scholarly journals Digitonin-permeabilized colonic cell layers. Demonstration of calcium-activated basolateral K+ and Cl- conductances.

1988 ◽  
Vol 92 (3) ◽  
pp. 281-306 ◽  
Author(s):  
D Chang ◽  
D C Dawson
Keyword(s):  
Ion Selectivity ◽  
Basolateral Membrane ◽  
Permeability Barrier ◽  
Buffer System ◽  
Bathing Solution ◽  
Marker Enzyme ◽  
Cell Layers ◽  
Mucosal Side ◽  
Apical Membranes ◽  
Conductive Properties

Sheets of isolated turtle colon were exposed to digitonin on the mucosal side to chemically remove the apical membrane as a permeability barrier. Increases in the mucosal uptake of 86Rb, [3H]mannitol, and 45Ca-EGTA, and the appearance of the cytosolic marker enzyme lactate dehydrogenase in the mucosal bath confirmed the permeabilizing effect of the detergent. Basolateral K+ and Cl- currents were generated by imposing transmural ion gradients, and cytosolic free Ca2+ was manipulated by means of a Ca2+-EGTA buffer system in the mucosal bathing solution. Raising the cytosolic free Ca2+ concentration from the nanomolar to the micromolar range activated basolateral conductances for K+ and Cl-. Differences in ion selectivity, blocker specificity, calcium activation kinetics, and divalent cation activation selectivity indicated that the Ca2+-induced increases in the K+ and Cl- conductances were due to separate populations of channels. The results are consistent with the notion that the apical membranes of turtle colon epithelial cells can be functionally removed under conditions that preserve some of the conductive properties of the basolateral membrane, specifically Ca2+-activated conductive pathways for K+ and Cl-. This permeabilized preparation should offer a means for the identification of macroscopic currents that are due to presumed Ca2+-activated channels, and may also provide a model system for the functional reconstitution of channel regulatory mechanisms.

scholarly journals Differentiation of two distinct K conductances in the basolateral membrane of turtle colon.

1986 ◽  
Vol 88 (2) ◽  
pp. 237-251 ◽  
Author(s):  
W J Germann ◽  
M E Lowy ◽  
S A Ernst ◽  
D C Dawson
Keyword(s):  
Physical Properties ◽  
Relative Abundance ◽  
Ion Selectivity ◽  
Basolateral Membrane ◽  
The Other ◽  
Tracer Kinetics ◽  
Present Evidence ◽  
K Channels ◽  
Cell Layers ◽  
Different Populations

The K conductance of the basolateral membrane of turtle colon was measured in amphotericin-treated cell layers under a variety of ionic conditions. Changing the composition of the bathing solutions changed not only the magnitude but also the physical properties of the basolateral K conductance. The results are consistent with the notion that altered ionic environments can lead to changes in the relative abundance of two different populations of K channels in the basolateral membrane, which can be differentiated on the basis of pharmacological specificity, ion selectivity, and tracer kinetics. In the following article (Germann, W. J., S. A. Ernst, and D. C. Dawson, 1986, Journal of General Physiology, 88:253-274), we present evidence consistent with the hypothesis that one of these conductances was due to the same channels that give rise to the normal resting basolateral K conductance of the transporting cells, while the other was associated with experimental maneuvers that led to extreme swelling of the epithelial cells.

Unique electrophysiological effects of dinitrophenol in Malpighian tubules

1992 ◽  
Vol 263 (3) ◽  
pp. R609-R614 ◽  
Author(s):  
T. L. Pannabecker ◽  
D. J. Aneshansley ◽  
K. W. Beyenbach
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Membrane Resistance ◽  
Malpighian Tubules ◽  
Membrane Voltage ◽  
Shunt Resistance ◽  
Electrophysiological Studies ◽  
Apical Membranes ◽  
Conductive Properties ◽  
Electrophysiological Effects

In the course of electrophysiological studies of Malpighian tubules of the mosquito Aedes aegypti, we have found unusual effects of 2,4-dinitrophenol (DNP) that offer new insights into the electrogenic and conductive properties of the tubule. DNP (10(-4)M) depolarized the basolateral membrane voltage from -58.0 to -3.3 mV, and it depolarized the apical membrane voltage from 110.6 to 8.9 mV. In parallel the transepithelial electrical resistance increased from 11.4 to 16.8 k omega.cm, and the fractional resistance of the apical membrane increased from 0.32 to 0.57. On the assumption that measures of transepithelial resistance in the presence of DNP approach the shunt resistance, the experimental results indicate the following characteristics for the equivalent circuit of the tubule: 1) a shunt resistance that is approximately one-half the transcellular resistance, 2) low and high electromotive forces, respectively, at the basolateral and apical membranes of principal cells, 3) an electrogenic pump at the apical membrane, and 4) a basolateral membrane voltage that is due mostly to the voltage developed by current flow across the basolateral membrane resistance.

scholarly journals Mouse retinal pigment epithelial cells exhibit a thiocyanate-selective conductance

2018 ◽  
Vol 315 (4) ◽  
pp. C457-C473 ◽  
Author(s):  
Xu Cao ◽  
Bikash R. Pattnaik ◽  
Bret A. Hughes
Keyword(s):  
Ion Selectivity ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Potential Gradient ◽  
Retinal Pigment Epithelial Cells ◽  
Transport Pathway ◽  
Intact Cells ◽  
Anion Conductance ◽  
Apical Membranes

The basolateral membrane anion conductance of the retinal pigment epithelium (RPE) is a key component of the transepithelial Cl− transport pathway. Although multiple Cl− channels have been found to be expressed in the RPE, the components of the resting Cl− conductance have not been identified. In this study, we used the patch-clamp method to characterize the ion selectivity of the anion conductance in isolated mouse RPE cells and in excised patches of RPE basolateral and apical membranes. Relative permeabilities ( PA/ PCl) calculated from reversal potentials measured in intact cells under bi-ionic conditions were as follows: SCN− >> ClO4− > [Formula: see text] > I− > Br− > Cl− >> gluconate. Relative conductances ( GA/ GCl) followed a similar trend of SCN− >> ClO4− > [Formula: see text] > I− > Br− ≈Cl− >> gluconate. Whole cell currents were highly time-dependent in 10 mM external SCN−, reflecting collapse of the electrochemical potential gradient due to SCN− accumulation or depletion intracellularly. When the membrane potential was held at −120 mV to minimize SCN− accumulation in cells exposed to 10 mM SCN−, the instantaneous current reversed at −90 mV, revealing that PSCN/ PCl is approximately 500. Macroscopic current recordings from outside-out patches demonstrated that both the basolateral and apical membranes exhibit SCN− conductances, with the basolateral membrane having a larger SCN− current density and higher relative permeability for SCN−. Our results suggest that the RPE basolateral and apical membranes contain previously unappreciated anion channels or electrogenic transporters that may mediate the transmembrane fluxes of SCN− and Cl−.

Suppression of gastric acid secretion by furosemide in isolated gastric mucosa of guinea pig

1980 ◽  
Vol 239 (6) ◽  
pp. G532-G535 ◽  
Author(s):  
A. Ayalon ◽  
A. Corcia ◽  
G. Klemperer ◽  
S. R. Caplan
Keyword(s):  
Guinea Pig ◽  
Acid Secretion ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Conductance ◽  
Short Circuit Current ◽  
Cl Transport ◽  
Consequent Reduction ◽  
Net Flux ◽  
Mucosal Side

The effect of furosemide on acid secretion and Cl- transport was studied in isolated fundic mucosa of the guinea pig. Furosemide (10(-3) M), applied to the serosal side produced an immediate effect on the short-circuit current (Isc), lowering it by 47 +/- 2%. Potential difference decreased by 29 +/- 3%, electrical conductance by 18 +/- 4%, acid secretion by 38 +/- 1%, and net flux of Cl- from serosal-to-mucosal side by 37%. Application of the drug to the mucosal side produced similar effects on acid secretion and on the electrical parameters. It is suggested that furosemide blocks the entrance of Cl-, by the Na+--Cl- cotransport mechanism, through the basolateral membrane of the secreting cell. The consequent reduction in electrogenic Cl- transport would cause Isc and acid secretion to decrease. A reduction of Cl- conductance of the apical membrane, upon mucosal application of the drug, would cause similar effects on acid secretion and Cl- transport.

Coupled transport of p-aminohippurate by rat kidney basolateral membrane vesicles

1988 ◽  
Vol 255 (4) ◽  
pp. F597-F604 ◽  
Author(s):  
J. B. Pritchard
Keyword(s):  
Rat Kidney ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Coupled System ◽  
Disulfonic Acid ◽  
Coupled Transport ◽  
Indirect Coupling ◽  
Negative Membrane Potential ◽  
Secretory Transport ◽  
Apical Membranes

p-Aminohippuric acid (PAH) transport by basolateral membrane (BLM) vesicles isolated from rat renal cortex was stimulated very little by a Na+ gradient (out greater than in). However, when micromolar concentrations of glutaric acid or alpha-ketoglutaric acid were added in the presence of a out greater than in Na+ gradient, PAH uptake was accelerated greater than 20-fold and an overshoot of greater than fivefold was produced. Other anions, e.g., fumarate, stimulated PAH uptake very modestly under these conditions (approximately 2-fold), and that stimulation was totally prevented by short circuiting, i.e., with K+ (in = out) and valinomycin. Glutarate-stimulated uptake was inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and probenecid and was slightly stimulated by the imposition of an inside-negative membrane potential. Furthermore, even in the absence of a Na+ gradient, glutarate-loaded vesicles exhibited a marked acceleration of PAH uptake (5-fold) and a modest overshoot (2.5-fold). These results suggest an indirect coupling of BLM PAH uptake to the Na+ gradient by a cyclic accumulation (Na+-dependent) of glutarate followed by its efflux from the vesicle in exchange for PAH. This coupled system was absent in apical membranes. Thus net secretory transport of PAH may entail Na+-dependent, glutarate-driven PAH uptake at the BLM, followed by the exit of PAH into the lumen down its electrochemical gradient, probably in exchange for other anions, e.g., Cl-, HCO3-, or OH-.

Localization of a novel 210 kDa protein in Xenopus tight junctions

1997 ◽  
Vol 110 (8) ◽  
pp. 1005-1012 ◽  
Author(s):  
C.S. Merzdorf ◽  
D.A. Goodenough
Keyword(s):  
Monoclonal Antibody ◽  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Basolateral Membrane ◽  
Immunofluorescent Staining ◽  
Junctional Complex ◽  
Permeability Barrier ◽  
Membrane Domains ◽  
Kidney Liver

The tight junction is the most apical member of the intercellular junctional complex. It functions as a permeability barrier between epithelial cells and maintains the integrity of the apical and basolateral membrane domains. In order to study tight junctions in Xenopus laevis, a polyclonal antibody was raised which recognized Xenopus ZO-1. Monoclonal antibody 19B1 (mAb 19B1) was generated in rats using a crude membrane preparation from Xenopus lung as antigen. mAb 19B1 gave immunofluorescent staining patterns identical to those seen with anti-ZO-1 on monolayers of Xenopus A6 kidney epithelial cells and on frozen sections of Xenopus kidney, liver, and embryos. Electron microscopy showed that the 19B1 antigen colocalized with ZO-1 at the tight junction. Western blotting and immunoprecipitation demonstrated that ZO-1 is an approximately 220 kDa protein in Xenopus, while mAb 19B1 identified an approximately 210 kDa antigen on immunoblots. Immunoprecipitates of ZO-1 were not recognized by mAb 19B1 by western analysis. The solubility properties of the 19B1 antigen suggested that it is a peripheral membrane protein. Thus, the antigen recognized by the new monoclonal antibody 19B1 is not ZO-1 and represents a different Xenopus tight junction associated protein.

Na+ pump inhibition downregulates an ATP-sensitive K+ channel in rabbit proximal convoluted tubule

1993 ◽  
Vol 264 (4) ◽  
pp. F760-F764 ◽  
Author(s):  
A. M. Hurst ◽  
J. S. Beck ◽  
R. Laprade ◽  
J. Y. Lapointe
Keyword(s):  
Basolateral Membrane ◽  
Proximal Convoluted Tubule ◽  
K Channel ◽  
Bathing Solution ◽  
Channel Activity ◽  
Open Probability ◽  
Functional Link ◽  
Tightly Coupled ◽  
Stimulation Of

In several epithelial and nonepithelial tissues a functional link between the basolateral Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) and a basolateral K+ conductance has been established. However, the nature of this link is unclear. We have previously identified a K+ channel on the basolateral membrane of the proximal convoluted tubule perfused in vitro, the activity of which is increased by stimulation of Na+ transport [J. S. Beck, A. M. Hurst, J.-Y. Lapointe, and R. Laprade. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F496-F501, 1993]. In the present study we investigate whether basolateral membrane K+ channel activity is tightly coupled to Na(+)-K(+)-ATPase activity. In cell-attached patches (150 mM K+ pipette), following stimulation of channel activity by addition of Na(+)-cotransported solutes to the tubule lumen, mean channel open probability (NPo) was reduced from 0.35 +/- 0.09 to 0.14 +/- 0.06 (n = 7, P < 0.05) by blocking the Na(+)-K(+)-ATPase with 100 microM strophanthidin. In excised patches the channel was reversibly blocked by 2 mM ATP from the cytosolic face of the patch, such that NPo fell to 20.1 +/- 7.0% (n = 5, P < 0.001) of control and recovered to 52.2 +/- 11.2% (n = 5, P < 0.05) after washout of ATP. Diazoxide, a putative opener of ATP-sensitive K+ channels, when added to the bathing solution of an unstimulated tubule (microperfused in the absence of Na(+)-cotransported solutes), increased NPo from 0.046 +/- 0.035 to 0.44 +/- 0.2 (n = 6, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Coupled NaCl entry into Necturus gallbladder epithelial cells

1982 ◽  
Vol 243 (3) ◽  
pp. C140-C145 ◽  
Author(s):  
A. C. Ericson ◽  
K. R. Spring
Keyword(s):  
Epithelial Cells ◽  
Cell Volume ◽  
Apical Membrane ◽  
Ionic Composition ◽  
Basolateral Membrane ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Bathing Solution ◽  
Parallel Operation ◽  
Solute Content

NaCl entry into Necturus maculosus gallbladder epithelial cells was studied by determination of the rate of fluid movement into the cell when the Na+-K+-ATPase was inhibited by 10(-4) M ouabain in the serosal bathing solution. The cell swelling was due to continuing entrance of NaCl into the cell across the apical membrane, which increased the solute content of the cell; the resultant rise in cell osmolality induced water flow and cell swelling. The rate of swelling was 4.3% of the cell volume per minute, equivalent to a volume flow across the apical membrane of 1.44 x 10(-6) cm/s, similar in magnitude to the normal rate of fluid absorption by the gallbladder. We determined the mechanism of NaCl entry by varying the ionic composition of the mucosal bath; when most of the mucosal Na+ or Cl- was replaced, cell volume did not increase during pump inhibition. The rate of NaCl entry was a saturable function of Na+ or Cl- in the mucosal bathing solution with K1/2 values of 26.6 mM for Na+ and 19.5 mM for Cl-. The mode of NaCl entry was probably not the parallel operation of Na+-H+ and Cl(-)-HCO-3 exchangers because of the lack of effect of bicarbonate removal or of the inhibitors amiloride and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. NaCl entry was reversibly inhibited by bumetanide in the mucosal bathing solution. Transepithelial NaCl and water absorption is the result of the coupled, carrier-mediated movement of NaCl into the cell across the apical membrane and the active extrusion of Na+ by the Na+-K+-ATPase in the basolateral membrane.

Barium inhibition of basolateral membrane potassium conductance in tracheal epithelium

1983 ◽  
Vol 244 (6) ◽  
pp. F639-F645 ◽  
Author(s):  
M. J. Welsh
Keyword(s):  
Driving Force ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Potassium Conductance ◽  
Short Circuit Current ◽  
Electrical Circuit ◽  
Tracheal Epithelium ◽  
Bathing Solution ◽  
K Permeability

Addition of barium ion, Ba2+, to the submucosal bathing solution of canine tracheal epithelium reversibly decreased the short-circuit current and increased transepithelial resistance. The decrease in short-circuit current represented a decrease in the net rate of Cl secretion with no change in the rate of Na absorption. Intracellular microelectrode techniques and an equivalent electrical circuit analysis were used to localize the effect of Ba2+ to an inhibition of the permeability of the basolateral membrane to K. Ba2+ (2 mM) doubled basolateral membrane resistance, decreased the equivalent electromotive force at the basolateral membrane, and decreased the magnitude of the depolarization of basolateral membrane voltage produced by increasing the submucosal K concentration. The inhibition of the basolateral K permeability depolarized the negative intracellular voltage, resulting in both a decrease in the driving force for Cl exit and an estimated increase in intracellular Cl concentration. These studies indicate that there is a Ba2+-inhibitable K conductance at the basolateral membrane of tracheal epithelial cells and that the K permeability plays an important role in the generation of the negative intracellular electrical potential that provides the driving force for Cl exit from the cell.

Microelectrode assessment of chloride-conductive properties of cortical collecting duct

1984 ◽  
Vol 247 (2) ◽  
pp. F291-F302 ◽  
Author(s):  
S. C. Sansom ◽  
E. J. Weinman ◽  
R. G. O'Neil
Keyword(s):  
Cell Membrane ◽  
Tight Junction ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Membrane Conductance ◽  
Cortical Collecting Duct ◽  
Apical Cell Membrane ◽  
Conductive Properties ◽  
Chloride Activity

The chloride-conductive properties of the isolated rabbit cortical collecting duct were assessed with microelectrode techniques. The transepithelial, apical, and basolateral membrane potential differences, Vte, Va, and Vb, respectively, were monitored continuously along with periodic measurements of the transepithelial conductance, Gte, and fractional resistance, fRa (ratio of apical to apical plus basolateral membrane resistance). Active transport was eliminated in all experiments by luminal addition of 50 microM amiloride in HCO3-free solutions. Upon reducing the chloride activity in the bath (gluconate replacement), there was a marked depolarization of Vb and decrease in Gte and fRa, demonstrating a major dependence of the basolateral membrane conductance on the bath chloride activity. However, a significant K+ conductance at that barrier was also apparent since raising the bath K+ concentration caused an increase in Gte and fRa and depolarization of Vb. Lowering the chloride activity of the perfusate caused a consistent decrease of Gte but not of fRa, effects consistent with a high C1- conductance of the tight junction and little, if any, apical membrane C1- conductance. By use of the C1- -dependent conductances, the C1- permeabilities at equilibrium were estimated to be near 1.0 X 10(-5) cm X s-1 for the tight junction, PtiC1, and 5 X 10(-5) cm X s-1 for the basolateral cell membrane, PbC1. It is concluded that the paracellular pathway provides a major route for transepithelial C1- transport. Furthermore, since the isotopically measured C1- permeability is severalfold greater than PtiC1, a significant transcellular flux of C1- must exist, implicating a neutral exchange mechanism at the apical cell membrane in series with the high basolateral membrane C1- conductance.

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