Effects of luminal salt concentration on electrical pathways in Necturus antrum

1987 ◽  
Vol 252 (1) ◽  
pp. G19-G27
Author(s):  
D. I. Soybel ◽  
S. W. Ashley ◽  
R. A. Swarm ◽  
C. D. Moore ◽  
L. Y. Cheung
Keyword(s):  
Cell Membranes ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Membrane Resistance ◽  
Paracellular Pathway ◽  
Surface Epithelium ◽  
Reversible Inhibitor ◽  
Apical Cell Membrane ◽  
Paracellular Pathways ◽  
Microelectrode Techniques

By use of microelectrode techniques the electrical resistances of the cell membranes and paracellular pathways of surface epithelium in Necturus antrum were determined under control conditions (Ringer solutions containing 106.6 mM Na+, 91.4 mM Cl-) and during exposure to mucosal solutions containing high Na+ and Cl- concentrations. Resistances were determined by briefly exposing tissues to mucosal solutions containing 10(-4) M amiloride, a reversible inhibitor of Na+ conductances. Under control conditions in eight tissues, measurements obtained by exposure to amiloride were not significantly different from those obtained by an independent method, intraepithelial cable analysis, thus indicating the validity of the measurements obtained by the amiloride method. In 10 tissues, high luminal NaCl concentrations (Ringer salts + 125 mM NaCl) increased the apical cell membrane resistance from 5,778 +/- 267 to 7,714 +/- 422 omega X cm2 (P less than 0.01) and the basolateral membrane resistance from 2,973 +/- 186 to 3,869 +/- 335 omega X cm2 (P less than 0.01). The resistance of the paracellular pathway decreased from 625 +/- 13 to 505 +/- 13 omega X cm2 (P less than 0.001). Similar alterations in these resistances were observed when Na+ or Cl- were increased individually, when added as salts of isethionate- and N-methyl-D-glucamine+, respectively. These effects were not attributable to increases in luminal osmolarity, since mucosal solutions made equally hyperosmotic with 250 mM sucrose elicited increases in paracellular pathway resistance and decreases in resistances of the cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Basolateral K+ conductances in surface epithelium of Necturus antrum: effects of Ca2+ and divalent cations

1992 ◽  
Vol 262 (4) ◽  
pp. G651-G659
Author(s):  
D. I. Soybel ◽  
S. W. Ashley ◽  
L. Y. Cheung
Keyword(s):  
Membrane Permeability ◽  
Divalent Cations ◽  
Basolateral Membrane ◽  
Membrane Resistance ◽  
Membrane Potentials ◽  
Time Dependent ◽  
Physiological Significance ◽  
Surface Epithelium ◽  
Antral Mucosa ◽  
Microelectrode Techniques

Intracellular microelectrode techniques were used to characterize basolateral membrane K+ conductances in isolated Necturus antral mucosa. Exposure of tissues to progressively higher levels of serosal K+ (4, 20, 40, or 60 mM) resulted in progressively greater depolarizations of basolateral membrane potentials and decreases in membrane resistance, consistent with the presence of a significant K+ conductance. Ba2+ (2 mM) partially blocks these conductances. Exposure of tissues to increased levels of serosal Ca2+ (from 1.8 to 6.8 mM) elicited significant hyperpolarization of basolateral potentials and decreases in basolateral resistance. These effects are also elicited by Sr2+ (5 mM), but not by Mg2+ (5 mM). Ba2+ (5 mM) elicits complex and time-dependent effects, but transiently elicits an effect similar to high Ca2+. Ion substitutions in the serosal perfusate suggest that the Ca(2+)-induced effects are due to enhancement of basolateral K+ conductances. Further work is necessary to identify the processes that mediate this increase in basolateral K+ conductance and to evaluate the physiological significance of this change in membrane permeability to K+.

Activation of CFTR Cl- conductance in polarized T84 cells by luminal extracellular ATP

1995 ◽  
Vol 268 (2) ◽  
pp. C425-C433 ◽  
Author(s):  
M. J. Stutts ◽  
E. R. Lazarowski ◽  
A. M. Paradiso ◽  
R. C. Boucher
Keyword(s):  
Adenosine Receptor ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Extracellular Atp ◽  
T84 Cells ◽  
Apical Cell Membrane ◽  
Cl Secretion ◽  
Cl Conductance

Luminal extracellular ATP evoked a bumetanide-sensitive short-circuit current in cultured T84 cell epithelia (90.2 +/- 18.2 microA/cm2 at 100 microM ATP, apparent 50% effective concentration, 11.5 microM). ATP appeared to increase the Cl- conductance of the apical membrane but not the driving force for Cl- secretion determined by basolateral membrane K+ conductance. Specifically, the magnitude of Cl- secretion stimulated by ATP was independent of basal current, and forskolin pretreatment abolished subsequent stimulation of Cl- secretion by ATP. Whereas ATP stimulated modest production of adenosine 3',5'-cyclic monophosphate (cAMP) by T84 cells, ATP caused smaller increases in intracellular Ca2+ and inositol phosphate activities than the Ca(2+)-signaling Cl- secretagogue carbachol. An inhibitor of 5'-nucleotidase, alpha,beta-methyleneadenosine 5'-diphosphate, blocked most of the response to luminal ATP. The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline blocked both the luminal ATP-dependent generation of cAMP and Cl- secretion when administered to the luminal but not submucosal bath. These results demonstrate that the Cl- secretion stimulated by luminal ATP is mediated by a A2-adenosine receptor located on the apical cell membrane. Thus metabolism of extracellular ATP to adenosine regulates the activity of cystic fibrosis transmembrane conductor regulator (CFTR) in the apical membrane of polarized T84 cells.

Characteristics of basolateral Cl- transport by gastric surface epithelium in Necturus antral mucosa

1993 ◽  
Vol 264 (5) ◽  
pp. G910-G920 ◽  
Author(s):  
D. I. Soybel ◽  
M. B. Davis ◽  
L. Y. Cheung
Keyword(s):  
Nutrient Solution ◽  
Cell Membranes ◽  
Basolateral Membrane ◽  
Membrane Potentials ◽  
Gastric Antrum ◽  
Ion Exchanger ◽  
Surface Epithelium ◽  
Antral Mucosa ◽  
Liquid Ion Exchanger ◽  
Electrochemical Equilibrium

Conventional and ion-selective microelectrodes were used to characterize transport of Cl- across the basolateral cell membranes of gastric surface epithelium in isolated preparations of gastric antrum of Necturus. Conventional, voltage-sensing electrodes were used to evaluate changes in membrane potentials and resistances during removal of Cl- from the nutrient perfusate. Liquid ion exchanger Cl(-)-selective microelectrodes were constructed and validated to measure intracellular Cl- activity (aiCl). Our data indicate that 1) aiCl (range 12-25 mM) is close to that predicted if Cl- is distributed across the cell membranes by electrochemical equilibrium, 2) aiCl is not influenced by changes in luminal Cl- content but is susceptible to changes in nutrient Cl- content, 3) Cl- conductances cannot be detected in the basolateral membrane and changes in membrane potentials do not influence aiCl, and 4) Cl- accumulation across the basolateral membrane depends on Na+ and the level of [K+] in the nutrient solution. Inhibition of K(+)-dependent Cl- accumulation, in the absence of nutrient Na+ or in the presence of the inhibitor bumetanide, was demonstrated. These findings suggest that basolateral Na(+)-K(+)-Cl- cotransport is important in regulating cell Cl- levels in surface cells of the gastric antrum in Necturus.

The collecting tubule of Amphiuma. I. Electrophysiological characterization

1987 ◽  
Vol 253 (6) ◽  
pp. F1263-F1272 ◽  
Author(s):  
M. Hunter ◽  
J. D. Horisberger ◽  
B. Stanton ◽  
G. Giebisch
Keyword(s):  
Cell Membrane ◽  
Cell Model ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Potassium Conductance ◽  
Sodium Reabsorption ◽  
Apical Cell Membrane ◽  
Cation Selectivity ◽  
Basolateral Cell Membrane ◽  
Collecting Tubules

Single collecting tubules of Amphiuma kidneys were perfused in vitro to characterize their electrophysiological properties. The lumen-negative potential (-24 mV) was abolished by amiloride in the lumen and by ouabain in the bath. Ion substitution experiments in the lumen demonstrated the presence of a large sodium conductance in the apical cell membrane, but no evidence was obtained for a significant potassium or chloride conductance. Ion substitutions in the bath solution and the depolarizing effect of barium on the basolateral membrane potential demonstrated the presence of a large potassium conductance in the basolateral cell membrane. Measurements of dilution potentials in amiloride-treated tubules revealed a modest cation selectivity of the paracellular pathway. These results support a cell model in which sodium reabsorption occurs by electrodiffusion across the apical cell membrane and active transport across the basolateral cell membrane. The absence of a detectable potassium conductance in the apical cell membrane suggests that secretion of this ion cannot take place by diffusion from cell to lumen.

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.

Conductive properties of the rabbit outer medullary collecting duct: inner stripe

1985 ◽  
Vol 248 (4) ◽  
pp. F500-F506 ◽  
Author(s):  
B. M. Koeppen
Keyword(s):  
Cell Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Rabbit Kidney ◽  
Apical Cell Membrane ◽  
Basolateral Cell Membrane ◽  
Medullary Collecting Duct ◽  
Conductive Properties

Segments of outer medullary collecting duct were dissected from the inner stripe of the rabbit kidney (OMCDi) and perfused in vitro. The conductive properties of the tubule epithelium and individual cell membranes were determined by means of cable analysis and intracellular voltage-recording microelectrodes. In 35 tubules the transepithelial voltage (VT) and resistance (RT) averaged 17.2 +/- 1.4 mV, lumen positive, and 58.6 +/- 5.3 k omega X cm, respectively. The basolateral membrane voltage, (Vbl) was -29.2 +/- 2.1 mV (n = 23). The apical cell membrane did not contain appreciable ion conductances, as evidenced by the high values of apical cell membrane fractional resistance (fRa = Ra/Ra + Rb), which approached unity (0.99 +/- 0.01; n = 23). Moreover, addition of amiloride or BaCl2 to the tubule lumen was without effect on the electrical characteristics of the cell, as was a twofold reduction in luminal [Cl-]. The conductive properties of the basolateral cell membrane were assessed with bath ion substitutions. A twofold reduction in bath [Cl-] depolarized Vbl by 14.7 +/- 0.4 mV (theoretical, 17 mV), while a 10-fold increase in bath [K+] resulted in only a 0.9 +/- 0.4 mV depolarization (theoretical, 61 mV). Substituting bath Na+ with tetramethylammonium (from 150 to 75 mM) was without effect. Reducing bath [HCO-3] from 25 to 5 mM (constant PCO2) resulted in a steady-state depolarization of Vbl of 8.4 +/- 0.4 mV that could not be attributed to conductive HCO-3 movement. Thus, the basolateral cell membrane is predominantly Cl- selective.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of Na+/H+ exchange on the apical side of surface colonocytes using BCECF

1994 ◽  
Vol 267 (1) ◽  
pp. G119-G128 ◽  
Author(s):  
G. G. King ◽  
W. E. Lohrmann ◽  
J. W. Ickes ◽  
G. M. Feldman
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Distal Colon ◽  
Transport Processes ◽  
Acetoxymethyl Ester ◽  
Apical Cell Membrane ◽  
Apical Side ◽  
Free Media ◽  
Phi Regulation

Colonocytes must regulate intracellular pH (pHi) while they transport H+ and HCO3-. To investigate the membrane transport processes involved in pHi regulation, colonocyte pHi was measured with 2,'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in intact segments of rat distal colon mounted on a holder that fits into a standard fluorometer cuvette and allows independent superfusion of mucosal and serosal surfaces. When NCECF-acetoxymethyl ester was in the mucosal solution only, BCECF loaded surface colonocytes with a high degree of selectivity. In HEPES-buffered solutions, basal pHi was 7.31 +/- 0.01 (n = 68), and pHi was dependent on extracellular Na+. Cells acidified in Na(+)-free solution, and pHi rapidly corrected when Na+ was returned. pHi recovered at 0.22 +/- 0.01 pH/min (n = 6) when Na+ was introduced into the mucosal solution and at 0.02 +/- 0.01 pH/min (n = 7) when Na+ was absent from the mucosal solution. The presence or absence of Na+ in the serosal solution did not affect pHi. This indicated that the Na(+)-dependent pHi recovery process is located in the apical cell membrane, but not in the basolateral membrane. Because amiloride (1 mM) inhibited Na(+)-dependent pHi recovery by 75%, Na+/H+ exchange appears to be present in the apical membrane. Because Na(+)-independent pHi recovery was not affected by K(+)-free media, 50 microM SCH-28080, 100 nM bafilomycin A1, or Cl(-)-free media, this transport mechanism does not involve a gastriclike H(+)-K(+)-ATPase, a vacuolar H(+)-ATPase, or a Cl-/base exchanger. In summary, pHi was selectively measured in surface colonocytes by this technique. In these cells, the Na+/H+ exchange activity involved in pHi regulation was detected in the apical membrane, but not in the basolateral membrane.

Electroneutral H+ secretion in distal tubule of Amphiuma

1987 ◽  
Vol 252 (4) ◽  
pp. F691-F699 ◽  
Author(s):  
B. Stanton ◽  
A. Omerovic ◽  
B. Koeppen ◽  
G. Giebisch
Keyword(s):  
Basolateral Membrane ◽  
Apical Cell ◽  
Distal Tubule ◽  
Cell Types ◽  
Disulfonic Acid ◽  
Type I ◽  
Cellular Mechanisms ◽  
Apical Cell Membrane ◽  
Membrane Type

This study examines the cellular mechanisms of acid secretion by the in vitro perfused late distal tubule of Amphiuma kidney. Acidification of tubule fluid occurred against an electrochemical gradient of 16 mV; thus H+ secretion was active. Amiloride (1 mM) or a reduction of sodium in the perfusion fluid (from 83.7 to 7.7 mM) partially reduced acidification. Amiloride, in the presence of low sodium, completely inhibited acidification. Furthermore, acetazolamide and ouabain in the bath solution (0.1 mM) also inhibited acidification. Conductive properties of the epithelium and of individual cell membranes were determined by means of cable analysis of the tubule and intracellular voltage recordings. The transepithelial voltage and resistance averaged -0.4 +/- 0.4 mV, lumen negative, and 7,147 +/- 845 omega X cm, respectively. Two functionally different cell types were identified by intracellular microelectrodes. Type I cells had a basolateral membrane voltage (Vbl) of -67.7 mV. As determined by ion substitution experiments, the basolateral membrane was conductive to K+ and Cl-. This cell also had a 4-acetamido-4'-isothiocyanostilbene-2-2'-disulfonic acid (SITS)-sensitive Na+-dependent HCO3- exit pathway in the basolateral membrane. Type II cells had a Vbl of -76.1 mV (P less than 0.05 vs. type I) and the basolateral membrane was conductive to K+ and Cl- but not to HCO3-. HCO3- movement across the basolateral membrane in this cell may occur by electroneutral Cl- -HCO3- exchange. The apical cell membrane of both cell types did not contain measurable ionic conductances, as evidenced by a high value of apical membrane fractional resistance (0.98 +/- 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular chloride activities in the isolated perfused shark rectal gland

1983 ◽  
Vol 245 (5) ◽  
pp. F640-F644
Author(s):  
M. J. Welsh ◽  
P. L. Smith ◽  
R. A. Frizzell
Keyword(s):  
Basolateral Membrane ◽  
Apical Cell ◽  
Electrical Potential ◽  
Electrochemical Potential ◽  
Potential Difference ◽  
Rectal Gland ◽  
Electrical Potential Difference ◽  
Apical Cell Membrane ◽  
Shark Rectal Gland ◽  
Electrochemical Equilibrium

The isolated, perfused shark rectal gland secretes Cl when stimulated with adenosine 3',5'-cyclic monophosphate (cAMP). To investigate the mechanism of secretion, we used Cl-selective and conventional (KCl-filled) microelectrodes to measure the intracellular Cl activity (aClc). Under nonsecreting conditions, the electrical potential difference across the basolateral membrane (psi b) was -78 m V and aClc was 57 mM, a value seven times greater than predicted for electrochemical equilibrium across the basolateral membrane. When theophylline and 8-bromo-cAMP were added to the perfusate, the transglandular electrical potential difference doubled and the rate of fluid secretion increased 20-fold; however, neither psi b nor aClc changed. During both nonsecreting and secreting conditions the intracellular accumulation of Cl results in an electrochemical potential difference favoring Cl exit across the apical cell membrane. The constancy of aClc despite the variation in secretion rate suggests that stimulation is associated with an equivalent enhancement of net Cl movement across both the apical and basolateral membranes. When stimulated glands were perfused with Na-free (choline) Ringer, secretion was abolished and aClc fell toward the value predicted for electrochemical equilibrium. These findings suggest that the "uphill" step in Cl secretion lies at the basolateral membrane, where cellular Cl accumulation probably involves secondary active transport; i.e., Cl entry is driven by an inwardly directed electrochemical potential difference for Na.

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