Neuroprotection of chloride channel blockers against NMDA-induced apoptosis of cultured rat hippocampal neurons / 南方医科大学学报

Activation of N-methyl-d-aspartic acid (NMDA) receptor plays an important role in neuronal apoptosis induced by cerebral ischemia but the underlying mechanisms are still unclear. The present study examined the neuroprotection of three chloride blockers in an in vitro cell model of cerebral ischemia established by treatment of cultured rat hippocampal neurons with NMDA. Hoechst 33258 staining and MTT assay were used to detect neuronal apoptosis and cell viability, respectively. The neuroprotective effects of chloride channel blockers on the cell viability and neuronal apoptosis were only observed when the blockers were applied before NMDA exposure. In comparison with DIDS, SITS showed more potent protective effect in a dose-dependent manner, whereas NPPB showed no significant neuroprotective effect. The results demonstrate that pretreatment with both SITS and DIDS have protective effect against neuronal apoptosis, which is achieved by blocking both NMDA receptor and chloride channel.

The effects of chloride channel blockers on thrombocytic cytoplasmic free calcium concentration and platelet aggregation / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of chloride channels on the regulation of platelet cytoplasmic free calcium concentration ([Ca2+]i) and platelet aggregation (PAG).</p><p><b>METHODS</b>Freshly separated platelets were activated by thrombin. Chloride channel blockers DIDS or NFA and calcium channel blockers SK&F96365 or nifedipine were added to study the effects on platelet [Ca2+]i and PAG by a single reagent or the combination of reagents and find out the interactions among DIDS, NFA, SK&F96365 and nifedipine.</p><p><b>RESULTS</b>Both DIDS and NFA could inhibit the thrombin (1 U/ml) induced PAG in a dose-dependent manner, whereas had little effect on resting [Ca2+]i. As compared with the control group, DIDS, SK&F96365 and Nifedipine could significantly reduce the PAG, Ca2+ release and Ca2+ influx in thrombin activated platelet (P < 0.05). The combination of DIDS and SK&F96365 had greater effects in reducing the PAG, Ca2+ release and Ca2+ influx than either reagent alone (P < 0.05). The combination of DIDS and nifedipine also had greater effect than each alone in reducing Ca2+ release (P < 0.05). The combination of NFA and SK&F96365 weakened each other's effect on Ca2+ release (P < 0.05), while NFA and nifedipine weakened each other's effects on PAG, Ca2+ release and Ca2+ influx in thrombin activated platelet (P < 0.05).</p><p><b>CONCLUSION</b>DIDS and NFA have no effect on the resting [Ca2+]i and the leak calcium influx of platelet. DIDS can inhibit the Ca2+ release, Ca2+ influx and PAG of platelet induced by thrombin, while NFA can only inhibit the Ca2+ release. The chloride channel and calcium channel blockers have interactions in affecting resting [Ca2+]i and PAG of platelet. The opening of chloride channel can influence the cellular calcium movement of platelet.</p>

Forward-mode Na+ - Ca2+ exchange during depolarization in the rat ventricular myocytes with high EGTA

During depolarization, extrusion of Ca2+ from sarcoplasmic reticulum through forward-mode Na+ - Ca2+ exchange was studied in the rat ventricular myocytes patch-clamped in whole-cell configuration. In order to confine the Ca2+ responses in a micro-domain by limiting the Ca2+ diffusion time, rat ventricular myocytes were dialyzed with high (14 mM) EGTA. K+ current was suppressed by substituting KCl with 105 mM CsCl and 20 mM TEA in the pipette filling solution and by omitting KCl in the external Tyrode solution. Cl- current was suppressed by adding 0.1 mM DIDS in the external Tyrode solution. During stimulation roughly mimicking action potential, the initial outward current was converted into inward current, 47+/-1% of which was suppressed by 0.1 mM CdCl2. 10 mM caffeine increased the remaining inward current after CdCl2 in a cAMP-dependent manner. This caffeine-induced inward current was blocked by 1 muM ryanodine, 10 muM thapsigargin, 5 mM NiCl2, or by Na+ and Ca2+ omission, but not by 0.1 muM isoproterenol. The IapprxV relationship of the caffeine-induced current elicited inward current from -45 mV to +3 mV with the peak at -25 mV. Taken together, it is concluded that, during activation of the rat ventricular myocyte, forward-mode Na+ - Ca2+ exchange extrudes a fraction of Ca2+ released from sarcoplasmic reticulum mainly by voltage-sensitive release mechanism in a micro-domain in the t-tubule, which is functionally separable from global Cai2+ by EGTA.

Effects of Cl- channel blockers on the cardiac ATP-sensitive K+ channel

To explore whether Cl- channel blockers interact with the ATP-sensitive K+ (KATP) channel, I have examined the effect of two common Cl- channel blockers on the KATP channel activity in isolated rat ventricular myocytes using patch clamp techniques. In inside-out patches, 4,4'-diisothio-cyanatostilbene-2,2'-disulfonic acid (DIDS) and niflumic acid applied to bath solution inhibited the KATP channel activity in a concentration-dependent manner with IC50 of 0.24 and 927 muM, respectively. The inhibitory action of DIDS was irreversible whereas that of niflumic acid was reversible. Furthermore, DIDS-induced block was not recovered despite exposure to ATP (1 mM). In cell-attached and inside-out patches, DIDS blocked the pinacidil- or 2,4-dinitrophenol (DNP)-induced KATP channel openings. In contrast, niflumic acid did not block the pinacidil-induced KATP channel openings in inside-out patches, but inhibited it in cell-attached patches. DIDS and niflumic acid produced additional block in the presence of ATP and did not affect current-voltage relationship and channel kinetics. All these results indicate that DIDS among Cl- channel blockers specifically blocks the cardiac KATP channel.

The Effect of Choloride Channel on Aqueous Humor Production

Regulating the cell volume is an important factorin secretory function of epithelial cells and regulatory volume decrease (RVD) phenomenon is involved in response to the changes of cell volume and osmolarity. RVD of epithelial cells reflects cellular release of K+ and C1- through channels and K+ and C1- channels were verified in the basal membrane of the non pigmented ciliary epithelial cells. Therefore, we attempted to observe the involvement of C1- channel in aqueous humor production by performing the fluorophotometry after administration of the DIDS(4.4-diisothiocyanatostilbene-2-2-disulfonic acid), the C1 channel blocker. Ten white New Zealand rabbits, 5 for tonometry and 5 for fluorophotometry, were used. One eye was injected 2x10-4M DIDS intravitreally using microsyringe and the other eye was injected normalsaline as a control in each rabbits. Tonometry was performed before the injection and every hour after dosing for 5 hours. Fluorophotometry was performed every 30 minutes for 3 hours starting 2 hours after injection. Wilcoxon`s signed rank test was used for statistical analysis. DIDS decreased intraocular pressure by 12.5%(P<0.05) and reduced aqueous humor flow rate by 28.5%(P0.05). In conclusion, it was observed.

H2O2-induced lipid peroxidation in mitochondria of pulmonary vascular smooth muscle tissue and its modification by DFO, DMTU and DIDS.

Treatment of bovine pulmonary artery smooth muscle tissue mitochondria with H2O2 stimulated iron release, hydroxyl radical (OH) production and lipid peroxidation. Pretreatment of mitochondria with deferoxamine (DFO) and dimethyl thiourea (DMTU) prevented OH production and markedly reduced lipid peroxidation without appreciably altering iron release caused by H2O2. Simultaneous treatment of either DFO or DMTU with H2O2 significantly reduced lipid peroxidation and also prevented OH production without causing marked decrease in iron release. In contrast, addition of DFO or DMTU even 2 min after treatment of the mitochondria with H2O2 did not significantly altered iron release, OH production and lipid peroxidation. Pretreatment of the mitochondria with 4,4'-dithiocyano-2,2'-disulfonic acid stilbene (DIDS) markedly reduced lipid peroxidation without appreciably altering the increase in OH production and iron release caused by H2O2.

Protective role of anion channel blocker in lipid peroxidation caused by H2O2 in microsomes of bovine pulmonary arterial smooth muscle tissue.

The role of hydroxyl radical (OH.) in H2O2-mediated stimulation of lipid peroxidation in microsomes of bovine pulmonary arterial smooth muscle tissue and the protective effects of DIDS, the anion channel blocker have been studied. Treatment of microsomes with H2O2 (1 mM) stimulate iron release, OH. production and lipid peroxidation. Pretreatment with DFO (an iron chelator) or DMTU (a hydroxyl radical scavenger) prevents OH. production and thereby reduces lipid peroxidation without any appreciable reduction of iron release. Simultaneous treatment of either DFO or DMTU with H2O2 significantly reduces lipid peroxidation and prevents OH. production without any significant reduction of iron release. However, addition of DFO or DMTU 2 min after treatment of the microsome with H2O2 does not produce any significant reduction of lipid peroxidation, OH production and iron release. Pretreatment of microsomes with DIDS markedly reduces the stimulation of lipid peroxidation without appreciably altering the increase in OH. production and iron release caused by H2O2.

Chronic myelogenous leukemia: alterations in red cell membrane band 3 and increased IgG binding.

Chronic myelogenous leukemia (CML) is a hematologic malignancy arising from an abnormal hemopoietic stem cell. Our earlier studies have identified defects in spectrin tetramer formation and organization of cytoskeletal proteins (Basu et al., Biochim. Biophys. Acta 1988, 121-126); and decreased ankyrin binding to ankyrin-depleted vesicles in CML patients. These may lead to clustering of band 3 and increased binding of autologous IgG. This has now been explored by studying the binding of 125I-protein A to normal and CML erythrocytes. There is increased binding of 125I-protein A in CML erythrocytes compared to normal erythrocytes. Since binding of autologous IgG is responsible for removal of erythrocytes from the circulation, the above findings suggest that CML erythrocytes are likely to be prematurely removed from the circulation, accounting for anemia.