[Inhibitory effects of tetraethylammonium on proliferation and voltage-gated potassium channels in human cervical carcinoma cell line SiHa].

BACKGROUND & OBJECTIVE: Various potassium channels are known to be involved in proliferation of many malignant cell lines. This study was to explore the role of voltage-gated potassium channels in proliferation of human cervical carcinoma cells through observing the effects of tetraethylammonium (TEA) on proliferation and outward potassium currents in human cervical carcinoma cell line SiHa. METHODS: SiHa cells were treated with TEA. The effect of TEA on proliferation of SiHa cells was assessed by MTT assay. Cell apoptosis and cell cycle were detected by flow cytometry with Hoechst 33258 staining. The outward potassium currents were recorded by patch clamp technique. RESULTS: TEA inhibited the proliferation of SiHa cells in dose-and time-dependent manners, and induced cell apoptosis. The cell cycle was arrested at G0/G1 phase after treatment with TEA. Exposure of SiHa cells to 10 mmol/L TEA reduced the peak outward potassium currents significantly from (260+/-12) pA to (58+/-6) pA (P<0.01). CONCLUSIONS: Voltage-gated potassium channels play an important role in regulating proliferation of cervical carcinoma SiHa cells. Blocking voltage-gated potassium channels could inhibit proliferation of SiHa cells.

Effects of amiloride on potassium and calcium currents in guinea pig ventricular myocytes.

AIM: To elucidate the possible mechanisms underlying antiarrhythmia of the non-selective Na+/H+ exchanger inhibitor--amiloride. METHODS: Single ventricular cells were isolated using a double-enzyme method. Effects of amiloride on voltage-dependent potassium and calcium currents in isolated guinea pig ventricular myocyte were recorded by using whole-cell patch clamp techniques. RESULTS: Exposure to amiloride (10 -100 micromol x L(-1)), the L-type and T-type calcium currents were depressed. Amiloride resulted in a concentration-dependent inhibition of peak (Ca,L), But amiloride did not change the shape of their I - V curves. It only decreased the amplitudes of the currents of the two types. When myocytes were incubated with 100 micromol x L(-1) amiloride, I(Kr) was slightly depressed and I(Ks) did not change. Amiloride (1 - 100 micromol x L(-10) depressed I(K1) in a concentration-dependent manner. CONCLUSION: Amiloride depressed potassium and calcium currents, which may give support to its uses in some diseases of the cardiovascular system.

Inhibitory effects of berberine on ion channels of rat hepatocytes.

AIM: To examine the effects of berberine, an isoquinoline alkaloid with a long history used as a tonic remedy for liver and heart, on ion channels of isolated rat hepatocytes. METHODS: Tight-seal whole-cell patch-clamp techniques were performed to investigate the effects of berberine on the delayed outward potassium currents (I(K)), inward rectifier potassium currents (I(K1)) and Ca(2+) release-activated Ca(2+) currents (I(CRAC)) in enzymatically isolated rat hepatocytes. RESULTS: Berberine 1-300 micromol/L reduced I(K) in a concentration-dependent manner with EC(50) of 38.86+/-5.37 micromol/L and n(H) of 0.82+/-0.05 (n = 8). When the bath solution was changed to tetraethylammonium (TEA) 8 mmol/L, I(K) was inhibited. Berberine 30 micromol/L reduced I(K) at all examined membrane potentials, especially at potentials positive to +60 mV (n = 8, P<0.05 or P<0.01 vs control). Berberine had mild inhibitory effects on I(K1) in rat hepatocytes. Berberine 1-300 micromol/L also inhibited I(CRAC) in a concentration-dependent fashion. The fitting parameters were EC(50) = 47.20+/-10.86 micromol/L, n(H) = 0.71+/-0.09 (n = 8). The peak value of I(CRAC) in the I-V relationship was decreased by berberine 30 micromol/L at potential negative to -80 mV (n = 8, P<0.05 vs control). But the reverse potential of I(CRAC) occurred at voltage 0 mV in all cells. CONCLUSION: Berberine has inhibitory effects on potassium and calcium currents in isolated rat hepatocytes, which may be involved in hepatoprotection.

Effects of berberine on potassium currents in acutely isolated CA1 pyramidal neurons of rat hippocampus.

The effects of berberine, an isoquinoline alkaloid with antiarrhythmic action, on voltage-dependent potassium currents were studied in acutely isolated CA1 pyramidal neurons of rat hippocampus by using the whole-cell patch-clamp techniques. Berberine blocked transient outward potassium current (IA) and delayed rectifier potassium current (IK) in a concentration-dependent manner with EC50 of 22.94+/-4.96 microM and 10.86+/-1.06 microM, Emax of 67.47+/-4.00% and 67.14+/-1.79%, n of 0.77+/-0.08 and 0.96+/-0.07, respectively. Berberine 30 microM shifted the steady-state activation curve and inactivation curve of IA to more negative potentials, but mainly affected the inactivation kinetics. Berberine 30 microM positively shifted the steady-state activation curve of IK. These results suggested that blockades on K+ currents by berberine are preferential for IK, and contribute to its protective action against ischemic brain damage.

Electrophysiological effects of anthopleurin-Q on rat hepatocytes.

AIM: To study the effects of AP-Q on CCl(4)-induced acute liver injury, delayed outward potassium current (I(K)), inward rectifier potassium current (I(K1)) and calcium release-activated calcium current (I(CRAC)) in isolated rat hepatocytes. METHODS: A single dose of CCl(4) (10 microg/mL, ip) was injected to induce acute liver injury in rats. Serum aminotransferase activities were determined. Whole cell patch-clamp techniques were used to investigate the effects of AP-Q on delayed outward potassium current (I(K)), inward rectifier potassium current (I(K1)) and calcium release-activated calcium current (I(CRAC)). RESULTS: AP-Q (3.5 and 7 microg/kg) pretreatment significantly reduced ALT and AST activities. AP-Q 0.1-100 nM produced a concentration-dependent increase of I(K) with EC(50) value of 5.55+/-1.8 nM (n=6). AP-Q 30 nM shifted the I-V curve of I(K) leftward and upward. CCl(4) 4 mM decreased I(K) current 28.6+/-6.5% at 140 mV. After exposure to CCl(4) for 5 min, AP-Q 30 nM attenuated the decrease of I(K) induced by CCl(4) close to normal amplitude. AP-Q 0.01-100 nM had no significant effect on either inward or outward components of I(K1) at any membrane potential examined. AP-Q 0.1-100 nM had no significant influence on the peak amplitude of I(CRAC), either, and did not affect the shape of its current voltage curve. CONCLUSION: AP-Q has a protective effect on CCl(4)-induced liver injury, probably through selectively increased I(K) in hepatocytes.

Perforated patch recording of L-type calcium current with beta-escin in guinea pig ventricular myocytes.

AIM: To establish a perforated patch recording (PPR) mode with beta-escin and compare L-type calcium current (I(Ca,L)) recorded under PPR and normal whole-cell recording (WCR) condition in isolated guinea-pig ventricular myocytes. METHODS: Single myocytes were dissociated by enzymatic dissociation method. beta-escin was added to the pipette solution to perforate the cell membrane and obtain PPR mode. I(Ca,L) was recorded using PPR and WCR techniques. RESULTS: beta-Escin 20, 25, and 30 micromol/L could permeabilize the cell membrane and obtain PPR mode. With beta-escin 25 micromol/L, the success rate was highest (16/17, 94 %) and the time required for permibilization was 2-15 (8+/-4) min. Run-down of I(Ca,L) was considerably slower in PPR than in WCR condition. The amplitude of I(Ca,L) was decreased by 36 % at 20 min after the formation of WCR, while it was slowly decreased by 8 % at 30 min after the formation of PPR. The current-voltage relation (I-V) curves, activation and inactivation curves of I(Ca,L) were not significantly different between WCR and PPR. The inactivation rate of ICa,L was slower in PPR than in WCR, the faster inactivation time constant (tau(f)) was longer in PPR than in WCR at membrane potentials of -20 mV -- +10 mV (n=6, P<0.05), and the slower time constant (tau(s)) was also longer in PPR than in WCR at membrane potentials of -10 mV to +10 mV (n=6, P<0.05). There was no significant difference between the activation rate in WCR and PPR. CONCLUSION: Using beta-escin 25 micromol/L can easily obtain stable PPR in isolated guinea-pig ventricular myocytes, and this method is useful in dealing with channels, which show run-down under normal WCR such as L-type Ca channel.

Effects of palmatine on potassium and calcium currents in isolated rat hepatocytes.

AIM: To study the effects of palmatine, a known inhibitor on delayed rectifier potassium current and L-type calcium current (I(Ca,L)) in guinea pig ventricular myocytes, on the potassium and calcium currents in isolated rat hepatocytes. METHODS: Tight-seal whole-cell patch-clamp techniques were performed to investigate the effects of palmatine on the delayed outward potassium currents (I(K)), inward rectifier potassium current (I(K1)) and Ca(2+) release-activated Ca(2+) current (I(CRAC)) in enzymatically isolated rat hepatocytes. RESULTS: Palmatine 0.3-100 microM reduced I(K) in a concentration-dependent manner with EC(50) of 41.62+/-10.11 microM and n(H), 0.48+/-0.07 (n=8). The effect of the drug was poorly reversible after washout. When the bath solution was changed to tetraethylammonium (TEA) 8 mM, IK was inhibited. Palmatine 10 microM and 100 microM shifted the I-V curves of I(K) downward, and the block of I(K) was voltage-independent. Palmatine 0.3-100 microM also inhibited I(CRAC) in a concentration-dependent manner. The fitting parameters were as follows: EC(50)=51.19+/-15.18 microM, and n(H)=0.46+/-0.07 (n=8). The peak value of I(CRAC) in the I-V relationship was decreased by palmatine 10 microM and 100 microM. But the reverse potential of I(CRAC) occurred at Voltage=0 mV in all cells. Palmatine 0.3-100 microM failed to have any significant effect on either inward or outward components of I(K1) at any membrane potential examined. CONCLUSION: The inhibitory effects on I(K) and I(CRAC) could be one of the mechanisms that palmatine exerts protective effect on hepatocytes.

Effects of tetrandrine on calcium and potassium currents in isolated rat hepatocytes.

AIM: To study the effects of tetrandrine (Tet) on calcium release-activated calcium current (I(CRAC)), delayed rectifier potassium current (I(K)), and inward rectifier potassium currents (I(K1)) in isolated rat hepatocytes. METHODS: Hepatocytes of rat were isolated by using perfusion method. Whole cell patch-clamp techniques were used in our experiment. RESULTS: The peak amplitude of I(CRAC) was -508+/-115 pA (n=15), its reversal potential of I(CRAC) was about 0 mV. At the potential of -100 mV, Tet inhibited the peak amplitude of I(CRAC) from -521+/-95 pA to -338+/-85 pA (P<0.01 vs control, n=5), with the inhibitory rate of 35 % at 10 micromol/L and from -504+/-87 pA to -247+/-82 pA (P<0.01 vs control, n=5), with the inhibitory rate of 49 % at 100 micromol/L, without affecting its reversal potential. The amplitude of I(CRAC) was dependent on extracellular Ca(2+) concentration. The peak amplitude of I(CRAC) was -205+/-105 pA (n=3) in tyrode's solution with Ca(2+) 1.8 mmol/L (P<0.01 vs the peak amplitude of I(CRAC) in external solution with Ca(2+) 10 mmol/L). Tet at the concentration of 10 and 100 micromol/L did not markedly change the peak amplitude of delayed rectifier potassium current and inward rectifier potassium current (P>0.05 vs control). CONCLUSION: Tet protects hepatocytes by inhibiting I(CRAC), which is not related to I(K) and I(K1).

[Effect of ouabain on the aortic rings of guinea pig and its interactions with Ca2+, norepinephrine].

AIM: To observe the effects of ouabain on vascular smooth muscle (VSM) of the guinea pig and its interactions with Ca2+ and norepinephrine (NE). METHODS: Using isolated thoracic aortic ring of the guinea pig, the degrees of contractile activity of drugs were recorded. RESULTS: Ouabain showed a direct contractile effect in a concentration-dependent manner on thoracic aortic ring of guinea pig. Ouabain shifted the NE dose-response curve to the left without changing in the maxium response. Ouabain shifted the CaCl2 dose-response curve to the left and upward, increased the maximum response to Ca2+; In Ca(2+)-free medium, the ouabain induced contraction was abolished, an increase in extracellular Ca2+ restored the response; nifedipine and verapamil abolished the ouabain induced contraction. CONCLUSION: The ouabain induced contraction is mainly dependent on the extracellular Ca2+ concentration, independent on the presence of endothelia of aorta, suggesting that Ca2+ antagonist may treat the hypertension induced by ouabain. Ouabain, NE and CaCl2 have synergetic contractile effects, suggesting that the synergetic contractile effects of ouabain and NE may contribute to the generation and development of hypertension.

[Effects of Rbl on action potentials and force of contraction in guinea pig ventricular papillary muscles].

OBJECTIVE: To evaluate the effects of Rbl on action potentials and force of contraction in guinea pig ventricular papillary muscles. METHOD: The ventricular papillary muscles of guinea pig were isolated regularly and immersed with Tyrode, s solution. The effects of Rbl (purified saponins of panaxnotoginseng) on the action potentials (AP), the slow action potentials and the force of contraction (FC) of the muscles were studied. The AP and FC were measured synchronously. RESULT: Rbl shortened the duration of AP, including APD2O and APD90, and reduced the FC(n = 5, P < 0.01), but didn't affect the rest potential (RP), the amplitude of action potential (APA), overshot (OS) and maximal upstrok velocity (Vmax). Rbl also decreased the APA of slow action potential, but quinidine had no such effects. CONCLUSION: Rbl may be a channel blocker.

Effects of tetrandrine on cardiovascular electrophysiologic properties.

Tetrandrine (Tet) is one of the best characterized Ca2+ channel blocker of plant origin. It can affect cardiovascular electrophysiologic properties in following field: inhibit the contractility, +/-dt/dpmax, and automaticity of myocardium, prolong the FRP, and exert concentration-dependent negative inotropic and chronotropic effects without altering cardiac excitability. Tet directly blocks both T-type and L-type calcium current in ventricular cells and vascular smooth muscle cells, but it does not shift the I-V relationship curve of ICa. All its effects would be beneficial in the treatment of angina, arrhythmias, and other cardiovascular disorders. Tet also directly inhibits the activity of BKCa channel in endothelial cell line and also inhibits Ca2+-release-activated channels in vessel endothelial cells, which might significantly contribute to the change of endothelial cell activity.

Effects of anthopleurin-Q on myocardial hypertrophy in rats and physiologic properties of isolated atria in guinea pigs.

AIM: To study effects of anthopleurin-Q (AP-Q) on myocardial hypertrophy in rats and isolated atria in guinea pigs. METHODS: Two myocardial hypertrophy models in rats were established, one introduced by levothyroxine, the other by stenosis of abdominal aorta. Cardiac myocytes morphometry and functional experiments were employed to investigate effects of AP-Q. RESULTS: Low dose of AP-Q (1 microg/kg/d, ip) reduced morphologic changes of myocardial hypertrophy in both rat models. While high dose of AP-Q (10 microg/kg/d, ip) did not, and caused mild hydropic degeneration in cardiomyocytes. High concentration of AP-Q (30 nmol/L) enhanced the contractility, raised automaticity, and prolonged the functional refractory period (FRP) in isolated left atria of guinea pigs; higher concentration (100 nmol/L) triggered arrhythmia in right atria; low concentration of AP-Q (1 nmol/L)did not affect any myocardial properties above. CONCLUSION: Low dose of AP-Q without inotropic effect can hinder the experimental myocardial hypertrophy in rats; high dose with positive inotropic effect may be responsible for its toxic reaction.

Effects of benzyltetrahydropalmatine on potassium currents in guinea pig and rat ventricular myocytes.

AIM: To investigate the effects of benzyltetrahydropalmatine (BTHP) on rapidly activating component (I(Kr)), slowly activating component (I(Ks)) of delayed rectifier potassium current, inward rectifier potassium current (I(K1)), and transient outward potassium current (I(to)) in single ventricular myocytes. METHODS: Whole-cell patch clamp technique was used to record ionic currents. RESULTS: (1) BTHP 30 micromol/L reduced I(Kr) and I(Kr,tail) by 31 %+/-4 % and 36 %+/-5 % (n=6, P <0.01), respectively and inhibited I(Ks) and I(Ks,tail) by 40 %+/-6 % and 45 %+/-5 % (n=7, P <0.01), respectively. I(Kr) and I(Ks) were blocked by BTHP 1-100 micromol/L in a concentration-dependent fashion (IC50 value was 13.5 micromol/L and 95 % confidence limit: 11.2-15.8 micromol/L for I(Kr), 9.3 micromol/L and 95 % confidence limit: 7.8-11.8 micromol/L for I(Ks), respectively). (2) BTHP 5 micromol/L inhibited I(to) by 63 %+/-6 % (n=6, P <0.01). BTHP 1-100 micromol/L reduced I(to) in a concentration-dependent manner (IC50 value was 3.6 micromol/L and 95 % confidence limit: 2.9-4.3 micromol/L). (3) BTHP 200 micromol/L did not affect I(K1). CONCLUSION: BTHP inhibited I(Kr), I(Ks), and I(to), but not I(K1). The antiarrhythmic effects of BTHP may be mainly due to its blockade on potassium channels.

[Modulation of protein kinase A and protein kinase C on the delayed rectifier potassium current in guinea pig ventricular myocytes].

AIM: To study modulation of protein kinase A (PKA) and protein kinase C (PKC) on the delayed rectifier potassium current (Ik)in guinea pig ventricular myocytes. METHODS: The delayed rectifier potassium current was recorded by using whole cell arrangement of the patch-clamp procedure. RESULTS: cAMP 150 micromol/L increased intracellularly Ik and Ik,tail(pA/pF) from 13.7 +/- 2.1 and 6.1 +/- 0.1 to 18.5 +/- 3.3 and 6.4 +/- 2.1 (P < 0.01, n=6). Ik and Ik,tail(pA/pF) were augmented by 8-CPT-cAMP 150 micromol/L extracellularly from 11.4 +/- 1.8 and 5.3 +/- 0.6 to 17.9 +/- 4.0 and 6.2 +/- 1.3. The half-maximal voltage of activation of Ik was shifted from + 23.3 mV to 18.7 mV by cAMP. Phorbol 12-myristate 13-acetate(PMA, 10.0 micromol/L) applied intracellularly caused an enhance effect on Ik, with increasing Ik and Ik,tail(pA/pF) from 12.9 +/- 1.8 and 5.0 +/- 1.7 to 23.7 +/- 2.8 and 7.5 +/- 1.1. With shifting position potential of depolarization, effect of PMA on Ik was gradually augmented. PMA resulted in shifting the slop of activation curve from +15.3 mV to +25.6 mV, with only a small effect on the half-maximal voltage of activation of Ik. CONCLUSION: Ik was increased by both PKA and PKC, with different characteristics of regulation.

Effects of benzyltetrahydropalmatine on the rapidly activating component of delayed rectifier potassium current in guinea pig ventricular myocytes.

AIM: To investigate the effect of benzyltetrahydropalmatine (BTHP) on the rapidly activating component of delayed rectifier K+ current (Ikr) in single guinea pig ventricular myocytes. METHODS: Whole-cell patch clamp technique was used to record Ikr. RESULTS: Ikr was blocked by 1-100 mumol.L-1 BTHP in concentration-, voltage-, and specifically frequency-dependent fashion, with IC50 of 13.5 mumol.L-1 (95% confidence range: 11.2-15.8 mumol.L-1). 30 mumol.L-1 BTHP reduced Ikr and Ikr.tail by (31 +/- 4)% and (36 +/- 5)% (n = 6, P < 0.01), respectively. The time constant for deactivation (tau') of the tail current was decreased by 30 mumol.L-1 BTHP from (238 +/- 16) ms to (196 +/- 14) ms, while drug had no any effect on the time constant for activation (tau) of Ikr,tail. CONCLUSION: BTHP inhibited Ikr in a frequency-dependent fashion.