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Effects of neferine on Kv4.3 channels expressed in HEK293 cells and ex vivo electrophysiology of rabbit hearts.

Wang C, Chen YF, Quan XQ, Wang H, Zhang R, Xiao JH, Wang JL, Zhang CT, Xiang JZ, Tang Q - Acta Pharmacol. Sin. (2015)

Bottom Line: Furthermore, neferine (10 μmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation.Neferine-induced blocking of Kv4.3 currents was frequency-dependent.Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD10 at Epi sites of rabbit heart.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

ABSTRACT

Aim: Neferine is an isoquinoline alkaloid isolated from seed embryos of Nelumbo nucifera (Gaertn), which has a variety of biological activities. In this study we examined the effects of neferine on Kv4.3 channels, a major contributor to the transient outward current (I(to)) in rabbit heart, and on ex vivo electrophysiology of rabbit hearts.

Methods: Whole-cell Kv4.3 currents were recorded in HEK293 cells expressing human cardiac Kv4.3 channels using patch-clamp technique. Arterially perfused wedges of rabbit left ventricles (LV) were prepared, and transmembrane action potentials were simultaneously recorded from epicardial (Epi) and endocardial (Endo) sites with floating microelectrodes together with transmural electrocardiography (ECG).

Results: Neferine (0.1-100 μmol/L) dose-dependently and reversibly inhibited Kv4.3 currents (the IC50 value was 8.437 μmol/L, and the maximal inhibition at 100 μmol/L was 44.12%). Neferine (10 μmol/L) caused a positive shift of the steady-state activation curve of Kv4.3 currents, and a negative shift of the steady-state inactivation curve. Furthermore, neferine (10 μmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation. Neferine-induced blocking of Kv4.3 currents was frequency-dependent. In arterially perfused wedges of rabbit LV, neferine (1, 3, and 10 μmol/L) dose-dependently prolonged the QT intervals and action potential durations (APD) at both Epi and Endo sites, and caused dramatic increase of APD10 at Epi sites.

Conclusion: Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD10 at Epi sites of rabbit heart.

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Frequency-dependent block of Kv4.3 channels by neferine. (A) Ten repetitive 200-ms depolarizing pulses of +50 mV from a holding potential of −80 mV were applied at three different frequencies, 0.5, 1 and 2 Hz, in the absence or presence of 10 μmol/L neferine. (B) The peak amplitudes of currents at each pulse were normalized to the peak amplitude of currents obtained at the first pulse (In/I1) and then plotted against the pulse numbers. (C) Neferine (10 μmol/L) induced changes (%) in the current amplitude relative to the control [(INef–ICont)/ICont]. The data are expressed as the mean±SEM. n=5. bP<0.05, cP<0.01 vs control. eP<0.05 vs 0.5 Hz.
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fig5: Frequency-dependent block of Kv4.3 channels by neferine. (A) Ten repetitive 200-ms depolarizing pulses of +50 mV from a holding potential of −80 mV were applied at three different frequencies, 0.5, 1 and 2 Hz, in the absence or presence of 10 μmol/L neferine. (B) The peak amplitudes of currents at each pulse were normalized to the peak amplitude of currents obtained at the first pulse (In/I1) and then plotted against the pulse numbers. (C) Neferine (10 μmol/L) induced changes (%) in the current amplitude relative to the control [(INef–ICont)/ICont]. The data are expressed as the mean±SEM. n=5. bP<0.05, cP<0.01 vs control. eP<0.05 vs 0.5 Hz.

Mentions: The frequency dependence of the effect of neferine was evaluated using 10 consecutive 200-ms depoplarizing pulses to +50 mV from a holding potential of −80 mV at 0.5, 1 and 2 Hz (Figure 5A). The amplitude elicited by the second to tenth episode of the train was normalized to that elicited by the first episode and plotted against the given frequency (Figure 5B). The reduction in the first depolarizing pulse was considered to be the tonic block, and the reduction in subsequent pulses was considered to be frequency-dependent inhibition22. The tonic blocks of 10 μmol/L neferine were 24.79%±1.47% at 0.5 Hz, 24.95%±1.25% at 1 Hz and 24.63%±1.46% at 2 Hz (n=5, P>0.05). Consistent with a previous observation1, significant decreases in the Kv4.3 current were observed at 1 and 2 Hz (compared with that of the first episode). The frequency-dependent inhibition of the Kv4.3 current by 10 μmol/L neferine was observed at a frequency ≥ 1 Hz. The block of Kv4.3 current at the tenth stimulus of the train increased from 26.98%±1.61% at 0.5 Hz to 28.96%± 1.49% at 1 Hz (n=5, P<0.05) and 33.08%±1.37% at 2 Hz (n=5, P<0.01) (Figure 5C).


Effects of neferine on Kv4.3 channels expressed in HEK293 cells and ex vivo electrophysiology of rabbit hearts.

Wang C, Chen YF, Quan XQ, Wang H, Zhang R, Xiao JH, Wang JL, Zhang CT, Xiang JZ, Tang Q - Acta Pharmacol. Sin. (2015)

Frequency-dependent block of Kv4.3 channels by neferine. (A) Ten repetitive 200-ms depolarizing pulses of +50 mV from a holding potential of −80 mV were applied at three different frequencies, 0.5, 1 and 2 Hz, in the absence or presence of 10 μmol/L neferine. (B) The peak amplitudes of currents at each pulse were normalized to the peak amplitude of currents obtained at the first pulse (In/I1) and then plotted against the pulse numbers. (C) Neferine (10 μmol/L) induced changes (%) in the current amplitude relative to the control [(INef–ICont)/ICont]. The data are expressed as the mean±SEM. n=5. bP<0.05, cP<0.01 vs control. eP<0.05 vs 0.5 Hz.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4816235&req=5

fig5: Frequency-dependent block of Kv4.3 channels by neferine. (A) Ten repetitive 200-ms depolarizing pulses of +50 mV from a holding potential of −80 mV were applied at three different frequencies, 0.5, 1 and 2 Hz, in the absence or presence of 10 μmol/L neferine. (B) The peak amplitudes of currents at each pulse were normalized to the peak amplitude of currents obtained at the first pulse (In/I1) and then plotted against the pulse numbers. (C) Neferine (10 μmol/L) induced changes (%) in the current amplitude relative to the control [(INef–ICont)/ICont]. The data are expressed as the mean±SEM. n=5. bP<0.05, cP<0.01 vs control. eP<0.05 vs 0.5 Hz.
Mentions: The frequency dependence of the effect of neferine was evaluated using 10 consecutive 200-ms depoplarizing pulses to +50 mV from a holding potential of −80 mV at 0.5, 1 and 2 Hz (Figure 5A). The amplitude elicited by the second to tenth episode of the train was normalized to that elicited by the first episode and plotted against the given frequency (Figure 5B). The reduction in the first depolarizing pulse was considered to be the tonic block, and the reduction in subsequent pulses was considered to be frequency-dependent inhibition22. The tonic blocks of 10 μmol/L neferine were 24.79%±1.47% at 0.5 Hz, 24.95%±1.25% at 1 Hz and 24.63%±1.46% at 2 Hz (n=5, P>0.05). Consistent with a previous observation1, significant decreases in the Kv4.3 current were observed at 1 and 2 Hz (compared with that of the first episode). The frequency-dependent inhibition of the Kv4.3 current by 10 μmol/L neferine was observed at a frequency ≥ 1 Hz. The block of Kv4.3 current at the tenth stimulus of the train increased from 26.98%±1.61% at 0.5 Hz to 28.96%± 1.49% at 1 Hz (n=5, P<0.05) and 33.08%±1.37% at 2 Hz (n=5, P<0.01) (Figure 5C).

Bottom Line: Furthermore, neferine (10 μmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation.Neferine-induced blocking of Kv4.3 currents was frequency-dependent.Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD10 at Epi sites of rabbit heart.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

ABSTRACT

Aim: Neferine is an isoquinoline alkaloid isolated from seed embryos of Nelumbo nucifera (Gaertn), which has a variety of biological activities. In this study we examined the effects of neferine on Kv4.3 channels, a major contributor to the transient outward current (I(to)) in rabbit heart, and on ex vivo electrophysiology of rabbit hearts.

Methods: Whole-cell Kv4.3 currents were recorded in HEK293 cells expressing human cardiac Kv4.3 channels using patch-clamp technique. Arterially perfused wedges of rabbit left ventricles (LV) were prepared, and transmembrane action potentials were simultaneously recorded from epicardial (Epi) and endocardial (Endo) sites with floating microelectrodes together with transmural electrocardiography (ECG).

Results: Neferine (0.1-100 μmol/L) dose-dependently and reversibly inhibited Kv4.3 currents (the IC50 value was 8.437 μmol/L, and the maximal inhibition at 100 μmol/L was 44.12%). Neferine (10 μmol/L) caused a positive shift of the steady-state activation curve of Kv4.3 currents, and a negative shift of the steady-state inactivation curve. Furthermore, neferine (10 μmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation. Neferine-induced blocking of Kv4.3 currents was frequency-dependent. In arterially perfused wedges of rabbit LV, neferine (1, 3, and 10 μmol/L) dose-dependently prolonged the QT intervals and action potential durations (APD) at both Epi and Endo sites, and caused dramatic increase of APD10 at Epi sites.

Conclusion: Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD10 at Epi sites of rabbit heart.

Show MeSH
Related in: MedlinePlus