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

The present study aimed to clarify the effect of berberine on the chloride channels in human colorectal carcinoma cells (SW480). The whole-cell patch clamp technique was used to detect the Cl(-) current activated by berberine. The physiological and pharmacological characteristics of the current were clarified by changing the osmotic pressure of extracellular perfusate and applying chloride channel blockers. The results showed that, under isotonic conditions, the background current of SW480 cells was weak and stable. A large current was induced by perfusing the cells with the isotonic solution containing berberine (10 nmol/L), current density being (85.8 ± 4.6) pA/pF at +80 mV, (-71.9 ± 3.5) pA/pF at -80 mV, with a latency of (115.6 ± 21.7) s. The chloride current showed weak outward rectification and negligible time- and voltage-dependent inactivation. The reversal potential (-5.5 mV ± 1.2 mV) of the current was close to the calculated equilibrium potential for Cl(-) (ECl = -0.9 mV). Experiments under different osmotic pressures showed that the properties of hypotonicity-activated current recorded in SW480 cells were similar to those of the current induced by berberine, and hypertonic solutions suppressed the berberine-induced current by (98.6 ± 2.3)%. On the other hand, berberine-induced Cl(-) current was significantly inhibited by the chloride channel blockers NPPB (100 µmol/L) and tamoxifen (20 μmol/L), with the inhibition ratios of (83.1 ± 3.6)% and (95.6 ± 1.2)% respectively. These results suggest that berberine can activate the chloride channels that are sensitive to NPPB and tamoxifen, as well as the changes of cell volume in human colorectal carcinoma cells.
Berberine ; pharmacology ; Cell Line, Tumor ; Chloride Channels ; drug effects ; Colorectal Neoplasms ; metabolism ; pathology ; Humans ; Nitrobenzoates ; pharmacology ; Osmotic Pressure ; drug effects ; Patch-Clamp Techniques ; Tamoxifen ; pharmacology

To characterize the background current in fetal human nasopharyngeal epithelial cells and clarify its relationship with volume activated Cl(-) currents (I(Cl,vol)), whole-cell patch clamp and cell imaging techniques were employed. Under isotonic conditions, a background current [(5.9+/-2.1) pA/pF at +80 mV, n=21] was detected. The current presented a weak outward rectification and a negligible time-dependent inactivation. The current-voltage relationship showed that the reversal potential of the background current [(-0.73+/-1.7) mV, n=21] was close to the calculated equilibrium potential for Cl(-)(-0.9 mV). Application of extracellular hypertonic stimulation (440 mOsmol/L) suppressed the current by (59.6+/-7.1)% and the inhibition was reversible after returned to isotonic conditions. Bathing the cells in hypotonic solution (160 mOsmol/L) induced a volume-sensitive Cl(-) current. The Cl(-) channel blockers, tamoxifen (20 micromol/L) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) (100 micromol/L), inhibited the background current by (74.0+/-5.2)% (P<0.01, n=5) and (60.9+/-8.9)% (P<0.01, n=6) at +80 mV and increased basal cell volume by (107.7+/-2.9)% (P<0.01, n=25) and (104.4+/-2.4)% (P<0.01, n=19), respectively. The data indicate that Cl(-) current is an important component of the background current in fetal human nasopharyngeal epithelial cells. The background Cl(-) current is involved in volume activated Cl(-) current and basal cell volume regulation.
Cells, Cultured ; Chloride Channels ; antagonists & inhibitors ; physiology ; Electrophysiology ; Epithelial Cells ; cytology ; metabolism ; physiology ; Fetus ; Humans ; Nasopharynx ; cytology ; Nitrobenzoates ; pharmacology ; Patch-Clamp Techniques ; Tamoxifen ; pharmacology

A series of 2-(3-butynoicamidophenyl)benzothiazole derivatives were synthesized starting from 4-fluoro-3-nitrobenzoic acid. Structures of all the synthesized compounds were confirmed by 1H NMR and HR-MS. Their antitumor activities against human tumor cells lines (HCT116, Mia-PaCa2, U87-MG, A549, NCI-H1975) were evaluated by MTT assay. The results revealed that most of the synthesized compounds showed potent activities against HCT116, Mia-PaCa2, U87-MG tumor cells lines. Particularly, compounds 14c and 14h exhibited better activity with IC50 values of 1 x 10(-8) mol x L(-1) against U87-MG and HCT116 respectively. The structure-activity relationship of compounds was also discussed preliminarily.
Antineoplastic Agents ; chemical synthesis ; pharmacology ; Benzothiazoles ; chemical synthesis ; pharmacology ; Cell Line, Tumor ; Drug Screening Assays, Antitumor ; Humans ; Nitrobenzoates ; Structure-Activity Relationship

Whole-cell patch clamp and cell volume measurement techniques were used to investigate the ATP-activated chloride current and the ATP effect on cell volume in nasopharyngeal carcinoma cells. Extracellular application of ATP in micromolar concentrations activated a current with the properties of modest outward rectification and negligible time-dependent inactivation in a dose-dependent manner. The current reversed at a potential [(-0.05+/-0.03) mV] close to the Cl- equilibrium potential (-0.9 mV). Substitution of Cl- with gluconate in the extracellular solution decreased the ATP-activated current and shifted the reversal potential positively. NPPB, one of the chloride channel blockers, inhibited the current by (81.03+/-9.36)%. The current was also depressed by the P2Y purinoceptor antagonist, reactive blue 2, by (67.39+/-5.06)%. ATP (50 micromol/L) decreased the cell volume under the isotonic condition. Depletion of extracellular and intracellular Cl- abolished the ATP effect on cell volume. The results suggest that extracellular ATP of micromolar scales can induce a chloride current associated with cell volume regulation by activation of chloride channel through binding to purinoceptor P2Y.
Adenosine Triphosphate ; physiology ; Cell Size ; drug effects ; Chloride Channels ; antagonists & inhibitors ; metabolism ; physiology ; Humans ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Nitrobenzoates ; pharmacology ; Patch-Clamp Techniques ; Tumor Cells, Cultured

AIMTo investigate the effects of NPPB, a chloride channel blocker, on proliferation of mesangial cells.

METHODSCell proliferation was determined by measuring cell number and 3H-thymidine incorporation. The LDH activity released from these cells was measured as evaluation of cell viability. The phase of cell cycle was detected by flow cytometry.

RESULTSCell proliferation assays showed that treatment with both NPPB (50 and 25 micromol x L(-1)) and in hypertonic media (100% increased osmolarity with D-mannitol ) significantly reduced the number of human MC and 3H-thymidine incorporation in a dose-dependent manner. But the LDH activity was not significantly altered in the treatment with 50 micromol x L(-1) NPPB. Flow cytometry experiments showed that 50 and 25 micromol x L(-1) NPPB arrested (84.2 +/- 2.4) % and (80.8 +/- 2.9) % of cells at G0/G1 stage, versus (70.5 +/- 1.4) % of control cells. Conclusion NPPB suppresses cell proliferation and produces growth arrest at G0/G1 phase in human MC by a mechanism probably associated with changes in cell volume.

Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chloride Channels ; antagonists & inhibitors ; Dose-Response Relationship, Drug ; Humans ; L-Lactate Dehydrogenase ; metabolism ; Mesangial Cells ; cytology ; metabolism ; Nitrobenzoates ; administration & dosage ; pharmacology

1) A beta agonist stimulated Na+ transport and decreased the intracellular Cl concentration ([Cl]c) associated with cell shrinkage via an increase in cytosolic cAMP level by activating adenylate cyclase in rat fetal distal lung epithelial (FDLE) cells. 2) Lowering [Cl-]c activated a 28-pS nonselective cation (NSC) channel by elongating the open time of the channel. 3) cAMP signals were converted to a protein tyrosine kinase (PTK)-mediated signal. 4) The PTK-mediated signal was involved in the cAMP-stimulated Na+ transport in rat FDLE cells.
Adrenergic beta-Agonists/pharmacology* ; Animal ; Biological Transport/physiology ; Biological Transport/drug effects ; Cell Size/physiology ; Chlorides/metabolism* ; Cyclic AMP/metabolism ; Cytosol/metabolism ; Enzyme Inhibitors/pharmacology ; Female ; Fetus/cytology ; Forskolin/pharmacology ; Nitrobenzoates/pharmacology ; Pregnancy ; Protein-Tyrosine Kinase/metabolism* ; Rats ; Rats, Wistar ; Respiratory Mucosa/enzymology* ; Respiratory Mucosa/embryology ; Respiratory Mucosa/cytology ; Sodium/metabolism* ; Tyrphostins/pharmacology

The transwell chamber migration assay and the patch-clamp technique were used to investigate the volume-activated Cl(-) current (I(Cl.vol)) in migrated nasopharyngeal carcinoma cells (CNE-2Z). 47% hypotonic solution activated a ICl.vol in the migrated CNE-2Z cells. Compared with the control cells (non-migrated), the properties of this current and the sensitivity to Cl(-) channel blockers were changed. The current density in migrated CNE-2Z cells was higher than that in non-migrated cells. The current was almost completely inhibited by extracellular application of adenosine-5'-triphosphate (ATP, 10 mmol/L), 5-nitro-2-3-phenylpropylamino benzoic acid (NPPB, 100 mmol/L) and tamoxifen (30 mmol/L) in all voltage steps applied. The inhibition of NPPB and tamoxifen on the current was stronger in migrated cells than that in non-migrated cells. The permeability sequence of the four anions was Br(-)>Cl(-)> I (-)>Gluconate. The sequence was different from that of the non-migrated cells (I(-)> Br(-)> Cl(-)> Gluconate). The results suggest that volume-activated chloride channels may be involved in the CNE-2Z cell migration.
Carcinoma ; drug therapy ; metabolism ; pathology ; Cell Cycle ; drug effects ; physiology ; Cell Division ; drug effects ; Cell Movement ; drug effects ; Cell Size ; drug effects ; Chloride Channels ; antagonists & inhibitors ; metabolism ; physiology ; Chlorides ; metabolism ; Humans ; Nasopharyngeal Neoplasms ; drug therapy ; metabolism ; pathology ; Nitrobenzoates ; pharmacology ; Patch-Clamp Techniques ; Tamoxifen ; pharmacology ; Tumor Cells, Cultured