Bufalin activates chloride channels in poorly differentiated nasopharyngeal carcinoma cells

AIM: To investigate the activation of chloride channels induced by bufalin and the properties of the channels in poorly differentiated nasopharyngeal carcinoma (CNE-2Z) cells. METHODS: The technique of whole-cell patch clamp was used to record the chloride currents and to analyze the characteristics of the currents in CNE-2Z cells.RESULTS: A chloride current was slowly activated by extracellular application of bufalin (1 μmol/L). The activation of the current was slower than that of the volume-activated chloride current, with an activation latency of(12.1±6.4) min. The reversal potential of the current was close to the calculated Cl^- equilibrium potential (E_Cl =-0.9 mV). The chloride current was outward-rectified and did not show significant time-dependent or voltage-dependent inactivation. The chloride channel blocker tamoxifen completely inhibited the outward and inward currents. The current was also completely inhibited by extra-cellular application of 47% hypertonic solution. CONCLUSION: Bufalin activates chloride channels and induces a chloride current in CNE-2Z cells. Compared with the volume-activated chloride current in CNE-2Z cells, the activation latency of the bufalin-induced current is longer and the outward rectification is more obvious.     

Chloride currents activated by cisplatin in poorly differentiated naso-pharyngeal carcinoma cells are not Ca2+-activated chloride currents

AIM：To investigate the type of chloride channel activated by cisplatin in poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells). METHODS：The technique of whole-cell patch-clamp was used to investigate the role of Ca2+ in the activation of cisplatin-activated chloride currents and to analyze the effect of hypertonic stress on these currents in CNE-2Z cells. RESULTS：Chloride currents were induced when the cells were exposed to the calcium-free cisplatin solution, showing the similar density to the currents induced by cisplatin with the presence of extracellular calcium. However, the latency and the peak time of cisplatin-activated currents in the absence of extracellular calcium were prolonged. The activation of cisplatin-activated chloride currents was insensitive to the depletion of intra- and extracellular calcium. Calcium channel antagonist nifedipine had no effect on the cisplatin-activated chloride currents, while hypertonic solution completely inhibited those currents. CONCLUSION：The cisplatin-activated chloride currents are independent on intra/extracellular calcium. The chloride channels activated by cisplatin are not calcium-activated chloride channels, but are probably volume-sensitive chloride channels.     

Inhibition of cell proliferation and arrest of cell cycle progression by blocking Cl- channels in nasopharyngeal carcinoma cells

AIM: The roles of Cl^-channels in regulatory volume decrease (RVD), cell proliferation and cell cycle progression in nasopharyngeal carcinoma cells (CNE-2Z) were investigated. METHODS: Image analysis of living cells was used to detect the volume changes following exposure to hypotonic solutions. Cell viability was determined by the trypan blue assay. MTT method was applied to detected cell proliferation. The effect of the blocker on the cell cycle distribution was monitored by the flow cytometry. RESULTS: 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) inhibited RVD and cell proliferation in a dose-dependent manner. NPPB at the concentration of 100 μmol/L arrested cells in G₁ phase (G₁ population increased from 54% to 71% at 48 h after treatments), but did not significantly alter cell viability. CONCLUSION: Block of chloride channels suppressed cell proliferation by arresting cells in G₁ phase. The results suggest that activation of Cl^-channels and RVD is necessary for facilitating cells to proceed to the S phase from G₁ phase and maintaining cell proliferation.     

Role of intracellular free Ca2+ concentration in the regulation of calcium-activated chloride channels in rat pulmonary artery smooth muscle cells

AIM: To investigate the role of intracellular free Ca²⁺ concentration (［Ca²⁺］_i) in the regulation of calcium-activated chloride (Cl_Ca) channels in pulmonary artery smooth muscle cells (PASMCs) of rats under normoxic, acute and chronic hypoxic conditions. METHODS: Acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe ［Ca²⁺］_i of rat PASMCs in normal and chronic hypoxic condition. The influences of Cl_Ca channels on PASMCs proliferation were assessed by MTT assay. RESULTS: (1) The Cl_Ca channel blockers niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) produced inhibitory effects on acute hypoxia-evoked contractions in pulmonary artery. (2) Under chronic hypoxic condition, ［Ca²⁺］_i was increased. In normoxic condition, ［Ca²⁺］_i was (123.63±18.98) nmol/L, and in hypoxic condition, ［Ca²⁺］_i was (281.75±16.48)nmol/L (P<0.01). (3) In normoxic condition, ［Ca²⁺］_i had no significant change and no effect on Cl_Ca channels was observed (P>0.05). (4) Chronic hypoxic increased ［Ca²⁺］_i which opened Cl_Ca channels. The NFA and IAA-94 blocked them and decreased ［Ca²⁺］_i from (281.75±16.48)nmol/L to (117.66±15.36)nmol/L (P<0.01). (5) MTT assay showed that in chronic hypoxic condition NFA and IAA-94 decreased the value of absorbing light degree (A value) from 0.459±0.058 to 0.224±0.025 (P<0.01). CONCLUSION: Hypoxia increased ［Ca²⁺］_i which opened Cl_Ca channels and had a positive-feedback to ［Ca²⁺］_i. This may play an important role in hypoxic pulmonary hypertension. In chronic hypoxic condition, Cl_Ca channel may play a role in the regulation of PASMCs proliferation.     

Altered expression of G protein-coupled inwardly rectifier potassium channels (GIRK) subunit 1 and 2 in hippocampus of chronic temporal epileptic rats induced by kainic acid

AIM: G protein-coupled inwardly rectifier potassium (GIRK) channel are distributed widely in mammalian brain. In CNS, GIRK 1/2 seems to be the predominant heterotetramers which play a pivot role in the regulation of the excitability of neurons and may contribute to the resting potential by leading to a hyperpolarization of membrane potential and reduction of the action potential frequency. In the context, the Weaver mouse is the first neurological abnormality directly linked to a genetic point mutation in the GIRK2 protein which includes spontaneous seizure. GIRK2 knock out mice showed normal development but more susceptible than normal mice to seizure induced by GABA antagonist. Here, we report that the mRNA and protein expression of GIRK subunit 2 is altered in kainic acid(KA)-induced epileptic rat hippocampus. METHODS: Rats were injected with kainate 14 mg/kg intraperitoneally to establish an acute and chronic temporal lobe epilepsy model. At chronic spontaneous seizure stage, by using of in situ hybridization, immunocytochemistry and Western blotting, the GIRK 1,2 mRNA and protein were analyzed quantitatively in the dentate gyreus, CA1, CA3 regions of hippocampus. RESULTS: GIRK1,2 mRNA and proteins were expressed abundantly in all regions of hippocampus. KA induced seizures and caused a significant increase in GIRK2 mRNA abundance and immunoreacitivity; only GIRK1 mRNA was increased significantly, but no difference was found by Western blotting protocol. CONCLUSION: GIRK1,2 mRNA and protein expression in the hippocampus of epileptic rat brain is up-regulated, which may be an adaptive response to over-excitability of neuron networks and prevent the over-excitability spread in hippocampus (DG-CA3-CA1).     

Effects of obstructive jaundice on enterocyte chloridion secretion in rats

AIM: To investigate the effects of obstructive jaundice (OJ) on enterocyte chloridion secretion in rats. METHODS: The OJ model of rats was set up. The rats were divided into OJ1 group and OJ2 group randomly. The animals in OJ1 group were sacrificed by exsanguination 7 days after operation and the rats in OJ2 group were sacrificed 14 days after operation. The Cl^- concentration in peripheral blood was detected. The epithelial cells in the terminal ileum mucosas were cultured in vitro. The concentration of intracellular Cl^- was detected by fluorospectrophotometry. The Cl^- current was measured by whole-cell patch clamp experiments. Western blotting was used to examine the change of voltage-gated Cl^- channel 2 protein (ClC-2) and the images were analyzed by image analysis system and statistical software quantitatively. RESULTS: The serum Cl^- concentration in OJ1 group and OJ2 group were obviously lower than that in control group . The intracellular Cl^- relative concentrations in OJ1 group and OJ2 group were higher than that in control group (3.14±0.38 and 3.55±0.47 vs 2.69±0.41,both P<0.05). The Cl^- current of normal epithelial cells displayed outward rectification, and transformed negative value into positive value following cell membrane depolarization. The Cl^- current was (-15.45±7.56) pA/pF and (5.85±0.81) pA/pF when voltage was -80 mV and 80 mV. The ranges of upgrade of absolute values of average electric current density in OJ groups were lower than those in control group (all P<0.05). Western blotting analysis showed that the ClC-2 protein generated a broad band about 90 kD. The average gradations of Western blotting images were lower in OJ groups than that in control group (0.20±0.04 and 0.19±0.06 vs 0.27±0.06, both P<0.05). However, no difference between the 2 OJ groups was observed (P>0.05). CONCLUSION: The chloridion secretion of intestinal epithelium is restrained in rats with OJ. The concentration gradient between exterior and interior of epithelial cells is decreased, and the Cl^- outward current is reduced. All of above are concerned with downregulation of ClC-2 protein in cell membrane.     

Effect of nitric oxide on adiponectin-induced inhibition of platelet aggregation in hyperlipidemia rats

AIM: To investigate the mechanism that adiponectin inhibits platelet aggregation via nitric oxide (NO) signaling pathway. METHODS: Adult rats were fed with normal or high-fat diet for 14 weeks. Their platelets were immediately isolated and treated with or without recombinant full-length adiponectin (rAPN). The platelet aggregation, NO and superoxide production, endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) expression, and antioxidant capacity were determined. RESULTS: Treatment with rAPN inhibited platelet aggregation induced by hyperlipidemia (P<0.05). Interestingly, total NO, a crucial molecule depressing platelet aggregate and thrombus formation, was significantly reduced, rather than increased in rAPN-treated platelets. Treatment with rAPN significantly decreased superoxide production by 62% (P<0.05) and increased antioxidant capacity by 38% (P<0.05) in hyperlipidemic platelets. Importantly, hyperlipidemia-induced reduction of eNOS phosphorylation and increase in iNOS expression were markedly reversed by rAPN treatment (P<0.05 and P<0.01, respectively). CONCLUSION: Adiponectin is an adipokine that inhibits platelet aggregation by enhancing eNOS activation and attenuating oxidative/nitrative stress including blockage of iNOS expression and superoxide production.     

氯化钙对始花期辣椒抗冷性的影响

用不同浓度的氯化钙溶液对始花期辣椒进行处理,研究了氯化钙对缓解始花期辣椒低温胁迫的影响。研究结果表明,低温环境下,氯化钙降低了辣椒叶片中的电解质渗透率和MDA含量,增加了POD、SOD活性和Pro含量,在一定程度上缓解了低温伤害,其中20mmol/L氯化钙的作用效果最好。     

Effect of anti-CD59 on CVF-induced platelet activation

AIM:To study the reactions of human platelet to active complement and the effects of anti-CD59 on human platelet activation induced by complement.METHODS:By applying CVF to activate complement, the platelet aggregation and release reactions induced by activated complement with or without appling anti-CD59 with different doses to block the complement modulative protein CD59 in healthy individuals, were observed.RESULTS:CVF induced platelet release and significant and lasting metamorphosis in healthy individuals, but platelet aggregation was not observed. CVF-induced platelet metamorphosis showed positive linear correlation to lg concentration of CVF (r=0.970. P<0.01. n=36). Anti-CD59 enhanced CVF-induced platelet shape change with a dose-dependent manner. The max enhancive ratio of platelet shape change was 1.36(P<0.01). Anti-CD59 enhanced platelet ATP release induces by CVF.CONCLUSION:Complement activated by CVF induces significant and lasting platelet metamorphosis and release reaction, but dose not induce platelet aggregation in healthy adult males. Anti-CD59 promotes the platelet reactions induced by active complement.     

Study on biological activity of thrombopoietinⅡ in vivo

AIM: To investigate the biological activity of thrombopoietin Ⅱ(TPOⅡ) in vivo, which consists of two new kinds of ligand binding with thrombopoietin receptor. METHODS: Purified ligandⅠof TPOⅡ, artificial compound ligandⅡ of TPOⅡand rhTPO were injected into purebred Babl/c mice respectively in 7 days by intraperitoneal injection once for a day. Then the biological activity of TPOⅡ was analyzed by measuring peripheral platelet counts by the end of the seventh day. RESULTS: On the seventh day, the platelet counts of mice treated by ligandⅠof TPOⅡ were higher than that in the negative control group(P＜0.05), while not significantly different from the platelet counts of mice treated by rhTPO(P＞0.05). On the fourteenth day, the platelet counts increased in two all experimental groups of TPOⅡcompared with negative control group(P＜0.01), while not significantly different from the platelet counts of mice treated by rhTPO too(P＞0.05). Moreover the platelet counts of mice in two experimental groups of TPOⅡ and the positive group showed increase with experimental days. CONCLUSION: The purified ligandⅠof TPOⅡ had obvious activity in increasing platelet production, which is not different from the effect of rhTPO.     

Effects of tamoxifen on volume-activated Cl- currents of nasopharyngeal carcinoma cells at different stages of the cell cycle

AIM:To investigate the inhibitory effects of Cl- channel blocker, tamoxifen, on volume-activated Cl- currents of nasopharyngeal carcinoma cells (CNE-2Z cells) in G₁ and S phases. METHODS:Highly synchronous cells in G₁ phase and S phase were obtained by the serum starvation and the double-block techniques. The whole-cell patch clamp technique was used to observe the effects of tamoxifen on volume-activated Cl- currents and to analyze the anion permeability of volume-activated Cl- channels. RESULTS:47% hypotonic stimulation activated a Cl- current in the nasopharngeal carcinoma cells at the cell cycle stage G₁ phase and S phase. Tamoxifen at concentration of 10 to 30 μmol/L completely inhibited the current. However, the time needed to completely inhibit the current was dose-dependent and was different between G₁ phase and S phase. The time needed to completely inhibit the current was shorter in G₁ cells than that in S phase cells. The anion permeability sequence of the volume-activated Cl- channel was I->Cl->gluconate in both G₁ phase and S phase cells. The permeability of G₁ phase cells to I- was higher than that in S phase cells, but to gluconate was lower than that in S phase cells. CONCLUSIONS:The density of the volume-activated Cl- current, the anion permeability of the channel and the sensitivity of the current to tamoxifen were different between the CNE-2Z cells in G₁ phase and those in S phase. The results suggest that the expression of tamoxifen-sensitive, volume-activated chloride channels is differentiated at different stages of the cell cycle.     

Potassium channels in vascular smooth muscle and essential hypertension

Essential hypertension (EH) is characterized by an increased total peripheral resistance. There are four types of potassium channels in vascular smooth muscle cells, including Kca, Kv, Kir, KATP, which play an important role in regulating the diameter of vascular. The change of potassium channels may have something to do with the pathogenesis of hypertension. This article reviews the characters of potassium channels and their roles in EH.     

Volume-activated chloride currents in human acute lymphoblastic leukemia Molt4 cells

AIM: To characterize the chloride current activated by extracellular hypotonic stress in human acute lymphoblastic leukemia Molt4 cells.METHODS: The technique of whole-cell patch clamp recording was used to detect the volume-activated Cl^- current in Molt4 cells. The characteristics of the current were investigated.RESULTS: The background Cl^- current was weak and stable under isotonic condition. However, a large Cl^- current was induced by exposure of the cells to 47% hypotonic solution. The current showed a characteristic of outward rectification. No voltage-dependent inactivation and time-dependent inactivation were observed. The current was sensitive to the change of cell volume and was inhibited by extracellular hypertonic solution. Extracellular tamoxifen, which is one of the chloride channel blockers, significantly inhibited the current. The effects of tamoxifen were almost equal for both inward and outward currents (P>0.05).CONCLUSION: There are volume-activated chloride channels on the cell membrane of Molt4 cells. Exposure of the cells to a hypotonic solution activates the chloride channels and induces a volume-activated chloride current. The volume-activated Cl^- channels are sensitive to tamoxifen in Molt4 cells.     

Role of Cl- in regulatory volume decrease of nasopharyngeal carcinoma cells

AIM:To clarify the role of Cl^- in regulatory volume decrease (RVD) of nasopharyngeal carcinoma cells (CNE-2Z).METHODS:Analysis of living cell images was used to detect the volume changes following exposure to hypotonic solution. Iron replacement and block of iron channels were also applied in the present study. RESULTS:Extracelluar hypotonic treatment made the cells swell and induced RVD. The RVD was correlated positively to the swelling in the range of 160-230 mOsmol/L. Substitution of gluconate for Cl^- in perfusing solutions markedly increased RVD. Depletion of cellular Cl^- abolished, and chloride channel blockers inhibited RVD. CONCLUSION:Cl^- is the key iron to establish the RVD in CNE-2Z cells. Activation of Cl^- channels and Cl^- efflux are the major mechanisms of RVD.     

外源氯对番茄幼苗生长及养分吸收、利用的影响

 研究了不同水平外源氯处理对番茄(Lycopersicon esculentum L. ) 干物质积累、叶绿素含量、Cl^- 、NO₃^- 、氮、磷、钾、钙和镁含量的影响。结果表明: 一定范围( 6.25～100 nmol·L ^{- 1} ) 的外源氯处理, 不降低番茄生物量甚至促进生物量, 并可明显提高番茄幼苗对钾、镁、磷的吸收和氮素利用效率, 而200、300 mmol·L ^{- 1} Cl^- 处理的幼苗干物质积累极显著下降。     

Molecular mechanism underlining ethanol-induced chloride currents in nasopharyngeal carcinoma cells

AIM：To study the effects and mechanisms of ethanol on chloride channels in poorly differentiated nasopharyngeal carcinoma CNE-2Z cells. METHODS：The effect of ethanol on the cell growth was analyzed by MTT assay. The technique of whole-cell patch-clamp was used to detect the chloride current. The characteristics of the chloride current were analyzed by using the chloride channel blockers. The siRNA technique was used to analyze the molecular basis of the ethanol-sensitive chloride channels. RESULTS：Under isotonic conditions, the background current was weak and stable. Ethanol at concentrations of 0.17~170 mmol/L activated a chloride current in a concentration-dependent manner (an inverted U-shape), with a maximum effect at the concentration of 17 mmol/L. The currents showed obviously outward rectification and were susceptible to extracellular hypertonicity and the chloride channel blocker, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). ClC-3 siRNA obviously decreased the currents activated by ethanol. CONCLUSION：Extracellular ethanol induces chloride currents through activating the ClC-3 chloride channels.     

Effect of polymorphonuclear leukocytes on washed platelet aggregation

AIM:To investigate the effect of non-activated or activated polymorphonuclear leukocytes(PMN) on washed platelet aggregation. METHODS:Born's method was used to determine platelet aggregation.RESULTS:non-activated PMN (5×10⁹ cells/L) significantly suppressed washed platelet aggregation induced by ADP or arachidonic acid. Aspirin enhanced this inhibition. N-formyl-methiongl-leucy-phenylalanine (fMLP)-or platelet-activating factor (PAF)-stimulated PMN strongly induced platelet aggregation, and the induction effect of PMN suspension was more active than that of PMN supernatant. Aspirin had no significant inhibitory effect on platelet aggregation induced by fMLP-or PAF-activated PMN. CONCLUSIONS:Different conditions of PMN (activated or non-activated) had the nearly opposite action on normal platelet reactivity. Briefly, non-activated-PMN inhibited platelet reactivity, whereas activated PMN stimulated it.     

The role of ATP- sensitive K+ channels in the cardiovascular system

15 years ago, Noma first applicated the patch clamp technique to CN-treated mamammalian heart cells, e-vealed specific K⁺ channels which were depressed by intracellular ATP at level greater than 1 mNl.He suggested the ATP-sensitive channels are important for regulating cellular energy metabolism in the control of membrane exitability. Recent studies supported the hypothesis and suggested that ATP- sensitive channels play an important role in hypoxia, myocardial ischemia/reperfusion and is chemic preconditioning     

Influence of Nao-xue-bao on blood coagulation and platelet aggregation in rats

AIM and METHODS:To study the effect of Nao-xue-bao at three different doses on blood coagulation,platelet aggregation by observing the changes in activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin-Ⅲ(AT-III), fibrinogen(Fng), plasminogen(Plg) and platelet aggregation(PAG). RESULTS:Compared with thrombosis group, the rats treated with Naoxuebao showed that the plasma APTT,PT were longer, and the activity of AT-III was increased. The content of Fng was reduced, TT was longer, there was a negative correlation between Fng and TT. Furthermore PAG-1, PAG-5 and PAG-M were reduced. CONCLUSION: Nao-xue-bao could inhibit thrombosis in different keys of blood coagulation.     

The influence of oral simvastatin and vitamine-E on serum LDL oxidation and platelet activation in coronary artery disease patients

AIM: Using simvastatin and vitamine E (Vit-E) treatment to coronary artery disease (CAD) patients with low HDL, to investigate the relationship between Ox-LDL, platelet activation and HDL. METHODS: 40 CAD patients with low HDL were divided into two groups (A and B): A group oral simvastatin, B group oral simvastatin and Vit-E. The level of serum Ox-LDL, TXB₂ and GMP-140 were measured before and after treatment. The relationship between Ox-LDL, TXB₂, GMP-140 and HDL were analysed. RESULTS:The level of serum HDL was significantly increased in A and B group after treatment and attained normal level. The level of serum Ox-LDL, TXB₂ and GMP-140 were decreased significantly after simvastatin and Vit-E treatment and neared normal. CONCLUSIONS:This study confirmed that HDL can effectively refrain LDL oxidation. It also revealed that Vit-E and simvastatin treatment were more effectively refrained platelet activation by increasement of HDL and decreasement of Ox-LDL.