4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and zinc-chloride

Hyperpolarization-activated inward currents were studied in single adult cockroach Periplaneta americana pacemaker neurosecretory cells, identified as dorsal unpaired median neurons using the whole-cell patch-clamp technique. Under current clamp, injection of negative current produced a hyperpolarization of the cell membrane with a sag in the membrane potential toward the resting value. Under voltage clamp, the whole-cell current-voltage relationship exhibited an unexpected biphasic aspect. The global hyperpolarization-activated inward current could be dissociated by means of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and tetraethylammonium chloride sensitivity, ionic selectivity, voltage dependence and activation threshold as inward potassium and calcium-sensitive chloride currents. The inward potassium current was activated around -80 mV. The reversal potential followed the potassium equilibrium potential when the extracellular potassium concentration was raised. This current was not dependent on the external sodium concentration and was sensitive to 10 mM tetraethylammonium chloride or 5 mM barium chloride. The hyperpolarization-activated inward calcium-sensitive chloride current was activated in a range of potential 20 mV more positive than the potassium current. The estimated reversal potential (-71 mV) was very close to the equilibrium potential for chloride ions ( 73 mV). Intracellularly applied 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and external application of 1 mM zinc chloride, calcium-free saline or high concentrations of intracellular 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetate blocked the inward chloride current. Current-clamp experiments indicated that the inward potassium current accounted for inward rectification of dorsal unpaired median neurons. Our findings report, for the first time in pacemaker neurosecretory cells, the co-existence of two distinct hyperpolarization-activated inward currents which have specialized function in pacemaker activity.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Barium Compounds; Calcium; Cells, Cultured; Cesium; Chelating Agents; Chloride Channels; Chlorides; Cockroaches; Egtazic Acid; Ganglia, Invertebrate; Ion Channel Gating; Magnesium; Male; Membrane Potentials; Neurons; Neurosecretory Systems; Patch-Clamp Techniques; Periodicity; Potassium; Tetraethylammonium; Zinc Compounds

Aqueous humor secretion is in part linked to HCO3- transport by nonpigmented ciliary epithelium (NPE) cells. During this process, the cells must maintain stable cytoplasmic pH (pHi). Because a recent report suggests that NPE cells have a plasma membrane-localized vacuolar H(+)-ATPase, the present study was conducted to examine whether vacuolar H(+)-ATPase contributes to pHi regulation in a rabbit NPE cell line. Western blot confirmed vacuolar H(+)-ATPase expression as judged by H(+)-ATPase 31-kDa immunoreactive polypeptide in both cultured NPE and native ciliary epithelium. pHi was measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Exposing cultured NPE to K(+)-rich solution caused a pHi increase we interpret as depolarization-induced alkalinization. Alkalinization was also caused by ouabain or BaCl2. Bafilomycin A1 (0.1 microM; an inhibitor of vacuolar H(+)-ATPase) inhibited the pHi increase caused by high K+. The pHi increase was also inhibited by angiotensin II and the metabolic uncoupler carbonyl cyanide m-chlorophenylhydazone but not by ZnCl2, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), omeprazole, low-Cl- medium, HCO3(-)-free medium, or Na(+)-free medium. Bafilomycin A1 slowed the pHi increase after an NH4Cl (10 mM) prepulse. However, no detectable pHi change was observed in cells exposed to bafilomycin A1 under control conditions. These studies suggest that vacuolar H(+)-ATPase is activated by cytoplasmic acidification and by reduction of the proton electrochemical gradient across the plasma membrane. We speculate that the mechanism might contribute to maintenance of acid-base balance in NPE.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Anti-Bacterial Agents; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line, Transformed; Cells, Cultured; Chlorides; Ciliary Body; Enzyme Inhibitors; Epithelial Cells; Humans; Hydrogen-Ion Concentration; Kidney; Kinetics; Macrolides; Omeprazole; Ouabain; Potassium; Proton-Translocating ATPases; Rabbits; Swine; Zinc Compounds

We recently described a large, multiple-conductance Cl- channel in excised patches from normal T lymphocytes. The properties of this channel in excised patches are similar to maxi-Cl- channels found in a number of cell types. The voltage dependence in excised patches permitted opening only at nonphysiological voltages, and channel activity was rarely seen in cell-attached patches. In the present study, we show that Cl- channels can be activated in intact cells at physiological temperatures and voltages and that channel properties change after patch excision. Maxi-Cl- channels were reversibly activated in 69% of cell-attached patches when the temperature was above 32 degrees C, whereas fewer than 2% of patches showed activity at room temperature. Upon excision, the same patches displayed large, multiple-conductance Cl- channels with characteristics like those we previously reported for excised patches. After patch excision, warm temperatures were not essential to allow channel activity; 37% (114/308) of inside-out patches had active channels at room temperature. The voltage dependence of the channels was markedly different in cell-attached recordings compared with excised patches. In cell-attached patches, Cl- channels could be open at cell resting potentials in the normal range. Channel activation was not related to changes in intracellular Ca2+ since neither ionomycin nor mitogens activated the channels in cell-attached patches, Ca2+ did not rise in response to warming and the Cl- channel was independent of Ca2+ in inside-out patches. Single-channel currents were blocked by internal or external Zn2+ (100-200 microM), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonate (SITS, 100-500 microM) and 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS, 100 microM). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Calcium; Chloride Channels; Chlorides; Electric Conductivity; Humans; Ion Channels; Ionomycin; Membrane Potentials; Membrane Proteins; Nitrobenzoates; T-Lymphocytes; Zinc; Zinc Compounds