Voltage-dependent potassium channels (Kv) are involved in proliferation and transformation in mammary epithelial cells. In previous studies, several groups have detected various potassium channels in breast cancer cells, and they assumed that potassium channels are related to the development of breast carcinoma, although the precise mechanisms are still unknown. We have previously reported that 4-aminopyridine (4-AP), one kind of potassium channel (K(+) channel) blocker, could affect the proliferation of MCF10A cells. The aim of the present study is to explore the expression and properties of K(+) channels in human mammary epithelial cells (MCF10A) and whether Kv channels are required for the proliferation of MCF10A cell. Electrophysiological, MTT analysis, PCR and Western blot methods were used to identify a K(+) conductance which is involved in tumorigenesis and not yet be described in MCF10A cells. A voltage-dependent, outward rectification and 4-AP-sensitive K(+) current was observed in these cells. The perfusion of 5 mmol/L 4-AP significantly decreased the amplitude of Kv current from (912.5+/-0.6) pA to (275+/-0.8) pA (n=5, P<0.01), when cells were recorded using 800 ms voltage steps from a holding potential of -60 mV to voltage ranging from -60 mV to +60 mV. PCR analysis demonstrated that Kv1.1, Kv1.2, Kv1.3, and Kv1.5 were all expressed in MCF10A and MCF7 cells. Furthermore, the expression of Kv1.5 was much higher in MCF10A than that in MCF7. Inhibitory effect of 4-AP on cell proliferation was dosage-dependent. Incubation with 5 mmol/L 4-AP reduced MCF10A cell proliferation to 25.29% in 48 h. Western blot analysis showed the activation of ERK1/2 which related to cell proliferation was enhanced, while p38 activation was decreased by 4-AP treatment for 10 min. These data provided the first evidence of the Kv channels expression in MCF10A cell and 4-AP could inhibit the proliferation of MCF10A through blocking the potassium channels, and the mechanism may be related to regulating the activity of different members of cell proliferation signaling pathway of MEK/ERK.
4-Aminopyridine ; pharmacology ; Cell Line ; Cell Proliferation ; Epithelial Cells ; physiology ; Humans ; Potassium Channel Blockers ; pharmacology ; Potassium Channels, Voltage-Gated ; physiology

In this paper, membrane current properties of the fully-grown oocytes from toad, Bufo bufo gargarizans, were studied by using two-microelectrode voltage clamp technique. Axion of adult female toad was destroyed, and then ovarian lobes containing oocytes in stage I to VI were removed and incubated in Ca(2+)-free ND96 solution with collagenase (1.5 mg/ml) for 1 h. Subsequently, the oocytes were washed in Ca(2+)-free ND96 solution for 10 min to completely remove the follicular layer. For the experiments only the oocytes in stage V and VI were selected and used during 1 to 5 d. The membrane was depolarized from a holding potential of -80 mV to +60 mV in 10 mV step. It was found that a sustained outward current was elicited by depolarization. Potassium channel blockers (tetraethylammonium chloride, TEA, 10 mmol/L and 4-aminopyridine, 4-AP, 10 mmol/L) reduced the outward current to (23.4+/-0.72)% of the maximum. However, further addition of chloride channel blocker (5-nitro-2, 3-phenypropylamino benzoate, NPPB, 30 micromol/L) could almost completely block the outward current to (2.1+/-0.08)% of the maximum. In the presence of TEA and 4-AP, removal of extracellular Ca(2+) or adding verapamil (40 micromol/L), could also reduce the outward current to (2.2+/-0.04) % and (3.1+/-0.15) % of the maximum, respectively. It is concluded that calcium-dependent chloride channels exist in plasma membrane of Bufo bufo gargarizans oocytes, besides potassium channels.
4-Aminopyridine ; toxicity ; Animals ; Bufo bufo ; Calcium ; metabolism ; Cell Membrane ; metabolism ; Chloride Channels ; drug effects ; physiology ; Female ; Nitrobenzoates ; pharmacology ; Oocytes ; metabolism ; Tetraethylammonium Compounds ; pharmacology ; Verapamil ; pharmacology

OBJECTIVETo study the effects of telmisartan on voltage dependant potassium channel (Kv) expression in lymphocytes from spontaneously hypertensive rat (SHR).

METHODSPeripheral blood was collected from male SHR aged 16 and 4 weeks. Peripheral lymphocytes were separated from heparinized whole blood by standard Ficoll-Hypaque density gradient centrifugation. The whole-cell Kv currents were recorded with patch-clamp technique in the absence and presence of telmisartan(10, 30, 100 µmol/L). Real-time PCR was used to determine Kv1.3 mRNA expression in lymphocytes.

RESULTS(1) The currents density of Kv was higher in lymphocytes from 16 weeks-old SHR [ (119.0 ± 9.6) pA/pF] than from 4 weeks-old SHR [(59.0 ± 7.2) pA/pF, P < 0.05]. (2) Currents density was positively correlated with systolic blood pressure in 16 weeks-old SHR (r = 0.837, P < 0.05). (3) The lymphocytes Kv 1.3 mRNA expression was significantly higher in 16-weeks-old SHR than in 4-weeks-old SHR (P < 0.05). (4) Telmisartan reduced the whole-cell Kv currents in a concentration-dependent manner (10.5 ± 3.4)% at 10 µmol/L, (45.8 ± 3.7)% at 30 µmol/L and (81.6 ± 4.2)% at 100 µmol/L, P < 0.01.

CONCLUSIONSThe lymphocyte Kv channel is upregulated in 16 weeks-old SHR suggesting a role of Kv in the pathophysiology of hypertension. Kv current in lymphocyte could be significantly blocked by telmisartan in a concentration dependent manner.

4-Aminopyridine ; pharmacology ; Animals ; Benzimidazoles ; pharmacology ; Benzoates ; pharmacology ; Lymphocytes ; drug effects ; metabolism ; Male ; Patch-Clamp Techniques ; Potassium Channels, Voltage-Gated ; drug effects ; Rats ; Rats, Inbred SHR ; metabolism

The purpose of this study was to clarify the characteristics of the pacemaker cells in the left ventricular outflow tract (aortic vestibule) and compare them with those of the cells in the sinoatrial node (SAN). By using conventional intracellular microelectrode technique to record their action potentials, some ionic channel blockers were used to observe their electrophysiological effects on the two types of pacemaker cells in the rabbit, especially on the ionic movement during phase 0 and phase 4. The results obtained are as follows. (1) Perfusion with 1 micromol/L verapamil (VER) resulted in a significant reduction in the amplitude of action potential (APA), maximal rate of depolarization (V(max)), absolute value of the maximal diastolic potential (MDP), velocity of diastolic depolarization (VDD) and rate of pacemaker firing (RPF), and also a prolongation of the 90% of the duration of action potential (APD(90)) in the pacemaker cells of the SAN and aortic vestibule (P<0.05). (2) Perfusion with 180 micromol/L nickel chloride (NiCl2) resulted in a decrease in VDD in the two types of the pacemaker cells (P<0.01). APA, V(max) and RPF fell notably, and the APD(90) prolonged in the sinoatrial node cells (P<0.05). (3) 2 mmol/L 4-aminopyridine (4-AP) led to a increase in VDD in both types of pacemaker cells (P<0.01). At the same time the absolute values of MDP, APA and V(max) decreased significantly, and APD(90) prolonged notably (P<0.05). During the perfusion, RPF in SAN increased markedly, while RPF in aortic vestibule exhibited no significant change. (4) 2 mmol/L cesium chloride (CsCl) led to a decrease in VDD and RPF in the two types of the pacemaker cells (P<0.05).These results suggested: (1) the ion currents in phase 0 and phase 4 of depolarization and repolarization of slow-response activity in aortic vestibule are similar to those in dominant pacemaker cells of sinoatrial node; (2) for the pacemaker cells in the left ventricular outflow tract, Ca(2+) current is the main depolarizing ion current of the phase 0, K(+) current is the main factor responsible for the repolarization. Attenuation of K(+) current is responsible for the phase 4 spontaneous depolarization. In addition, it seems that I(Ca-T), I(Ca-L) and I(f ) play some role in the pacemaker currents.
4-Aminopyridine ; pharmacology ; Action Potentials ; drug effects ; Animals ; Aorta, Thoracic ; cytology ; physiology ; Female ; Male ; Nickel ; pharmacology ; Periodicity ; Rabbits ; Sinoatrial Node ; cytology ; physiology ; Verapamil ; pharmacology

The study aimed to investigate the age-related changes and drug reactions of transient outward potassium current (Ito) of ventricular myocytes. Twenty-eight Sprague Dawley rats were divided into young (3-5 months), adult (13-15 months) and aged (22-24 months) groups, and Ito currents of isolated myocytes from each group were recorded respectively by patch-clamp. The perfusion of 2.0 mmol/L 4-AP or 1.0 μmol/L isoproterenol was added respectively in each group, and the changes of Ito were observed. In comparison with young and adult groups, Ito densities of ventricular myocytes in aged group was significantly increased, the curve of steady-state activation of Ito shifted to the left, the close-state inactivation rate significantly decreased, and recovery rate from the steady-state inactivation became quicker. However, no significant changes could be detected for the Ito steady-state inactivation of ventricular myocytes in aged group. The similar responsiveness to 4-AP was observed in all three groups, but the responsiveness to isoproterenol was weaker in the aged group (55.9%) than in the other two groups (127.5% and 125.8%). In conclusion, the results show that Ito of rat ventricular myocyte of aging heart has increased current density and decreased response to isoproterenol.
4-Aminopyridine ; pharmacology ; Aging ; Animals ; Cells, Cultured ; Heart Ventricles ; cytology ; Isoproterenol ; pharmacology ; Myocytes, Cardiac ; physiology ; Potassium Channels ; physiology ; Rats ; Rats, Sprague-Dawley

AIMTo discover new regulators of potassium channel, an in vitro assay based on DiBAC4 (3) to determine the fluorescence was established for high throughput screening.

METHODSA cell-based 96-well format fluorescence assay using DiBAC4 (3) in cultured PC12 cells was described. Cells were loaded with 5 mumol.L-1 DiBAC4 (3) and incubated at 37 degrees C for 30 min before adding KCl or several known potassium channel regulators. The cellular DiBAC4 (3) fluorescence responce was then detected. The fluorescence changes can be used to evaluate membrane potential changes, which are determined mainly by potassium channels.

RESULTSExtracellular high K(+)-induced depolarization and several potassium channel blockers including 4-AP, TEA, E-4031, glibenclamide, quinidine and nifedipine all evoked increases in DiBAC4 (3) fluorescence response. The potassium channel opener, cromakalim, evoked decrease in DiBAC4 (3) fluorescence response. The fluorescence changes of 4-AP, TEA, glibenclamide, nifedipine and cromakalim were in a concentration-dependent manner. In 76 compounds screened by using the established DiBAC4 (3)-based assay, 9 compounds were found to change the fluorescence dose-dependently. Patch clamp technique is needed to further testify and screen their actions on potassium currents.

CONCLUSIONThe DiBAC4 (3)-based assay is easily operated, economical and repeatable. So, it can be performed by high throughput screening for potassium channel regulators.

4-Aminopyridine ; pharmacology ; Animals ; Barbiturates ; chemistry ; Calcium Channel Blockers ; pharmacology ; Cromakalim ; pharmacology ; Isoxazoles ; chemistry ; Membrane Potentials ; drug effects ; Nifedipine ; pharmacology ; PC12 Cells ; Patch-Clamp Techniques ; Piperidines ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; drug effects ; Pyridines ; pharmacology ; Quinidine ; pharmacology ; Rats

Intracellular recordings in cerebellar slice preparation showed that applications of 4-AP altered the pattern of oscillatory firing activity in Purkinje cells (PCs), especially yielding pronounced changes in action potential shape. 4-AP increased the amplitude and duration of action potential significantly and decreased the spike frequency. After 4-AP application, the duration of bursting was prolonged and the duration of after-burst hyperpolarization was progressively shortened. In all PCs tested, the rhythmicity of oscillatory firing activity was abolished completely at the steady state. These results suggest that 4-AP-sensitive currents determine the shape and frequency of individual Ca(2+)-dependent action potentials as well as maintaining oscillatory firing activity in PCs.
4-Aminopyridine/pharmacology* ; Action Potentials/drug effects* ; Animal ; Calcium/physiology* ; Electrophysiology ; In Vitro ; Oscillometry ; Purkinje Cells/physiology* ; Purkinje Cells/drug effects* ; Rats ; Rats, Sprague-Dawley

Neuronal firing is crucial to the information processing in the nervous system. In order to make a further study of bifurcation scenarios, experiments were performed on neural pacemakers formed at the injured site of rat sciatic nerve subjected to chronic ligatures. We chose the conductance of voltage-dependent potassium ion channels as conditional parameter, and the extracellular calcium concentration as bifurcation parameter, to give a demonstration of how the firing pattern of neural pacemaker responses to dual parameter adjusting. Among 28 preparations observed, 21 were insensitive to dual parameter adjusting since no change of bifurcation scenario structure was detected. On the contrary, the residual 7 preparations showed dramatic bifurcation scenario shifting corresponding to different dual parameter configuration. Briefly, when concentration of 4-aminopyridine (4-AP), a voltage-dependent potassium ion channels blocker, was kept at different level and extracellular Ca2+ concentration was decreased gradually, different bifurcation scenarios of firing patterns were exhibited in an identical neural pacemaker. The two-parameter bifurcation scenarios of experimental neural pacemaker with different parameter configuration were also different. The results show that neural firing pattern is different when the parameter configuration is different, and the bifurcation scenario is a fundamental framework to identify the transitions between firing patterns.
4-Aminopyridine ; pharmacology ; Action Potentials ; physiology ; Animals ; Calcium ; metabolism ; Male ; Neurons ; physiology ; Periodicity ; Potassium Channels ; physiology ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; injuries ; physiopathology

AIMTo investigate the role of three K+ channels-delayed rectifier K+ channel (Kv), large-conductance Ca(2+)-activated K+ channel (BK(Ca)) and ATP-sensitive K+ channel (K(ATP)) in the regulation of the resting and contracting tone of rat bronchial smooth muscle (BSM).

METHODSBy measuring the isometric tone of bronchial strips in vitro, the regulating effects of the three K+ channels on the tone of rat BSM were observed.

RESULTS(1) Kv blocker 4-aminopyridine (4-AP) caused concentration dependent contraction in resting bronchial strips, but BK(Ca) blocker tetraethylammonium (TEA) and K(ATP) blocker glibenclamide (Glib) had no such effects. (2) The bronchial epithelium had no effects on the contraction induced by 4-AP, but nifedipine, a Ca2+ channel blocker, significantly suppressed it. (3) Before or after treatment with 0.1 mmol/L histamine or 50 mmol/L KCl, administration of TEA (1 or 5 mmol/L) or 0.1 mmol/L 4-AP could significantly increase the contraction induced by histamine and KCl in bronchial strips. But Glib (10 micromol/L) had no effect on it.

CONCLUSIONNot BK(Ca) and K(ATP) but Kv participated in the regulation of the resting tone in rat BSM. The closure of BK(Ca) or Kv increased the contracting tone induced by histamine or KCl in rat BSM in vitro, but K(ATP) had no such effect on it.

4-Aminopyridine ; pharmacology ; Animals ; Bronchi ; drug effects ; In Vitro Techniques ; Male ; Membrane Potentials ; physiology ; Myocytes, Smooth Muscle ; physiology ; Patch-Clamp Techniques ; Potassium Channels ; physiology ; Rats ; Rats, Wistar

AIMTo investigate the effects of K+ channel blockers on arsenic trioxide-induced HeLa cell death.

METHODSViability of HeLa cells was assessed by mitochondrial dehydrogenase activity using colorimetric MTT assay and the voltage-dependent K+ currents were recorded by using patch-clamp technique.

RESULTSExposure of As2O3 (5 micromol x L(-1)) for 24 h caused marked HeLa cell death. The rest living cells after As2O3 24 h-incubation showed significant increase of K+ currents densities. At +80 mV, the densities of K+ currents (61 +/- 18) pA/10 pF (n = 8) in As2O3 24 h-incubation group were significantly more than that in the control group (38 +/- 10) pA/10 pF (n = 8, P < 0.05). The HeLa cells were prevented partially from As2O3-induced cell death by co-application for 24 h with typical voltage-dependent K+ channel blockers, 4-aminopyridine (3 mmol x L(-1)) or tetraethylammonium (5 mmol x L(-1)). 4-Aminopyridine (3 mmol x L(-1)) or tetraethylammonium (5 mmol x L(-1)) did not show any toxic effects on HeLa cells.

CONCLUSIONChronic treatment with As2O3 increased voltage-dependent K+ currents in HeLa cells and the cell death induced by As2O3 was reduced partially by voltage-dependent K+ channel blockers, 4-aminopyridine or tetraethylammonium.

4-Aminopyridine ; pharmacology ; Arsenicals ; antagonists & inhibitors ; pharmacology ; Cell Death ; drug effects ; HeLa Cells ; Humans ; Oxides ; antagonists & inhibitors ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Potassium Channels, Voltage-Gated ; drug effects ; Tetraethylammonium ; pharmacology