The inhibitory effects of levo-tetrahydropalmatine on rat Kv1.5 channels expressed in HEK293 cells.

Levo-tetrahydropalmatine (l-THP) exerts various pharmacological effects on neural and cardiac tissues and K+ channel can be one of its multiple targets. The rapidly activating Kv1.5 channel is expressed in a variety of tissues including atrial cells and hippocampal neurons, and has an essential role in tuning the action potential and excitability in those cells. The aim of current study is to explore whether there are the possible effects of l-THP on Kv1.5 channels expressed in HEK293 cells. Superfusion of l-THP led to a dose-dependent blockage of Kv1.5 currents with an IC50 value of 53.2µM. This blocking effect was substantially attenuated in mutant H452G rather than R476V and R476Y, suggesting a specific binding site in the outer mouth region. In addition, the properties of Kv1.5 channel kinetics were markedly altered by l-THP. Treatment with l-THP resulted in a potential left shift of the inactivation curve, with the half-maximum inactivation potential (V1/2) of 4.5mV in control and -12.8mV in 50µM l-THP. Our data reveal that l-THP can exert an inhibitory effect on the delayed rectifier Kv1.5 channels expressed in HEK293 cells. These lines of evidence provided an insight to understand the possible effects exerted by l-THP on relative tissues.

[Effect of Ginsenoside Rg2 and Its Stereoisomers on Oxygen-Glucose Deprivation and Reperfusion Induced Cortical Neuronal Injury Model].

OBJECTIVE: To observe the effect of natural type ginsenoside Rg2 (Rg2) and its stereoisomers [20 (R)-Rg2 and 20 (S)-Rg2] at different concentrations on oxygen-glucose deprivation/ reperfusion (OGD/R) induced cortical neuronal injury model in vitro, and to explore the mechanism, and compare their differences of action. METHODS: Cortical neurons after 7-day culture were randomly divided into 5 groups, i.e., the control group, the model group, the Rg2 group, 20 (R) -Rg2 group, and 20 (S) - Rg2 group. Cortical neurons in the Rg2 group, 20 (R)-Rg2 group, and 20(S)-Rg2 group were pretreated with 20, 40, and 80 µmol/L Rg2, 20 (R) -Rg2, and 20 (S) -Rg2 for 24 h to prepare OGD/R model. The cell survival rate, the activity of Caspase-3, the intracellular Ca2+ concentration, contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected 24 h later. RESULTS: Compared with the control group, cell survival rates and activities of SOD obviously decreased, the activity of Caspase-3, Ca2+ fluorescent optical gray value, and contents of MDA significantly increased with statistical difference (P < 0.05). Compared with the model group, cell survival rates and activities of SOD obviously increased, the activity of Caspase-3, Ca2+ fluorescent optical gray value, and contents of MDA significantly decreased in 20 µmol/L Rg2 group, 40 µmol/L 20 (R) -Rg2 group, and 80 µmol/L 20 (S) -Rg2 group (P < 0.05). Compared with 20(S)-Rg2 group, cell survival rates increased and contents of MDA significantly decreased in 20, 40, and 80 µmol/L Rg2 and 20 (R)-Rg2 groups (P < 0.05). The activity of Caspase-3 decreased and contents of SOD increased in 80 µmol/L 20 (R)-Rg2 group, and 40, 80 µmol/L Rg2 groups (P < 0.05). Ca2+ fluorescent optical gray value decreased in 40, 80 µmol/L Rg2 and 20 (R)-Rg2 groups (P < 0.05). Compared with 20 (R)-Rg2 group, Ca2+ fluorescent optical gray value decreased in 80 µmol/L Rg2 group (P < 0.05); contents of SOD increased in 40 and 80 µmol/L Rg2 groups (P < 0.05); contents of MDA decreased in 20, 40, and 80 µmol/L Rg2 groups (P < 0.05). CONCLUSIONS: Rg2 and its stereoisomers could improve cell vitality of cortical neurons against OGD/R induced injury. This might be related to improving anti-apoptotic capacities and antioxidant abilities, and reducing Ca2+ inflow. Besides, the neuroprotective effect of 20 (R) -Rg2 was better than that of 20 (S) -Rg2, but inferior to that of Rg2.

Voltage­gated K+ channel blocker quinidine inhibits proliferation and induces apoptosis by regulating expression of microRNAs in human glioma U87­MG cells.

Accumulating evidence has proved that potassium channels (K+ channels) are involved in regulating cell proliferation, cell cycle progression and apoptosis of tumor cells. However, the precise cellular mechanisms are still unknown. In the present study, we investigated the effect and mechanisms of quinidine, a commonly used voltage-gated K+ channel blocker, on cell proliferation and apoptosis of human glioma U87-MG cells. We found that quinidine significantly inhibited the proliferation of U87-MG cells and induced apoptosis in a dose-dependent manner. The results of caspase colorimetric assay showed that the mitochondrial pathway was the main mode involved in the quinidine-induced apoptotic process. Furthermore, the concentration range of quinidine, which inhibited voltage-gated K+ channel currents in electrophysiological assay, was consistent with that of quinidine inhibiting cell proliferation and inducing cell apoptosis. In U87-MG cells treated with quinidine (100 µmol/l), 11 of 2,042 human microRNAs (miRNAs) were upregulated and 16 were downregulated as detected with the miRNA array analysis. The upregulation of miR-149-3p and downregulation of miR-424-5p by quinidine treatment were further verified by using quantitative real-time PCR. In addition, using miRNA target prediction program, putative target genes related to cell proliferation and apoptosis for two differentially expressed miRNAs were predicted. Taken together, these data suggested that the anti-proliferative and pro-apoptosis effect of voltage-gated K+ channel blocker quinidine in human glioma cells was mediated at least partly through regulating expression of miRNAs, and provided further support for the mechanisms of voltage-gated K+ channels in mediating cell proliferation and apoptosis.

Voltage-gated and ATP-sensitive K+ channels are associated with cell proliferation and tumorigenesis of human glioma.

Increasing evidence indicates that potassium (K+) channels play important roles in the growth and development of human cancer. In the present study, we investigated the contribution of and the mechanism by which K+ channels control the proliferation and tumor development of U87-MG human glioma cells. A variety of K+ channel blockers and openers were used to differentiate the critical subtype of K+ channels involved. The in vitro data demonstrated that selective blockers of voltage-gated K+ (K(V)) channels or ATP-sensitive K+ (K(ATP)) channels significantly inhibited the proliferation of U87-MG cells, blocked the cell cycle at the G0/G1 phase and induced apoptosis. In the U87-MG xenograft model in nude mice, K(V) or K(ATP) channel blockers markedly suppressed tumor growth in vivo. Furthermore, electrophysiological results showed that KV or KATP channel blockers inhibited K(V)/K(ATP) channel currents as well as cell proliferation and tumor growth over the same concentration range. In contrast, iberiotoxin, a selective blocker of calcium-activated K+ channels, had no apparent effect on the cell proliferation, cell cycle or apoptosis of U87-MG cells. In addition, the results of fluorescence assays indicated that blockers of K(V) or K(ATP) channels attenuated intracellular Ca2+ signaling by blocking Ca2+ influx in U87-MG cells. Taken together, these data suggest that K(V) and K(ATP) channels play important roles in the proliferation of U87-MG cells and that the influence of K(V) and K(ATP) channels may be mediated by a Ca2+-dependent mechanism.