Nitric oxide-dependent modulation of the delayed rectifier K+ current and the L-type Ca2+ current by ginsenoside Re, an ingredient of Panax ginseng, in guinea-pig cardiomyocytes

Abstract

1 Ginsenoside Re, a major ingredient of Panax ginseng, protects the heart against ischemia-reperfusion injury by shortening action potential duration (APD) and thereby prohibiting influx of excessive Ca2+. Ginsenoside Re enhances the slowly activating component of the delayed rectifier K+ current (IKs) and suppresses the L-type Ca2+ current (I(Ca,L)), which may account for APD shortening. 2 We used perforated configuration of patch-clamp technique to define the mechanism of enhancement of IKs and suppression of I(Ca,L) by ginsenoside Re in guinea-pig ventricular myocytes. 3 S-Methylisothiourea (SMT, 1 microm), an inhibitor of nitric oxide (NO) synthase (NOS), and N-acetyl-L-cystein (LNAC, 1 mm), an NO scavenger, inhibited IKs enhancement. Application of an NO donor, sodium nitroprusside (SNP, 1 mm), enhanced IKs with a magnitude similar to that by a maximum dose (20 microm) of ginseonside Re, and subsequent application of ginsenoside Re failed to enhance IKs. Conversely, after IKs had been enhanced by ginsenoside Re (20 microm), subsequently applied SNP failed to further enhance IKs. 4 An inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microm), barely suppressed IKs enhancement, while a thiol-alkylating reagent, N-ethylmaleimide (NEM, 0.5 mm), clearly suppressed it. A reducing reagent, di-thiothreitol (DTT, 5 mm), reversed both ginsenoside Re- and SNP-induced IKs enhancement. 5 I(Ca,L) suppression by ginsenoside Re (3 microm) was abolished by SMT (1 microm) or LNAC (1 mm). NEM (0.5 mm) did not suppress I(Ca,L) inhibition and DTT (5 mm) did not reverse I(Ca,L) inhibition, whereas in the presence of ODQ (10 microm), ginsenoside Re (3 microm) failed to suppress I(Ca,L). 6 These results indicate that ginsenoside Re-induced IKs enhancement and I(Ca,L) suppression involve NO actions. Direct S-nitrosylation of channel protein appears to be the main mechanism for IKs enhancement, while a cGMP-dependent pathway is responsible for I(Ca,L) inhibition.

Figure 1

Effects of ginsenoside Re on I_Ks amplitude and the voltage dependence of its activation. (a) Representative superimposed current traces in the drug-free condition or in the presence of ginsenoside Re at a concentration of 0.3, 1, or 3 μM. In this and following figures, current traces of I_Ks are shown only at a V_t of +50 mV. (b) The concentration–response curve. The fractional increases in the amplitude of I_Ks,tail relative to the value in the absence of drug are averaged from five experiments, and are plotted against the concentration of ginsenoside Re. In this and following figures, GS represents ginsenoside Re. Continuous line is the result of Hill's plotting in formula (1) in the text. (c) Activation curves are constructed by plotting I_Ks,tail amplitude normalized to the maximum amplitude (at +50 mV) against the V_t at various concentrations of ginsenoside Re. Continuous curves are result of least-squares fit of Boltzmann distribution in formula (2) in the text. (d) The V_1/2 values at each concentration of ginsenoside Re. ^*P<0.05 vs control.

Figure 2

Reversible enhancement of I_Ks by ginsenoside Re. The left panels in this and the following figures represent averaged amplitude of I_Ks,tail normalized to the value elicited by the first pulse against the time after start of experiments. The timing of the drug applications is shown on the top. Standard error bars are omitted to avoid crowdedness. The right panel depicts representative superimposed current traces recorded at the timing indicated by italic lower-case alphabets in the left panel.

Figure 3

Effects of propranolol, isoproterenol, a PKA inhibitor, H89, and a PKC inhibitor, chelerythrine. (a) Effects of a blocker of β-adrenergic receptor, propranolol (10 μM), on the enhancement of I_Ks by ginsenoside Re. (b) Effects of a stimulator of β-adrenergic receptor, isoproterenol (iso, 30 nM), on the I_Ks that had been enhanced by ginsenoside Re. (c) Effects of a PKA inhibitor, H89 (0.1 μM), on the enhancement of I_Ks by ginsenoside Re. (d) Effects of a PKC inhibitor, chelerythrine (5 μM), on the enhancement of I_Ks by ginsenoside Re.

Figure 4

Effects of SNP. (a) Representative superimposed current traces in the drug-free condition or in the presence of SNP at concentrations of 0.1, 0.3, 1, or 3 mM. (b) The concentration–response curve. The fractional increases in the amplitude of I_Ks,tail relative to the value in the absence of drug are averaged from five experiments, and are plotted against the concentration of SNP. Continuous line is the result of Hill's plotting in the form of (1) in the text. The maximum response (A) was 0.59±0.05, n was 1.6±0.9, and EC₅₀ was 0.18±0.17 mM. (c) Effects of an NO donor, SNP (1 mM), on the enhancement of I_Ks by a maximum dose (20 μM) of ginsenoside Re. (d) Effects of SNP (1 mM) on the I_Ks that had been enhanced by maximum dose (20 μM) of ginsenoside Re.

Figure 5

Effects of SMT and LNAC. (a) Effects of an NOS inhibitor, SMT (1 μM), on the enhancement of I_Ks by ginsenoside Re. (b) Effects of an NO scavenger, LNAC (1 mM), on the enhancement of I_Ks by ginsenoside Re.

Figure 6

Effects of ODQ. (a) Effects of guanylate cyclase inhibitor, ODQ (10 μM), on the enhancement of I_Ks by ginsenoside Re. (b) Effects of guanylate cyclase inhibitor, ODQ (10 μM), on the enhancement of I_Ks by SNP.

Figure 7

Effects of NEM and DTT. (a) Effects of a thiol-alkylating reagent, NEM (0.5 mM), on the enhancement of I_Ks by ginsenoside Re. (b) Effects of a reducing reagent, DTT (5 mM), on the I_Ks that had been enhanced by ginsenoside Re. (c) Effects of DTT (5 mM) on the I_Ks that had been enhanced by 1 mM SNP. (d) Effects of DTT (5 mM) alone on the I_Ks in the control state.

Figure 8

NO-dependent inhibition of I_Ca,L by ginsenoside Re. Left panels in the figure represent the averaged amplitude of peak inward current of I_Ca,L normalized to the value elicited by the first pulse plotted against the time after start of experiments. (a) Effects of ginsenode Re (3 μM). (b) Effects of an NOS inhibitor, SMT (1 μM), on the inhibition of I_Ca,L by ginsenoside Re. (c) Effects of an NO scavenger, LNAC (1 mM), on the inhibition of I_Ca,L by ginsenoside Re. (d) Effects of a NO-dependent guanylate cyclase inhibitor, ODQ (10 μM), on the inhibition of I_Ca,L by ginsenoside Re. (e) Effects of a thiol-alkylating reagent, NEM (0.5 mM), on the inhibition of I_Ca,L by ginsenoside Re. (f) Effects of a reducing reagent, DTT (5 mM), on the I_Ca,L that had been inhibited by ginsenoside Re.