1 Ginsenoside Re, a major ingredient of Panax ginseng, protects the heart against ischemiareperfusion injury by shortening action potential duration (APD) and thereby prohibiting influx of excessive Ca 2 þ . Ginsenoside Re enhances the slowly activating component of the delayed rectifier K þ current (I Ks ) and suppresses the L-type Ca 2 þ 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 I Ks and suppression of I Ca,L by ginsenoside Re in guinea-pig ventricular myocytes. 3 S-Methylisothiourea (SMT, 1 mM), an inhibitor of nitric oxide (NO) synthase (NOS), and N-acetyl-L-cystein (LNAC, 1 mM), an NO scavenger, inhibited I Ks enhancement. Application of an NO donor, sodium nitroprusside (SNP, 1 mM), enhanced I Ks with a magnitude similar to that by a maximum dose (20 mM) of ginseonside Re, and subsequent application of ginsenoside Re failed to enhance I Ks . Conversely, after I Ks had been enhanced by ginsenoside Re (20 mM), subsequently applied SNP failed to further enhance I Ks . 4 An inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 mM), barely suppressed I Ks 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 I Ks enhancement. 5 I Ca,L suppression by ginsenoside Re (3 mM) was abolished by SMT (1 mM) 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 mM), ginsenoside Re (3 mM) failed to suppress I Ca,L . 6 These results indicate that ginsenoside Re-induced I Ks enhancement and I Ca,L suppression involve NO actions. Direct S-nitrosylation of channel protein appears to be the main mechanism for I Ks enhancement, while a cGMP-dependent pathway is responsible for I Ca,L inhibition.

Intracellular Ca2+ augments delayed rectifier K' current (IK) in cardiac myocytes, which may play a major modulatory role in repolarization of action potentials. We investigated subcellular mechanisms for Ca2`-induced enhancement of IK in large-pipette inside-out membrane patches excised from isolated guinea pig ventricular myocytes. When [Ca2+]`was raised from 10`8 to 10`6 mol/L, the amplitude of IK measured at +80 (Circ Res. 1994;74:96-104.) Key Words * delayed rectifier K' current * calmodulin . Ca2+Despite its possible pathophysiological importance, the subcellular mechanism underlying regulation of IK by Ca2' remains unclarified. The major difficulty comes from the lack of methods for obtaining stable singlechannel recordings of IK in excised patches. The regulation of IK by Ca2+ has been studied exclusively in the whole-cell clamp mode using dialyzed cells.2-4 However, in the dialyzed whole-cell clamp configuration, [Ca24]i cannot be controlled precisely, and it is nearly impossible to test numbers of different intracellular solutions because of the limited efficacy of intracellular dialysis. Recently, the major component of IK was reported to be due to the activity of high-density extremely-low-conductance K4 channels, and this current could be recorded in excised membrane patches.6-8 By this method, IK can be recorded under the conditions in which various intracellular solutions can be changed rapidly and easily. In the present study, we used the excised membrane patch to explore the mechanism of subcellular regulation of IK by Ca24. Materials and Methods PreparationEnzymatic dissociation of single ventricular myocytes from guinea pig hearts was done using essentially the same method as previously reported from our laboratory.9 SolutionsThe internal (bath) solution contained (mmol/L) potassium aspartate, 140; MgCl2, 1; CaCl2, 1; EGTA, 11; ATP, 5; and HEPES, 5; the pH was adjusted to 7.3 with KOH. Concentrations of free Ca24 (pCa 9 to 5) and free Mg 24 (0.8 mmol/L) in the internal (bath) solution were estimated on the basis of by guest on May 12, 2018

Endothelin-1 (ET-1), a 21-amino acid vasoconstrictive peptide, increases intracellular Ca2' level and has hypertrophic action on ventricular myocytes. To elucidate a possible role of Ca2+ entry through sarcolemmal Ca2+ channels on this ET-1 action, we examined effects of ET-1 on L-type (ICa,L) and T-type (ICaT) Ca2+ currents in cultured neonatal rat ventricular myocytes using the patch-clamp technique. ET-1 at a concentration of 10 nM increased the maximum current density of ICa,T from -3.0± 1.4 pA/cm2 in the control condition to -4.4+ 1.6 gA/cm2 (p<0.01). Although the peak amplitude of IC8,L was decreased during ET-1 application (from -9.7±+1.9 ,gA/cm2 in the control condition to -5.0±

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(Heart 1999;82:75-81)