abstract/Free Full Text]
Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, and Masaki T (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411-415.[CrossRef][Medline]
Yasunari K, Maeda K, Nakamura M, and Yoshikawa J (2002) Pressure promotes angiotensin II-mediated migration of human coronary smooth muscle cells through increase in oxidative stress. Hypertension 39: 433-437.[Abstract/Free Full Text]

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