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.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid ; pharmacology ; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid ; pharmacology ; Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Bisbenzimidazole ; chemistry ; Cell Survival ; drug effects ; Cells, Cultured ; Chloride Channels ; antagonists & inhibitors ; Hippocampus ; cytology ; Microscopy, Fluorescence ; N-Methylaspartate ; pharmacology ; Neurons ; chemistry ; cytology ; drug effects ; Neuroprotective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley

AIMTo study the structure and crystal forms of chlorobenzylidine.

METHODSKarl Fischer titrimetry, FTIR, thermal analysis, single and powder X-ray diffraction were used for the studies of the structure of chlorobenzylidine and for the identification of two forms of chlorobenzylidine.

RESULTSChlorobenzylidine and its diastereoisomer have been studied in this article. They can be distinguished by their different melting points. Two crystal forms of chlorobenzylidine (form A and form B) have also been detected and studied. Form A was studied by single-crystal X-ray diffraction, it crystallized in the triclinic system, space group P1(-), with two formula units per cell, is monohydrate. Karl Fischer titrimetry, FTIR, thermal analysis and powder X-ray diffraction were used for identification of the two forms.

CONCLUSIONThe studies of structure and crystal forms of chlorobenzylidine are very useful for the clinical research and the selection of recrystallization process.

Benzylidene Compounds ; Crystallization ; Crystallography, X-Ray ; Differential Thermal Analysis ; Molecular Conformation ; Molecular Structure ; Polycyclic Compounds ; chemistry ; Stereoisomerism

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.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid ; Aqueous Humor* ; Cell Size ; Epithelial Cells ; Fluorophotometry ; Intraocular Pressure ; Manometry ; Membranes ; Osmolar Concentration ; Rabbits

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.
2,4-Dinitrophenol ; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid ; Adenosine Triphosphate ; Animals ; Baths ; Inhibitory Concentration 50 ; Kinetics ; Muscle Cells ; Niflumic Acid ; Patch-Clamp Techniques ; Rats

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.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid ; Action Potentials ; Animals ; Cadmium Chloride ; Caffeine ; Diffusion ; Egtazic Acid* ; Isoproterenol ; Muscle Cells* ; Rats* ; Ryanodine ; Sarcoplasmic Reticulum ; Tea ; Thapsigargin

BACKGROUND: It is generally accepted that morphine affords cardioprotection against ischemia/reperfusion injury. Inhibition of the mitochondrial permeability transition pore (MPTP) is considered an end target for cardioprotection. The aim of this study was to investigate the involvement of opioid receptors (OR) and MPTP in morphine-induced postconditioning (M-Post). METHODS: Isolated rat hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Hearts were treated with 1 microM morphine, with or without the OR antagonists or a MPTP opener at early reperfusion. Infarct size was measured with 2,3,5-triphenyltetrazolium chloride staining. RESULTS: There were no significant differences in cardiodynamic variables except a decrease in heart rate in the M-Post group (P < 0.01 vs. control) after reperfusion. M-Post dramatically reduced infarct-risk volume ratio (9.8 +/- 2.5%, P < 0.001 vs. 30.0 +/- 3.7% in control). This beneficial effect on infarct volume by M-Post was comparable with ischemic postconditioning (11.9 +/- 2.2%, P > 0.05). The nonspecific OR antagonist naloxone (25.7 +/- 1.9%, P < 0.01), the delta-OR antagonist naltrindole (27.8 +/- 4.3%, P < 0.05) and delta1-OR antagonist 7-benzylidenenaltrexone (24.7 +/- 3.7%, P < 0.01) totally abrogated the anti-infarct effect of M-Post. In addition, the anti-infarct effect by M-Post was also totally blocked by the MPTP opener atractyloside (26.3 +/- 5.2%, P < 0.05). CONCLUSIONS: M-Post effectively reduces myocardial infarction. The anti-infarct effect by M-Post is mediated via activation of delta-OR, especially delta1-OR, and inhibition of the MPTP opening.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Atractyloside ; Benzylidene Compounds ; Heart ; Heart Rate ; Ischemia ; Ischemic Postconditioning ; Mitochondrial Membrane Transport Proteins ; Morphine ; Myocardial Infarction ; Naloxone ; Naltrexone ; Permeability ; Rats ; Receptors, Opioid ; Reperfusion ; Reperfusion Injury ; Tetrazolium Salts

OBJECTIVETo explore the effects of chloride channels on the regulation of platelet cytoplasmic free calcium concentration ([Ca2+]i) and platelet aggregation (PAG).

METHODSFreshly 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.

RESULTSBoth 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).

CONCLUSIONDIDS 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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid ; pharmacology ; Adult ; Blood Platelets ; cytology ; drug effects ; metabolism ; Calcium ; metabolism ; Calcium Channel Blockers ; pharmacology ; Cells, Cultured ; Chloride Channels ; antagonists & inhibitors ; physiology ; Cytoplasm ; drug effects ; metabolism ; Drug Interactions ; Humans ; Imidazoles ; pharmacology ; Nifedipine ; pharmacology ; Niflumic Acid ; pharmacology ; Platelet Aggregation ; drug effects ; Thrombin ; pharmacology

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid ; pharmacology ; Adolescent ; Adult ; Blood Platelets ; metabolism ; Calcium ; metabolism ; Chloride Channels ; antagonists & inhibitors ; Cytosol ; metabolism ; Dose-Response Relationship, Drug ; Female ; Humans ; Male ; Niflumic Acid ; pharmacology ; Oligopeptides ; pharmacology ; Platelet Aggregation ; drug effects ; Platelet Glycoprotein GPIIb-IIIa Complex ; antagonists & inhibitors

Toremifene is an anti-estrogen which has been shown to be effective in the treatment of breast cancer, and is thought to be a less uterotrophic agent than tamoxifen. The risk assessment concerning endometrial cancer has been inconclusive because of its rare use up to the mid-1990s. We report a case of an adenosarcoma, which is a very rare type of uterine malignancy, after toremifene treatment for 5 years in a breast cancer patient. After 1 year of toremifene use, the patient had a benign Mullerian adenofibroma. After an additional 4 years of toremifene treatment, the endometrial polypoid lesion was transformed into a Mullerian adenosarcoma. Although toremifene is a promising anti-estrogenic agent in the treatment of breast cancer patients, clinicians should not neglect the possibility of a uterine malignancy.
Adenofibroma ; Adenosarcoma ; Breast ; Breast Neoplasms ; Endometrial Neoplasms ; Female ; Humans ; Risk Assessment ; Tamoxifen ; Toremifene

Antiestrogens have been widely used in the treatment of breast cancer patients. Although tamoxifen is one of the most prevalent antiestrogens, some reported its hepatocarcinogenic effects and the long-term treatment may increase the risk of endometrial and gastrointestinal cancer. Toremifene is an interesting new antiestrogen and have a similar antitumor efficacy as tamoxifen, with less side-effect including less uterotrophic effect compared to tamoxifen, in mice. we report a case of endometrial polyp which were associated with toremifene use, in postmenopausal woman with breast cancer, with a brief review of literature.
Animals ; Breast Neoplasms ; Estrogen Receptor Modulators ; Female ; Gastrointestinal Neoplasms ; Humans ; Mice ; Polyps* ; Tamoxifen ; Toremifene