Effect of changes in extracellular Cl on intracellular Cl activity in frog skin

1988 ◽  
Vol 254 (1) ◽  
pp. F95-F104 ◽  
Author(s):  
K. Drewnowska ◽  
T. U. Biber
Keyword(s):  
Frog Skin ◽  
Rana Pipiens ◽  
Initial Rate ◽  
Apical Cell ◽  
Disulfonic Acid ◽  
Free Solution ◽  
Granular Cells ◽  
Apical Side ◽  
Basolateral Side ◽  
Cl Permeability

Intracellular Cl activity was measured in isolated frog skin (Rana pipiens) with double-barrel microelectrodes. The initial rate of Cl uptake was measured in Cl-depleted cells on reexposure to Cl on apical or basolateral side. In skins with high and low conductance, cell CL activity increased 1.33 and 0.14 mM/s with apical reexposure and 5.03 and 0.30 mM/s with basolateral reexposure, respectively. The initial Cl uptake was reduced on the apical side by 93% with 10(-3) M DIDS (4,4'-diisothiocyanostilbene-2,2ߗ-disulfonic acid) and on the basolateral side by 99% with 10(-3) M SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) plus 10(-5) M bumetanide. The initial rate of Cl loss was measured when Cl was removed from the bath: addition of HCO3 to Cl- and HCO3-free solution caused an acceleration of Cl loss in absence but not in presence of DIDS on apical side. In contrast, Cl loss across the basolateral side was not enhanced by HCO3. In conclusion, Na-transporting cells have a substantial Cl permeability on both sides. HCO3-stimulated Cl loss provides evidence for Cl-HCO3 exchange and permits localization of this process in apical cell membranes of granular cells.

pH in principal cells of frog skin (Rana pipiens): dependence on extracellular Na+

1988 ◽  
Vol 255 (5) ◽  
pp. F930-F935 ◽  
Author(s):  
K. Drewnowska ◽  
E. J. Cragoe ◽  
T. U. Biber
Keyword(s):  
Frog Skin ◽  
Rana Pipiens ◽  
Apical Cell ◽  
Ringer Solution ◽  
Feedback Mechanism ◽  
Open Circuit ◽  
Apical Cell Membrane ◽  
Principal Cells ◽  
Apical Side ◽  
Basolateral Side

Measurements of intracellular pH (pHi) and of apical cell membrane potential (Va) were made in principal cells of frog skin (Rana pipiens) with double-barrel microelectrodes under open-circuit conditions. The tissues were pretreated with stilbenes (10(-3) M) and bathed in HCO3- -free NaCl Ringer solution that was buffered with 6 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.8). Substitution of extracellular Na+ on both sides of the epithelium with N-methyl-D-glucamine caused intracellular acidification by 0.27 pH units. Restoration of Na+ on the apical side alone or on both sides caused a pHi recovery of 0.24 and 0.28 pH units, respectively, whereas return of Na+ on the basolateral side caused no recovery. Recovery of pHi on restoration of Na+ to the apical side was prevented by 10(-5) M 5-(N-ethyl-N-isopropyl)-amiloride. In individual preparations there was no correlation between pHi recovery due to return of apical Na+ and changes in Va. The average change in pHi was several times greater than the one expected from voltage clamp-induced changes in Va at constant extracellular Na+. The results suggest the presence of a Na+-H+ exchange on the apical side of principal cells. Such a process could be part of a negative feedback mechanism for regulation of Na+ entry via apical Na+ channels into principal cells.

pH in principal cells of frog skin (Rana pipiens): effects of amiloride and potential

1988 ◽  
Vol 255 (5) ◽  
pp. F922-F929 ◽  
Author(s):  
K. Drewnowska ◽  
T. U. Biber
Keyword(s):  
Cell Membrane ◽  
Voltage Clamp ◽  
Frog Skin ◽  
Rana Pipiens ◽  
Apical Cell ◽  
Disulfonic Acid ◽  
Intracellular Acidification ◽  
Apical Cell Membrane ◽  
Principal Cells ◽  
Apical Side

Intracellular pH (pHi) and apical cell membrane potential (Va) were determined in principal cells of frog skin (Rana pipiens) with double-barrel micro-electrodes. In the Northern and Southern varieties, respectively, pHi is 0.38 and 0.26 pH units below bath pH. Amiloride, applied apically, causes reversible intracellular acidification at concentrations of 10(-5) M or higher. Voltage clamp-induced hyperpolarization and depolarization of Va result in intracellular acidification and alkalinization, respectively. This response of pHi is inhibited or abolished when the apical side is treated with 10(-3) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Amiloride-induced intracellular acidification is not exclusively due to the hyperpolarization of Va that accompanies amiloride treatment since 1) amiloride causes greater acidification than equivalent voltage clamp-induced hyperpolarization of Va, 2) amiloride-induced acidification persists in DIDS-treated tissues, and 3) there is no correlation between hyperpolarization of Va and intracellular acidification occurring after amiloride. We conclude that pHi is below the extracellular pH. Amiloride causes intracellular acidification that may be in part connected with hyperpolarization of Va. However, a major component of amiloride-induced acidification is due to other factors, possibly inhibition of apical Na+-H+ exchange. The inhibitory effect of apically applied DIDS suggests that the voltage dependent changes in pHi are related to movement of HCO3 (or OH) ions across the apical cell membrane.

Intracellular Cl activity changes of frog skin

1985 ◽  
Vol 249 (3) ◽  
pp. F432-F438
Author(s):  
T. U. Biber ◽  
K. Drewnowska ◽  
C. M. Baumgarten ◽  
R. S. Fisher
Keyword(s):  
Membrane Potential ◽  
Frog Skin ◽  
Ringer Solution ◽  
Free Solution ◽  
Intracellular Potential ◽  
Apical Side ◽  
Basolateral Side ◽  
Significant Difference ◽  
Electrochemical Equilibrium ◽  
Cl Conductance

The intracellular Cl activity and potential were determined in short-circuited frog skin with single-barrel microelectrodes. With NaCl Ringer solution on the apical and basolateral side, the intracellular Cl activity was 15.5 +/- 0.5 mM and the intracellular potential was -90 +/- 1.0 mV, indicating that the intracellular Cl activity was above electrochemical equilibrium. When the solution on the apical side was changed to a Cl-free solution (Cl replaced by methanesulfonate), no significant difference was observed in intracellular Cl activity. However, when the skins were Cl-depleted by replacing the NaCl Ringer solution on both sides with a Cl-free solution, the intracellular Cl activity decreased to 1.7 +/- 0.1 mM and the intracellular potential fell to -66.7 +/- 1.3 mV. Addition of Cl (i.e., NaCl Ringer solution) to the apical side of Cl-depleted skins caused a significant increase in intracellular Cl activity to 6.3 mM. This increase was prevented by amiloride (10(-4) M) added on the apical side simultaneously with Cl. Restoration of Cl on the basolateral side of Cl-depleted tissues also raised the intracellular Cl activity to about the same level as when Cl was added on the apical side (6.8 mM). Changes in membrane potential occurred in a delayed fashion over a period of 15 min or more when Cl was added or removed on either side of the skin. The absence of an immediate membrane potential response indicates that Cl conductance is not detectable. We conclude, therefore, that the Cl transfer across the apical and basolateral cell membrane occurs primarily via electroneutral mechanisms.

scholarly journals Intracellular pH controls cell membrane Na+ and K+ conductances and transport in frog skin epithelium.

1988 ◽  
Vol 92 (6) ◽  
pp. 767-791 ◽  
Author(s):  
B J Harvey ◽  
S R Thomas ◽  
J Ehrenfeld
Keyword(s):  
Intracellular Ph ◽  
Frog Skin ◽  
Basolateral Membrane ◽  
Specific Conductance ◽  
Intracellular Acidosis ◽  
Skin Epithelium ◽  
Frog Skin Epithelium ◽  
Apical Side ◽  
Basolateral Side ◽  
Intracellular Alkalinization

We determined the effects of intracellular respiratory and metabolic acid or alkali loads, at constant or variable external pH, on the apical membrane Na+-specific conductance (ga) and basolateral membrane conductance (gb), principally due to K+, in the short-circuited isolated frog skin epithelium. Conductances were determined from the current-voltage relations of the amiloride-inhibitable cellular current pathway, and intracellular pH (pHi) was measured using double barreled H+-sensitive microelectrodes. The experimental set up permitted simultaneous recording of conductances and pHi from the same epithelial cell. We found that due to the asymmetric permeability properties of apical and basolateral cell membranes to HCO3- and NH+4, the direction of the variations in pHi was dependent on the side of addition of the acid or alkali load. Specifically, changing from control Ringer, gassed in air without HCO3- (pHo = 7.4), to one containing 25 mmol/liter HCO3- that was gassed in 5% CO2 (pHo = 7.4) on the apical side caused a rapid intracellular acidification whereas when this maneuver was performed from the basolateral side of the epithelium a slight intracellular alkalinization was produced. The addition of 15 mmol/liter NH4Cl to control Ringer on the apical side caused an immediate intracellular alkalinization that lasted up to 30 min; subsequent removal of NH4Cl resulted in a reversible fall in pHi, whereas basolateral addition of NH4Cl produced a prolonged intracellular acidosis. Using these maneouvres to change pHi we found that the transepithelial Na+ transport rate (Isc), and ga, and gb were increased by an intracellular alkalinization and decreased by an acid shift in pHi. These variations in Isc, ga, and gb with changing pHi occurred simultaneously, instantaneously, and in parallel even upon small perturbations of pHi (range, 7.1-7.4). Taken together these results indicate that pHi may act as an intrinsic regulator of epithelial ion transport.

Mechanism of adrenergic stimulation of anion secretion in cultured rat epididymal epithelium

1988 ◽  
Vol 254 (1) ◽  
pp. F121-F133 ◽  
Author(s):  
P. Y. Wong
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Dependent Manner ◽  
Adrenergic Stimulation ◽  
Anion Secretion ◽  
Apical Side ◽  
Basolateral Side ◽  
Cauda Epididymidis

Primary monolayer cultures of the epithelial cells from the rat cauda epididymidis had a basal transepithelial potential of 1.24 +/- 0.11 mV, apical side negative, a short-circuit current (SCC) of 2.42 +/- 0.17 microA/cm2, and a transepithelial resistance of 503 +/- 29.6 omega/cm2. Epinephrine (0.23 microM) added to the basolateral side caused a rise in the SCC (inward flowing) that was partially reversed by addition of amiloride, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), bumetanide, and acetazolamide to the basolateral side. In the absence of Cl or HCO3, the SCC response to epinephrine was reduced by 65 and 66%, respectively. Removal of K reduced the SCC response by 40%, whereas Na removal completely abolished it. A23187 and phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated the SCC when added to the apical side. Addition of the Cl channel blockers, anthracene-9-carboxylate and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) to the apical side reduced the SCC in a dose-dependent manner. It is concluded that anion secretion by the epididymis shares common mechanisms with other Cl-secreting epithelia.

scholarly journals Comparison of α-Tocopherol, α-Tocopherol Acetate, and α-Tocopheryl Polyethylene Glycol Succinate 1000 Absorption by Caco-2 TC7 Intestinal Cells

Nutrients ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 129
Author(s):  
Charlotte Cuerq ◽  
Claire Bordat ◽  
Charlotte Halimi ◽  
Emilie Blond ◽  
Marion Nowicki ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
High Performance ◽  
Lipid Extraction ◽  
Free Form ◽  
Glycol Succinate ◽  
Tocopherol Acetate ◽  
Apical Side ◽  
Basolateral Side ◽  
Human Enterocyte ◽  
First Time

(1) Background: vitamin E is often supplemented in the form of tocopherol acetate, but it has poor bioavailability and can fail to correct blood tocopherol concentrations in some patients with severe cholestasis. In this context, α-tocopheryl polyethylene glycol succinate 1000 (TPGS) has been of value, but very little is known about the mechanisms of its absorption. The aim of our work was to evaluate the mechanisms of absorption/secretion of TPGS compared to tocopherol acetate (TAC) and α-tocopherol by human enterocyte-like Caco-2 TC7 cells. (2) Methods: two weeks post-confluence Caco-2 cells were incubated with tocopherol- or TAC- or TPGS-rich mixed micelles up to 24 h and, following lipid extraction, TAC and tocopherol amounts were measured by high performance liquid chromatography (HPLC) in apical, cellular, and basolateral compartments. (3) Results: at equivalent concentrations of tocopherol in the apical side, the amounts of tocopherol secreted at the basolateral pole of Caco-2 cells are (i) significantly greater when the tocopherol is in the free form in the micelles; (ii) intermediate when it is in the TAC form in the micelles (p < 0.001); and (iii) significantly lower with the TPGS form (p < 0.0001). Interestingly, our results show, for the first time, that Caco-2 cells secrete one or more esterified forms of the vitamin contained in TPGS at the basolateral side.

Luminal flow rate regulates proximal tubule H-HCO3 transporters

1992 ◽  
Vol 262 (1) ◽  
pp. F47-F54 ◽  
Author(s):  
P. A. Preisig
Keyword(s):  
Proximal Tubule ◽  
Flow Rate ◽  
Initial Rate ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Rate Dependence ◽  
Unstirred Layer ◽  
Disulfonic Acid ◽  
Luminal Flow

In vivo microperfusion was used to examine the mechanism of luminal flow rate dependence of proximal tubule acidification. Luminal flow rate was acutely changed between 5 and 40 nl/min, while luminal and peritubular capillary composition were held constant. With inhibition of basolateral membrane base transport by peritubular 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), cell pH (pHi) provides a sensitive index of apical membrane H secretory activity. At a luminal perfusate [HCO3] of 25 mM, progressive increases in luminal flow rate (5----15----25----40 nl/min) caused progressive increases in pHi. This effect was of a smaller magnitude with a luminal perfusate [HCO3] of 60 mM and was further decreased at a luminal perfusate [HCO3] of 100 mM. This pattern of diminished flow rate dependence at higher luminal [HCO3] is consistent with the presence of a luminal unstirred layer, whose composition can be modified by luminal flow rate. The activity of the apical membrane Na-H antiporter, assayed as the initial rate of pHi recovery from an acid load in the presence of peritubular DIDS, was faster at 40 compared with 5 nl/min. Basolateral membrane Na-3HCO3 symporter activity, assayed as the initial rate of pHi recovery from an alkali load in the absence of luminal and peritubular chloride, was faster at 40 compared with 5 nl/min. This effect was eliminated by luminal amiloride, suggesting an indirect effect of flow mediated by changes in pHi secondary to flow rate-dependent changes in apical membrane Na-H antiporter activity. In summary, increases in luminal flow rate directly increase apical membrane H secretion, possibly by modification of a luminal unstirred layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular pH transients in rat diaphragm muscle measured with DMO

1978 ◽  
Vol 235 (1) ◽  
pp. C49-C54 ◽  
Author(s):  
A. Roos ◽  
W. F. Boron
Keyword(s):  
Intracellular Ph ◽  
Weak Acid ◽  
Diaphragm Muscle ◽  
Disulfonic Acid ◽  
Free Solution ◽  
Rat Diaphragm ◽  
Control Value ◽  
Acid Load ◽  
Acid Extrusion ◽  
Acid Loading

Changes of the intracellular pH of rat diaphragm muscle were monitored at 30-min intervals with the weak acid DMO (5,5-dimethyl-2,4-oxazolidinedione). Transferring the muscle from a CO2-containing to a CO2-free solution caused intracellular pH (pHi) to rise by an average of 0.18 during the first 30 min and then to level off at a slightly lower value over the next 60-90 min. Transferring the muscle from a CO2-free to a CO2-containing solution caused pHi to fall by 0.18 during the first 30 min and then to recover by 0.05 over the next 90 min. Subsequent return to the CO2-free solution caused pHi to overshoot the control value by 0.10. Both the recovery and the overshoot can be accounted for by an acid-extruding pump. Intracellular acid loading with 118 mM DMO similarly caused pHi to fall initially, to recover slowly during the acid loading, and then to overshoot the control pHi on removal of the acid load. In the absence of HCO3-/CO2, acid extrusion was reduced by about a fifth. SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) had no effect. The absence of either Na+ or Cl- from HCO3-/CO2- free solution reduced acid extrusion by about a half.

K(+)-induced alkalinization in mouse cerebral astrocytes mediated by reversal of electrogenic Na(+)-HCO3- cotransport

1994 ◽  
Vol 267 (6) ◽  
pp. C1633-C1640 ◽  
Author(s):  
N. Brookes ◽  
R. J. Turner
Keyword(s):  
Intracellular Ph ◽  
Primary Cultures ◽  
Atmospheric Co2 ◽  
Disulfonic Acid ◽  
Free Solution ◽  
High Affinity ◽  
Alkaline Shift ◽  
Net Flux ◽  
K Concentration ◽  
The Relationship

Raising extracellular K+ concentration ([K+]o) induces an alkaline shift of intracellular pH (pHi) in astrocytes. The mechanism of this effect was examined using the fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in primary cultures of mouse cerebral astrocytes. Raising [K+]o from 3 to 12 mM increased pHi by 0.28 pH units in 26 mM HCO(3-)-buffered solution. In nominally HCO(3-)-free solution (containing approximately 95 microM HCO3-), the alkalinization fell to 0.21 pH units and further to 0.08 pH units on removal of atmospheric CO2, suggesting a process with high affinity for HCO3-. This effect was Na+ dependent, Cl- independent, and inhibited by 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, indicating the involvement of Na(+)-HCO3- cotransport. The relationship between pHi and log[K+]o was found to be linear and to predict a stoichiometry of at least two HCO3- transported with each Na+. After removal of exogenous CO2/HCO3-, the direction of changes in pHi elicited by adding 1 mM HCO3- showed that net flux of HCO3- via the Na(+)-HCO3- cotransporter was outward at rest and was reversed by depolarization.

Bovine pancreatic duct cells express cAMP- and Ca(2+)-activated apical membrane Cl- conductances

1997 ◽  
Vol 273 (1) ◽  
pp. G204-G216 ◽  
Author(s):  
L. al-Nakkash ◽  
C. U. Cotton
Keyword(s):  
Epithelial Cells ◽  
Cell Membrane ◽  
Pancreatic Duct ◽  
Apical Membrane ◽  
Apical Cell ◽  
Primary Cultures ◽  
Apical Cell Membrane ◽  
Anion Secretion ◽  
Duct Epithelium ◽  
Cl Permeability

Secretion of salt and water by the epithelial cells that line pancreatic ducts depends on activation of apical membrane Cl- conductance. In the present study, we characterized two types of Cl- conductances present in the apical cell membrane of bovine pancreatic duct epithelial cells. Primary cultures of bovine main pancreatic duct epithelium and an immortalized cell line (BPD1) derived from primary cultures were used. Elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) or Ca2+ in intact monolayers of duct epithelium induced sustained anion secretion. Agonist-induced changes in plasma membrane Cl- permeability were accessed by 36 Cl- efflux, whole cell current recording, and measurements of transepithelial Cl- current across permeabilized epithelial monolayers. Elevation of intracellular cAMP elicited a sustained increase in Cl- permeability, whereas elevation of intracellular Ca2+ induced only a transient increase in Cl- permeability. Ca(2+)- but not cAMP-induced increases in Cl- permeability were abolished by preincubation of cells with the Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl) ester (BAPTA-AM). N-phenylanthranilic acid (DPC; 1 mM) and glibenclamide (100 microM), but not 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 500 microM), inhibited the cAMP-induced increase in Cl- permeability. In contrast, DPC and DIDS, but not glibenclamide, inhibited the Ca(2+)-induced increase in Cl- permeability. We conclude from these experiments that bovine pancreatic duct epithelial cells express at least two types of Cl- channels, cAMP and Ca2+ activated, in the apical cell membrane. Because the Ca(2+)-activated increase in Cl- permeability is transient, the extent to which this pathway contributes to sustained anion secretion by the ductal epithelium remains to be determined.

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