The Analysis of Leontopodium leontopodioides (Willd.) Beauv. Chemical Composition by GC/MS and UPLC-Q-Orbitrap MS.

Leontopodium leontopodioides (Willd.) Beauv. (L. leontopodioides.) has been used to treat lung diseases in traditional Chinese medicine (TCM). However, a systematic analysis of its chemical components has not been reported so far. In this study, UPLC-Q-Orbitrap MS and GC-MS were applied to investigate the chemical composition of the water extracts and essential oils of L. leontopodioides. UPLC-Q-Orbitrap MS adopts a heating electrospray ionization source, collecting primary and secondary mass spectrometry data in positive and negative ions, respectively, and uses Compound Discoverer 3.2 software to analyze the collected raw data. As a result, a total of 39 compounds were identified from their high-resolution mass spectra in both positive and negative ionization modes, including 13 flavonoids and their glycosides, 15 phenolic acids, 4 oligosaccharides and glycosides, 4 pentacyclic triterpenoids, and 3 other compounds. Among them, 18 chemical components have not been reported in L. leontopodioides. In the GC-MS section, two common organic solvents (n-hexane and diethyl ether) were used to extract essential oils, and the mass spectra were recorded at 70 eV (electron impact) and scanned in the range of 35∼450 m/z. Compounds were identified using NIST (version 2017), and the peak area normalization method was used to calculate their relative amounts. Finally, 17 components were identified in the volatile oil extracted with n-hexane, accounting for 80.38% of the total volatile oil, including monoterpenoids, phenylpropene, fatty acids, and aliphatic hydrocarbons. In the volatile oil extracted with diethyl ether, 16 components were identified, accounting for 73.50% of the total volatile oil, including phenylpropene, aliphatic hydrocarbons, monoterpenoids, fatty acids, and esters. This study was the first to conduct a comprehensive analysis of the chemical composition of the L. leontopodioides water extract and its essential oil, and a comprehensive chemical composition spectrum was constructed, to lay a foundation for its further pharmacodynamic material basis and quality evaluation.

Preliminary discussion on the mechanism of active components of Radix Aconiti kusnezoffii in the treatment of migraine based on network pharmacology.

To investigate the mechanism of action of Cao Wu in the treatment of migraine from the perspective of network pharmacology. The Swiss Target Prediction Database and CTD database were used to predict the target information of Cao Wu. Human Genome Database gene cards were used to find migraine-related target genes. The target protein interaction network and the "active ingredient-target" network were obtained by combining Cytoscape 3.7.1 software and R language. Enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes pathway and gene function analysis (GO) were performed using the R language to preliminarily explore the multiple pharmacological mechanisms of Radix Aconiti kusnezoffii. Forty-three indicators were identified. A total of 176 migraine targets were identified through the genecards database and OMIM database. Radix Aconiti kusnezoffii targets were compared with migraine targets and 12 overlapping targets were extracted. The protein interaction network of the overlapping targets was analyzed to identify the key targets for the drug to affect the disease. In addition, Kyoto Encyclopedia of Genes and Genomes pathway and go function enrichment analyses were performed on the overlapping targets to explore the therapeutic mechanism of migraine. The treatment of migraine with herbal woo is characterized by multi-component, multi-target, and multi-channel, which exerts complex network regulation through the interaction between different targets, providing a new idea and basis for further exploring the mechanism of action of herbal woo in the treatment of migraine.

The polycystin complex mediates Wnt/Ca(2+) signalling.

WNT ligands induce Ca(2+) signalling on target cells. PKD1 (polycystin 1) is considered an orphan, atypical G-protein-coupled receptor complexed with TRPP2 (polycystin 2 or PKD2), a Ca(2+)-permeable ion channel. Inactivating mutations in their genes cause autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases. Here, we show that WNTs bind to the extracellular domain of PKD1 and induce whole-cell currents and Ca(2+) influx dependent on TRPP2. Pathogenic PKD1 or PKD2 mutations that abrogate complex formation, compromise cell surface expression of PKD1, or reduce TRPP2 channel activity suppress activation by WNTs. Pkd2(-/-) fibroblasts lack WNT-induced Ca(2+) currents and are unable to polarize during directed cell migration. In Xenopus embryos, pkd1, Dishevelled 2 (dvl2) and wnt9a act within the same pathway to preserve normal tubulogenesis. These data define PKD1 as a WNT (co)receptor and implicate defective WNT/Ca(2+) signalling as one of the causes of ADPKD.

[Effect of Bear Bile Powder and Its Succedaneum on Peripheral Blood and Bone Marrow Megakaryocytes in Mouse Model with Thrombocytopenia Induced by Cytosine Arabinoside].

OBJECTIVE: To investigate the effect of bear bile powder and ursodesxy cholic acid (UDCA) on peripheral blood, bone marrow megakaryocyte and immune organs in mouse model with thrombocytopenia, so as to provide a reference for studying the curative effects of bear bile powder and its succedaneum on thrombocytopenic purpura (TP). METHODS: The mouse model with thrombocytopenia indued by cytosine arabinoside (Ara-C) was established, a total of 70 mice were randomly divided into normal group, model group, prednisone group, bear bile (middle and high dose) powder group and UDCA (middle and high dose) group. From the first day of making model mice in the each group, 0.4 ml/(20 g·d) corresponding drug was administered by infusion. At day 10 after treatment the peripheral blood, spleen and thymus organ index, the number of bone marrow megakaryocyte in each group were compared. RESULTS: compared with the normal group, the Plt, WBC and megakaryocyte counts in model group decreased, the spleen index increased obviously (P<0.05), but the WBC count returned to normal by 10 days; after treatment, compared with model group, the Plt, WBC and megakaryocyte counts of treated groups increased, spleen index decreased significantly (P<0.05), but the WBC count in prednisone group decreased, which in bear bile powder (high) group and UDCA (high) group were particularly significant. CONCLUSION: The bear bile powder and UDCA have been confirmed to have therapeutical effect on thrombocytopenia models induced by Ara-C, UDCA can substitute bear bile powder as a treatment drug for thrombocytopenic purpura.

Flavonol glycosides in the petal of Rosa species as chemotaxonomic markers.

Thirteen flavonol glycosides were isolated from the petals of Rosa species belonging to the section Gallicanae, and their structures were identified from their spectroscopic data. These flavonol glycosides, along with two flavonol glycosides isolated from Rosa rugosa, in the petals of 31 Rosa species belonging to sections Gallicanae, Cinnamomeae, Caninae, and Synstylae were quantitatively analyzed by UPLC. The results indicated that the species belonging to these sections could be classified into four types (Type A, B, C and D) based on the pattern of flavonol glycoside contents, whereas the R. rugosa flavonol glycosides were detected only in section Cinnamomeae. A principal components analysis (PCA) calculated from the 15 flavonol glycosides contained in these samples supported the presence of four types. The distribution of the species in Type D (a group of Cinnamomeae) was shown to reflect close interrelationships, but species in Type B (one group of Gallicanae) could be subdivided into two groups, one of which contained species in section Synstylae. Moreover, the flavonol glycosides were grouped by sugar moieties: a disaccharide composed of two hexoses (S1), a hexose (S2), including a hexose with galloyl group, a pentose (S3), and a disaccharide composed of a hexose and a pentose (S4). The ratios of the amounts of S1-S4 to total flavonol glycoside content indicated that differences among the four sections were more distinctive than the amounts of the 15 flavonol glycosides. The 31 samples were divided into Type B, composed of one type of Gallicanae and Synstylae, Type A+C, composed of another type of Gallicanae and Caninae, and Type D, composed of Cinnamomeae. The R. rugosa flavonol glycosides were shown to be important chemotaxonomic markers for the classification of species in Cinnamomeae, and this method of using flavonol glycosides as chemotaxonomic markers could be useful for the identification of Rosa species belonging to sections Gallicanae, Cinnamomeae, Caninae, and Synstylae.

A TRPC1 protein-dependent pathway regulates osteoclast formation and function.

Ca(2+) signaling is essential for bone homeostasis and skeletal development. Here, we show that the transient receptor potential canonical 1 (TRPC1) channel and the inhibitor of MyoD family, I-mfa, function antagonistically in the regulation of osteoclastogenesis. I-mfa null mice have an osteopenic phenotype characterized by increased osteoclast numbers and surface, which are normalized in mice lacking both Trpc1 and I-mfa. In vitro differentiation of pre-osteoclasts derived from I-mfa-deficient mice leads to an increased number of mature osteoclasts and higher bone resorption per osteoclast. These parameters return to normal levels in osteoclasts derived from double mutant mice. Consistently, whole cell currents activated in response to the depletion of intracellular Ca(2+) stores are larger in pre-osteoclasts derived from I-mfa knock-out mice compared with currents in wild type mice and normalized in cells derived from double mutant mice, suggesting a cell-autonomous effect of I-mfa on TRPC1 in these cells. A new splice variant of TRPC1 (TRPC1ε) was identified in early pre-osteoclasts. Heterologous expression of TRPC1ε in HEK293 cells revealed that it is unique among all known TRPC1 isoforms in its ability to amplify the activity of the Ca(2+) release-activated Ca(2+) (CRAC) channel, mediating store-operated currents. TRPC1ε physically interacts with Orai1, the pore-forming subunit of the CRAC channel, and I-mfa is recruited to the TRPC1ε-Orai1 complex through TRPC1ε suppressing CRAC channel activity. We propose that the positive and negative modulation of the CRAC channel by TRPC1ε and I-mfa, respectively, fine-tunes the dynamic range of the CRAC channel regulating osteoclastogenesis.

Identification and functional characterization of an N-terminal oligomerization domain for polycystin-2.

Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited cause of kidney failure, is caused by mutations in either PKD1 (85%) or PKD2 (15%). The PKD2 protein, polycystin-2 (PC2 or TRPP2), is a member of the transient receptor potential (TRP) superfamily and functions as a non-selective calcium channel. PC2 has been found to form oligomers in native tissues suggesting that it may form functional homo- or heterotetramers with other subunits, similar to other TRP channels. Our experiments unexpectedly revealed that PC2 mutant proteins lacking the known C-terminal dimerization domain were still able to form oligomers and co-immunoprecipitate full-length PC2, implying the possible existence of a proximal dimerization domain. Using yeast two-hybrid and biochemical assays, we have mapped an alternative dimerization domain to the N terminus of PC2 (NT2-1-223, L224X). Functional characterization of this domain demonstrated that it was sufficient to induce cyst formation in zebrafish embryos and inhibit PC2 surface currents in mIMCD3 cells probably by a dominant-negative mechanism. In summary, we propose a model for PC2 assembly as a functional tetramer which depends on both C- and N-terminal dimerization domains. These results have significant implications for our understanding of PC2 function and disease pathogenesis in ADPKD and provide a new strategy for studying PC2 function.

Mutant polycystin-2 induces proliferation in primary rat tubular epithelial cells in a STAT-1/p21-independent fashion accompanied instead by alterations in expression of p57KIP2 and Cdk2.

BACKGROUND: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in genes PKD1 and PKD2 account for nearly all cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1 regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-1, polycystin-2 can act as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2. METHODS: Here we utilized different kidney cell-lines expressing wild-type and mutant PKD2 as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation. RESULTS: Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation. On the other hand, expression of mutated PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of the STAT-1/p21 pathway. On the contrary, multiple approaches revealed unequivocally that expression of the cyclin-dependent kinase inhibitor, p57KIP2, is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels. CONCLUSION: Our results indicate the probable involvement of p57KIP2 on epithelial cell proliferation in ADPKD implicating a new mechanism for mutant polycystin-2 induced proliferation. Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.

Activation of TRPP2 through mDia1-dependent voltage gating.

The TRPP2 cation channel is directly responsible for approximately 15% of all cases of autosomal dominant polycystic kidney disease. However, the mechanisms underlying fundamental properties of TRPP2 regulation, such as channel gating and activation, are unknown. We have shown that TRPP2 was activated by EGF and physically interacted with the mammalian diaphanous-related formin 1 (mDia1), a downstream effector of RhoA. Now, we show that mDia1 regulates TRPP2 by specifically blocking its activity at negative but not positive potentials. The voltage-dependent unblock of TRPP2 by mDia1 at positive potentials is mediated through RhoA-induced molecular switching of mDia1 from its autoinhibited state at negative potentials to its activated state at positive potentials. Under physiological resting potentials, EGF activates TRPP2 by releasing the mDia1-dependent block through the activation of RhoA. Our data reveal a new role of mDia1 in the regulation of ion channels and suggest a molecular basis for the voltage-dependent gating of TRP channels.

Acute effects of oestrogen on the guinea pig and human IKr channels and drug-induced prolongation of cardiac repolarization.

Female gender is a risk factor for drug-induced arrhythmias associated with QT prolongation, which results mostly from blockade of the human ether-a-go-go-related gene (hERG) channel. Some clinical evidence suggests that oestrogen is a determinant of the gender-differences in drug-induced QT prolongation and baseline QT(C) intervals. Although the chronic effects of oestrogen have been studied, it remains unclear whether the gender differences are due entirely to transcriptional regulations through oestrogen receptors. We therefore investigated acute effects of the most bioactive oestrogen, 17beta-oestradiol (E2) at its physiological concentrations on cardiac repolarization and drug-sensitivity of the hERG (I(Kr)) channel in Langendorff-perfused guinea pig hearts, patch-clamped guinea pig cardiomyocytes and culture cells over-expressing hERG. We found that physiological concentrations of E2 partially suppressed I(Kr) in a receptor-independent manner. E2-induced modification of voltage-dependence causes partial suppression of hERG currents. Mutagenesis studies showed that a common drug-binding residue at the inner pore cavity was critical for the effects of E2 on the hERG channel. Furthermore, E2 enhanced both hERG suppression and QT(C) prolongation by its blocker, E4031. The lack of effects of testosterone at its physiological concentrations on both of hERG currents and E4031-sensitivity of the hERG channel implicates the critical role of aromatic centroid present in E2 but not in testosterone. Our data indicate that E2 acutely affects the hERG channel gating and the E4031-induced QT(C) prolongation, and may provide a novel mechanism for the higher susceptibility to drug-induced arrhythmia in women.

Formation of a new receptor-operated channel by heteromeric assembly of TRPP2 and TRPC1 subunits.

Although several protein-protein interactions have been reported between transient receptor potential (TRP) channels, they are all known to occur exclusively between members of the same group. The only intergroup interaction described so far is that of TRPP2 and TRPC1; however, the significance of this interaction is unknown. Here, we show that TRPP2 and TRPC1 assemble to form a channel with a unique constellation of new and TRPP2/TRPC1-specific properties. TRPP2/TRPC1 is activated in response to G-protein-coupled receptor activation and shows a pattern of single-channel conductance, amiloride sensitivity and ion permeability distinct from that of TRPP2 or TRPC1 alone. Native TRPP2/TRPC1 activity is shown in kidney cells by complementary gain-of-function and loss-of-function experiments, and its existence under physiological conditions is supported by colocalization at the primary cilium and by co-immunoprecipitation from kidney membranes. Identification of the heteromultimeric TRPP2/TRPC1 channel has implications in mechanosensation and cilium-based Ca(2+) signalling.

Progesterone regulates cardiac repolarization through a nongenomic pathway: an in vitro patch-clamp and computational modeling study.

BACKGROUND: Female sex is an independent risk factor for torsade de pointes in long-QT syndrome. In women, QT interval and torsade de pointes risk fluctuate dynamically during the menstrual cycle and pregnancy. Accumulating clinical evidence suggests a role for progesterone; however, the effect of progesterone on cardiac repolarization remains undetermined. METHODS AND RESULTS: We investigated the effects of progesterone on action potential duration and membrane currents in isolated guinea pig ventricular myocytes. Progesterone rapidly shortened action potential duration, which was attributable mainly to enhancement of the slow delayed rectifier K+ current (I(Ks)) under basal conditions and inhibition of L-type Ca2+ currents (I(Ca,L)) under cAMP-stimulated conditions. The effects of progesterone were mediated by nitric oxide released via nongenomic activation of endothelial nitric oxide synthase; this signal transduction likely takes place in the caveolae because sucrose density gradient fractionation experiments showed colocalization of the progesterone receptor c-Src, phosphoinositide 3-kinase, Akt, and endothelial nitric oxide synthase with KCNQ1, KCNE1, and Ca(V)1.2 in the caveolae fraction. We used computational single-cell and coupled-tissue action potential models incorporating the effects of progesterone on I(Ks) and I(Ca,L); the model reproduces the fluctuations of cardiac repolarization during the menstrual cycle observed in women and predicts the protective effects of progesterone against rhythm disturbances in congenital and drug-induced long-QT syndrome. CONCLUSIONS: Our data show that progesterone modulates cardiac repolarization by nitric oxide produced via a nongenomic pathway. A combination of experimental and computational analyses of progesterone effects provides a framework to understand complex fluctuations of QT interval and torsade de pointes risks in various hormonal states in women.

Ginsenoside Re, a main phytosterol of Panax ginseng, activates cardiac potassium channels via a nongenomic pathway of sex hormones.

Ginseng root is one of the most popular herbs throughout the world and is believed to be a panacea and to promote longevity. It has been used as a medicine to protect against cardiac ischemia, a major cause of death in the West. We have previously demonstrated that ginsenoside Re, a main phytosterol of Panax ginseng, inhibits Ca(2+) accumulation in mitochondria during cardiac ischemia/reperfusion, which is attributable to nitric oxide (NO)-induced Ca(2+) channel inhibition and K(+) channel activation in cardiac myocytes. In this study, we provide compelling evidence that ginsenoside Re activates endothelial NO synthase (eNOS) to release NO, resulting in activation of the slowly activating delayed rectifier K(+) current. The eNOS activation occurs via a nongenomic pathway of each of androgen receptor, estrogen receptor-alpha, and progesterone receptor, in which c-Src, phosphoinositide 3-kinase, Akt, and eNOS are sequentially activated. However, ginsenoside Re does not stimulate proliferation of androgen-responsive LNCaP cells and estrogen-responsive MCF-7 cells, implying that ginsenoside Re does not activate a genomic pathway of sex hormone receptors. Fluorescence resonance energy transfer experiments with a probe, SCCoR (single cell coactivator recruitment), indicate that the lack of genomic action is attributable to failure of coactivator recruitment. Thus, ginsenoside Re acts as a specific agonist for the nongenomic pathway of sex steroid receptors, and NO released from activated eNOS underlies cardiac K(+) channel activation and protection against ischemia-reperfusion injury.

Nontranscriptional regulation of cardiac repolarization currents by testosterone.

BACKGROUND: Women have longer QTc intervals than men and are at greater risk for arrhythmias associated with long QTc intervals, such as drug-induced torsade de pointes. Recent clinical and experimental data suggest an important role of testosterone in sex-related differences in ventricular repolarization. However, studies on effects of testosterone on ionic currents in cardiac myocytes are limited. METHODS AND RESULTS: We examined effects of testosterone on action potential duration (APD) and membrane currents in isolated guinea pig ventricular myocytes using patch-clamp techniques. Testosterone rapidly shortened APD, with an EC50 of 2.1 to 8.7 nmol/L, which is within the limits of physiological testosterone levels in men. APD shortening by testosterone was mainly due to enhancement of slowly activating delayed rectifier K+ currents (IKs) and suppression of L-type Ca2+ currents (I(Ca,L)), because testosterone failed to shorten APD in the presence of an IKs inhibitor, chromanol 293B, and an I(Ca,L) inhibitor, nisoldipine. A nitric oxide (NO) scavenger and an inhibitor of NO synthase 3 (NOS3) reversed the effects of testosterone on APD, which suggests that NO released from NOS3 is responsible for the electrophysiological effects of testosterone. Electrophysiological effects of testosterone were reversed by a blocker of testosterone receptors, a c-Src inhibitor, a phosphatidylinositol 3-kinase inhibitor, and an Akt inhibitor. Immunoblot analysis revealed that testosterone induced phosphorylation of Akt and NOS3. CONCLUSIONS: The nontranscriptional regulation of IKs and I(Ca,L) by testosterone is a novel regulatory mechanism of cardiac repolarization that can potentially contribute to the control of QTc intervals by androgen.

Role of nitric oxide in Ca2+ sensitivity of the slowly activating delayed rectifier K+ current in cardiac myocytes.

Sarcolemmal Ca2+ entry is a vital step for contraction of cardiomyocytes, but Ca2+ overload is harmful and may trigger arrhythmias and/or apoptosis. To maintain the amount of Ca2+ entry within an appropriate range, cardiomyocytes have feedback systems that tightly regulate ion channel activities in response to the changes in intracellular Ca2+ concentration ([Ca2+]i), thereby regulating Ca2+ entry. In guinea pig ventricular myocytes, Ca2+ ionophore, A23187, induced suppression of the L-type Ca2+ currents (I(Ca,L)) and enhancement of the slowly activating delayed rectifier K(+) currents (I(Ks)). At a low stimulation rate, I(Ca,L) suppression and I(Ks) enhancement contributed to the A23187-induced APD shortening with a similar magnitude, whereas at a high stimulation rate, I(Ks) enhancement dominantly contributed to APD shortening. I(Ks) enhancement induced by A23187 was attributable to actions of nitric oxide (NO), because they were inhibited by an inhibitor of NO synthase (NOS) and by a NO scavenger. A23187-induced alterations of APD and I(Ks) were strongly suppressed by a NOS3 inhibitor, but barely affected by a NOS1 inhibitor, suggesting that NOS3 was responsible for NO release in this phenomenon. Inhibition of calmodulin (CaM), but not Akt, blocked the enhancement of I(Ks) by A23187. Thus, CaM-dependent NOS3 activation confers the selective Ca2+-sensitivity on I(Ks). Ca2+-induced I(Ks) enhancement and resultant APD shortening potentially act as a physiological regulatory mechanism of Ca2+ recycling, because they were observed at a physiological range of [Ca2+]i in cardiac myocytes and are induced by physiologically relevant Ca2+ loading, such as digitalis application and rise in extracellular Ca2+ concentration.

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.

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.

Electrophysiological effects of ginseng and ginsenoside Re in guinea pig ventricular myocytes.

Panax ginseng is a folk medicine with various cardiovascular actions; however, its underlying mechanisms of action are not well known. In the present study, we examined the effects of ginseng and its main component, ginsenoside Re, on action potentials and membrane currents recorded from isolated guinea pig ventricular myocytes with the whole-cell patch clamp technique. Ginseng (1 mg/ml) shortened the action potential duration in a rate-dependent manner. Ginseng depressed the L-type Ca2+ current (I(Ca-L)) in a mode of both tonic block and use-dependent block, and enhanced the slowly activating component of the delayed rectifier K+ current (I(Ks)). Ginsenoside Re 3 microM exhibited similar electrophysiological effects to those of 1 mg/ml ginseng, but of slightly smaller magnitude. Inhibition of I(Ca,L) and enhancement of I(Ks) by ginsenoside Re appear to be one of the main electrophysiological actions of ginseng in the heart, although contributions from other ingredients should be considered.

Involvement of local anesthetic binding sites on IVS6 of sodium channels in fast and slow inactivation.

Local anesthetics (LAs) block Na(+) channels with a higher affinity for the fast or slow inactivated state of the channel. Their binding to the channel may stabilize fast inactivation or induce slow inactivation. We examined the role of the LA binding sites on domain IV, S6 (IVS6) of Na(+) channels in fast and slow inactivation by studying the gating properties of the mutants on IVS6 affecting LA binding. Mutation of the putative LA binding site, F1579C, inhibited fast and slow inactivation. Mutations of another putative LA binding site, Y1586C, and IVS6 residue involved in LA access and binding, I1575C, both enhanced fast and slow inactivation. None of the mutations affected channel activation. These results suggest that the LA binding site on IVS6 is involved in slow inactivation as well as fast inactivation, and these two gatings are coupled at the binding site.