Endothelin-1 regulates cardiac L-type calcium channels via NAD(P)H oxidase-derived superoxide

Abstract

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 microM)] 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 microM)]. Pretreatment of cells with the ROS scavenger tempol (100 microM), polyethylene glycol-superoxide dismutase (SOD, 25 U/ml), or the NAD(P)H-oxidase inhibitor gp91ds-tat ([H]RKKRRQRRR-CSTRIRRQL[NH(3)]) (5 microM) 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 Ca(2+) channels via stimulation of NAD(P)H-derived superoxide production in cardiac myocytes.

Fig. 1

Effects of ET-1 and Bay K 8644 on I_CaL recorded in cell-attached patches of isolated cardiomyocytes. A, representative tracings showing I_CaL activity recorded in a cardiomyocyte before and after application of ET-1 (10 nM). The bars show the baseline (I_CaL 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 I_CaL 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 I_CaL 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.

Fig. 2

Role of ET_A receptor, ET_B receptor, and superoxide in ET-1-induced increases in I_CaL in isolated cardiomyocytes. A, bar graphs showing the effect of an ET_A receptor antagonist, BQ-123 (BQ123, 1 μM), and an ET_B receptor antagonist, BQ-788 (BQ788, 1 μM), on ET-1-induced increases in I_CaL 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 I_CaL 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.

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).

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).

Fig. 5

Identification of ROS involved in ET-1-induced increases in I_CaL activity. A, role of superoxide. I_CaL 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. I_CaL 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.