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

Total crypt volume has been estimated by analysis of photographic images of intact viable crypts isolated from guinea-pig small intestine. Exposing these crypts to a hypotonic medium, led to transient swelling followed by regulatory volume decrease (RVD) in 12-20 min. RVD was blocked by inhibitors of K+ and Cl- conductance, suggesting that it occurs by activation of K+ and Cl- permeability pathways and loss of these ions.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Alkaline Phosphatase; Animals; Anthracenes; Barium; Barium Compounds; Chloride Channels; Chlorides; Guinea Pigs; Intestinal Mucosa; Intestine, Small; Ion Channels; Membrane Proteins; Microvilli; Potassium; Potassium Channels; Thymidine Kinase

Synaptic membranes from rat brain were incorporated into planar lipid bilayers, and the characteristics of two types of anion-selective channels (type I and type II) were investigated. In asymmetric BaCl2 buffers (cis, 100 mM/trans, 25 mM), single channel conductances at -40 mV were 70 pS (type I) and 120 pS (type II). Permeability ratios (PNa:PBa:PCl) calculated from the Goldman-Hodgkin-Katz current equation for type I and type II channels were 0.23:0.04:1 and 0.05:0.03:1, respectively. Both channels exhibited characteristic voltage-dependent bursting activities. Open probability for type I channels had a maximum of approximately 0.7 at about 0 mV and decreased to zero at greater transmembrane potentials of either polarity. Type II channels were relatively voltage independent at negative voltages and were inactivated at highly positive voltages. Type I channels showed spontaneous irreversible inactivation often preceded by sudden transition to subconducting states. DIDS blocked type I channels only from the cis side, while it blocked type II channels from either side.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Barium; Barium Compounds; Cell Membrane; Cell Membrane Permeability; Chlorides; Electric Conductivity; Electric Stimulation; Female; Ion Channels; Lipid Bilayers; Rats; Rats, Inbred Strains; Sodium Chloride; Synaptosomes

Regulatory volume decrease after exposure to hypo-osmotic media in MDCK epithelial cells results from activation of both K+ and Cl- conductances. Swelling-stimulated 86Rb(K) losses were observed only across the basal-lateral membrane and were relatively insensitive to 10 mM Ba2+. The effect of hypo-osmotic media upon MDCK epithelia mounted in Ussing chambers has been investigated. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a transient stimulation of inward short-circuit current (Isc) followed by inhibition of inward Isc in both control layers and in layers where inward current (due to transepithelial Cl- secretion) was first stimulated by 5 microM prostaglandin E1 (PGE1). The transient stimulation of inward current by hypo-osmotic media was not markedly attenuated by 10 mM Ba2+ in PGE1-stimulated layers. After stimulation of inward (Cl(-)-secretory) current to high levels by 10 microM adrenaline, the predominant effect of basal-lateral exposure to hypo-osmotic media was an inhibition of the inward current. This inhibition was partially reversed by 40 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS). The stimulation, then inhibition, of inward Isc is likely to be the result of separate swelling-induced K+ and Cl- conductances (respectively) at the basal-lateral membrane. The swelling-stimulated Cl- conductance is distinct from the apical Cl- conductance regulated by PGE1 or adrenaline.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Alprostadil; Animals; Barium; Barium Compounds; Biological Transport, Active; Cells, Cultured; Chlorides; Epinephrine; Epithelium; Ions; Kidney; Osmolar Concentration; Potassium; Rubidium

Volume regulatory decrease (VRD) by Necturus gallbladder epithelial cells in Cl Ringer was unaffected by the addition of 5 mM BaCl2 to apical perfusates but was inhibited by the addition of 5 mM BaCl2 and 50 or 3 microM phencyclidine (PCP) to serosal perfusates, suggesting that K channels in the basolateral membrane were activated during VRD. VRD was unaffected by replacement of Cl with NO3 or SCN, suggesting that Cl-dependent Na-K-Cl and K-Cl cotransport were not involved. In SCN Ringer, VRD was inhibited by the addition of 0.1 mM bumetanide to serosal perfusates, suggesting that bumetanide-sensitive anion channels in the basolateral membrane were also activated. A transient 10-mV hyperpolarization of the membrane potential was associated with VRD. The channel blockers that inhibited VRD had little or no effect on the hyperpolarization, suggesting that the changes in membrane potential were unrelated to the changes in cell volume. Perfusion of the apical surface of the epithelium with isotonic solutions containing 10 mM D-glucose resulted in a variable increase in cell volume followed by a variable shrinkage to normal, suggesting that VRD was also activated during organic solute absorption. The increase in cell volume was blocked by the addition of 0.01 or 1 mM phlorizin to mucosal perfusates. The reduction in cell volume was inhibited by the addition of 0.1 mM bumetanide, but not BaCl2 or PCP, to serosal perfusates, indicating the the shrinkage mechanism secondary to glucose addition differed from that seen after exposure to hypotonic perfusates.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Barium; Barium Compounds; Biological Transport, Active; Bumetanide; Chlorides; Epithelial Cells; Epithelium; Gallbladder; Glucose; Homeostasis; In Vitro Techniques; Ion Channels; Kinetics; Membrane Potentials; Necturus; Perfusion; Phencyclidine; Phlorhizin; Solutions; Thiocyanates

The factors which influence the exocytosis of mucins are not well characterized. Since the physical properties of mucins may be affected significantly by the co-secretion of electrolytes and water, we studied the relationship between ion movement and mucin secretion in T84 cells, a human colonic adenocarcinoma cell line which has been well characterized with respect to apical chloride secretion. Secretion of mucin was assessed by immunoassay of mucin appearing in the medium within 30 min of stimulation. Cells were grown on plastic in DMEM/Ham's F12 medium and experiments were carried out at 70% confluence. Mucin secretion was stimulated by the calcium ionophore A23187, or A23187 plus vasoactive intestinal polypeptide. Stimulated mucin secretion was not affected by loop diuretics (furosemide (1 x 10(-3) M) or bumetanide (1 x 10(-4) M)), with or without the addition of ouabain (5 x 10(-5) M) and amiloride (1 x 10(-5) M), making it unlikely that transcellular chloride movements in necessary for mucin secretion. However, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; (1 x 10(-5) and 5 x 10(-5) M) and three potassium channel blockers BaCl2 (1 x 10(-3) and 5 x 10(-3) M), tetraethylammonium chloride (1 x 10(-2) M) and quinine (5 x 10(-4) M) inhibited mucin secretion. A DIDS-sensitive chloride channel or chloride/bicarbonate exchanger and a Ca2(+)-dependent potassium channel may play important roles in mucin secretion. Since plasma membranes are sparingly permeable to DIDS, the DIDS-sensitive site is likely to be on the apical plasma membrane, perhaps at an initiation locus for exocytosis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenocarcinoma; Amiloride; Barium; Barium Compounds; Calcimycin; Cell Line; Chlorides; Colonic Neoplasms; Furosemide; Humans; Kinetics; Mucins; Ouabain; Potassium Channels; Quinine; Stilbenes; Tetraethylammonium; Tetraethylammonium Compounds; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The effects of succinate on the intracellular potential difference, PDI, were measured in isolated rabbit ciliary processes. Concentration-dependent increases in the hyperpolarization of PDI occurred between 1 and 15 mM succinate in NaCl Ringers. With 5 mM succinate, there was a 6 mV hyperpolarization. Even though the hyperpolarization of PDI was comparable with 10 and 15 mM succinate, it was more sustained at the latter two concentrations. Succinate also elicited comparable hyperpolarizations of PDI in either Cl(-)-free or HCO3(-)-free Ringers. Similarly, following incubation with either 0.1 mM DIDS or 3 mM BaCl2 the effect of succinate on PDI was unchanged. Five mM succinate had no effect if it was added after 5 mM malonate. Malonate (5 mM) rapidly reversed a 5 mM succinate-induced hyperpolarization of PDI which also suggests a metabolically mediated effect on PDI. An isosmotic substitution of Na+ with NMDG Ringers depolarized PDI, whereas PDI depolarized biphasically during exposure to 0.1 mM ouabain. The addition of 5 mM succinate had no effect on either the time course or the magnitude of the depolarization of PDI during blocking of the Na+:K+ pump with either Na(+)-free Ringers or ouabain. Taken together, these results show that succinate selectively stimulates the Na+:K+ pump, but has no effect on any Cl-, HCO3- or a Ba2(+)-sensitive K+ conductance.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Barium; Barium Compounds; Biological Transport, Active; Chlorides; Ciliary Body; Epithelium; Female; In Vitro Techniques; Male; Malonates; Membrane Potentials; Ouabain; Potassium; Rabbits; Sodium; Succinates

The mechanism of chloride uptake at the basal membrane (stromal side) of rabbit corneal epithelium was examined by observing the effects of ion transport inhibitors and ion concentrations on the stimulated epithelial short-circuit current (Isc). Loop diuretics inhibited the theophylline-stimulated peak and sustained Isc. Treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 0.2 mM) and/or 5-(N,N-dimethyl)amiloride (0.1 mM) as well as the potent anion exchange inhibitor, 5c(+)[(2,3,9,9a-tetrahydro-1H-fluoren-7-yl)oxy]acetic acid (0.01 mM), had no significant effect on Isc. These results are consistent with Cl- uptake by a Na+-Cl- or Na+-K+-2Cl- cotransport mechanism rather than Cl(-)-HCO3(-)-OH- exchange coupled to Na+-H+ exchange. Incubation in low [Na+] or [Cl-] before stimulation with forskolin (0.1 mM) reduced both peak and sustained Isc, and saturation kinetics were exhibited. Hill coefficients for [Na+] and [Cl-] were 0.99 and 1.04, respectively, for peak Isc and 0.66 and 1.18, respectively, for sustained Isc. Apparent ion affinities for Na+ and Cl- were 13.5 and 18 mM, respectively, for peak Isc and 15 and 22 mM, respectively, for sustained Isc. These results favor Cl- uptake by a 1 Na+:1 Cl- cotransport mechanism for the rabbit corneal epithelium, but involvement of K+ in this process has not been eliminated.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Barium; Barium Compounds; Bicarbonates; Biological Transport, Active; Cell Membrane; Chlorides; Colforsin; Cornea; Epithelium; Furosemide; In Vitro Techniques; Kinetics; Potassium; Rabbits; Sodium; Theophylline

The behaviour of intracellular pH (pHi) was studied with micro-electrodes in frog semitendinosus muscle which was superfused with Ringer solution and with depolarizing solutions. The electrodes were introduced into the depolarized muscle about 40 min after contracture had subsided. All studies were done at external pH (pHo) of 7.35 and at 22 degrees C. The pHi in normal Ringer solution buffered with HEPES was 7.18 +/- 0.03 (S.E. of mean) (n = 10); the membrane potential, Vm, was -88 +/- 1.8 mV. When pHi was lowered to about 6.8 by replacing the HEPES by 5% CO2, 24 mM-HCO3 (constant pHo), it recovered at a very slow rate of 0.025 +/- 0.011 delta pHi h-1 (n = 6). When all the Na was replaced by N-methyl-D-glucamine (initial pHi 7.20 +/- 0.04, initial Vm -89 +/- 1.5 mV, n = 8), this slow alkalinization was converted into a slow acidification at a rate of 0.069 +/- 0.024 delta pHi h-1. In muscle depolarized in 15 mM-K (Vm approximately -50 mV), the rate of recovery from CO2 acidification was not increased above that in normal Ringer solution (2.5 mM-K). When, however, the muscle was depolarized in 50 mM-K to about -20 mV, the rate of recovery increased to 0.33 +/- 0.07 delta pHi h-1 (n = 6) when external Cl was kept constant, or to 0.21 +/- 0.03 (n = 9) when [K]. [Cl] product was kept constant. In the absence of Na, pHi recovery rate in 50 mM-K was reduced by at least 90%. Enhanced recovery from CO2-induced acidification was also observed in 2.5 mM-K when the fibres were depolarized to about -20 mV in one of two ways: (a) by previous exposure for 60 min to 50 mM-K at constant Cl, or (b) by reduction of external Cl to 5.9 mM in the presence of 0.5 mM-Ba. When pHi of depolarized fibres (50 mM-K) was lowered to about 6.8 by the weak acid dimethyl-2,4-oxazolidinedione (DMO), it recovered at a rate of 0.12 delta pHi h-1 in two experiments. In fibres depolarized in 50 mM-K and constant Cl, either 0.1 mM-SITS or 0.5 mM-amiloride slowed pHi recovery from CO2 exposure by about 50%. When the depolarization was achieved at constant [K]. [Cl] product, amiloride slowed pHi recovery by about 50%, while SITS had, at most, only a slight effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Animals; Barium; Barium Compounds; Buffers; Carbon Dioxide; Chlorides; Hydrogen-Ion Concentration; In Vitro Techniques; Isotonic Solutions; Membrane Potentials; Muscles; Potassium Chloride; Rana pipiens; Ringer's Solution; Sodium; Time Factors