Effects of ClC-3 over-expression on structure and function of thyroid in mice / 中国病理生理杂志

AIM:To study the effect of ClC-3 gene over-expression on thyroid structure and function in mice. METHODS:Three-months-old FVB mice were used to study the difference of thyroid structure and function between wild-type(WT)mouse and ClC-3 transgene mice.The expression and distribution of ClC-3 in the thyroid of mice were deter-mined by the methods of qPCR,Western blot and immunofluorescence.Behavioral monitoring was performed on the daily activities of mice.Serum concentrations of total triiodothyronine(TT3), total thyroxine(TT4)and thyrotropin(TSH) were measured by ELISA.RESULTS:Compared with the WT group,the expression of ClC-3 in the thyroid of ClC-3 trans-gene group was significantly increased(P<0.05).The thyroid gland showed obvious hyperplasia and the folliculi glandu-lae thyreoideae was significantly bigger in ClC-3 transgene mice(P<0.05).The weight loss was increased in ClC-3 trans-gene mice(P<0.05).The expression of TT3 and TT4 were significantly higher than that of WT group(P<0.05),but the change of TSH was not obvious.CONCLUSION:ClC-3 over-expression results in thyroid hyperplasia and thyroid hor-mone secretion.This study suggests that ClC-3 is likely to be involved in the synthesis of thyroid hormones.

Role of acetylcholine in gelsenicine-induced death in mice / 生理学报

The aim of this study was to investigate the relationship between the acetylcholine concentration in the blood and gelsenicine-induced death in mice. Kunming mice were given intraperitoneal injections of normal saline, gelsenicine or different doses of acetylcholine chloride. Atropine was given to the mice which received gelsenicine or medium dose acetylcholine chloride injection. The blood was sampled immediately when the mice died or survived for 20 min after injection. The acetylcholine concentration and acetylcholinesterase activity in the blood were measured by the testing kits, and the mortality was calculated and analyzed. The results showed that half lethal dose of gelsenicine (0.15 mg/kg) reduced the acetylcholinesterase activity and increased the blood acetylcholine concentration. The blood acetylcholine concentration of the dead mice in the gelsenicine group was increased to 43.0 μg/mL (from 31.1 μg/mL in the control), which was lower than that (53.9 μg/mL) of the dead mice in the medium dose acetylcholine chloride group, but almost equal to that (42.7 μg/mL) of the survival mice in the medium dose acetylcholine chloride group. Atropine could successfully rescue the mice from acetylcholine poisoning, but its efficiency of rescuing the mice from gelsenicine intoxication was weak. These results suggest that gelsenicine can inhibit acetylcholinesterase activity and increase blood acetylcholine concentration, but the accumulation of acetylcholine may not be the only or main cause of the death induced by gelsenicine in mice.

Fingerprint of X-ray diffraction of Tibetan medicine dairy Nanhanshuishi and its application in processing by microwave / 中国中药杂志

The characteristic fingerprint of conventional dairy Nanhanshuishi was established by X-ray diffraction (XRD), based on similarity of caculation on public peaks by MATLAB software, and the feasibility of new dairy technology of microwave method was explored between XRD and the dissolution rate in artificial simulation gastric juices. The result showed that similarity of shared peak in XRD of conventional dairy Nanhanshuishi was > 95%, This XRD characteristic fingerprint of conventional dairy Nanhanshuishi had strong specificity, could be used to provide a reference for identification and quality evaluation. This study also showed that the similarity of microware dairy products and conventional dairy products was good, and the sample of microwave 15 min was the best, and new dairy method by the microwave could replace the traditional method.

Ursolic acid activates chloride channels and decreases cell volume in poorly differentiated nasopharyngeal carcinoma cells / 生理学报

The present study aimed to investigate the effects of ursolic acid on the chloride channels and cell volume in nasopharyngeal carcinoma cells (CNE-2Z). The whole-cell patch clamp technique was used to detect the current, and cell imaging technique was applied to measure cell volume. The properties of the currents induced by ursolic acid were investigated by changing the extracellular osmotic pressure, replacing the extracellular anions and applying chloride channel blockers. The results showed that, under isotonic conditions, the background current was weak and stable. When perfusing the cells with ursolic acid (100 nmol/L), a large current (-59.86 pA/pF ± 4.86 pA/pF at -80 mV, 78.92 pA/pF ± 6.39 pA/pF at +80 mV) was induced. The chloride current showed outward rectification and negligible time- and voltage-dependent inactivation. The reversal potential (-4.83 mV ± 0.30 mV) of the current was close to the calculated equilibrium potential for Cl⁻ (-0.9 mV). The permeabilities of the channel to different anions were ranked in order as follows: Cl⁻ = I⁻ > Br⁻ > gluconate. Hypertonic solutions inhibited the current induced by ursolic acid. The chloride channel blockers, tamoxifen (20 μmol/L) and 5-nitro-2-(3-phenylpro-pylamino) benzoic acid (NPPB, 100 μmol/L), suppressed the current. Furthermore, ursolic acid decreased the cell volume by (11.78 ± 1.20)% in 1 h, and the effect was inhibited by NPPB. These results suggest that ursolic acid can activate chloride channels, resulting in outflow of Cl⁻ and decrease of cell volume in nasopharyngeal carcinoma cells.

ClC-3 siRNA inhibits regulatory volume decrease in nasopharyngeal carcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of ClC-3 chloride channels in regulatory volume decrease (RVD) of nasopharyngeal carcinoma (NPC) CNE-2Z cells.?</p><p><b>METHODS</b>ClC-3 siRNA was transfected into CNE-2Z cells in the presence of the transfection reagent HiPerFect Reagent(TM). The transfection efficiency of ClC-3 siRNA was detected by flow cytometry. The expression of ClC-3 protein was detected by Western blotting, and the changes of cell volume in 160 mOsmol/L hypotonic solution were determined by image analysis.</p><p><b>RESULTS</b>The transfection efficiency of ClC-3 siRNA was (63.8∓3.8)% (n=3, P<0.01), and compared with the control group, ClC-3 siRNA transfection resulted in a reduction of ClC-3 expression by (60.9∓4.0)% (n=3, P<0.01). The hypotonic challege (160 mOsmol/L) caused cell swelling and induced RVD. In the control group, hypotonic solution bath for 35 min resulted in a RVD of (42.6∓2.8)% (n=20), which was significantly decreased to (10.5∓4.8)% (n=16) in ClC-3 siRNA-transfected cells, demonstrating a reduction of RVD capacity by 75.4% (P<0.01).?</p><p><b>CONCLUSION</b>The capacity of RVD is significantly reduced in CNE-2Z cells by ClC-3 chloride channel protein knock-down via ClC-3 siRNA transfection, indicating an important role of ClC-3 chloride channels in the RVD of CNE-2Z cells.</p>

Berberine activates volume-sensitive chloride channel in human colorectal carcinoma cells / 生理学报

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.

Role of chloride channels in gambogic acid-induced apoptosis of poorly differentiated nasopharyngeal carcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of chloride channels in the apoptosis of poorly differentiated nasopharyngeal carcinoma CNE-2Z cells induced by gambogic acid (GA).</p><p><b>METHODS</b>MTT assay was applied to detect the proliferation of CNE-2Z cells after GA treatment, and the cell apoptosis was detected by Hoechst 33342 staining. Whole-cell patch clamp technique was employed to record GA-activated Cl(-) currents in the cells.</p><p><b>RESULTS</b>GA inhibited the cell proliferation in a time- and concentration-dependent manner with an IC(50) of 3.1 µmol/L for a 48-h treatment. The apoptosis-inducing effect of 8 µmol/L GA was attenuated by the chloride channel blocker NPPB (100 µmol/L) and tamoxifen (20 µmol/L). GA induced an outward-rectified Cl(-) current in the cells, which was significantly inhibited by NPPB.</p><p><b>CONCLUSION</b>GA suppresses cell proliferation and induces apoptosis by activating Cl(-) channels in CNE-2Z cells, suggesting the important role of Cl(-) channels in GA-induced apoptosis.</p>

Construction of cyclin D1 recombinant plasmids and its expression in human nasopharyngeal carcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct the eukaryotic expression vectors of human cyclin D1 gene and express them in poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells).</p><p><b>METHODS</b>The full-length cyclin D1 was cloned from CNE-2Z cells by RT-PCR. The cDNA fragments were inserted into pIRES2-EGFP plasmids and pEGFP-C2 plasmids and confirmed by restriction enzyme digestion, PCR and sequencing. The recombinant vectors were transfected into CNE-2Z cells via Lipofectamine 2000, and the expression of cyclin D1 in the cells was examined by immunofluorescence and Western blotting.</p><p><b>RESULTS</b>Agarose gel electrophoresis showed a 918 bp band of the RT-PCR products, which matched the expected size. Restriction enzyme digestion, PCR and sequencing demonstrated successful construction of the recombinant vectors. CNE-2Z cells transfected with the recombinant vectors expressed cyclin D1 protein or cyclin D1-GFP protein as were verified by immunofluorescence and Western blotting.</p><p><b>CONCLUSION</b>We have cloned cyclin D1 gene and constructed its eukaryotic expression vectors that can be expressed in nasopharyngeal carcinoma cells, which may facilitate the study of the role of cyclin D1 in the development of nasopharyngeal carcinoma.</p>

Ca(2+)-dependent potassium channels play important roles in regulatory volume decrease in human nasopharyngeal carcinoma cells / 生理学报

It has been shown that cell volume regulation mechanisms play important roles in various cell functions. We demonstrated previously that volume-activated chloride channels were involved in cell volume regulation. The present study aimed to clarify the roles of various types of potassium channels in regulatory volume decrease (RVD) induced by hypotonic challenges in human nasopharyngeal carcinoma cells (CNE-2Z cells). The whole-cell patch clamp technique was used to record hypotonic challenge-induced potassium currents. During current recordings, cells were held at 0 mV and stepped to +/-46 and +/-92 mV, repeatedly. The cell volume was computed from cell diameters. The changes of cell volume were monitored and analyzed by the time-lapse imaging technique. The results showed that the exposure to 160 mOsm/L hypotonic solution caused the cells to swell by (144.5+/-4.2)%, activated a potassium current (59.2 pA/pF+/-13.3 pA/pF at 92 mV), and induced RVD. Cell volume was recovered from hypotonic challenge-induced swelling by (48.9+/-4.6)% after 20 min. The potassium current (at 92 mV) and RVD were inhibited by the calcium-dependent potassium channel blocker, clotrimazole (100 mumol/L), by (98.5+/-2.8)% and (89.3+/-4.9)%, respectively. Depletion of extracellular calcium prevented the activation of the hypotonic challenge-induced potassium current and inhibited the process of RVD. The voltage-gated potassium channel blocker, 4-AP (5 mmol/L), partially inhibited the hypotonic challenge-activated potassium currents by (66.6+/-5.3)% (at 92 mV). These results suggest that the Ca(2+)-dependent potassium channel is the main component of volume-activated potassium channels and plays an important role in volume regulation of CNE-2Z cells. The voltage-gated potassium channels may also contribute in part to the formation of the volume-activated potassium current.

Background chloride currents in fetal human nasopharyngeal epithelial cells / 生理学报

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.

Volume-activated Cl- current in migrated nasopharyngeal carcinoma cells / 生理学报

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.