18β-Glycyrrhetinic acid preferentially blocks late Na current generated by ΔKPQ Nav1.5 channels

Aim:

To compare the effects of two stereoisomeric forms of glycyrrhetinic acid on different components of Na⁺ current, HERG and Kv1.5 channel currents.

Methods:

Wild-type (WT) and long QT syndrome type 3 (LQT-3) mutant ΔKPQ Nav1.5 channels, as well as HERG and Kv1.5 channels were expressed in Xenopus oocytes. In addition, isolated human atrial myocytes were used. Two-microelectrode voltage-clamp technique was used to record the voltage-activated currents.

Results:

Superfusion of 18β-glycyrrhetinic acid (18β-GA, 1–100 μmol/L) blocked both the peak current (I_Na,P) and late current (I_Na,L) generated by WT and ΔKPQ Nav1.5 channels in a concentration-dependent manner, while 18α-glycyrrhetinic acid (18α-GA) at the same concentrations had no effects. 18β-GA preferentially blocked I_Na,L (IC₅₀=37.2±14.4 μmol/L) to I_Na,P (IC₅₀=100.4±11.2 μmol/L) generated by ΔKPQ Nav1.5 channels. In human atrial myocytes, 18β-GA (30 μmol/L) inhibited 47% of I_Na,P and 87% of I_Na,L induced by Anemonia sulcata toxin (ATX-II, 30 nmol/L). Superfusion of 18β-GA (100 μmol/L) had no effects on HERG and Kv1.5 channel currents.

Conclusion:

18β-GA preferentially blocked the late Na current without affecting HERG and Kv1.5 channels.     

The new antihypertensive drug iptakalim activates ATP-sensitive potassium channels in the endothelium of resistance blood vessels

Aim:

To investigate the mechanisms underlying the activation of ATP-sensitive potassium channels (K_ATP) by iptakalim in cultured rat mesenteric microvascular endothelial cells (MVECs).

Methods:

Whole-cell K_ATP currents were recorded in MVECs using automated patch clamp devices. Nucleotides (ATP, ADP and UDP) were added to the internal perfusion system, whereas other drugs were added to the cell suspension on NPC-1 borosilicate glass chips.

Results:

Application of iptakalim (10 and 100 μmol/L) significantly increased the whole-cell K_ATP currents, which were prevented by the specific K_ATP blocker glibenclamide (1.0 μmol/L). The opening of K_ATP channels by iptakalim depended upon the intracellular concentrations of ATP or NDPs: iptakalim activated K_ATP channels when the intracellular ATP or NDPs were at 100 or 1000 μmol/L, and was ineffective when the non-hydrolysable ATP analogue ATPγS (1000 μmol/L) was infused into the cells. In contrast, the K_ATP opener pinacidil activated K_ATP channels when the intracellular concentrations of ATP or NDPs ranged from 10 to 5000 μmol/L, and even ATPγS (1000 μmol/L) was infused into the cells.

Conclusion:

Iptakalim activates K_ATP channels in the endothelial cells of resistance blood vessels with a low metabolic status, and this activation is dependent on both ATP hydrolysis and ATP ligands.     

Tanshinone II-A sodium sulfonate (DS-201) enhances human BKCa channel activity by selectively targeting the pore-forming α subunit

Aim:

Tanshinone II-A sodium sulfonate (DS-201), a water-soluble derivative of Tanshinone II-A, has been found to induce vascular relaxation and activate BK_Ca channels. The aim of this study was to explore the mechanisms underlying the action of DS-201 on BK_Ca channels.

Methods:

Human BK_Ca channels containing α subunit alone or α plus β1 subunits were expressed in HEK293 cells. BK_Ca currents were recorded from the cells using patch-clamp technique. The expression and trafficking of BK_Ca subunits in HEK293 cells or vascular smooth muscle cells (VSMCs) were detected by Western blotting, flow cytometry and confocal microscopy.

Results:

DS-201 (40–160 μmol/L) concentration-dependently increased the total open probability of BK_Ca channels in HEK293 cells, associated with enhancements of Ca²⁺ and voltage dependence as well as a delay in deactivation. Coexpression of β1 subunit did not affect the action of DS-201: the values of EC₅₀ for BK_Ca channels containing α subunit alone and α plus β1 subunit were 66.6±1.5 and 62.0±1.1 μmol/L, respectively. In both HEK293 cells and VSMCs, DS-201 (80 μmol/L) markedly increased the expression of α subunit without affecting β1 subunit. In HEK293 cells, DS-201 enriched the membranous level of α subunit, likely by accelerating the trafficking and suppressing the internalization of α subunit. In both HEK293 cells and VSMCs, DS-201 (≥320 μmol/L) induced significant cytotoxicity.

Conclusion:

DS-201 selectively targets the pore-forming α subunit of human BK_Ca channels, thus enhancing the channel activities and increasing the subunit expression and trafficking, whereas the β1 subunit does not contribute to the action of DS-201.     

Magnesium lithospermate B dilates mesenteric arteries by activating BKca currents and contracts arteries by inhibiting Kv currents

Aim:

To examine the involvement of K⁺ channels and endothelium in the vascular effects of magnesium lithospermate B (MLB), a hydrophilic active component of Salviae miltiorrhiza Radix.

Methods:

Isolated rat mesenteric artery rings were employed to investigate the effects of MLB on KCl- or norepinephrine-induced contractions. Conventional whole-cell patch-clamp technique was used to study the effects of MLB on K⁺ currents in single isolated mesenteric artery myocytes.

Results:

MLB produced a concentration-dependent relaxation in mesenteric artery rings precontracted by norepinephrine (1 μmol/L) with an EC₅₀ of 111.3 μmol/L. MLB-induced relaxation was reduced in denuded artery rings with an EC₅₀ of 224.4 μmol/L. MLB caused contractions in KCl-precontracted artery rings in the presence of N-nitro-L-arginine methyl ester (L-NAME) with a maximal value of 130.3%. The vasodilatory effect of MLB was inhibited by tetraethylammonium (TEA) in both intact and denuded artery rings. In single smooth muscle cells, MLB activated BK_Ca currents (EC₅₀ 156.3 μmol/L) but inhibited K_V currents (IC₅₀ 26.1 μmol/L) in a voltage- and concentration-dependent manner.

Conclusion:

MLB dilated arteries by activating BK_Ca channels in smooth muscle cells and increasing NO release from endothelium, but it also contracted arteries precontracted with KCl in the presence of L-NAME.     

The protease inhibitor atazanavir blocks hERG K+ channels expressed in HEK293 cells and obstructs hERG protein transport to cell membrane

Aim:

Atazanavir (ATV) is a HIV-1 protease inhibitor for the treatment of AIDS patients, which is recently reported to provoke excessive prolongation of the QT interval and torsades de pointes (TdP). In order to elucidate its arrhythmogenic mechanisms, we investigated the effects of ATV on the hERG K⁺ channels expressed in HEK293 cells.

Methods:

hERG K⁺ currents were detected using whole-cell patch clamp recording in HEK293 cells transfected with EGFP-hERG plasmids. The expression of hERG protein was measured with Western blotting. Two mutants (Y652A and F656C) were constructed in the S6 domain within the inner helices of hERG K⁺ channels that were responsible for binding of various drugs. The trafficking of hERG protein was studied with confocal microscopy.

Results:

Application of ATV (0.01–30 μmol/L) concentration-dependently decreased hERG K⁺ currents with an IC₅₀ of 5.7±1.8 μmol/L. ATV (10 μmol/L) did not affect the activation and steady-state inactivation of hERG K⁺ currents. Compared with the wild type hERG K⁺ channels, both Y652A and F656C mutants significantly reduced the inhibition of ATV on hERG K⁺ currents. Overnight treatment with ATV (0.1–30 μmol/L) concentration-dependently reduced the amount of fully glycosylated 155 kDa hERG protein without significantly affecting the core-glycosylated 135 kDa hERG protein in the cells expressing the WT-hERG protein. Confocal microscopy studies confirmed that overnight treatment with ATV obstructed the trafficking of hERG protein to the cell membrane.

Conclusion:

ATV directly blocks hERG K⁺ channels via binding to the residues Y652 and F656 in the S6 domain, and indirectly obstructs the transport of the hERG protein to the cell membrane.     

Effects of docosahexaenoic acid on large-conductance Ca2+-activated K+ channels and voltage-dependent K+ channels in rat coronary artery smooth muscle cells

Aim:

To investigate the effects of docosahexaenoic acid (DHA) on large-conductance Ca²⁺-activated K⁺(BK_Ca) channels and voltage-dependent K⁺ (K_V) channels in rat coronary artery smooth muscle cells (CASMCs).

Methods:

Rat CASMCs were isolated by an enzyme digestion method. BK_Ca and K_V currents in individual CASMCs were recorded by the patch-clamp technique in a whole-cell configuration at room temperature. Effects of DHA on BK_Ca and K_V channels were observed when it was applied at 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L.

Results:

When DHA concentrations were greater than 10 μmol/L, BK_Ca currents increased in a dose-dependent manner. At a testing potential of +80 mV, 6.1%±0.3%, 76.5%±3.8%, 120.6%±5.5%, 248.0%±12.3%, 348.7%±17.3%, 374.2%±18.7%, 432.2%±21.6%, and 443.1%±22.1% of BK_Ca currents were increased at the above concentrations, respectively. The half-effective concentration (EC₅₀) of DHA on BK_Ca currents was 37.53±1.65 μmol/L. When DHA concentrations were greater than 20 μmol/L, K_V currents were gradually blocked by increasing concentrations of DHA. At a testing potential of +50 mV, 0.40%±0.02%, 1.37%±0.06%, 11.80%±0.59%, 26.50%±1.75%, 56.50%±2.89%, 73.30%±3.66%, 79.70%±3.94%, and 78.1%±3.91% of K_V currents were blocked at the different concentrations listed above, respectively. The EC₅₀ of DHA on K_V currents was 44.20±0.63 μmol/L.

Conclusion:

DHA can activate BK_Ca channels and block K_V channels in rat CASMCs, and the EC₅₀ of DHA for BK_Ca channels is lower than that for K_V channels; these findings indicate that the vasorelaxation effects of DHA on vascular smooth muscle cells are mainly due to its activation of BK_Ca channels.     

Discovery of a retigabine derivative that inhibits KCNQ2 potassium channels

Aim:

Retigabine, an activator of KCNQ2-5 channels, is currently used to treat partial-onset seizures. The aim of this study was to explore the possibility that structure modification of retigabine could lead to novel inhibitors of KCNQ2 channels, which were valuable tools for KCNQ channel studies.

Methods:

A series of retigabine derivatives was designed and synthesized. KCNQ2 channels were expressed in CHO cells. KCNQ2 currents were recorded using whole-cell voltage clamp technique. Test compound in extracellular solution was delivered to the recorded cell using an ALA 8 Channel Solution Exchange System.

Results:

A total of 23 retigabine derivatives (HN31-HN410) were synthesized and tested electrophysiologically. Among the compounds, HN38 was the most potent inhibitor of KCNQ2 channels (its IC₅₀ value=0.10±0.05 μmol/L), and was 7-fold more potent than the classical KCNQ inhibitor XE991. Further analysis revealed that HN38 (3 μmol/L) had no detectable effect on channel activation, but accelerated deactivation at hyperpolarizing voltages. In contrast, XE991 (3 μmol/L) did not affect the kinetics of channel activation and deactivation.

Conclusion:

The retigabine derivative HN38 is a potent KCNQ2 inhibitor, which differs from XE991 in its influence on the channel kinetics. Our study provides a new strategy for the design and development of potent KCNQ2 channel inhibitors.     

Comparison of the effects of antiarrhythmic drugs flecainide and verapamil on fKv1.4ΔN channel currents in Xenopus oocytes

Aim:

To study the effects of Na⁺ channel blocker flecainide and L-type Ca²⁺ channel antagonist verapamil on the voltage-gated fKv1.4ΔN channel, an N-terminal-deleted mutant of the ferret Kv1.4 K⁺ channel.

Methods:

fKv1.4ΔN channels were stably expressed in Xenopus oocytes. The K⁺ currents were recorded using a two-electrode voltage-clamp technique. The drugs were administered through superfusion.

Results:

fKv1.4ΔN currents displayed slow inactivation, with a half-inactivation potential of −41.74 mV and a slow recovery from inactivation (τ=1.90 s at −90 mV). Flecainide and verapamil blocked the currents with IC₅₀ values of 512.29±56.92 and 260.71±18.50 μmol/L, respectively. The blocking action of the drugs showed opposite voltage-dependence: it was enhanced with depolarization for flecainide, and was attenuated with depolarization for verapamil. Both the drugs exerted state-dependent blockade on fKv1.4ΔN currents, but verapamil showed a stronger use-dependent blockage compared with flecainide. Flecainide accelerated the C-type inactivation rate without affecting the recovery kinetics and the steady-state activation. Verapamil also accelerated the inactivation kinetics of the currents, but unlike flecainide, it affected both the recovery and the steady-state activation, causing slower recovery of fKv1.4ΔN channel and a depolarizing shift of the steady-state activation curve.

Conclusion:

The results demonstrate that widely used antiarrhythmic drugs flecainide and verapamil substantially inhibit fKv1.4ΔN channels expressed in Xenopus oocytes by binding to the open state of the channels. Therefore, caution should be taken when these drugs are administered in combination with K⁺ channel blockers to treat arrhythmia.     

Anti-hypoxic effect of ginsenoside Rbl on neonatal rat cardiomyocytes is mediated through the specific activation of glucose transporter-4 ex vivo

Aim:

The aim of this study was to investigate whether Gs-Rbl relieves the CoCl₂-induced apoptosis of hypoxic neonatal rat cardiomyocytes and in which the role of glucose transporter-4 (GLUT-4).

Methods:

Gs-Rbl (0, 10, 50, 100, 200, 400, and 500 μmol/L), adenine 9-β-D-arabinofuranoside (ara A, 500 μmol/L; AMPK inhibitor) and wortmannin (0.5 μmol/L; PI3K inhibitor) only in combination with 200 μmol/L Gs-Rbl were administered in hypoxic cardiomyocytes, which were induced by 500 μmol/L CoCl₂ for 12 h. Then, the apoptotic rate (AR), 2-[³H]-deoxy-D-glucose (2-[³H]-DG) uptake, and the expression of GLUT-4 (including in plasma membrane, PM), phospho-AMPKα (Thr172), AMPKα and Akt in cells were assayed.

Results:

Compared with simple hypoxia (0 μmol/L Gs-Rbl), Gs-Rb1 greater than 10 μmol/L significantly decreased the apoptotic rate (P<0.01) and significantly increased 2-[³H]-DG uptake (P<0.01), GLUT-4 content in cells and PM (P<0.01), AMPK activity (P<0.01) and Akt (P<0.01) levels in a dose-dependent manner. AMPK activity was completely suppressed by ara-A, just as Akt was suppressed by wortmannin. The AR, glucose uptake and GLUT-4 levels in cells and PM were partly down-regulated by ara-A or wortmannin.

Conclusion:

Gs-Rb1 may protect neonatal rat cardiomyocytes from apoptosis induced by CoCl₂. The anti-apoptotic effect of Gs-Rb1 may occur by improving glucose uptake, in which GLUT-4 translocation and expression played a key role. Both the AMPK and the PI3K/Akt pathways may take part in the anti-hypoxic efficacy of Gs-Rb1.     

Effects of diltiazem and propafenone on the inactivation and recovery kinetics of fKv1.4 channel currents expressed in Xenopus oocytes

Aim:

To investigate the effects of diltiazem, an L-type calcium channel blocker, and propafenone, a sodium channel blocker, on the inactivation and recovery kinetics of fKv1.4, a potassium channel that generates the cardiac transient outward potassium current.

Methods:

The cRNA for fKv1.4ΔN, an N-terminal deleted mutant of the ferret Kv1.4 potassium channel, was injected into Xenopus oocytes to express the fKv1.4ΔN channel in these cells. Currents were recorded using a two electrode voltage clamp technique.

Results:

Diltiazem (10 to 1000 μmol/L) inhibited the fKv1.4ΔN channel in a frequency-dependent, voltage-dependent, and concentration-dependent manner, suggesting an open channel block. The IC₅₀ was 241.04±23.06 μmol/L for the fKv1.4ΔN channel (at +50 mV), and propafenone (10 to 500 μmol/L) showed a similar effect (IC₅₀=103.68±10.13 μmol/L). After application of diltiazem and propafenone, fKv1.4ΔN inactivation was bi-exponential, with a faster drug-induced inactivation and a slower C-type inactivation. Diltiazem increased the C-type inactivation rate and slowed recovery in fKv1.4ΔN channels. However, propafenone had no effect on either the slow inactivation time constant or the recovery.

Conclusion:

Diltiazem and propafenone accelerate the inactivation of the Kv1.4ΔN channel by binding to the open state of the channel. Unlike propafenone, diltiazem slows the recovery of the Kv1.4ΔN channel.     

Potent vasorelaxant activity of the TMEM16A inhibitor T16Ainh-A01

Background and Purpose

T16A_inh-A01 is a recently identified inhibitor of the calcium-activated chloride channel TMEM16A. The aim of this study was to test the efficacy of T16A_inh-A01 for inhibition of calcium-activated chloride channels in vascular smooth muscle and consequent effects on vascular tone.

Experimental Approach

Single channel and whole cell patch clamp was performed on single smooth muscle cells from rabbit pulmonary artery and mouse thoracic aorta. Isometric tension studies were performed on mouse thoracic aorta and mesenteric artery as well as human abdominal visceral adipose artery.

Key Results

In rabbit pulmonary artery myocytes T16A_inh-A01 (1–30 μM) inhibited single calcium (Ca²⁺)-activated chloride (Cl⁻) channels and whole cell currents activated by 500 nM free Ca²⁺. Similar effects were observed for single Ca²⁺-activated Cl⁻ channels in mouse thoracic aorta, and in both cell types, channel activity was abolished by two antisera raised against TMEM16A but not by a bestrophin antibody. The TMEM16A potentiator, F_act (10 μM), increased single channel and whole cell Ca²⁺-activated Cl⁻ currents in rabbit pulmonary arteries. In isometric tension studies, T16A_inh-A01 relaxed mouse thoracic aorta pre-contracted with methoxamine with an IC₅₀ of 1.6 μM and suppressed the methoxamine concentration–effect curve. T16A_inh-A01 did not affect the maximal contraction produced by 60 mM KCl and the relaxant effect of 10 μM T16A_inh-A01 was not altered by incubation of mouse thoracic aorta in a cocktail of potassium (K⁺) channel blockers. T16A_inh-A01 (10 μM) also relaxed human visceral adipose arteries by 88 ± 3%.

Conclusions and Implications

T16A_inh-A01 blocks calcium-activated chloride channels in vascular smooth muscle cells and relaxes murine and human blood vessels.     

Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit in vitro

Aim:

To examine the effects of anisomycin on glioma cells and the related mechanisms in vitro.

Methods:

The U251 and U87 human glioblastoma cell lines were tested. The growth of the cells was analyzed using a CCK-8 cell viability assay. Apoptosis was detected using a flow cytometry assay. The expression of proteins and phosphorylated kinases was detected using Western blotting.

Results:

Treatment of U251 and U87 cells with anisomycin (0.01–8 μmol/L) inhibited the cell growth in time- and concentration-dependent manners (the IC₅₀ values at 48 h were 0.233±0.021 and 0.192±0.018 μmol/L, respectively). Anisomycin (4 μmol/L) caused 21.5%±2.2% and 25.3%±3.1% of apoptosis proportion, respectively, in U251 and U87 cells. In the two cell lines, anisomycin (4 μmol/L) activated p38 MAPK and JNK, and inactivated ERK1/2. However, neither the p38 MAPK inhibitor SB203580 (10 μmol/L) nor the JNK inhibitor SP600125 (10 μmol/L) prevented anisomycin-induced cell death. On the other hand, anisomycin (4 μmol/L) reduced the level of PP2A/C subunit (catalytic subunit) in a time-dependent manner in the two cell lines. Treatment of the two cell lines with the PP2A inhibitor okadaic acid (100 nmol/L) caused marked cell death.

Conclusion:

Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit. The regulation of PP2A/C exression by anisomycin provides a clue to further study on its role in glioma therapy.     

Non-genomic vasorelaxant effects of 17β-estradiol and progesterone in rat aorta are mediated by L-type Ca2+ current inhibition

Aim:

The sex hormones 17β-estradiol (βES) and progesterone (PRG) induce rapid non-genomic vasodilator effects which could be protective for the cardiovascular system. The purpose of this study was to analyze the mechanisms underlying their vasodilator effect in rat aortic smooth muscle preparations.

Methods:

Endothelium-denuded aorta artery rings were prepared from male Wistar rats and incubated in an organ bath. The contractions of the preparation were recorded through isometric transducers. The effects of the hormones on K⁺ current and L-type Ca²⁺ current (LTCC) were analyzed by using the whole cell voltage-clamp technique in A7r5 cells.

Results:

Both βES and PRG (1–100 μmol/L) concentration-dependently relaxed the endothelium-denuded aortic rings contracted by (–)-Bay K8644 (0.1 μmol/L) or by KCl (60 mmol/L). The IC₅₀ values of the two hormones were not statistically different. The K_V channel blocker 4-aminopyridine (2 mmol/L), BK_Ca channel blocker tetraethylammonium (1mmol/L) and K_ATP channel blocker glibenclamide (10 μmol/L) did not significantly modify the relaxant effect of the hormones. On the other hand, the blockage of the intracellular βES and PRG receptors with estradiol receptor antagonists ICI 182,780 (1 μmol/L) and PRG receptor antagonist mifepristone (30 μmol/L), respectively, did not significantly modify the relaxant action of the hormones. In A7r5 cells, both the hormones (1–100 μmol/L) rapidly and reversibly inhibited the basal and BAY-stimulated LTCC. However, these hormones had no effect on the basal K⁺ current.

Conclusion:

The vasorelaxant effects of βES and PRG are due to the inhibition of LTCC. The K⁺ channels are not involved in the effects.     

Metergoline inhibits the neuronal Navl.2 voltage- dependent Na^＋ channels expressed in Xenopus oocytes

Aim： Metergoline is an ergot-derived psychoactive drug that acts as a ligand for serotonin and dopamine receptors. The aim of this study was to investigate the regulatory effects of metergoline on the neuronal Nav1.2 voltage-dependent Na^＋ channels in vitro. Methods： Xenopus oocytes were injected with cRNAs encoding rat brain Nav1.2 α and β1 subunits. Voltage-activated Na^＋ currents were recorded using two-electrode voltage clamp technique. Drugs were applied though perfusion. Results： Both metergoline and lidocaine reversibly and concentration-dependently inhibited the peak of Na^＋ currents with IC50 values of 3.6±4.2 and 916.9±98.8 μmol/L, respectively. Metergoline （3 pmol/L） caused a 6.8±1.2 mV depolarizing shift of the steady-state activation curve of the Na^＋ currents, and did not alter the inactivation curve. In contrast, lidocaine （3 μmol/L） caused a 12.7±1.2 mV hyperpolarizing shift of the inactivation curve of the Na^＋ currents without changing the steady-state activation curve. Both metergoline and lidocaine produced tonic and use-dependent inhibition on the peak of Na^＋ currents. Conclusion： Metergoline exerts potent inhibition on the activity of neuronal Nav1.2 channels, which may contribute to its actions on the central nervous system.     

Novel microtubule-targeted agent 6-chloro-4-(methoxyphenyl) coumarin induces G2-M arrest and apoptosis in HeLa cells

Aim:

To identify a novel coumarin analogue with the highest anticancer activity and to further investigate its anticancer mechanisms.

Methods:

The viability of cancer cells was investigated using the MTT assay. The cell cycle progression was evaluated using both flow cytometric and Western blotting analysis. Microtubule depolymerization was observed with immunocytochemistry in vivo and a tubulin depolymerization assay in vitro. Apoptosis was demonstrated using Annexin V/Propidium Iodide (PI) double-staining and sub-G₁ analysis.

Results:

Among 36 analogues of coumarin, 6-chloro-4-(methoxyphenyl) coumarin showed the best anticancer activity (IC₅₀ value about 200 nmol/L) in HCT116 cells. The compound had a broad spectrum of anticancer activity against 9 cancer cell lines derived from colon cancer, breast cancer, liver cancer, cervical cancer, leukemia, epidermoid cancer with IC₅₀ value of 75 nmol/L–1.57 μmol/L but with low cytotocitity against WI-38 human lung fibroblasts (IC₅₀ value of 12.128 μmol/L). The compound (0.04–10 μmol/L) induced G₂-M phase arrest in HeLa cells in a dose-dependent manner, which was reversible after the compound was removed. The compound (10–300 μmol/L) induced the depolymerization of purified porcine tubulin in vitro. Finally, the compound (0.04–2.5 μmol/L) induced apoptosis of HeLa cells in dose- and time-dependent manners.

Conclusion:

6-Chloro-4-(methoxyphenyl) coumarin is a novel microtubule-targeting agent that induces G₂–M arrest and apoptosis in HeLa cells.     

Bigelovin inhibits STAT3 signaling by inactivating JAK2 and induces apoptosis in human cancer cells

Aim:

To study the function and mechanism of bigelovin, a sesquiterpene lactone from the flower of Chinese herb Inula hupehensis, in regulating JAK2/STAT3 signaling and cancer cell growth.

Methods:

HepG2 cells stably transfected with the STAT3-responsive firefly luciferase reporter plasmid (HepG2/STAT3 cells), and a panel of human cancer cell lines were used to identify active compounds. Cell viability was measured using MTT assay. Western blotting was used to detect protein expression and phosphorylation. Kinase assays were performed and the reaction between bigelovin and thiol-containing compounds was analyzed with LC-MS.

Results:

Bigelovin (1–50 μmol/L) dose-dependently inhibited the IL-6-induced STAT3 activation in HepG2/STAT3 cells (IC₅₀=3.37 μmol/L) and the constitutive STAT3 activation in A549 and MDA-MB-468 cells. Furthermore, bigelovin dose-dependently inhibited JAK2 phosphorylation in HeLa and MDA-MB-468 cells, as well as the enzymatic activity of JAK2 in vitro (IC₅₀=44.24 μmol/L). Pretreatment of the cells with DTT (500 μmol/L) or GSH (500 μmol/L) eliminated the inhibitory effects of bigelovin on the IL-6-induced and the constitutive STAT3 activation. The results in LC-MS analysis suggested that bigelovin might react with cysteine residues of JAK2 leading to inactivation of JAK2. Bigelovin (5 and 20 μmol/L) had no effects on the signaling pathways of growth factors EGF, PDGF or insulin. Finally, bigelovin suppressed the cell viability and induced apoptosis in 10 different human cancer cell lines, particularly those with constitutively activated STAT3.

Conclusion:

Bigelovin potently inhibits STAT3 signaling by inactivating JAK2, and induces apoptosis of a variety of human cancer cells in vitro.     

Atorvastatin attenuates homocysteine-induced apoptosis in human umbilical vein endothelial cells via inhibiting NADPH oxidase-related oxidative stress-triggered p38MAPK signaling

Aim:

To examine the effect of atorvastatin on homocysteine (Hcy)-induced reactive oxygen species (ROS) production and apoptosis in human umbilical vein endothelial cells (HUVECs).

Methods:

HUVECs were cultured with Hcy (0.1−5 mmol/L) in the presence or absence of atorvastatin (1−100 μmol//L) or various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI, 10 μmol/L), the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 (10 μmol/L) and antioxidants N-acetyl cysteine (NAC, 1 mmol/L). Cell apoptosis was evaluated by Annexin V/propidium iodide staining and flow cytometry. ROS were detected by 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFH-DA). NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence. Hcy-induced expression of p38MAPK protein was measured by Western blotting analysis.

Results:

Atorvastatin inhibited endothelial cell apoptosis induced by 1 mmol/L Hcy in a dose-dependent manner and the maximal inhibitory effect was reached at 100 μmol/L. Atorvastatin (10 μmol/L) significantly suppressed Hcy (1 mmol/L for 30 min) induced ROS accumulation (3.17±0.33 vs 4.34±0.31, P<0.05). Atorvastatin (10 μmol/L) also antagonized Hcy (1 mmol/L for 30 min) induced activation of NADPH oxidase (2.57±0.49 vs 3.33±0.6, P<0.05). Furthermore, atorvastatin inhibited Hcy-induced phosphorylation of p38 MAPK (1.7±0.1 vs 2.22±0.25, P<0.05), similar effects occurred with DPI, NAC and SB203580.

Conclusion:

Atorvastatin may inhibit Hcy-induced ROS accumulation and endothelium cell apoptosis through an NADPH oxidase and/or p38MAPK-dependent mechanisms, all of which may contribute to atorvastatin-induced beneficial effect on endothelial function.     

Modulation of K2P2.1 and K2P10.1 K+ channel sensitivity to carvedilol by alternative mRNA translation initiation

Background and Purpose

The β-receptor antagonist carvedilol blocks a range of ion channels. K_2P2.1 (TREK1) and K_2P10.1 (TREK2) channels are expressed in the heart and regulated by alternative translation initiation (ATI) of their mRNA, producing functionally distinct channel variants. The first objective was to investigate acute effects of carvedilol on human K_2P2.1 and K_2P10.1 channels. Second, we sought to study ATI-dependent modulation of K_2P K⁺ current sensitivity to carvedilol.

Experimental Approach

Using standard electrophysiological techniques, we recorded currents from wild-type and mutant K_2P2.1 and K_2P10.1 channels in Xenopus oocytes and HEK 293 cells.

Key Results

Carvedilol concentration-dependently inhibited K_2P2.1 channels (IC_50,oocytes = 20.3 μM; IC_50,HEK = 1.6 μM) and this inhibition was frequency-independent. When K_2P2.1 isoforms generated by ATI were studied separately in oocytes, the IC₅₀ value for carvedilol inhibition of full-length channels (16.5 μM) was almost 5-fold less than that for the truncated channel variant (IC₅₀ = 79.0 μM). Similarly, the related K_2P10.1 channels were blocked by carvedilol (IC_50,oocytes = 24.0 μM; IC_50,HEK = 7.6 μM) and subject to ATI-dependent modulation of drug sensitivity.

Conclusions and Implications

Carvedilol targets K_2P2.1 and K_2P10.1 K⁺ channels. This previously unrecognized mechanism supports a general role of cardiac K_2P channels as antiarrhythmic drug targets. Furthermore, the work reveals that the sensitivity of the cardiac ion channels K_2P2.1 and K_2P10.1 to block was modulated by alternative mRNA translation initiation.     

Inhibitory effects of tetrandrine on the Na+ channel of human atrial fibrillation myocardium

Aim:

Tetrandrine (Tet) is a Ca²⁺ channel blocker and has antiarrhythmic effects. Less information exists with regard to the mechanisms underlying its antiarrhythmic action other than blocking Ca²⁺ channels. In this study, the effects of Tet on the Na⁺ current (I_Na) in the atrial myocardium of patients in atrial fibrillation (AF) and sinus rhythm (SR) were investigated, and the characteristics of the Na⁺ current were synchronously compared between the AF and SR patients.

Methods:

Na⁺ currents were recorded using the whole-cell patch clamp technique in single atrial myocyte of the AF and the normal SR groups. The effects of Tet (40–120 μmol/L) on the Na⁺ current in the two groups were then observed.

Results:

Tet (60–120 μmol/L) decreased I_Na density in a concentration-dependent manner and made the voltage-dependent activation curve shift to more positive voltages in the SR and AF groups. After exposure to Tet, the voltage-dependent inactivation curve of I_Na was shifted to more negative voltages in the two groups. Tet delayed the time-dependent recovery of I_Na in a concentration dependent manner in both AF and SR cells; however, there were no differences in the effects of Tet on I_Na density and properties in the two groups. The I_Na density of AF patients did not differ from that of the SR patients.

Conclusion:

Tet can block sodium channels with slow recovery kinetics, which may explain the mechanisms underlying the antiarrhythmic action of Tet. The decreased conduction velocity (CV) in AF patients is not caused by the Na⁺ current.     

Blocking effect of methylflavonolamine on human NaV1.5 channels expressed in Xenopus laevis oocytes and on sodium currents in rabbit ventricular myocytes

Aim:

To investigate the blocking effects of methylflavonolamine (MFA) on human Na_V1.5 channels expressed in Xenopus laevis oocytes and on sodium currents (I_Na) in rabbit ventricular myocytes.

Methods:

Human Na_V1.5 channels were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. I_Na and action potentials in rabbit ventricular myocytes were studied using the whole-cell recording.

Results:

MFA and lidocaine inhibited human Na_V1.5 channels expressed in Xenopus oocytes in a positive rate-dependent and concentration-dependent manner, with IC₅₀ values of 72.61 μmol/L and 145.62 μmol/L, respectively. Both of them markedly shifted the steady-state activation curve of I_Na toward more positive potentials, shifted the steady-state inactivation curve of I_Na toward more negative potentials and postponed the recovery of the I_Na inactivation state. In rabbit ventricular myocytes, MFA inhibited I_Na with a shift in the steady-state inactivation curve toward more negative potentials, thereby postponing the recovery of the I_Na inactivation state. This shift was in a positive rate-dependent manner. Under current-clamp mode, MAF significantly decreased action potential amplitude (APA) and maximal depolarization velocity (V_max) and shortened action potential duration (APD), but did not alter the resting membrane potential (RMP). The demonstrated that the kinetics of sodium channel blockage by MFA resemble those of class I antiarrhythmic agents such as lidocaine.

Conclusion:

MFA protects the heart against arrhythmias by its blocking effect on sodium channels.