The Effects of Glycoprotein IIb/IIIa Antagonists and Chloride Channel Blockers on Platelet Cytoplasmic Free Calcium.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3939-3939
Author(s):  
Songmei Yin ◽  
Yiqing Li ◽  
Shuangfeng Xie ◽  
Danian Nie ◽  
Haiming Li ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Fluorescence Intensity ◽  
Chloride Channel ◽  
Disulfonic Acid ◽  
Free Calcium ◽  
Channel Blockers ◽  
Combined Effects ◽  
Inhibition Rate ◽  
Platelet Suspension ◽  
Cytoplasmic Free Calcium

Abstract Objective To explore the effects and interactions of GPIIb/IIIa antagonists and chloride channel blockers on the platelet cytoplasmic free calcium ([Ca2+]i ). Methods We washed and suspended fresh platelets with Hepes buffer containing 0.1% bovine serum albumin (BSA), then loaded platelets with 5μmol/L Fura-3/AM. Then RGDS, the GPIIb/IIIa antagonists, and the chloride channel blockers 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene(DIDS) or niflumic acid(NFA) were added to the platelet suspension. After 2 minutes incubating, we observed the effects and interactions of GPIIb/IIIa antagonists and chloride channel blockers on platelet [Ca2+]i by measuring the Fura-3 fluorescence intensity. Results 1. Effects of GPIIb/IIIa antagonists and chloride channel blockers on platelet [Ca2+]i. The fluorescence intensity of resting platelet [Ca2+]i were 369.6±62.2, 381.9±72.4, 392.8 ±69.9 after adding RGDS(250 μmol/L), DIDS(100 μmol/L) or NFA( 100 μmol/L) respectively. every agent had no significant effect on resting [Ca2+]i (p>0.05). After thrombin(0.03 U/ml) stimulating and adding RGDS(250 μmol/L), DIDS(100 μmol/L) or NFA( 100 μmol/L), the platelet [Ca2+]i were 883.9±107.0, 789.8±99.8, 564.1±79.4. Compare with the control(977.9±108.8), the three agents could inhibit the elevation of [Ca2+]i stimulated by thrombin (p<0.05). The inhibiting rates were (9.37±7.5)%, (18.7±10.4)% and (41.8±10.1)% respectively. 2. Combined effects of GPIIb/IIIa antagonists and chloride channel blockers The fluorescence intensity of resting platelet [Ca2+]i was 383.9±67.9 after incubated with RGDS and DIDS. That had no significant effects. When platelets were stimulated by thrombin (0.03 U/ml), the combined inhibition rate was (24.4±10.8)%, RGDS and DIDS couldn’t weaken or enhance each other on thrombin-induced elevation of [Ca2+]i (p>0.05). Neither RGDS nor NFA had significant combined effects on resting [Ca2+]i(p>0.05). The combined inhibition rate was (46.0±7.3)%, they had no interactions too(p>0.05). Conclusion The GPIIb/IIIa antagonists RGDS and the chloride channel blockers DIDS or NFA have no effect on resting platelet [Ca2+]i. All of them can inhibit the elevation of platelet [Ca2+]i induced by thrombin. There are no interactions between GPIIb/IIIa antagonists RGDS and chloride channel blockers (DIDS or NFA) in resting platelet [Ca2+]i and elevation of platelet [Ca2+]i induced by thrombin, and their effects were independent.

The Effects of Chloride Channel Blockers on Platelet Cytoplasmic Free Calcium Concentration and Platelet Aggregation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3879-3879
Author(s):  
Songmei Yin ◽  
Xiaolin Chen ◽  
Danian Nie ◽  
Shuangfeng Xie ◽  
Liping Ma ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Calcium Channel ◽  
Calcium Channel Blockers ◽  
Chloride Channel ◽  
Calcium Concentration ◽  
Disulfonic Acid ◽  
Free Calcium ◽  
Channel Blockers ◽  
Free Calcium Concentration ◽  
Cytoplasmic Free Calcium

Abstract Objective To explore the effects of chloride channels on the regulations of platelet cytoplasmic free calcium concentration ([Ca2+]i) and platelet aggregation (PAG). Methods Platelet were separated freshly and then activated by thrombin; The chloride channel blockers 4,4′-diisothiocyano-2, 2′-disulfonic acid stilbene (DIDS) or niflumic acid (NFA), and calcium channel blockers 1-{β-[3-(4-methoxyphenyl)propoxy]- 4-methoxyphenethyl}- 1H - imidazole hydrochloride (SK&F96365) or Nifedipine were added to react with the activated platelets. The effects of each agent on platelet [Ca2+]i and PAG were detected. The combine effects and the interactions among chloride channel blockers (DIDS, NFA) and calcium channel blockers (SK&F96365, Nifedipine) were also investigated. Results Both DIDS and NFA [the concentration were12.5, 25, 50, 100 and 200μmol•L−1 respectively] could inhibit the PAG induced by thrombin (1U/ml) and the effect was dose-dependent. Compared with the control, they had no significant effects on resting [Ca2+]i. Compare with the control group, DIDS (100μmol•L−1), SK&F96365 (100μmol•L−1) and Nifedipine (100μmol•L−1) could significantly reduce the PAG, Ca2+ release and Ca2+ influx activated by thrombin in platelet (P<0.05). DIDS (100μmol•L−1) and SK&F96365 (100μmol•L−1) could enhance each other’s effect on reducing the PAG, Ca2+ release and Ca2+ influx (P<0.05). DIDS (100μmol•L−1) and Nifedipine (100μmol•L−1) could enhance each other’s effect on reducing Ca2+ release (P<0.05). NFA (100μmol•L−1) and SK&F96365 (100μmol•L−1) could weaken each other’s effect on Ca2+ release (P<0.05). NFA (100μmol•L−1) and Nifedipine (100μmol•L−1) could weaken each other’s effect on PAG, Ca2+ release and Ca2+ influx activated by thrombin in platelet (P<0.05). Conclusion The chloride channel blockers 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 of platelet induced by thrombin. There are interactions between chloride channel blockers and calcium channel blockers in resting [Ca2+]i and PAG of platelet. The opening of chloride channel can influence the cellular calcium movement of platelet.

ClC-3 is a candidate of the channel proteins mediating acid-activated chloride currents in nasopharyngeal carcinoma cells

2012 ◽  
Vol 303 (1) ◽  
pp. C14-C23 ◽  
Author(s):  
Liwei Wang ◽  
Wenbo Ma ◽  
Linyan Zhu ◽  
Dong Ye ◽  
Yuan Li ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Chloride Channel ◽  
Cell Function ◽  
Extracellular Ph ◽  
Carcinoma Cells ◽  
Chloride Current ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Chloride Currents ◽  
Channel Proteins

Acid-activated chloride currents have been reported in several cell types and may play important roles in regulation of cell function. However, the molecular identities of the channels that mediate the currents are not defined. In this study, activation of the acid-induced chloride current and the possible candidates of the acid-activated chloride channel were investigated in human nasopharyngeal carcinoma cells (CNE-2Z). A chloride current was activated when extracellular pH was reduced to 6.6 from 7.4. However, a further decrease of extracellular pH to 5.8 inhibited the current. The current was weakly outward-rectified and was suppressed by hypertonicity-induced cell shrinkage and by the chloride channel blockers 5-nitro-2–3-phenylpropylamino benzoic acid (NPPB), tamoxifen, and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS). The permeability sequence of the channel to anions was I− > Br− > Cl− > gluconate−. Among the ClC chloride channels, ClC-3 and ClC-7 were strongly expressed in CNE-2Z cells. Knockdown of ClC-3 expression with ClC-3 small interfering (si)RNA prevented the activation of the acid-induced current, but silence of ClC-7 expression with ClC-7 siRNA did not significantly affect the current. The results suggest that the chloride channel mediating the acid-induced chloride current was volume sensitive. ClC-3 is a candidate of the channel proteins that mediate or regulate the acid-activated chloride current in nasopharyngeal carcinoma cells.

scholarly journals Evidence for a Superoxide Permeability Pathway in Endosomal Membranes

2008 ◽  
Vol 28 (11) ◽  
pp. 3700-3712 ◽  
Author(s):  
Davis R. Mumbengegwi ◽  
Qiang Li ◽  
Canhui Li ◽  
Christine E. Bear ◽  
John F. Engelhardt
Keyword(s):  
Chloride Channel ◽  
Anion Channel ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Luminescent Probes ◽  
Redox Activation ◽  
Early Endosomes ◽  
Signaling Events ◽  
Endosomal Membranes ◽  
Immunoaffinity Isolation

ABSTRACT The compartmentalized production of superoxide (·O2 −) by endosomal NADPH oxidase is important in the redox-dependent activation of NF-κB following interleukin 1β (IL-1β) stimulation. It remains unclear how ·O2 − produced within endosomes facilitates redox-dependent signaling events in the cytoplasm. We evaluated ·O2 − movement out of IL-1β-stimulated endosomes and whether SOD1 at the endosomal surface mediates redox-signaling events required for NF-κB activation. The relative outward permeability of NADPH-dependent ·O2 − from fractionated endosomes was assessed using membrane-permeable (luminol and lucigenin) and -impermeable (isoluminol) luminescent probes for ·O2 −. In these studies, ∼60% of ·O2 − efflux out of endosomes was inhibited by treatment with either of two anion channel blockers, 4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) or niflumic acid (NFA). Furthermore, radioisotopic electrodiffusion flux assays on endomembrane proteoliposomes suggested that ·O2 − and Cl− are transported through the same DIDS-sensitive channel(s). Rab5-based immunoaffinity isolation of IL-1β-stimulated early endosomes demonstrated SOD1 recruitment to endosomes harboring the IL-1 receptor. Finally, SOD1-deficient cells were found to be defective in their ability to activate NF-κB following IL-1β stimulation. Together, these results suggest that ·O2 − exits endosomes through a DIDS-sensitive chloride channel(s) and that SOD1-mediated dismutation of ·O2 − at the endosomal surface may produce the localized H2O2 required for redox-activation of NF-κB.

Hormonally controlled chloride movement across Drosophila tubules is via ion channels in stellate cells

1998 ◽  
Vol 274 (4) ◽  
pp. R1039-R1049 ◽  
Author(s):  
Michael J. O’Donnell ◽  
Mark R. Rheault ◽  
Shireen A. Davies ◽  
Phillipe Rosay ◽  
Brian J. Harvey ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Stellate Cells ◽  
Fluid Secretion ◽  
Physiological Effect ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Anion Conductance ◽  
Cation Conductance

Anion conductance across the Drosophila melanogaster Malpighian (renal) tubule was investigated by a combination of physiological and transgenic techniques. Patch-clamp recordings identified clusters of 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-sensitive “maxi-chloride” channels in a small domain of the apical membrane. Fluid secretion assays demonstrated sensitivity to the chloride channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid, diphenylamine-2-carboxylate, anthracene-9-carboxylic acid, and niflumic acid. Electrophysiological analysis showed that the calcium-mediated increase in anion conductance was blocked by the same agents. Vibrating probe analysis revealed a small number of current density hot spots, coincident with “stellate” cells, that were abolished by low-chloride saline or the same chloride channel blockers. GAL-4-targeted expression of an aequorin transgene revealed that the neurohormone leucokinin elicits a rapid increase in intracellular calcium levels in stellate cells that precedes the fastest demonstrable physiological effect. Taken together, these data show that leucokinins act on stellate cells through intracellular calcium to increase transcellular chloride conductance through channels. As electrogenic cation conductance is confined to principal cells, the two pathways are spatially segregated in this tissue.

Electrophysiological properties of cultured outer medullary collecting duct cells

1992 ◽  
Vol 263 (6) ◽  
pp. F1004-F1010 ◽  
Author(s):  
C. A. Pappas ◽  
B. M. Koeppen
Keyword(s):  
Collecting Duct ◽  
Extracellular Fluid ◽  
Outward Current ◽  
Membrane Voltage ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Dependent Manner ◽  
Whole Cell ◽  
Electrophysiological Properties ◽  
Medullary Collecting Duct

Whole cell patch-clamp techniques were used to characterize the electrophysiological properties of cells from the inner stripe portion of the rabbit outer medullary collecting duct (OMCDi) grown in primary culture. With pipette and bathing solutions mimicking intracellular and extracellular fluid, the resting membrane voltage was -30 to -40 mV. The whole cell conductance exhibited slight outward rectification, and at the resting membrane voltage the cell conductance averaged 2.58 +/- 0.49 nS (n = 17). The major conductive ion species was Cl-. The Cl- conductance was also found to have a significant permeability to HCO3- and was inhibited by the Cl(-)-channel blockers diphenylamine carboxylic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. A small K+ conductance was also present, but no Na+ conductance was detected. Current generated by the H(+)-adenosinetriphosphatase (H(+)-ATPase) was quantitated. This current was dependent on the presence of ATP in the pipette. Dicyclohexylcarbodiimide, N-ethylmaleimide, and bafilomycin A1, inhibitors of the vacuolar H(+)-ATPase, also reduced this outward current in an ATP-dependent manner. The inhibitor-sensitive component of the outward current, a measure of the current generated by the H(+)-ATPase, was in the range of 35-100 pA/cell.

A new congenital defect of platelet secretion: impaired responsiveness of the platelets to cytoplasmic free calcium

1983 ◽  
Vol 53 (4) ◽  
pp. 543-557 ◽  
Author(s):  
R. M. Hardisty ◽  
S. J. Machin ◽  
T. J. C. Nokes ◽  
T. J. Rink ◽  
Sharon W. Smith
Keyword(s):  
Congenital Defect ◽  
Free Calcium ◽  
Cytoplasmic Free Calcium ◽  
Platelet Secretion
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