4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and thiocyanate

Proteins of the CLCA gene family including the human ClCa1 (hClCa1) have been suggested to constitute a new family of chloride channels mediating Ca(2+)-dependent Cl- currents. The present study examines the relationship between the hClCa1 protein and Ca(2+)-dependent Cl- currents using heterologous expression of hClCa1 in HEK293 and NCIH522 cell lines and whole cell recordings. By contrast to previous reports claiming the absence of Cl- currents in HEK293 cells, we find that HEK293 and NCIH522 cell lines express constitutive Ca(2+)-dependent Cl- currents and show that hClCa1 increases the amplitude of Ca(2+)-dependent Cl- currents in those cells. We further show that hClCa1 does not modify the permeability sequence but increases the Cl- conductance while decreasing the G(SCN-)/G(Cl-) conductance ratio from approximately 2-3 to approximately 1. We use an Eyring rate theory (two barriers, one site channel) model and show that the effect of hClCa1 on the anionic channel can be simulated by its action on lowering the first and the second energy barriers. We conclude that hClCa1 does not form Ca(2+)-dependent Cl- channels per se or enhance the trafficking/insertion of constitutive channels in the HEK293 and NCIH522 expression systems. Rather, hClCa1 elevates the single channel conductance of endogenous Ca(2+)-dependent Cl- channels by lowering the energy barriers for ion translocation through the pore.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Calcium; Cell Line; Cell Line, Tumor; Cell Membrane; Chloride Channels; Chlorides; Electric Stimulation; Electrophysiological Phenomena; Gene Expression; Gluconates; Humans; Membrane Potentials; Models, Molecular; Niflumic Acid; Permeability; Thermodynamics; Thiocyanates; Transfection

The transepithelial transport of inorganic carbon to endolymph and its subsequent deposition on otoliths were pharmacologically examined by incubating the sacculus containing an otolith with NaH(14)CO(3). Calcium incorporation was also studied. Carbon incorporation into endolymph and otoliths was saturated with increased concentrations of bicarbonate ions in the incubation medium and was followed by the Michaelis-Menten equation with a K(m) of 26.3 mM and 0.4 mM, respectively. Carbon incorporation decreased with an increase in chloride concentrations in the medium. Calcium incorporation was not affected by chloride and bicarbonate ions up to 10 mM. Higher concentrations of bicarbonate ions reduced calcium incorporation into both fractions. Carbon incorporation into endolymph and otoliths was inhibited by acetazolamide, disulfonate stilbenes (DIDS and SITS), thiocyanate, and ouabain. Calcium incorporation was not affected by these inhibitors. Amiloride inhibited carbon incorporation into otoliths alone. These results suggest that HCO(3)(-)-ATPase and Cl(-)/HCO(3)(-)-exchangers are involved in the transepithelial transport of bicarbonate ions to the endolymph. Carbonic anhydrase was also suggested to play a role in carbonate production for otolith calcification.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Bicarbonates; Calcium; Carbon; Chlorides; Endolymph; Enzyme Inhibitors; In Vitro Techniques; Ion Transport; Kinetics; Otolithic Membrane; Ouabain; Salmon; Thiocyanates

1. Zinc is taken up into human red cells by two mechanisms that depend upon the presence of anions. One of these requires bicarbonate ions, is inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and appears to be catalysed by the anion exchanger. The second occurs in the presence of thiocyanate or salicylate ions and may represent transport of a neutral complex with Zn2+. 2. The initial rate of Zn2+ uptake via the anion exchanger is 64 +/- 13 mumol (10(13) cells x h)-1 microM-1 external Zn2+, in the presence of 5 mM-bicarbonate at pH 7.4 and 37 degrees C (+/- S.D.). This is about 1/250 of the corresponding rate of Pb2+ uptake by the anion exchanger. 3. The variation of transport with Zn2+ concentration, HCO3- concentration and pH suggests that the transported species may be ZnCO3Cl- or Zn(HCO3)Cl.OH-. 4. Zinc efflux could not be observed by either of the above routes. This observation suggests that the intracellular free Zn2+ concentration is below 3 nM.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anions; Bicarbonates; Cations, Divalent; Cations, Monovalent; Erythrocyte Membrane; Humans; Lead; Salicylates; Salicylic Acid; Thiocyanates; Zinc

The transapical Cl- influx and transepithelial Na+ transport were measured in rabbit gallbladder. Only 11.7% of the transported Na+ was found to be accompanied by HCO3-. 10(-4) M SITS eliminated the HCO3- dependent fraction of Cl- influx (50%) but did not significantly alter intracellular Na+ activity and Na+ transport. Exposure to HCO3-free salines or to 10(-4) M acetazolamide about halved Cl- influx and Na+ transport. 25 mM SCN- reduced Cl- influx to zero, decreased intracellular Na+ activity, but only halved Na+ transport which under these conditions was abolished only in the absence of HCO3-. Exposure to a Cl- -free saline produced effects similar to those caused by SCN-. These results suggest that when Cl-/HCO3- exchange is inhibited at the apical membrane, Na+/H+ exchange and transepithelial Na+ transfer are unmodified if HCO3- is available for transport. The permanent uncoupling of the exchangers and the elevated transepithelial transport of Na+ are not due to an increased activity of the parallel Na+-Cl- cotransport but to a redirection of HCO3- flux toward the basolateral side.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Animals; Bicarbonates; Chlorides; Gallbladder; In Vitro Techniques; Rabbits; Sodium; Thiocyanates

The uptake of chloride was studied in gill plasma membrane vesicles from low-salinity-adapted blue crabs (Callinectes sapidus). Cl- uptake was not Na+ dependent. However, when a HCO-3 gradient (in greater than out) was imposed across the membrane, a transient overshoot of about 2.5-fold was produced. Approximately 90% of the Cl- uptake reflected entry into the osmotically active intravesicular space. Cl- itself, nitrate, hydroxyl, and sulfite could substitute for HCO-3. The HCO-3/Cl- exchange process appeared to saturate at higher concentrations of either HCO-3 or Cl-. The apparent Km for Cl- was 15 mM. HCO-3-dependent Cl- uptake was significantly inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and thiocyanate but not by amiloride, furosemide, or ouabain. Alterations in membrane potential had no effect on Cl- uptake. Addition of Cl- or HCO-3 to the external medium also accelerated efflux of 36Cl- and H14CO-3 from preloaded vesicles. These results indicate that the uptake of Cl- by the crab gill plasma membrane is a carrier-mediated Na+-independent anion exchange process.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetazolamide; Animals; Anions; Bicarbonates; Brachyura; Cell Membrane; Chlorides; Dialysis; Gills; Ion Exchange; Kinetics; Membrane Potentials; Thiocyanates