Electrodiffusion, barrier, and gating analysis of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin.

Current-voltage curves for DIDS-insensitive Cl- conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous C1- conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K+ and Cl- were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at approximately 65% above 1 microM DIDS at both 25 degrees C and 37 degrees C. The nonlinear current-voltage curves for DIDS-insensitive net Cl- effluxes, induced by valinomycin or gramicidin at varied [K+] o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K+]o. Under our experimental conditions (pH 7.5, 23 degrees C, 1-3 microM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio PK/PCl is 74 +/- 9 with 10 microM DIDS, corresponding to 73% inhibition of PCl. Fitting the constant field current-voltage equation to the measured Cl- currents yields PCl = 0.13 h-1 with DIDS, compared to 0.49 h-1 without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl- current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 +/- 0.005 S/g Hb, half the channels are open at -27 +/- 2 mV, and the equivalent gating charge is -1.2 +/- 0.3.

Odontogenic tumors that mimic a dentigerous cyst.

Hyperplastic follicular tissue and a dentigerous cyst are the two most common entities associated with the crown of an impacted tooth. However, other diagnostic possibilities, including odontogenic tumors should also be considered in the differential diagnosis of a pericoronal radiolucency. This article presents three cases of radiolucent lesions that were histologically diagnosed as an odontogenic tumor, and stresses the importance of histological evaluation of all pericoronal radiolucencies.

Voltage dependence of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin.

Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS-insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS-insensitive Cl conductance turns on at an Em of approximately -40 mV.

Proton (or hydroxide) fluxes and the biphasic osmotic response of human red blood cells.

Upon exposure of human red blood cells to hypertonic sucrose, the fluorescence of the potentiometric indicator 3,3'-dipropylthiadicarbocyanine iodide, denoted diS-C3(5), displays a biphasic time course indicating the rapid development of an inside-positive transmembrane voltage, followed by a slow DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene)-sensitive decline of the voltage. In addition to monitoring membrane potential, proton (or hydroxide) fluxes were measured by a pH stat method, cell volume was monitored by light scattering, and cell electrolytes were measured directly when red cells were shrunken either with hypertonic NaCl or sucrose. Shrinkage by sucrose induced an initial proton efflux (or OH- influx) of 5.5 mu eq/g Hb.min and a Cl shift of 21-31 mu eq/g Hb in 15 min. Upon shrinkage with hypertonic NaCl, the cells are initially close to Donnan equilibrium and exhibit no detectable shift of Cl or protons. Experiments with the carbonic anhydrase inhibitor ethoxzolamide demonstrate that for red cell suspensions exposed to air and shrunken with sucrose, proton fluxes mediated by the Jacobs-Stewart cycle contribute to dissipation of the increased outward Cl concentration gradient. With maximally inhibitory concentrations of ethoxzolamide, a residual proton efflux of 2 mu eq/g Hb.min is insensitive to manipulation of the membrane potential with valinomycin, but is completely inhibited by DIDS. The ethoxzolamide-insensitive apparent proton efflux may be driven against the electrochemical gradient, and is thus consistent with HCl cotransport (or Cl/OH exchange). The data are consistent with predictions of equations describing nonideal osmotic and ionic equilibria of human red blood cells. Thus osmotic equilibration after shrinkage of human red blood cells by hypertonic sucrose occurs in two time-resolved steps: rapid equilibration of water followed by slower equilibration of chloride and protons (or hydroxide). Under our experimental conditions, about two-thirds of the osmotically induced apparent proton efflux is mediated by the Jacobs-Stewart cycle, with the remainder being consistent with mediation via DIDS-sensitive HCl cotransport (or Cl/OH exchange).

Two mechanisms by which fluorescent oxonols indicate membrane potential in human red blood cells.

Optical potentiometric indicators have been used to monitor the transmembrane electrical potential (Em) of many cells and organelles. A better understanding of the mechanisms of dye response is needed for the design of dyes with improved responses and for unambiguous interpretation of experimental results. This paper describes the responses to delta Em of 20 impermeant oxonols in human red blood cells. Most of the oxonols interacted with valinomycin, but not with gramicidin. The fluorescence of 15 oxonols decreased with hyperpolarization, consistent with an "on-off" mechanism, whereas five oxonols unexpectedly showed potential-dependent increases in fluorescence at less than 2 microM [dye]. Binding curves were determined for two dyes (WW781, negative response and RGA451, positive response) at 1 mM [K]o (membrane hyperpolarized with gramicidin) and at 90 mM [K]o (delta Em = 0 with gramicidin). Both dyes showed potential-dependent decreases in binding. Changes in the fluorescence of cell suspensions correlated with changes in [dye]bound for WW781, in accordance with the "on-off" mechanism, but not for RGA451. Large positive fluorescence changes (greater than 30%) dependent on Em were observed between 0.1 and 1.0 microM RGA451. A model is suggested in which RGA451 moves between two states of different quantum efficiencies within the membrane.

Calcium, cell shrinkage, and prolytic state of human red blood cells.

The effects of intracellular calcium, or Cac, on the Na permeability of human red blood cells were examined during 3-h incubations with the Ca ionophore A23187 and varied external Ca, Cao. Above 3 microM Cao, Nac increased significantly as ATP decreased. Maintenance of normal ATP with vanadate did not prevent the gain of Nac. Similar amounts of Nac were gained in 3 h by ouabain-treated cells exposed to the K ionophore valinomycin or by cells osmotically shrunken. Cells shrunken with sucrose also exhibited partial loss of Kc. When the cells with increased Nac were subsequently transferred to Na-free, high-K medium, the Nac and Kc that had changed slowly over 3 h returned toward normal within 10 min. The development of irreversible high cation permeability in shrunken cells was not prevented by a variety of transport inhibitors. These observations and cell volume distributions suggest that prolonged shrinkage induces a subpopulation of cells to become highly cation permeable, or "prolytic". The major effect of Cac on Na permeability appears to be an indirect consequence of cell shrinkage due to KCl loss.

Relationship between the shape and the membrane potential of human red blood cells.

Microscopic observations of isotonic suspensions of human red blood cells demonstrate that cell shape is unaltered when the transmembrane electrical potential, or Em, is set in the range -85 to + 10 mV with valinomycin at varied external K+, or Ko X Em was measured with the fluorescent potentiometric indicator, diS-C3(5), as calibrated by a delta pH method. Repeating Glaser's experiments in which echinocytosis was attributed to hyperpolarization, we found that at low ionic strength the pH-dependent effects of amphotericin B appear to be unrelated to Em. The effects of increased intracellular Ca2+, or Cac, on echinocytosis and on Em are separable. With Ca ionophore A23187 half-maximal echinocytosis occurs at greater Cao than that which induces the half-maximal hyperpolarization associated with Ca-induced K+ conductance (Gardos effect). Thus, cells hyperpolarized by increased Cac remain discoidal when Ca is below the threshold for echinocytosis. With A23187 and higher Cao, extensive echinocytosis occurs in cells which are either hyperpolarized or at their resting potential. The Ca-activation curve for echinocytosis is left-shifted by low Ko, a new observation consistent with increased DIDS-sensitive uptake of 45Ca by hyperpolarized cells. These results support the following conclusions: (1) the shape and membrane potential of human red blood cells are independent under the conditions studied; (2) in cells treated with A23187, the Gardos effect facilitates echinocytosis by increasing Cac.

Membrane potentials associated with Ca-induced K conductance in human red blood cells: studies with a fluorescent oxonol dye, WW 781.

A divalent anionic dye, bis-[3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-pentamethine oxonol (WW 781) is a rapidly responding fluorescent indicator of KCl diffusion potentials induced in human red blood cells with valinomycin, gramicidin, and with the Ca ionophore A 23187 in the presence of external Ca. WW 781 has a sensitivity of 0.13% delta F/mV, a detection limit of 10 mV, a response time of less than 1 sec, and exhibits a decrease in fluorescence intensity upon hyperpolarization without detectable shifts in absorption or emission peaks. This dye does not perturb the normal resting potential, and unlike the slow permeant cyanine dyes, does not inhibit Ca-induced K conductance in human red blood cells. However, WW 781 does stimulate Ca-induced unidirectional Rb efflux. With Ca plus A 23187, the initial rapid change in dye fluorescence is sensitive to [Ca]o and to [A 23187], is reversible with excess EGTA, and is inhibited by quinine, oligomycin, and by trifluoperazine. A biphasic dependence of hyperpolarization on Ko is evident at pH 6, where the ionic selectivity of activation is K, Rb greater than Cs greater than Na and that of conductance is K, Rb greater than Cs. Conditions were defined which permitted continuous monitoring of Em for at least 10 min, and the time dependence of the Ca-induced potentials was characterized. Since the properties of the Ca-induced changes in dye fluorescence correlate well with the known characteristics of Ca-induced K permeability, we conclude that WW 781 is a useful indicator of changes in Em, provided that sufficient controls are employed to separate direct effects of Ca on dye fluorescence from the effects of Em on fluorescence.