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

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Anthracenes ; pharmacology ; Chloride Channels ; metabolism ; Chlorides ; agonists ; antagonists & inhibitors ; metabolism ; Culture Media ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; metabolism ; Humans ; Hypotonic Solutions ; metabolism ; pharmacology ; Indoles ; pharmacology ; Ion Transport ; drug effects ; Maleimides ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; pharmacology ; Primary Cell Culture ; Protein Kinase C ; metabolism

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

OBJECTIVETo study the salt stress tolerance of Hongxinju, Huangju and F1 seedlings from orthogonal and reciprocal cross under different salt treatments. Grope for transmissibility of salt tolerance between parents and F1 seedlings, and relativity between flavone, chlorogenic acid contents and salt tolerance.

METHODThe materials were put in 5 different concentrations of Hoagland nutrient solution (0, 40, 80, 120, 160 mmol x L(-1)) containing NaCl, keeping grads while raising the consistency of NaCl day by day. The injured leaf area per plant, proline, betaine, MDA, flavones and chlorogenic acid contents were measured and analyzed after treatment.

RESULTAs NaCl concentration was below 120 mmol x L(-1), the salt tolerance of Hongxinju was higher than that of Huangju, the salt tolerance of Hongxinju x Huangju higher than that of parents, the salt tolerance of Huangju x Hongxinju was at the level of parents. As NaCl concentration between 120 to 160 mmol x L(-1), the salt tolerance of Huangju was higher than that of Hongxinju, the salt tolerance of Huangju x Hongxinju higher than that of parents and the salt tolerance of Hongxinju x Huangju was at the level of parents.

CONCLUSIONSalt tolerance of F1 is more influenced by female parent, relativity showed between flavonoids, chlorogenic acid contents and salt tolerance.

Breeding ; China ; Chlorides ; metabolism ; Chrysanthemum ; genetics ; growth & development ; physiology ; Salt-Tolerance ; Seedlings ; genetics ; growth & development ; physiology

OBJECTIVETo explore the effect of MnCl(2) on the mitochondrial function of human lung cells, and to study the changes of protein expression level of nuclear respiratory factor-1 (NRF-1) in mitochondrial dysfunction induced by MnCl(2).

METHODSThe effects of MnCl(2) on cell survival rate were assessed by the reductions of tetrazolium dye (MTT) in cultured cell lines 16HBE and A549 cells. All tested16HBE and A549 cells were incubated with different concentrations of MnCl(2). The permeability transition pore (PTP) of mitochondria, mitochondrial membrane potential and the inhibition rate of mitochondrial enzymes as indicators of mitochondrial damage were measured by fluorescent spectrometry and MTT assay, respectively. Apoptosis was determined by flow cytometry. Protein levels of NRF-1 and mtTFA were measured by Western blot assay.

RESULTSMnCl(2) decreased the survival rate of the two cell lines. The IC(50) of 16HBE and A549 cells were 1.91 mmol/L and 1.98 mmol/L, respectively. MnCl(2) caused a concentration-dependent decrease of mitochondrial enzymes and the inhibition rate of mitochondrial enzymes of the two cell lines induced by 1.00 mmol/L MnCl(2) were (52.8 ± 5.4)% and (50.6 ± 2.2)%, respectively. The PTP opening increased in MnCl(2)-treated cells in a dose- and time-dependent manner. Compared with the control group, mitochondrial membrane potential in the two cell lines was decreased by MnCl(2), by (7.9 ± 3.0)%, (26.2 ± 2.2)% and (27.8 ± 4.1)% in the 16HBE cells, and (4.7 ± 1.0)%, (14.9 ± 2.4)% and (27.5 ± 1.2)% in the A549 cells. Increased apoptosis rates of the two cell lines were induced by 1.00 mmol/L MnCl(2), (12.3 ± 1.9)% and (6.0 ± 0.4)%, respectively. The results of Western blot assay revealed that the protein levels of NRF-1 and mtTFA were decreased in manganese-treated cells in a dose-dependent manner, with a significant difference compared with that of the control cells (P < 0.05).

CONCLUSIONMnCl(2) induces mitochondrial dysfunction in 16HBE and A549 cells, and decreases the expression level of nuclear respiratory factor-1 (NRF-1), indicating that NRF-1 may play an important role in mitochondrial dysfunction.

Apoptosis ; drug effects ; Bronchi ; cytology ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cells, Cultured ; Chlorides ; administration & dosage ; toxicity ; DNA-Binding Proteins ; metabolism ; Dose-Response Relationship, Drug ; Epithelial Cells ; cytology ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Manganese Compounds ; administration & dosage ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; Mitochondrial Proteins ; metabolism ; Nuclear Respiratory Factor 1 ; metabolism ; Transcription Factors ; metabolism

The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein-mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-transfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors, encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.
Animals ; Cells, Cultured ; Chlorides ; metabolism ; Codon ; genetics ; Cricetinae ; Cystic Fibrosis ; therapy ; Cystic Fibrosis Transmembrane Conductance Regulator ; genetics ; metabolism ; DNA, Complementary ; genetics ; Dependovirus ; genetics ; Genetic Therapy ; Genetic Vectors ; Humans ; Inteins ; physiology ; Kidney ; cytology ; Protein Processing, Post-Translational ; Recombinant Proteins ; genetics ; metabolism ; Trans-Splicing ; Transfection

The effect of NaCl plus 3% chitosan on the systolic blood pressure of spontaneously hypertensive rats (SHR) were evaluated and compared with NaCl plus KCl (NaCl, 49.36% + KCl 49.36%) and chitosan or NaCl treatment alone. In SHR, administration of NaCl plus chitosan (44 mM Na/day) for two months significantly decreased the systolic blood pressure greater than of NaCl plus KCl and NaCl alone. NaCl plus chitosan resulted, though not statistically significant, in decreased urinary Na+ excretion and decreased blood urea nitrogen levels. Urinary creatinine of NaCl plus chitosan was slightly decreased compared to 3 treated groups. Serum electrolytes levels, however, remained unchanged. The combination of NaCl and chitosan may be superior to the conventional use of NaCl plus KCl or NaCl alone in the prevention of hypertension. Even though these supplementary diets have demonstrated potential anti-hypertensive effects in the experimental animal model, further research is needed before any recommendations can be made.
Angiotensin I/blood ; Angiotensin II/biosynthesis ; Animals ; Blood Pressure/*drug effects/physiology ; Blood Urea Nitrogen ; Body Weight/drug effects ; Chitosan/*administration & dosage ; Chlorides/blood/urine ; Creatinine/urine ; Heart/physiology ; Histocytochemistry ; Hypertension/*prevention & control ; Kidney/physiology ; Male ; Potassium/blood/urine ; Potassium Chloride/administration & dosage ; Random Allocation ; Rats ; Rats, Inbred SHR ; Sodium/blood/urine ; Sodium Chloride, Dietary/*administration & dosage ; Systole/drug effects/physiology

gamma -aminobutyric acid (GABA) is an inhibitory neurotransmitter in adult mammalian central nervous system (CNS). During CNS development, the role of GABA is switched from an excitatory transmitter to an inhibitory transmitter, which is caused by an inhibition of calcium influx into postsynaptic neuron derived from release of GABA. The switch is influenced by the neuronal chloride concentration. When the neuronal chloride concentration is at a high level, GABA acts as an excitatory neurotransmitter. When neuronal chloride concentration decreases to some degree, GABA acts as an inhibitory neurotransmitter. The neuronal chloride concentration is increased by Na+-K+-Cl(-)-Cl(-) cotransporters 1 (NKCC1), and decreased by K+-Cl(-) cotransporter 2 (KCC2).
Animals ; Calcium Signaling ; physiology ; Cell Differentiation ; physiology ; Central Nervous System ; cytology ; embryology ; metabolism ; Chlorides ; metabolism ; Humans ; Neural Inhibition ; physiology ; Neurons ; cytology ; metabolism ; Synapses ; metabolism ; Synaptic Transmission ; physiology ; gamma-Aminobutyric Acid ; metabolism ; physiology

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

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