High Capability of Pentagalloylglucose (PGG) in Inhibiting Multiple Types of Membrane Ionic Currents

Figures

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  Figure 1. Inhibitory effect of PGG on the amplitude of delayed-rectifier K+ current (IK(DR)) measured from pituitary tumor (GH3) cells. In these whole-cell experiments, we kept cells to be bathed in Ca2+-free Tyrode’s solution containing 1 μM tetrodotoxin, and the recording pipette was filled with K+-containing solution. The composition of these solutions is elaborated in the Materials and Methods. (A) Representative IK(DR) density evoked during 1-sec square-voltage command from −50 to +50 mV (indicated in the Inset). a: control; b: 1 μM PGG; c: 3 μM PGG; d: 10 μM PGG. (B) Concentration-dependent inhibition of IK(DR) density produced by the presence of different PGG concentrations (mean ± SEM; n = 8). The continuous sigmoidal line over which the data points (○) are overlaid is appropriately fitted by the three-parameter logistic model (i.e., modified Hill equation). The vertical dashed line is placed at the IC50 value (i.e., 3.6 μM). (C) Time course for the PGG-induced inhibition of IK(DR) density in a GH3 cell. Current density was obtained at the end of 1-sec depolarizing pulse from −50 to +50 mV. The horizontal bar shown above indicates the application of PGG (3 μM).

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  Figure 2. Mean current density versus voltage relationship of IK(DR) obtained without or with the addition of PGG. The examined cells were voltage-clamped at −50 mV with different voltage commands ranging between −60 and +50 mV (representing in inset of the upper part). (A) Representative IK(DR) densities achieved in the control (i.e., the absence of PGG; upper part) or during GH3-cell exposure to 10 μM PGG (lower part). (B) Mean current density versus voltage relationship of IK(DR) in the absence (■) and presence (□) of 10 μM PGG (mean ± SEM; n = 7). Current density was measured at the end of each step command. The statistical analyses were made by ANOVA-2 for repeated measures, p (factor 1) < 0.05, p (factor 2) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05. * indicates a significant difference from controls obtained at the same level of membrane potential (p < 0.05).

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  Figure 3. Inhibitory effect of PGG on erg-mediated K+ current (IK(erg)) identified in GH3 cells. In these whole-cell recordings, we bathed cells in a high-K+, Ca2+-free solution, and the pipette was filled up with K+-containing solution. (A) Representative IK(erg) density obtained in the control (i.e., in the absence of PGG; upper) or during the exposure to 10 μM PGG (lower). The uppermost part denotes the voltage protocols applied. (B) Mean IK(erg) density versus voltage relationships obtained in the absence (■) or presence (○) of 10 μM PGG (mean ± SEM; n = 8 for each point). Current density was measured at the start of each voltage step. The statistical analyses were made by ANOVA-2 for repeated measures, p (factor 1) < 0.05, p (factor 2) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05. * indicates a significant difference from controls measured at the same level of the hyperpolarizing step (p < 0.05). (C) Time course of IK(erg) density obtained during cell exposure to 10 μM PGG. Each point (□) was measured at the start of a 1-sec hyperpolarizing pulse from −10 to −100 mV, and the horizontal bar shown above indicates the…

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  Figure 4. Biophysical and pharmacological properties of the proton-induced Cl− current (IPAC) density identified in GH3 cells. The experiments were undertaken in cells which were kept bathed in Ca2+-free Tyrode’s solution, and the recording electrode was filled up with Cs+-containing solution. (A) Representative IPAC densities obtained in cells exposed to the control condition (i.e., pH 7.4, upper) or to the acidic solution (i.e., pH 4.0, lower). The voltage protocol delivered is illustrated in the uppermost part. (B) Mean current density versus voltage relationships of IPAC (mean ± SEM; n = 8 for each point). ■: control (pH 7.4); □: in the presence of acidic solution (pH 4.0); ○: in the presence of acidic solutions plus 10 μM niflumic acid. The statistical analyses were made by ANOVA-2 for repeated measures, p (factor 1) < 0.05, p (factor 2) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05. * and ** indicate a significant difference from the controls (i.e., pH 7.4) (p < 0.05 or p < 0.01, respectively) measured at the same level of the membrane potential, while † indicates a significant difference from the acidic solution (i.e., pH…

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  Figure 5. Concentration-dependent inhibitory effect of PGG on IPAC density (A,B) and comparisons in effects of PGG, chlorotoxin, hesperetin, kaempferol, morin, hydroxychloroquine and iberiotoxin on IPAC density (C). In these experiments, cells were kept immersed in acidic (i.e., pH 4.0) and Ca2+-free Tyrode’s solution, and the electrode was filled up with Cs+-containing solution. (A) Representative IPAC density taken in the control (i.e., in the absence of PGG, but still exposed to acidic extracellular solution with pH 4.0) and during cell exposure to 3 μM PGG (b) or 10 μM PGG (c), but still in the presence of acidic solution. (B) Concentration–response relationship for the PGG-induced inhibition of IPAC density measured at the end of the 500-msec square potential command to +50 mV from a holding potential of −50 mV (mean ± SEM; n = 8–9 for each point). A smooth sigmoidal line is drawn according to the three-parameter logistic model (i.e., modified Hill equation) as described in the Materials and Methods. A vertical dashed line indicates the IC50 value of this compound needed for its inhibition of IPAC density. (C) Comparison between the effect of PGG and those of chlorotoxin, hesperetin,…

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  Figure 6. Effect of PGG on the voltage-dependent hysteresis of IPAC density identified from GH3 cells. Cells were bathed in acidic (pH = 7.4), Ca2+-free Tyrode’s solution. (A) Representative IPAC density in the absence (a) or presence (b) of 3 μM PGG. Current densities were elicited in response to 2-sec long isosceles-triangular ramp voltage command (indicated in the Inset). The arrows denote the direction of IPAC density in which time passes. (B) Summary bar graph showing the effect of PGG (1 or 3 μM) or niflumic acid (3 or 10 μM) on the Δarea (indicated in the shaded area of (A)) of the voltage hysteresis (mean ± SEM; n = 8 for each bar). The statistical analyses were done by ANOVA-1, p < 0.05, followed by a post-hoc Fisher’s least-significant difference test, p < 0.05. * indicates a significant difference from control (p < 0.05), † indicates significant difference from the PGG (1 μM)-alone group (p < 0.05), and ‡ indicates a significant difference from the niflumic acid (3 μM)-alone group (p < 0.05).

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  Figure 7. Inability of PGG to perturb the hyperpolarization-activated cation current (Ih) identified from GH3 cells. In this set of experiments, cells were bathed in a Ca2+-free Tyrode’s solution that contained 1 μM tetrodotoxin, and the electrode was filled up with K+-containing solution. (A) Representative Ih densities obtained in the control, and during the exposure to 10 μM PGG or 10 μM PGG plus 3 μM zatebradine (ZAT). In the experiments on PGG plus zatebradine, as the cells were continually exposed to PGG, further addition of zatebradine was applied. (B) Summary bar graph showing the effects of PGG and PGG plus zatebradine (ZAT) on the density of hyperpolarization-evoked Ih (mean ± SEM; n = 7 for each bar). Each current density in (A,B) was taken at the end of 2-sec hyperpolarizing step to −120 mV from a holding potential of −40 mV. The statistical analyses were done by ANOVA-1, p < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05. * indicates a significant difference from control (p < 0.05). (C) Time course of effect of PGG (10 μM) and PGG (10 μM) plus ZAT (3 μM) on Ih density. Ih density was taken at the end of 2-sec hyperpolarizing step from −40 to…

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  Figure 8. Inhibitory effect of PGG on the density of voltage-gated Na+ current (INa) in GH3 cells. The experiments were conducted in cells kept to be bathed in Ca2+-free Tyrode’s solution containing 10 mM tetraethylammonium chloride and 0.5 mM CdCl2, and we filled up the recording electrode by using Cs+-containing solution. (A) Representative INa densities obtained in the control (a) and during the exposure to 10 μM PGG (b) or 10 μM PGG plus 10 μM sesamin (SSM) (c). The voltage protocol delivered is presented in the upper part, while inset indicates an expanded record from the dashed box. Of note, the voltage-clamp profile used in the present study was applied to measure transient and deactivating densities of INa. (B) Summary bar graph revealing effects of PGG, PGG plus sesamin (SSM), and PGG plus tetrodotoxin (TTX) on the density of depolarization-evoked INa (mean ± SEM; n = 8). The density of peak INa was taken at the start of brief depolarization from −80 to −10 mV. The statistical analyses were done by ANOVA-1, p < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05. * indicates a significant difference from control (p < 0.05), and † significantly different…

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  Figure 9. Inhibitory effectiveness of PGG in the density of ramp voltage-induced IK(DR) recorded from human cardiac myocytes (HCM). In this set of whole-cell current recordings, we kept HCM in Ca2+-free Tyrode’s solution and then filled up the electrode by using K+-enriched solution. (A) Ramp-induced IK(DR) density obtained in the absence (a) or presence of 3 μM PGG (b). The inset indicates the linear ramp protocol delivered to the cell. (B) Summary bar graph showing effect of PGG and PGG plus tetraethylammonium chloride (TEA) on IK(DR) density in HCM (mean ± SEM; n = 8 for each bar). Current density was measured at the level of +100 mV. The statistical analyses were done by ANOVA-1, p < 0.05, followed by post-hoc Fisher’s least-significant difference test, p < 0.05. * indicates a significant difference from control (p < 0.05), † indicates a significant difference from the PGG (1 μM)-alone group (p < 0.05), and ‡ indicates a significant difference from the PGG (3 μM)-alone group (p < 0.05).