Abstract
Cilobradine (CIL, DK-AH269), an inhibitor of hyperpolarization-activated cation current (Ih), has been observed to possess pro-arrhythmic properties. Whether and how CIL is capable of perturbing different types of membrane ionic currents existing in electrically excitable cells, however, is incompletely understood. In this study, we intended to examine possible modifications by it or other structurally similar compounds of ionic currents in pituitary tumor (GH3) cells and in heart-derived H9c2 cells. The standard whole-cell voltage-clamp technique was performed to examine the effect of CIL on ionic currents. GH3-cell exposure to CIL suppressed the density of hyperpolarization-evoked Ih in a concentration-dependent manner with an effective IC50 of 3.38 μM. Apart from its increase in the activation time constant of Ih during long-lasting hyperpolarization, the presence of CIL (3 μM) distinctly shifted the steady-state activation curve of Ih triggered by a 2-s conditioning pulse to a hyperpolarizing direction by 10 mV. As the impedance-frequency relation of Ih was studied, its presence raised the impedance magnitude at the resonance frequency induced by chirp voltage. CIL also suppressed delayed-rectifier K+ current (IK(DR)) followed by the accelerated inactivation time course of this current, with effective IC50 (measured at late IK(DR)) or KD value of 3.54 or 3.77 μM, respectively. As the CIL concentration increased 1 to 3 μM, the inactivation curve of IK(DR) elicited by 1- or 10-s conditioning pulses was shifted to a hyperpolarizing potential by approximately 10 mV, and the recovery of IK(DR) inactivation during its presence was prolonged. The peak Na+ current (INa) during brief depolarization was resistant to being sensitive to the presence of CIL, yet to be either decreased by subsequent addition of A-803467 or enhanced by that of tefluthrin. In cardiac H9c2 cells, unlike the CIL effect, the addition of either ivabradine or zatebradine mildly led to a lowering in IK(DR) amplitude with no conceivable change in the inactivation time course of the current. Taken together, the compound like CIL, which was tailored to block hyperpolarization-activated cation (HCN) channels effectively, was also capable of altering the amplitude and gating of IK(DR), thereby influencing the functional activities of electrically excitable cells, such as GH3 cells.
Highlights
Cilobradine (CIL, DK-AH269) has been previously demonstrated to suppress hyperpolarization-activated cyclic nucleotide-gated (HCN) channels with an IC50 (The concentration of CIL for half-maximal inhibition) of 0.62 μM in mouse sinoatrial node cells (Stieber et al, 2006)
We initially tested whether this compound had any perturbations on the amplitude and gating of this current identified in these cells
The effective IC50 required for the CIL-mediated inhibition of include hyperpolarization-activated cation current (Ih) was calculated to be 3.38 μM (Figure 1D), a value that tends to be higher than those for either its suppression of HCN channels identified in mouse sinoatrial cells (i.e., 0.62 μM) [2] and in cardiac Purkinje fibers [17] or the slowing of heart rate (i.e., 0.023 μM) [35–37]
Summary
Cilobradine (CIL, DK-AH269) has been previously demonstrated to suppress hyperpolarization-activated cyclic nucleotide-gated (HCN) channels with an IC50 (The concentration of CIL for half-maximal inhibition) of 0.62 μM in mouse sinoatrial node cells (Stieber et al, 2006). CIL could slow the heart rate by decreasing the spontaneous firing rate of the sinoatrial node in the heart (Bois et al, 2011). In telemetric ECG recordings in mice, this compound has been able to reduce heart rate in a dose-dependent fashion with effective half-maximal dose for the decrease of heart rate (ED50) of 1.2 mg/kg [1,2]. There is evidence to reveal that the presence of CIL possesses a positive inotropic action in the mammalian heart [3]. Previous observations have revealed its effectiveness in modifying hyperpolarization-elicited hyperpolarization-activated cation current (Ih) in pancreatic α- or β-cells, thereby influencing hormonal secretion [4–6]. To what extent this compound is capable of resulting in any perturbations on various other types of ionic currents in different types of endocrine cells, such as pituitary cells, has not been thoroughly elaborated
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