Outwardly rectifying swelling-activated chloride conductance (ICl,Swell) in rabbit heart plays a critical role in cardioprotection following ischemic preconditioning (IP). But the functional characterization and molecular basis of this chloride conductance in rabbit heart ventricular myocytes is not clear. Candidate chloride channel clones (e.g. ClC-2, ClC-3, ClC-4 and ClC-5) were determined using RT-PCR and Western blot analysis. Whole cell ICl,Swell was recorded from isolated rabbit ventricular myocytes using patch clamp techniques during hypo-osmotic stress. The inhibitory effects of 4,4' isothiocyanato-2,2-disulfonic acid (DIDS), 5-nitro-2(3-phenylroylamino) benzoic acid (NPPB) and indanyloxyacetic acid 94 (IAA-94) on ICl,Swell were examined. The expected size of PCR products for ClC-2, ClC-3 and ClC-4 but not for ClC-5 was obtained. ClC-2 and ClC-3 expression was confirmed by automated fluorescent DNA sequencing. RT-PCR and Western blot showed that ClC-4 was expressed in abundance and ClC-2 was expressed at somewhat lower levels. The biological and pharmacological properties of I(Cl,Swell), including outward rectification, activation due to cell volume change, sensitivity to DIDS, IAA-94 and NPPB were identical to those known properties of ICl,Swell in exogenously expressed systems and other mammals hearts. It was concluded that ClC-3 or ClC-4 might be responsible for the outwardly rectifying part of ICl,Swell and may be the molecular targets of cardioprotection associated with ischemic preconditioning or hypo-osmotic shock.
Biophysics/methods ; Chlorides/*chemistry ; Chlorides/metabolism ; DNA Primers/chemistry ; Electrophysiology/methods ; Gene Expression Regulation ; Glycolates/pharmacology ; Heart Ventricles/*cytology ; Ischemic Preconditioning ; Muscle Cells/*cytology ; Osmosis ; Sequence Analysis, DNA

Aluminum Compounds ; pharmacology ; Animals ; Bone and Bones ; drug effects ; metabolism ; Chlorides ; pharmacology ; Decalcification Technique ; methods ; Formates ; chemistry ; Humans

Amino Acid Substitution ; Arsenicals ; pharmacology ; Cell Line ; Chlorides ; pharmacology ; Drug Resistance, Neoplasm ; genetics ; Humans ; Mutation, Missense ; Oncogene Proteins, Fusion ; genetics ; metabolism

OBJECTIVETo explore the effect of terbutaline on sodium transport in rat alveolar type I (ATI) and type II (ATII) cells of rats.

METHODSThe whole cell currents were recorded from ATII cells isolated from rat lungs perfused with or without amiloride (inhibitor of epithelial sodium channel) and ZnCl(2) (inhibitor of cyclic nucleotide-gated cation channel) in the whole cell recording mode using the patch-clamp technique. The effect of terbutaline on the currents was examined.

RESULTSThe main currents recorded from ATII cells were amiloride-sensitive and Zn(2+)-sensitive. The amiloride-sensitive and Zn(2+)-sensitive current shared a similar proportion (P>0.05). Both currents could be significantly increased by terbutaline (P<0.05), and the proportion of amiloride-sensitive current was 1.7 times that of Zn(2+)-sensitive current (P<0.05).

CONCLUSIONThere are functional epithelial sodium channels (ENaC) and cyclic nucleotide-gated cation channels (CNG) on freshly isolated ATII cells, both serving as the main channels for sodium transport. Terbutaline increases the absorption of alveolar fluid primarily by increasing sodium transport of ENaC and CNG on ATI and AT II cells.

Amiloride ; pharmacology ; Animals ; Chlorides ; pharmacology ; Cyclic Nucleotide-Gated Cation Channels ; antagonists & inhibitors ; drug effects ; Male ; Peptides ; pharmacology ; Pulmonary Alveoli ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sodium ; metabolism ; Sodium Channels ; drug effects ; Terbutaline ; pharmacology ; Zinc Compounds ; pharmacology

The gating mechanism of ClC-1 chloride channel was studied in this paper by heteroexpression of rat wild type ClC-1 gene in Xenopus oocytes and by two-electrode voltage clamping technique. The deactivation gating kinetic parameters were obtained by applying two exponential fitting of the deactivating currents at various extracellular chloride concentrations. It was found that decrease in extracellular chloride concentration increased the fractional amplitude of fast deactivating component, and depressed the fractional amplitude of slow deactivating component accompanied by a decrease in fast and slow deactivating time constants. These results demonstrate that the deactivation kinetic parameters of ClC-1 are largely dependent on the extracellular chloride concentration, which induces changes in channel gating.
Animals ; Chloride Channels ; drug effects ; physiology ; Chlorides ; pharmacology ; Electrophysiology ; Female ; In Vitro Techniques ; Ion Channel Gating ; drug effects ; physiology ; Oocytes ; physiology ; Rats ; Xenopus

This study was designed to identify and characterize Na+ -activated K+ current (I(K(Na))) in guinea pig gastric myocytes under whole-cell patch clamp. After whole-cell configuration was established under 110 mM intracellular Na+ concentration ([Na+]i) at holding potential of -60 mV, a large inward current was produced by external 60 mM K+([K+] degree). This inward current was not affected by removal of external Ca2+. K+ channel blockers had little effects on the current (p>0.05). Only TEA (5 mM) inhibited steady-state current to 68+/-2.7% of the control (p<0.05). In the presence of K+ channel blocker cocktail (mixture of Ba2+, glibenclamide, 4-AP, apamin, quinidine and TEA), a large inward current was activated. However, the amplitude of the steadystate current produced under [K+]degree (140 mM) was significantly smaller when Na+ in pipette solution was replaced with K+ - and Li+ in the presence of K+ channel blocker cocktail than under 110 mM [Na+]i. In the presence of K+ channel blocker cocktail under low Cl- pipette solution, this current was still activated and seemed K+ -selective, since reversal potentials (E(rev)) of various concentrations of [K+]degree-induced current in current/voltage (I/V) relationship were nearly identical to expected values. R-56865 (10-20 microgram), a blocker of IK(Na), completely and reversibly inhibited this current. The characteristics of the current coincide with those of IK(Na) of other cells. Our results indicate the presence of IK(Na) in guinea pig gastric myocytes.
Tetraethylammonium Compounds/pharmacology ; Stomach/*physiology ; Sodium/metabolism/*pharmacology ; Potassium Channels/*physiology ; Potassium Channel Blockers/pharmacology ; Myocytes, Smooth Muscle/*physiology ; Membrane Potentials ; Male ; Guinea Pigs ; Female ; Chlorides/pharmacology ; Calcium/metabolism ; Animals

No abstract available.
Adenosine Triphosphate/pharmacology ; Anions/metabolism ; Bicarbonates/metabolism* ; Chlorides/metabolism* ; Cyclic AMP/pharmacology ; Cystic Fibrosis Transmembrane Conductance Regulator/physiology* ; Electric Conductivity ; Electrophysiology ; Gluconates/pharmacology ; Human ; Membrane Potentials/physiology ; Membrane Potentials/drug effects ; Potassium/pharmacology ; Sweat Glands/metabolism*

OBJECTIVETo investigate the role of autophagy in MnCl2-induced apoptosis in human bronchial epithelial 16HBE cells.

METHODSCell proliferation was measured by MTT assay. Mitochondrial membrane potential (MMP) and apoptosis were measured by flow cytometry. Autophagic vacuoles were detected by fluorescence microscopy. Cellular levels of apoptosis and autophagy-related proteins were measured by western blotting.

RESULTS16HBE cell proliferation was inhibited by MnCl2 in a dose- and time-dependent manner. MnCl2-induced 16HBE cell growth inhibition was related to MMP depolarization prior to the induction of apoptosis. Our data revealed that MnCl2-induced apoptosis in 16HBE cells was mediated by decreased expression of Bcl-2 and increased levels of cleaved caspase-3. It was observed that when we exposed 16HBE cells to MnCl2 in a dose-dependent manner, the formation of autophagic vacuoles and the levels of LC-3B-II were elevated. RNA interference of LC3B in these MnCl2-exposed cells demonstrated that MMP loss and apoptosis were enhanced. Additionally, the pan-caspase inhibitor Z-VAD-FMK increased the cellular levels of Bcl-2 and decreased apoptosis, but did not affect the cellular levels of LC3B in MnCl2-treated 16HBE cells.

CONCLUSIONMnCl2 dose- and time-dependently inhibits 16HBE cell proliferation and induces MMP loss and apoptosis. Autophagy acts in a protective role against MnCl2-induced apoptosis in 16HBE cells.

Amino Acid Chloromethyl Ketones ; pharmacology ; Apoptosis ; drug effects ; Autophagy ; drug effects ; physiology ; Bronchi ; Cell Line ; Chlorides ; pharmacology ; Down-Regulation ; Epithelial Cells ; drug effects ; Gene Expression Regulation ; drug effects ; Humans ; Manganese Compounds ; pharmacology

OBJECTIVETo investigate the effects of inward rectifier potassium channel blockers (BaCl2, CsCl) on the functions of endothelial progenitor cells (EPCs).

METHODSDensity gradient centrifugation-isolated rat hone marrow mononuclear cells were cultured in vitro. EPCs were harvested and seeded on six culture dish when cells grew to 3-5 passages. Before testing the EPCs were synchronized with M199, which contain 2% fetal calf serum. In the end, EPCs were treated with different intervention. The experiment mainly included two parts: (1) BaCl2 (100 micromol/L) and free BaC2 of Tyrodes solution; (2) CsCl (1 mmol/L) and control. Cell pretreated with blockers above mentioned for 12 h, then the gene expression of stromal cell-derived factor-1 (SDF-1), epoprotenol (PGI2) were assessed, beyond that the ability of adhesion, migration were assayed with different tests. In addition, the medium was collected when EPCs were treated for 3 days. The levels of SDF-1 were measured by sandwich enzyme-linked immunosorbent assay (ELISA). Going even further, EPCs were treated with the signal pathway blockers in advance, after repeat the above steps, in order to analyze the change of SDF-1 and then discuss its mechanism.

RESULTSCompared with control group, BaCl2, CsCl could increase EPC adhesion and migration to same extent. Moreover, the gene expression of SDF-1, PGI2 was significantly up-regulated and the production of SDF-1 increased evidently. Furthermore, the mechanism of SDF-1 secretion increasing mainly was associated with eNOS signaling pathways.

CONCLUSIONBa2+ and Cs+ play important roles in increasing EPCs functions, such as adhesion, migration and secretion.

Animals ; Barium Compounds ; pharmacology ; Cells, Cultured ; Cesium ; pharmacology ; Chemokine CXCL12 ; metabolism ; Chlorides ; pharmacology ; Endothelial Cells ; cytology ; Enzyme-Linked Immunosorbent Assay ; Potassium Channels, Inwardly Rectifying ; antagonists & inhibitors ; physiology ; Rats ; Stem Cells ; cytology

AIMTo study the effect of ZD7288 on synaptic transmission in the pathway from perforant pathway (PP) fibers to CA3 region in rat hippocampus.

METHODSThe extracellular recording technique in vivo was used to record the CA3 region field potentials. High-performance liquid chromatography (HPLC) with fluorescence detection was applied to measure the content of amino acids in hippocampal tissues. The effect of ZD7288 and CsCl on the amplitudes of population spike (PS) in CA3 region evoked by stimulation (0.5 Hz) of the perforant pathway (PP) fibers, and the content of amino acids in hippocampal tissue were observed.

RESULTSMicroinjection of ZD7288 (20, 100 and 200 nmol) and CsCl (1, 5 and 10 micromol) into CA3 region decreased the population spike (PS) amplitudes in a dose-dependent manner. The inhibitory effects appeared at 5 min after microinjection and lasted at least 90 min. In those rats treated with ZD7288 (100 nmol), the contents of glutamate, aspartate, glycine and GABA decreased significantly as compared to those of saline control (all P < 0.01, except P < 0.05 for that of glycine). A similar decrease in the contents of amino acids was observed when the rats were microinjected with CsCl (5 micromol). CONCLUSION; ZD7288 could obviously inhibit synaptic transmission in the pathway from PP fibers to CA3 region in rat hippocampus, and this action of ZD7288 may be associated with altered contents of amino acids.

Amino Acids ; metabolism ; Animals ; Cesium ; pharmacology ; Chlorides ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; Hippocampus ; metabolism ; physiology ; Male ; Microinjections ; Perforant Pathway ; physiology ; Pyrimidines ; administration & dosage ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Synaptic Transmission ; drug effects