Transport properties of toad kidney epithelia in culture

1981 ◽  
Vol 241 (3) ◽  
pp. C154-C159 ◽  
Author(s):  
F. M. Perkins ◽  
J. S. Handler
Keyword(s):  
Electrical Resistance ◽  
Hormonal Regulation ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Transepithelial Electrical Resistance ◽  
Apical Surface ◽  
Osmotic Water ◽  
Sodium Flux ◽  
Regulation Of Transport ◽  
Osmotic Water Flow

The characteristics of a continuous line of toad kidney epithelial cells (A6) are described. These cells form a monolayer epithelium of high transepithelial electrical resistance (about 5,000 omega . cm2). The cells generate a transepithelial potential difference (apical surface negative) of about 9 mV. The short-circuit current is equivalent to net sodium flux. Net sodium flux is stimulated by aldosterone and by analogues of cAMP. The stimulation is readily reversible. Neither urea permeability nor osmotic water flow is altered by analogues of cAMP. Amiloride eliminates 90% of the short-circuit current. Thus A6 cells form an epithelium with several differentiated properties including hormonal regulation of transport.

Protamine increases the permeability of cultured epithelial monolayers

1990 ◽  
Vol 68 (1) ◽  
pp. 220-227 ◽  
Author(s):  
M. W. Peterson ◽  
D. Gruenhaupt
Keyword(s):  
Actin Cytoskeleton ◽  
Electrical Resistance ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Transepithelial Electrical Resistance ◽  
Apical Surface ◽  
Type Ii ◽  
Epithelial Monolayers ◽  
Organ Specific ◽  
Darby Canine Kidney

Polycations, including protamine, have been reported to decrease the barrier integrity of cultured rat pulmonary type II epithelial monolayers. In contrast, protamine has been reported to increase the transepithelial electrical resistance of gallbladder epithelium. The present study was done using Madin Darby canine kidney epithelial cells (MDCK) to determine whether the effect of protamine on type II epithelial monolayers was species or organ specific or was dependent on the presence of nonepithelial cells and to investigate the effect of protamine on the actin cytoskeleton. Exposure of MDCK monolayers to protamine resulted in decreased transepithelial electrical resistance (Rt), increased short-circuit current (Isc) across the monolayers, and increased mannitol permeability (Pmann) of the monolayers. The decrease in Rt and increase in Isc was seen only after the addition of protamine to the apical surface of the cells. The importance of charge in this action was supported by the fact that exposure of the monolayer to the polycation poly-L-lysine also resulted in increased Pmann, and both the decreased Rt and increased Pmann seen after the addition of protamine were prevented if the monolayers were exposed in the presence of the polyanions heparin or sulfated dextran. The increase in Pmann appeared to be the result of increased permeability in the paracellular pathway, because increased mannitol uptake by the cells represented only a fraction of the increase in Pmann. Subtle changes in the actin cytoskeleton were seen after exposure of the monolayers to protamine.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Amiloride-sensitive trypsinization of apical sodium channels. Analysis of hormonal regulation of sodium transport in toad bladder.

1983 ◽  
Vol 81 (6) ◽  
pp. 785-803 ◽  
Author(s):  
H Garty ◽  
I S Edelman
Keyword(s):  
Hormonal Regulation ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Toad Urinary Bladder ◽  
Apical Surface ◽  
Na Channels ◽  
Na Transport ◽  
Na Permeability ◽  
Trypsin Inhibition

Incubation of the mucosal surface of the toad urinary bladder with trypsin (1 mg/ml) irreversibly decreased the short-circuit current to 50% of the initial value. This decrease was accompanied by a proportionate decrease in apical Na permeability, estimated from the change in amiloride-sensitive resistance in depolarized preparations. In contrast, the paracellular resistance was unaffected by trypsinization. Amiloride, a specific blocker of the apical Na channels, prevented inactivation by trypsin. Inhibition of Na transport by substitution of mucosal Na, however, had no effect on the response to trypsin. Trypsinization of the apical membrane was also used to study regulation of Na transport by anti-diuretic hormone (ADH) and aldosterone. Prior exposure of the apical surface to trypsin did not reduce the response to ADH, which indicates that the ADH-induced Na channels were inaccessible to trypsin before addition of the hormone. On the other hand, stimulation of short-circuit current by aldosterone or pyruvate (added to substrate-depleted, aldosterone-repleted bladders) was substantially reduced by prior trypsinization of the apical surface. Thus, the increase in apical Na permeability elicited by aldosterone or substrate involves activation of Na channels that are continuously present in the apical membrane in nonconductive but trypsin-sensitive forms.

scholarly journals Acute ENaC Stimulation by cAMP in a Kidney Cell Line is Mediated by Exocytic Insertion from a Recycling Channel Pool

2004 ◽  
Vol 125 (1) ◽  
pp. 81-101 ◽  
Author(s):  
Michael B. Butterworth ◽  
Robert S. Edinger ◽  
John P. Johnson ◽  
Raymond A. Frizzell
Keyword(s):  
Hormonal Regulation ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Membrane Capacitance ◽  
Apical Surface ◽  
Cortical Collecting Duct ◽  
Ussing Chambers ◽  
Camp Stimulation

Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na+ transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, MPKCCD14) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (ISC), membrane capacitance (CT), and conductance (GT) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 ± 6% (n = 31) increase in ISC and a 15.3 ± 1.5% (n = 15) increase in CT. Selective membrane permeabilization demonstrated that the CT increase was due to an increase in apical membrane capacitance. ISC and CT declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (∼1 h each cycle) resulted in response fatigue; ΔISC decreased ∼10% per stimulation–recovery cycle. When channel production was blocked by cycloheximide, ΔISC decreased ∼15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated ISC, and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the ISC responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation.

Effects of prostacyclin on short-circuit current and water flow in the toad urinary bladder

1983 ◽  
Vol 244 (3) ◽  
pp. F270-F277
Author(s):  
T. Pohlman ◽  
J. Yates ◽  
P. Needleman ◽  
S. Klahr
Keyword(s):  
Urinary Bladder ◽  
Water Transport ◽  
Water Flow ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Toad Bladder ◽  
Camp Production ◽  
Acid Product ◽  
Osmotic Water ◽  
Osmotic Water Flow

The effects of prostaglandins of the E series on sodium and water transport have been studied extensively. PGE2 has been shown to inhibit the increase in osmotic water flow produced by vasopressin and to stimulate short-circuit current (SCC) in the toad bladder. On the other hand, the effects of prostacyclin (PGI2), an arachidonic acid product, on sodium and water transport have not been extensively evaluated. The present studies describe the effects of PGI2 on basal and vasopressin-stimulated osmotic water flow and on SCC in the urinary bladder of the toad. Studies were performed in the absence or presence of indomethacin. PGI2 in the absence of indomethacin had no effect on basal or vasopressin-stimulated osmotic water flow. When indomethacin was present, thereby eliminating intrinsic prostaglandin biosynthesis, PGI2 inhibited basal but not vasopressin-stimulated osmotic water flow. PGI2 increased SCC in the presence or absence of indomethacin. 6-keto PGF1 alpha, the stable metabolite of PGI2, had no effect on SCC. PGI2 stimulated cAMP production in isolated toad bladder epithelial cells. 2',5'-Dideoxyadenosine, an inhibitor of cAMP production, blocked the increase in SCC produced by PGI2, suggesting that the effects of this compound on SCC are mediated via cAMP.

8-BrcAMP-induced capacitance and transport of H2O and Na in skin and urinary bladder of urodele amphibians

1989 ◽  
Vol 256 (6) ◽  
pp. C1145-C1152 ◽  
Author(s):  
R. B. Silver ◽  
L. G. Palmer
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Constant Current ◽  
Voltage Response ◽  
Tiger Salamander ◽  
Current Pulses ◽  
Osmotic Water ◽  
Transepithelial Voltage ◽  
Mucosal Side ◽  
Osmotic Water Flow

The effects of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) on capacitance (C), osmotic water flow (Jv), and amiloride-sensitive short-circuit current (INa) were studied in bladder and skin derived from the tiger salamander (aquatic and postmetamorphosed terrestrial phase). 8-BrcAMP-dependent increases in C, measured from the transepithelial voltage response to constant current pulses, occurred in aquatic (delta C = 44%) and terrestrial (delta C = 61%) bladders and terrestrial skin (delta C = 19%). Jv (200-mosM gradient, mucosal side hypotonic) was observed in the bladders and was further enhanced by addition of 8-BrcAMP [10(-3) M; delta Jv = 0.42 microliter.min-1.microF-1 (aquatic) and 0.32 microliter.min-1.microF-1 (terrestrial)]. The aquatic and terrestrial skins were relatively impermeable to water, but the terrestrial skin showed a small response to 8-BrcAMP (delta Jv = 0.04 microliter.min-1.microF-1). 8-BrcAMP-mediated natriferic responses were observed in aquatic bladder (delta INa = 62%) and terrestrial skin (delta INa = 105%). Antidiuretic hormone (ADH)-induced Jv was also observed in the aquatic bladder (delta Jv = 0.33 microliter.min-1.microF-1) and was similar to the 8-BrcAMP-mediated Jv measured in this tissue. The terrestrial bladder displayed a more vigorous response to 8-BrcAMP than to ADH (delta JvADH = 0.09 microliter.min-1.microF-1 and delta Jv8-BrcAMP = 0.32 microliter.min-1.microF-1), suggesting that diminished sensitivity to ADH accompanies the transition from water to land in this species.

scholarly journals Interleukin-4 and Interleukin- 13 Act on Glomerular Visceral Epithelial Cells

2000 ◽  
Vol 11 (3) ◽  
pp. 413-422
Author(s):  
JOSÉ G. VAN DEN BERG ◽  
JAN ATEN ◽  
M. ANWAR CHAND ◽  
NIKE CLAESSEN ◽  
LISETTE DIJKINK ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Epithelial Cells ◽  
Electrical Resistance ◽  
Structural Changes ◽  
Cell Structure ◽  
Short Circuit ◽  
Transepithelial Electrical Resistance ◽  
Interleukin 4 ◽  
Direct Effects ◽  
Direct Role

Abstract. In minimal change nephrosis (MCN), proteinuria is associated with structural changes of the glomerular visceral epithelial cells (GVEC). The occurrence of MCN has been associated with T-helper2 lymphocyte-dependent conditions. To examine a direct role for type 2 cytokines in GVEC injury, the expression of interleukin (IL)-4/IL-13 receptors by GVEC and direct effects of IL-4 and IL-13 on GVEC were studied. Reverse transcription-PCR showed that isolated human and rat glomeruli and cultured human and rat GVEC expressed mRNA for IL-4Rα, IL-13Rα1, and IL-13Rα2. Protein expression of IL-4Rα and IL-13Rα2 by GVEC in human kidney biopsies and by cultured human GVEC was detected by immunohistochemistry. Western blotting demonstrated phosphorylation of STAT6 in cultured GVEC upon incubation with IL-4 or IL-13. This indicated signal transduction via the heterodimeric receptor complex IL-4R2, which is composed of the IL-4Rα and the IL-13Rα1. Direct effects on GVEC function were examined in monolayer experiments. IL-4 and IL-13 dose-dependently decreased transepithelial electrical resistance of monolayers of rat GVEC to approximately 30 and 40% of baseline values, respectively. The transepithelial electrical resistance decrease was associated with a significant increase in short-circuit current, whereas no changes were observed in the transmonolayer flux of the macromolecules horseradish peroxidase (molecular weight, 44 kD) and 14C-mannitol (molecular weight, 182 Da). No changes in cell structure were observed with electron microscopy. It is concluded that by binding to specific IL-4/IL-13 receptors, IL-4 and IL-13 can exert specific effects on GVEC function, which could be of pathogenetic relevance for glomerular injury in MCN.

Na+-sugar cotransport system as a polarization marker during organization of epithelial membrane

1988 ◽  
Vol 255 (6) ◽  
pp. C745-C753 ◽  
Author(s):  
C. Lasheras ◽  
J. A. Scott ◽  
C. A. Rabito
Keyword(s):  
Electrical Resistance ◽  
Apical Membrane ◽  
Sugar Transport ◽  
Cell Polarization ◽  
Transepithelial Electrical Resistance ◽  
Apical Surface ◽  
Binding Studies ◽  
Occluding Junctions ◽  
Basolateral Side ◽  
Epithelial Membranes

The present study analyzed the changes in Na+-dependent sugar transport and transepithelial electrical resistance as LLC-PK1 cells reorganize into epithelial membranes. Sugar influx increased to reach a maximum 9 h after plating. The increase in the transepithelial electrical resistance, however, showed a significant delay, reaching steady state 15 h after plating. No changes in the electrochemical Na+ gradient were observed during the reorganization of the epithelial membranes. Kinetic analysis and [3H]phlorizin-binding studies showed that the increase in sugar influx resulted from an increase in the number of carriers. Unidirectional sugar influx measurements indicated that the sugar transporters were primarily located at the apical surface of the epithelial cells. These observations are consistent with the hypothesis that the sorting of native proteins occurs intracellularly before their insertion in the apical membrane, or as an alternative that they are randomly inserted, but then immediately sorted such as any carrier could be detected in the basolateral side during the reorganization process. In addition, the results suggest that the functional development of the apical membrane may occur before the complete sealing of the intercellular space during the development of the occluding junctions. Furthermore, development of the sugar transport system and occluding junctions was inhibited by cycloheximide and puromycin but not by actinomycin D, suggesting that the expression of epithelial cell polarization is probably a posttranslational event in the protein synthesis.

Selective effects of bumetanide on chloride transport in bullfrog cornea

1978 ◽  
Vol 234 (4) ◽  
pp. F297-F301
Author(s):  
O. A. Candia ◽  
H. F. Schoen
Keyword(s):  
Electrical Resistance ◽  
Loop Diuretic ◽  
Chloride Transport ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Selective Inhibitor ◽  
O2 Consumption ◽  
Na Transport ◽  
Bathing Medium

Frog corneas were mounted in a modified Ussing chamber and short-circuit current (SCC) and unidirectional Cl fluxes were measured. Bumetanide, a loop diuretic, at concentrations as low as 10(-7) M, reduced the SCC 29%. At 10(-5) M, bumetanide reduced the SCC 96% and increased transcorneal electrical resistance 20-51%. The forward Cl flux declined from 0.71 +/- 0.04 to 0.20 +/- 0.03 mueq/h.cm2 (n, 7), while, in separate experiments, the backward Cl flux did not change significantly (from 0.22 +/- 0.03 to 0.23 +/- 0.04; n, 7). When corneas were mounted in Cl-free Ringer and the net Na transport was stimulated with amphotericin B, 10(-5) M bumetanide had no effect on the SCC. In separate experiments the effect of 10(-5) M bumetanide on the O2 consumption was measured in a stirrer bath assembly. Bumetanide decreased the O2 consumption from 352 +/- 14 to 297 +/- 19 microliter/h.cm2 (significantly different from sham-treated controls). This decrease was similar to that obtained with furosemide or when Cl was removed from the bathing medium. We infer from these results that bumetanide is a selective inhibitor of active Cl transport in the bullfrog cornea.

Effects of ammonium ion and ammonia on function and morphology of in vitro frog gastric mucosa

1993 ◽  
Vol 265 (2) ◽  
pp. G277-G288 ◽  
Author(s):  
A. Yanaka ◽  
H. Muto ◽  
S. Ito ◽  
W. Silen
Keyword(s):  
Gastric Mucosa ◽  
Electrical Resistance ◽  
Rana Catesbeiana ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Ammonium Ion ◽  
Gastric Epithelial Cells ◽  
Sudden Drop ◽  
Oxynticopeptic Cells

The effects of ammonium ion (NH+4) and ammonia (NH3) on function and morphology of gastric epithelial cells were studied in intact sheets of in vitro frog (Rana catesbeiana) gastric mucosa. Luminal 115 mM NH4Cl at luminal pH 8.0 (calculated [NH3] 2.7 mM), but not at 5.0 (calculated [NH3] 3 microM) induced 1) an increase in intracellular pH (pHi) in oxynticopeptic cells (OPC) and decreases in transmucosal potential difference (PD) and electrical resistance (R) in resting tissues, 2) a decrease in histamine-stimulated H+ secretion and an increase in H+ backdiffusion after removal of luminal NH4Cl, and 3) augmented acidification of OPC during luminal acidification. Serosal 30 mM NH4Cl at serosal pH 7.2 (calculated [NH3] 0.47 mM) induced 1) an increase in pHi in OPC and inhibition of the alkalinization of OPC after removal of ambient Cl-, 2) a decrease in PD associated with the increase in R and decrease in short-circuit current, effects attenuated by serosal 15 mM K+, accentuated by 0.2 mM Ba2+, and abolished by removal of ambient Cl-, 3) a sudden drop of PD in resting, but not in stimulated tissues, effects prevented by high serosal pH (7.8), serosal HCO3-, or removal of luminal Cl-, 4) a decrease in histamine-stimulated H+ secretion and an increase in H+ backdiffusion after removal of NH4Cl, and 5) augmented acidification of OPC during luminal acidification. These results suggest that 1) luminal NH3, but not NH+4, increases backdiffusion of H+ from the lumen to the mucosa, 2) serosal NH3 and/or NH+4 induces depolarization of OPC and decreases electrogenic Cl- transport, thereby attenuating the activity of the basolateral Cl(-)-HCO3- exchanger in OPC, and 3) both of these effects contribute to the augmented acidification of OPC during exposure to high luminal [H+].

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