Combination of Sildenafil and Ba2+ at a Low Concentration Show a Significant Synergistic Inhibition of Inward Rectifier Potassium Current Resulting in Action Potential Prolongation

Figures

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  FIGURE 1. Sildenafil inhibits inward rectifier potassium current (I K1). (A) The experimental protocol (upper panel) and representative current traces in control conditions (C), under the effect of 1 µM sildenafil (S) and 100 µM Ba2+ (B) at −50 mV and −110 mV (lower panel); inset: current traces at −50 mV in detail; arrows indicate the approximate time of analysis of I K1 magnitude. (B) Changes of I K1 at −50 mV and −110 mV during subsequent applications of 1 µM sildenafil and 100 µM Ba2+ (left panel) and the highest sildenafil concentration 100 µM (right panel). (C) Average current-voltage relationship in control and under the effect of 1 µM sildenafil (n = 5/3).

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  FIGURE 2. Average effects of sildenafil on I K1. (A) Average I K1 density in control and under the effect of sildenafil between 0.1 and 100 μM at −110 mV (left graph) and −50 mV (right graph); paired t-test, *, ** and ***—statistically significant difference at p < 0.05, 0.01 and 0.001, respectively, (no significant differences among control values at the respective voltage). (B) Concentration dependence of the inhibition by sildenafil was present at both −50 mV and −110 mV; no significant difference between effects at both tested voltages was observed.

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  FIGURE 3. Synergistic inhibitory effect of sildenafil and Ba2+ at a low concentration of 0.1 µM. (A) Changes of I K1 in the presence of sildenafil and Ba2+, applied both separately and in combination; paired t-test, *, ** and ***—statistically significant difference at p < 0.05, 0.01 and 0.001, respectively. (B) Massive inhibition of I K1 appeared after combined application of both sildenafil and Ba2+. The inhibition was significantly more potent than a simple sum of separate effects of sildenafil and Ba2+. The effect was not significantly different between voltages.

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  FIGURE 4. Combined action of sildenafil (S) and Ba2+ (B) at selected concentrations. (A) The synergy between sildenafil and Ba2+ was observed only in the case of combination of 0.1 µM sildenafil + 0.1 µM Ba2+ (n = 7–11/4−8); ***—statistical significance at p < 0.001. (B) Paired comparison of the effect of 0.1 µM Ba2+, 0.1 µM sildenafil + 0.1 µM Ba2+, 0.3 µM Ba2+, and 0.1 µM sildenafil + 0.3 µM Ba2+ (left panel–representative recording; right panel–average data, n = 4/1); *—statistical significance at p < 0.05.

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  FIGURE 5. Changes of action potential duration (APD) under the effect of 0.1 µM sildenafil alone and in combination with Ba2+ (0.1 µM); APD was evaluated at 90% repolarization (APD 90). (A) Representative AP waveforms in control and under the effect of the drugs alone and in combination at 23°C. (B) Average APD 90 changes at 23°C (n = 7/3). (C) Values of APD 90 and resting membrane potential (RMP) in a representative cell measured both at 23 and 37°C. (D) Comparison of the average relative APD 90 changes under the effect of sildenafil in the absence and presence of Ba2+ at 23 and 37°C (n = 4/2); * and **—statistical significance at p < 0.05 and 0.01, respectively.