The Evidence for Sparsentan-Mediated Inhibition of INa and IK(erg): Possibly Unlinked to Its Antagonism of Angiotensin II or Endothelin Type a Receptor

Figures

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  Figure 1. Effect of sparsentan (Spar) on voltage-gated Na+ current (INa) recorded from pituitary GH3 cells. In this set of experiments, we placed cells in Ca2+-free Tyrode’s solution containing 10 mM tetraethylammonium chloride (TEA) and 0.5 mM CdCl2, and the recording pipettes were filled up with a Cs+-containing solution. Under voltage-clamp conditions (as indicated in top panel), the INa was evoked. (A) Representative current traces achieved in the control period (a, sparsentan was not present), and during cell exposure to 1 μM sparsentan (b) or 3 μM sparsentan (c). Current traces in the right upper, middle or lower panel (indicated on a faster time scale) demonstrate the expanded records from dashed box in the left side of (A). a: control; b: 1 μM sparsentan; c: 3 μM sparsentan. (B) Trajectories of INa activated by brief step depolarization. Black circle represents the digitized trace in the control period (a), while red circle is one taken in the presence of 3 μM sparsentan (c). Current trajectory achieved in the absence (a) and presence (b) of 3 μM sparsentan was optimally fitted by single exponential function with 3.37 and 1.72 ms, respectively. (C) Positive linear relationship of…

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  Figure 2. Effect of sparsentan on the current versus voltage (I-V) relationship (A,B) or inactivation curve of peak INa identified in GH3 cells. In these experiments, cells were bathed in Ca2+-free Tyrode’s solution, and we filled up the electrode with Cs+-containing solution. (A) Representative current trances achieved in the control period (i.e., sparsentan was not present; top panel) or during exposure to 10 μM sparsentan (bottom panel). The uppermost panel indicates the voltage-clamp protocol applied. (B) Averaged I-V relationship of peak INa in the absence (solid squares) or presence (open squares) of 10 μM sparsentan (mean ± SEM; n = 8 for each point). (C) Steady-state inactivation curve (i.e., I/Imax versus the conditioning potential) of peak INa without (solid squares) or with (open squares) the application of 10 μM sparsentan (mean ± SEM; n = 8 for each point). In these experiments, as the tested cell was maintained at −80 mV, a test pulse to −10 mV, the conditioning voltage pulses with a duration of 40 ms to varying membrane potentials ranging between −100 and +30 mV were imposed to it. The sigmoidal smooth curves obtained in the absence or presence of 10 μM sparsentan were…

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  Figure 3. Effect of sparsentan on the recovery of INa block. In these experiments, as whole-cell configuration was firmly established, two-step voltage protocol was imposed on the examined cells. Inset indicates the voltage-clamp protocol applied. The relationship of interpulse pulse versus the relative amplitude achieved in the absence (solid squares) and presence (open squares) of 10 μM sparsentan (mean ± SEM; n = 8 for each point). The relative amplitude in the ordinate achieved obtained as the second amplitude activated by 50 ms depolarizing pulse from −80 to −10 mV was divided by the first one. The smooth curve in the absence and presence of 10 μM sparsentan was fit to single exponential function with time constant of 5.2 and 9.3 ms, respectively. Of note, the existence of sparsentan can result in a prolongation of recovery in INa block.

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  Figure 4. Effect of sparsentan (Spar) on window INa (INa(W)) evoked by the upsloping ramp pulse. In these experiments, the examined cell was held at −80 mV, the ascending ramp pulse from −100 to +50 mV with a duration of 30 or 60 ms was imposed on it. (A) Representative current trance achieved by ramp pulse of a duration of 30 ms (black color) or 60 ms (red color) during the control period (top panel) or in the existence of 10 μM sparsentan (bottom panel). Inset in the right lower corner of panel (A) shows the voltage-clamp protocol used, while the downward deflection signifies inward current. The black or red color in each trace corresponds to that activated by voltage-clamp protocol shown in Inset. In (B,C), the total charges of instantaneous INa(W) evoked by the upsloping ramp pulse with different duration (30, 60 or 90 ms) were, respectively, demonstrated in the absence and presence of 10 μM sparsentan (mean ± SEM; n = 7 for each bar). Statistical analyses in (B,C) were performed by one-way ANOVA (p < 0.05). * Significantly different from those taken with the duration of 30 ms (p < 0.5), and ** significantly different from those with the duration of 60 ms (p < 0.05).

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  Figure 5. Effect of sparsentan (Spar) on resurgent INa (INa(R)) identified in GH3 cells. These experiments were conducted in cells bathed in Ca2+-free Tyrode’s solution, and we filled up the electrode with Cs+-enriched solution. The examined cell was held at −80 mV, and to ensure complete recovery of INa, the voltage preceding depolarizing pulse to +30 mV was set at −100 mV for 30 ms; thereafter, a descending ramp pulse from +30 to −100 ms with a duration of 60 ms was applied to evoke INa(R). (A) Representative current traces obtained in the control period (a) and during cell exposure to 10 μM tefluthrin (b, Tef) or to 10 μM Tef plus 3 μM sparsentan. The top panel shows the voltage-clamp protocol imposed, and downward deflections signify occurrence of inward current. (B) Relationship of instantaneous INa(R) versus membrane potential. a: control; b: 10 μM tefluthrin; c: 10 μM tefluthrin plus 3 μM sparsentan. (C) Summary bar graph showing effect of tefluthrin and tefluthrin plus sparsentan (1 or 3 μM) on the amplitude of INa(R) in GH3 cells (mean ± SEM; n = 7 for each bar). INa(R) amplitude activated by the 60 ms downsloping ramp pulse from +30 to −100 mV was measured at the level of −50 mV.…

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  Figure 6. Effect of tefluthin, β-pompilidoxin, tefluthrin plus sparsentan, β-pompilidotoxin plus sparsentan on INa in GH3 cells. The voltage-clamp experiments were undertaken, as cells were voltage-clamped at −80 mV and the depolarizing pulse to −10 mV was imposed to them. (A) Representative current traces obtained in the control period (a, sparsentan was not present), and during exposure to 3 μM sparsentan (b) or to 3 μM sparsentan plus 3 μM tefluthrin (c). The top panel indicates the voltage-clamp protocol applied. Of note, the deactivating INa was additionally detected as membrane potential was returned to −50 mV with a duration of 40 ms. (B) Summary bar graph demonstrating effect of 3 μM tefluthrin, 3 μM β-pompilidotoxn, 3 μM tefluthrin plus 3 μM sparsentan and 3 μM β-pompilidotoxin plus 3 μM sparsentan on the peak amplitude of INa (mean ± SEM; n = 8 for each bar). The peak amplitude was measured at the start of 40 ms depolarizing command voltage from −80 to −10 mV. Tef: tefluthrin; Pomp: β-pompilidotoxin; Spar, sparsentan. Statistical analysis was performed by one-way ANOVA (p < 0.05). * Significantly different from controls (p < 0.05) and ** significantly different from Tef (3 μM) or…

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  Figure 7. Effect of sparsentan, sparsentan plus endothelin 1 and sparsentan plus angiotensin II on INa in GH3 cells. The voltage-clamp experiments were performed in the tested cells, which were depolarized from −100 to −10 mV. (A) Representative current traces achieved in the control (a) and during exposure to 3 μM sparsentan (b) or to 3 μM sparsentan plus 1 μM endothelin 1. The upper part shows the voltage-clamp protocol used. In (B), current traces denote an expanded record from the dashed box in (A). a: control; b: sparsentan (3 μM); c: sparsentan (3 μM) plus endothelin 1 (1 μM). (C) Summary bar graph showing effect of sparsentan (3 μM), sparsentan (3 μM) plus endothelin 1 (1 μM) or sparsentan (3 μM) plus angiotensin II (1 μM) on the peak amplitude of INa (mean ± SEM; n = 7 for each bar). Current amplitude was measured at the beginning of depolarizing pulse from −100 to −10 mV. * Significantly different from control (p < 0.05). (D) Representative current traces taken in the control (a), and during exposure to 1 μM endothelin 1 (b) or 1 μM endothelin 1 plus 3 μM sparsentan (c). Current traces were evoked by depolarizing pulse from −80 to −10 mV with a duration of 50 ms. a: control; b:…

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  Figure 8. Effect of sparsentan (Spar) on erg-mediated K+ (IK(erg)) identified in GH3 cells. In this set of experiments, we bathed cells in high-K+, Ca2+-free solution containing 1 μM TTX and 0.5 mM CdCl2, and the recording pipette was filled with a K+-enriched solution. (A) Representative current traces evoked by the voltage-clamp protocol (indicated in the uppermost part). Current traces in top panel are controls and those in bottom panel were obtained in the presence of 3 μM sparsentan. The colors shown in current traces correspond to those in voltage-clamp protocol. In (B,C), averaged I-V relationships of the peak (square symbols) or late (circle symbols) component of IK(erg) achieved in the control period (filled symbols) and during cell exposure to 3 μM sparsentan (open symbols), respectively. Each point in (B,C) indicates the mean ± SEM (n = 8 for each point). Statistical analyses in (B,C) were made by two way-ANOVA for repeated measures, P (factor 1, groups among data [peak versus sustained IK(erg)] taken at different level of membrane potentials) < 0.05, P (factor 2, groups between the absence and presence of sparsentan) < 0.05, P (interaction) < 0.05, followed post hoc Duncan’s…

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  Figure 9. Effect of sparsentan (Spar) on delayed-rectifier K+ current (IK(DR)) recorded from GH3 cells. These experiments were performed in cells bathed in Ca2+-free Tyrode’s solution containing 1 μM TTX and 0.5 mM CdCl2, and the electrode was filled with K+-containing solution. (A) Representative current traces obtained in the control period (top panel) and during cell exposure to 10 μM sparsentan (bottom panel). The uppermost part shows the voltage-clamp protocol applied. (B) Averaged I-V relationships of IK(DR) achieved in the absence (filled squares) and presence (open squares) of 10 μM sparsentan (mean ± SEM; n = 8 for each point). Current amplitude with or without sparsentan was achieved at the end of each voltage step. Statistical analyses were made by two way-ANOVA for repeated measures, P (factor 1, groups among data taken at different level of membrane potentials) < 0.05, P (factor 2, groups between the absence and presence of sparsentan) < 0.05, P (interaction) < 0.05, followed post hoc Duncan’s multiple-range comparisons (p < 0.05). (C) Summary bar graph showing effect of sparsentan (Spar, 3 and 10 μM) and sparsentan (10 μM) plus tetraethylammonium chloride (TEA, 10 mM) on the…