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

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

Histamine activates phospholipase C (PLC) in a number of cell-types including those of neuronal and neuroendocrine origin. We report here that Cl(-)-channel antagonists of the niflumic acid-, but not stilbene disulphonic acid-class, produced a concentration-dependent inhibition of histamine-stimulated PLC activity in bovine adrenal medullary chromaffin cells. Low extracellular [Cl-] (10 mM) produced a similar degree of inhibition. While the mechanism(s) responsible for this inhibition are not resolved it may be significant that low extracellular Cl- also reduced the magnitude of the histamine-induced Ca2+ signal. Thus, PLC inhibition may be secondary to a reduction in Ca2+-inflow, a conclusion consistent with the known actions of niflumic acid-type compounds and the previously reported importance of Ca2+-influx in supporting histamine-stimulated PLC activity.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adrenal Medulla; Animals; Calcium; Cattle; Chloride Channels; Chlorides; Enzyme Activation; Extracellular Space; Gluconates; Histamine; Ion Transport; Niflumic Acid; Nitrates; Phosphatidylinositol Diacylglycerol-Lyase; Sulfates; Type C Phospholipases

By using the patch-clamp technique we have shown that, in hypotonic extracellular solutions, the mouse neuroblastoma cells Neuro2A (N2A) develop ionic currents mediated by a chloride-selective channel which is also permeable to other anions in accordance with the permeability sequence: I->Br->Cl->gluconate->glutamate-. The currents persist for several hours when Mg-ATP is present in the recording pipette but occur only transiently in the absence of Mg-ATP. Typical blockers of anions channels such as La3+ and Zn2+ do not affect the hypotonicity-activated channel; conversely, the stilbene sulfonate-derivatives, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), reversibly inhibit the channel in a voltage-dependent manner. Also intact cells exposed to hyposmotic solutions activate volume-regulation mechanisms which decrease the transient volume increase that develops immediately after the application of the hyposmotic challenge. Since N2A neurons have been used as an expression system of exogenous channels, the presence of osmolarity-regulated channels in these cells is an important aspect that deserves the attention of researchers who may wish to express and study the properties of transport proteins in this cell line.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine Triphosphate; Animals; Bromine; Chloride Channels; Chlorides; Gluconates; Glutamic Acid; Hypertonic Solutions; Iodine; Ion Channel Gating; Magnesium; Membrane Potentials; Mice; Neuroblastoma; Neurons; Patch-Clamp Techniques; Sodium Chloride; Tumor Cells, Cultured; Water-Electrolyte Balance

Non-Mg(2+)-loaded rat erythrocytes with a physiological level of Mg(2+)(i) exhibited Mg(2+) efflux when incubated in nominally Mg(2+)-free media. Two types of Mg(2+) efflux were shown: (1) An Na(+)-dependent Mg(2+) efflux in NaCl and Na gluconate medium, which was inhibited by amiloride and quinidine, as was Na(2+)/Mg(2+) antiport in Mg(2+)-loaded rat erythrocytes; and (2) an Na(+)-independent Mg(2+) efflux in sucrose medium and choline Cl medium, which may be differentiated into SITS-sensitive Mg(2+) efflux at low Cl(-)(o) (in sucrose) and into SITS-insensitive Mg(2+) efflux at high Cl(-)(o) (in 150 mmol/l choline Cl).

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Animals; Antiporters; Bucladesine; Cations, Divalent; Cells, Cultured; Choline; Erythrocytes; Furosemide; Gluconates; Magnesium; Male; Quinidine; Rats; Rats, Wistar; Sodium Chloride; Sucrose

A novel kinetic approach was used to measure monovalent anion binding to better define the mechanistic basis for competition between stilbenedisulfonates and transportable anions on band 3. An anion-induced acceleration in the release of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) from its complex with band 3 was measured using monovalent anions of various size and relative affinity for the transport site. The K1/2 values for anion binding were determined and correlated with transport site affinity constants obtained from the literature and the dehydrated radius of each anion. The results show that anions with ionic radii of 120-200 pm fall on a well-defined correlation line where the ranking of the K1/2 values matched the ranking of the transport site affinity constants (thiocyanate < nitrate approximately bromide < chloride < fluoride). The K1/2 values for the anions on this line were about 4-fold larger than expected for anion binding to inhibitor-free band 3. Such a lowered affinity can be explained in terms of allosteric site-site interactions, since the K1/2 values decreased with increasing anionic size. In contrast, iodide, with an ionic radius of about 212 pm, had a 10-fold lower affinity than predicted by the correlation line established by the smaller monovalent anions. These results indicate that smaller monovalent anions have unobstructed access to the transport site within the band 3 / DBDS binary complex, while iodide experiences significant steric hindrance when binding. The observation of steric hindrance in iodide binding to the band 3 / DBDS binary complex, but not in the binding of smaller monovalent anions, suggests that the stilbenedisulfonate binding site is located at the outer surface of an access channel leading to the transport site.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Allosteric Regulation; Anion Exchange Protein 1, Erythrocyte; Anions; Chlorides; Gluconates; Kinetics; Protein Binding

1. Non-linear membrane ionic current was studied in highly stretched cut frog twitch fibres in a double Vaseline-gap voltage clamp chamber, with the internal solution containing 0.1 mM EGTA and the external solution containing Cl- as the major anion. After the Na+ currents was abolished by TTX in the external solution and the K+ currents were suppressed by external TEA+ and Rb+ and internal Cs+, a delayed outward ionic current with a time course similar to that of the delayed rectifier current was observed during depolarization. 2. The delayed outward ionic current was resistant to 1 mM 3,4-diaminopyridine (3,4-DAP) in the external solution and was unaltered when a fraction of the internal Cs+ was replaced by K+ or Na+, suggesting that the current was not carried by cations flowing through the delayed rectifiers. 3. The delayed outward ionic current was greatly reduced by replacing the external Cl- with CH3SO3-,SO4(2-), glutamate or gluconate, indicating strongly that the current was carried by Cl- flowing through anion channels. The current was also suppressed by 1 mM external 9-anthracenecarboxylic acid (9-ACA). 4. The delayed outward ionic current was reduced by blockers of calcium-dependent Cl- channels, such as SITS and frusemide (furosemide), in a dose- and voltage-dependent manner and by increasing intracellular [EGTA] to 20 mM, suggesting that part of the Cl- current in the muscle fibres could be calcium dependent. 5. The total Cl- current could be dissected into calcium-dependent and calcium-independent components. Each component accounted for roughly half of the total Cl- current. The maximum slope conductance of the calcium-dependent Cl- channels was 60.9 +/- 6.0 microS microF-1 (mean +/- S.E.M., n = 4).

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4-Aminopyridine; Amifampridine; Animals; Anions; Calcium; Cesium; Chloride Channels; Egtazic Acid; Furosemide; Gluconates; Glutamic Acid; Ion Channels; Muscle, Skeletal; Rana temporaria; Rubidium; Tetradecanoylphorbol Acetate; Tetrodotoxin; Time Factors

Relationships between short-circuit current (Isc), cell Cl and the mechanism(s) of Cl accumulation in toad bladder epithelial cells were investigated. In serosal Cl-free gluconate Ringer, 80% of the cell Cl (measured by x-ray microanalysis) was lost over 30-60 min with an associated decrease in cell water content. concomitantly, Isc fell to 20% of its initial value within 10 min but then recovered to 45% of its initial value despite continued Cl loss. With the reintroduction of Cl, cell Cl and Isc both recovered within 10 min. Serosal SITS (4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonate; 0.5 mM) plus bumetanide (0.1 mM), did not prevent the fall in Isc or the loss of cell Cl in gluconate medium, although they did inhibit subsequent recovery of Isc in this medium. They also prevented the recovery of Isc in Cl medium but not the reaccumulation of Cl by the cells. Although SITS and bumetanide did not prevent the loss or recovery of Cl, they modified the pattern of the ion changes. In their absence, changes in cellular Cl were twice that of the changes in measured cellular cations implicating basolateral Cl/HCO3 exchange in Cl movement. With SITS plus bumetanide present, changes of similar magnitude in Cl were associated with equivalent changes in cation, consistent with the inhibition of Cl/HCO3 exchange.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Animals; Antiporters; Bendroflumethiazide; Bufo marinus; Bumetanide; Cell Size; Chloride-Bicarbonate Antiporters; Chlorides; Culture Media; Electron Probe Microanalysis; Epithelial Cells; Epithelium; Gluconates; Hydrochlorothiazide; Intracellular Fluid; Membrane Potentials; Nitrobenzoates; Sodium; Urinary Bladder

The involvement of K+ on the volume regulatory process in astrocytes was investigated by characterizing the hyposmolarity-induced efflux of K+ using 86Rb as a tracer. About 70 and 30% of the intracellular content of 86Rb was released after reductions in osmolarity from 320 to 160 or 220 mosM, respectively, during the time in which cells exhibit a volume regulatory response subsequent to swelling. No significant increase in 86Rb efflux was observed with lower reductions in osmolarity. The 86Rb efflux was Ca2+ independent and insensitive to temperature. It was inhibited by furosemide but not by bumetanide and was unaffected when nitrate, but not gluconate, replaced intracellular Cl-. The efflux was markedly inhibited by quinidine and by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Quinidine also prevented the volume regulatory decrease of cells, and this effect was overcome when a large cation permeability was imposed by gramicidin. In isosmotic conditions 86Rb efflux was not activated by N-ethylmaleimide, but this drug strongly inhibited the hyposmolarity-activated release. These findings suggest that 86Rb efflux from astrocytes associated to cell swelling is not mediated by an electroneutral cotransporter and rather favor the implication of a conductive exit pathway that may be a Ca(2+)-independent K+ channel.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Astrocytes; Biological Transport, Active; Bumetanide; Cells, Cultured; Cerebellum; Chlorides; Culture Media; Ethylmaleimide; Furosemide; Gluconates; Gramicidin; Hypotonic Solutions; Kinetics; Nitrates; Osmolar Concentration; Quinidine; Rats; Rubidium; Temperature

1. The efflux of chloride (using 36Cl) from lactating rat mammary tissue slices has been investigated. 2. Chloride efflux was found to be temperature dependent; lowering the temperature of the incubation medium reduced the fractional efflux. 3. The stilbene derivatives DIDS was without effect on the fractional release of Cl when studied at 20 degrees C. However, DIDS was found to attenuate the increase in efflux found upon transferring the tissue from a medium maintained at 4 degrees C to one at 20 degrees C. 4. The loop-diuretic furosemide, also reduced the temperature-sensitive portion of Cl efflux. 5. Chloride efflux was transiently increased when tissue slices were transferred from a medium containing gluconate as the principal anion to one containing Cl. 6. The results appear to confirm that mammary Cl transport is mediated via anion exchange and via (Na + K + Cl) cotransport.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Chlorides; Female; Furosemide; Gluconates; In Vitro Techniques; Lactation; Mammary Glands, Animal; Rats; Temperature

1. Intracellular pH (pHi) was measured in single rat cerebellar Purkinje cells maintained in primary culture using microspectrofluorescence analysis of the intracellularly trapped pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF). 2. The ratio of the fluorescence signals measured at 530 nm in response to an alternating excitation at 450 and 490 nm was calibrated using the K(+)-H+ ionophore nigericin. This calibration gave a steady-state pHi of 7.06 +/- 0.02 (S.E.M., n = 17) when cells were perfused by a 5% CO2-25 mM-HCO3(-)-buffered solution at an external pH of 7.40 at 37 degrees C. 3. Replacement of external chloride with gluconate in the presence of bicarbonate induced a cytoplasmic alkalinization of about 0.3 pH unit. This alkalinization was independent of external sodium and was greatly reduced by 0.5 mM-DIDS, indicating the presence of a chloride-bicarbonate exchange. 4. In bicarbonate-free (HEPES-buffered) solution the steady-state pHi was 7.37 +/- 0.02 (n = 19), significantly higher than in bicarbonate-buffered solution. Recovery from an intracellular acid load brought about by the ammonium chloride pre-pulse technique was blocked by the removal of external sodium or the addition of 1.5 mM-amiloride, indicating the presence of a sodium-hydrogen exchange. 5. In bicarbonate-buffered solution pHi recovery after an acid load was also completely blocked by addition of 1.5 mM-amiloride indicating the absence of a bicarbonate-dependent acid extrusion mechanism. 6. Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) induced an amiloride-sensitive alkalinization of about 0.3 pH unit in bicarbonate-buffered solution but had no effect in HEPES-buffered solution. This observation suggests that in cultured Purkinje cells the sodium-hydrogen exchanger could be activated through a protein kinase C pathway only when pHi is maintained at a low physiological value by the activity of the chloride-bicarbonate exchange.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Bicarbonates; Cells, Cultured; Chlorides; Fluoresceins; Gluconates; HEPES; Hydrogen-Ion Concentration; Ion Exchange; Purkinje Cells; Rats; Sodium; Spectrometry, Fluorescence; Tetradecanoylphorbol Acetate

The relationships between extracellular pH (pHo), intracellular pH (pHi), and loss of cell viability were evaluated in cultured rat hepatocytes after ATP depletion by metabolic inhibition with KCN and iodoacetate (chemical hypoxia). pHi was measured in single cells by ratio imaging of 2',7'-biscarboxy-ethyl-5,6-carboxyfluorescein (BCECF) fluorescence using multiparameter digitized video microscopy. During chemical hypoxia at pHo of 7.4, pHi decreased from 7.36 to 6.33 within 10 min. pHi remained at 6.1-6.5 for 30-40 min (plateau phase). Thereafter, pHi began to rise and cell death ensued within minutes, as evidenced by nuclear staining with propidium iodide and coincident leakage of BCECF from the cytoplasm. An acidic pHo produced a slightly greater drop in pHi, prolonged the plateau phase of intracellular acidosis, and delayed the onset of cell death. Inhibition of Na+/H+ exchange also prolonged the plateau phase and delayed cell death. In contrast, monensin or substitution of gluconate for Cl- in buffer containing HCO3- abolished the pH gradient across the plasma membrane and shortened cell survival. The results indicate that intracellular acidosis after ATP depletion delays the onset of cell death, whereas reduction of the degree of acidosis accelerates cell killing. We conclude that intracellular acidosis protects against hepatocellular death from ATP depletion, a phenomenon that may represent a protective adaptation against hypoxic and ischemic stress.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acidosis; Amiloride; Animals; Bicarbonates; Carrier Proteins; Cell Survival; Cells, Cultured; Chlorides; Fluoresceins; Gluconates; Hydrogen-Ion Concentration; Liver; Male; Monensin; Oxygen; Rats; Rats, Inbred Strains; Sodium-Hydrogen Exchangers

We have measured the intracellular pH (pHi) of single isolated rat pancreatic acini using the fluorescent dye dicyano-hydroquinone (DCH). Steady-state pHi was unaffected by either acetylcholine (10 microM), caerulein (10 nM) or secretin (1 nM), or by the anion exchange inhibitor DIDS (1 mM) and only changed by 0.32 units when extracellular pH was varied over the range from 6.8 to 8.4. However, replacing extracellular chloride with gluconate, such that the final bath chloride concentration was less than 20 mM, caused a marked alkalinization of pHi to a new steady-state value. The rate of pHi alkalinization under these conditions was bicarbonate-dependent, blocked by pre-incubation of the acini with 1 mM-DIDS, but unaffected by removal of extracellular sodium. These observations are consistent with the presence of a Cl- -HCO3- exchanger on rat pancreatic acinar cells. Since DIDS had no effect on steady-state pHi the anion exchanger is unlikely to play a role in pHi regulation. However, rat pancreatic acini secrete a NaCl-rich fluid and the Cl- -HCO3- a exchanger might function in parallel with Na+-H+ exchangers to provide a mechanism for chloride accumulation across the basolateral membrane of the acinar cells.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Bicarbonates; Carrier Proteins; Chloride-Bicarbonate Antiporters; Chlorides; Gluconates; Hydrogen-Ion Concentration; Kinetics; Pancreas; Potassium; Rats; Sodium

Porcine gallbladder, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasma-like Ringer solution generates a serosal positive transepithelial potential of 4-7 mV and a short-circuit current (Isc) of 50-120 microA/cm2. Substitution of Cl with gluconate or HCO3 with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) results in a 50% decrease in Isc. Treatment with 1 mM amiloride (mucosal side) or 0.1 mM acetazolamide (both sides) causes 25-27% inhibition of the Isc. Mucosal addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits the Isc by 17%. Serosal addition of 0.1 mM bumetanide inhibits the Isc by 28%. Amiloride (1 mM) inhibits the net transepithelial fluxes of Na and Cl by 55 and 41%, respectively. Substitution of Cl with gluconate inhibits the net Na flux by 50%, whereas substitution of HCO3 with HEPES inhibits 85-90% of the net Na flux and changes Cl absorption to net secretion. Based on these results, it is hypothesized that Na and Cl transport across the apical membrane is mediated by two pathways, Na-H/Cl-HCO3 exchange and Na-HCO3 cotransport. Partial recycling of Cl and HCO3 presumably occurs through a Cl conductive pathway and Cl-HCO3 exchange, respectively, in the apical membrane. This results in net Na absorption, which accounts for most of the Isc observed under basal conditions. The effect of bumetanide on the basolateral membrane and the fact that Cl secretion occurs when HCO3 is absent suggests that Cl secretion involves a basolateral NaCl or Na-K-Cl cotransport system arranged in series with a Cl conductive pathway in the apical membrane.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acetazolamide; Amiloride; Animals; Bicarbonates; Biological Transport; Bumetanide; Cell Membrane; Chlorides; Electric Conductivity; Epithelium; Gallbladder; Gluconates; Membrane Potentials; Microscopy, Electron; Sodium; Swine

Cell-mediated cytotoxicity can be inhibited by the replacement of chloride with ions that are incapable of passing through chloride channels or by the presence of stilbene disulfonate derivatives known to interfere with chloride flux. We show that the stilbene disulfonate (4,4-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) inhibits lysis of YAC-1 targets by the cloned cell line NKB61A2. Inhibition of lysis occurs on the level of the effector cell inasmuch as preincubation of effectors but not of targets interferes with subsequent lysis. Moreover, inhibition of chloride flux in the target does not interfere with target cell lysis by cytotoxic granules isolated from killer cells. Target cell binding takes place in the presence of DIDS or absence of external chloride, suggesting that events that follow target cell binding require chloride flux. We show that reorientation of the Golgi apparatus, which occurs subsequent to target cell binding in the effector cell, occurs under conditions that interfere with chloride flux. It is therefore suggested that events in the effector cell taking place subsequent to the Golgi apparatus reorientation reaction are inhibited and that delivery of the lethal hit is a stimulus-induced secretory event that requires transmembrane chloride flux. Delivery of the lethal hit is shown to be independent of the release of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) serine esterase, suggesting that cytolytic components and BLT serine esterase are likely packaged in different vesicles.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Cell Line; Cell Membrane; Cell Survival; Chlorides; Clone Cells; Culture Media; Cytoplasmic Granules; Cytotoxicity, Immunologic; Gluconates; Golgi Apparatus; Granzymes; Immunosuppressive Agents; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Serine Endopeptidases; Serine Proteinase Inhibitors

Large voltage-dependent outward currents are recorded with the whole-cell patch-clamp technique from rat cultured astrocytes under conditions where an outward movement of potassium ions is excluded (either by blockage of the potassium channels pharmacologically or by replacement of the internal potassium by the impermeant large organic cation N-methyl-(+)-glucamine). The current, which is activated at potentials more positive than -40 to -50 mV, is normally carried by an inward movement of chloride ions. Its reversal potential is the same as the chloride equilibrium potential. With depolarization to +60 mV (for 225 ms) little or no inactivation of the current occurs: with depolarizations to +90 to +110 mV a time-dependent decay is seen. The current, which is often not marked immediately after formation of the whole-cell clamp, generally increases over a period of a few minutes to a maximum (after which it usually declines), as if some as yet unknown intracellular factor keeping the channels closed were being washed away from the membrane. The time course of this phenomenon is not affected by changing of the internal free calcium concentration (from 10(-8)M to 10(-6)M) or by an intracellular mixture of cyclic AMP (1 mM), ATP (4 mM) and Mg+ (2 mM). The conductance is slightly increased when the chloride of the bathing medium is replaced by bromide; is much reduced on replacement by methylsulphate, sulphate, isethionate, or acetate; and is virtually abolished on replacement by the large anion gluconate. The outward current is inhibited by the disulphonate stilbenes DIDS and SITS; this blocking action was initially partly reversible, although never completely so. It is suggested that the chloride conductance plays a role in the spatial buffering of potassium by astrocytes.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Astrocytes; Cells, Cultured; Chlorides; Electric Conductivity; Gluconates; Ion Channels; Membrane Potentials; Rats; Rats, Inbred Strains