Effectiveness of Columbianadin, a Bioactive Coumarin Derivative, in Perturbing Transient and Persistent INa

Abstract

Columbianadin (CBN) is a bioactive coumarin-type compound with various biological activities. However, the action of CBN on the ionic mechanism remains largely uncertain, albeit it was reported to inhibit voltage-gated Ca2+ current or to modulate TRP-channel activity. In this study, whole-cell patch-clamp current recordings were undertaken to explore the modifications of CBN or other related compounds on ionic currents in excitable cells (e.g., pituitary GH3 cells and HL-1 atrial cardiomyocytes). GH3-cell exposure to CBN differentially decreased peak or late component of voltage-gated Na+ current (INa) with effective IC50 of 14.7 or 2.8 µM, respectively. The inactivation time course of INa activated by short depolarization became fastened in the presence of CBN with estimated KD value of 3.15 µM. The peak INa diminished by 10 µM CBN was further suppressed by subsequent addition of either sesamin (10 µM), ranolazine (10 µM), or tetrodotoxin (1 µM), but it was reversed by 10 µM tefluthrin (Tef); however, further application of 10 µM nimodipine failed to alter CBN-mediated inhibition of INa. CBN (10 µM) shifted the midpoint of inactivation curve of INa to the leftward direction. The CBN-mediated inhibition of peak INa exhibited tonic and use-dependent characteristics. Using triangular ramp pulse, the hysteresis of persistent INa enhanced by Tef was noticed, and the behavior was attenuated by subsequent addition of CBN. The delayed-rectifier or erg-mediated K+ current was mildly inhibited by 10 µM CBN, while it also slightly inhibited the amplitude of hyperpolarization-activated cation current. In HL-1 atrial cardiomyocytes, CBN inhibited peak INa and raised the inactivation rate of the current; moreover, further application of 10 µM Tef attenuated CBN-mediated decrease in INa. Collectively, this study provides an important yet unidentified finding revealing that CBN modifies INa in electrically excitable cells.

Characterization of Direct Perturbations on Voltage-Gated Sodium Current by Esaxerenone, a Nonsteroidal Mineralocorticoid Receptor Blocker

“…In these whole-cell current recordings, the examined cell was voltage-clamped at −50 mV and a set of upright isosceles-triangular ramp pulses ranging between −100 and +50 mV with varying durations at a rate of 0.05 Hz was gingerly applied to it through digitalto-analog conversion (Figure 6A). In accordance with previous observations [25,38], when GH 3 cells were exposed to 10 µM Tef alone, the amplitude of I Na(P) at high or low threshold respectively activated by the upsloping (forward, ascending) or downsloping (backward, descending) end of upright triangular ramp voltage was raised and a striking figure-ofeight hysteresis (i.e., ∞) in the instantaneous I-V relationship of I Na(P) was concurrently revealed (Figure 6B,C). For example, as the isosceles-triangular ramp pulse with a duration of 0.8 s (or ramp speed of ±0.38 mV/ms) was applied, in the presence of 10 µM Tef, the peak I Na(P) amplitude measured at the level of −30 mV (i.e., high-threshold I Na(P) ) during the ascending phase of triangular ramp pulse was conceivably raised to 62 ± 9 pA (n = 7, p < 0.05) from a control value (measured at the same level of membrane potential) of 21 ± 4 pA (n = 7).…”

“…As the ramp speed decreased, the area of such hysteretic loop progressively reduced. Therefore, findings from the present observations revealed that the triangular pulse-induced I Na(P) was observed to undergo hysteretic change in the voltage dependence found in GH 3 cells [29,30,38].…”

“…The voltage sensor energetically coupled to channel activation is supposed to be a conformationally flexible region of the protein. It is; therefore, tempting to speculate that such I Na(P) , particularly during exposure to Tef, could intrinsically and dynamically possess "memory" of previous or past events, or that mode shift occurs with respect to the voltage sensitivity of gating charge movement, which depends on the previous state (or conformation) of the Na V channel [27,29,38]. Such unique type of hysteretic behavior inherently in Na V channels would potentially play substantial role in influencing electrical behavior, Na + overload due to an excessive Na + influx, or hormonal secretion in different types of excitable cells during exposure to pyrethroid insecticides (e.g., tefluthrin or cypermethrin) [23,25,37,40].…”

Effective Accentuation of Voltage-Gated Sodium Current Caused by Apocynin (4′-Hydroxy-3′-methoxyacetophenone), a Known NADPH-Oxidase Inhibitor

“…In the first stage of measurements, we kept cells immersed in a Ca 2+ -free Tyrode’s solution containing 0.5 mM CdCl 2 , the composition of which was stated in Materials and Methods, and we filled up the pipette by using the Cs + -containing solution. As the whole-cell configuration was firmly established, we voltage-clamped the tested cell at the level of −80 mV and a brief step depolarization to −10 mV was delivered to activate I Na with a rapid activation and inactivation [ 23 , 25 , 26 ]. Of interest, one minute after cells were continually exposed to aPO , the peak amplitude of I Na was progressively increased, and the concomitant inactivation time course of the current slowed ( Figure 1 A).…”

“…The statistical analyses were undertaken by ANOVA-2 for repeated measures, p (factor 1, groups among data ken at different interpulse intervals) < 0.05, p (factor 2, groups between the absence and presence of aPO) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher's least-significant difference test, p < 0.05). Tef, a type-I pyrethroid insecticide, was reported to be an activator of I Na [23][24][25]27], Ran is recognized as a late I Na blocker as well as an inhibitor of NOX activity [26,[28][29][30], and RFM, known to be an antiepileptic agent, was previously demonstrated to perturb I Na inactivation [31,32]. For these reasons, we further examined and then compared the effects of these agents on peak I Na identified in GH 3 cells.…”