Chloride transport in rabbit esophageal epithelial cells

2002 ◽  
Vol 282 (4) ◽  
pp. G663-G675 ◽  
Author(s):  
Solange Abdulnour-Nakhoul ◽  
Nazih L. Nakhoul ◽  
Canan Caymaz-Bor ◽  
Roy C. Orlando
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Chloride Transport ◽  
Ussing Chamber ◽  
Basolateral Membrane ◽  
Rate Of Change ◽  
Luminal Cell ◽  
Transport Pathways ◽  
Basolateral Membranes ◽  
Esophageal Epithelial Cells

We investigated Cl− transport pathways in the apical and basolateral membranes of rabbit esophageal epithelial cells (EEC) using conventional and ion-selective microelectrodes. Intact sections of esophageal epithelium were mounted serosal or luminal side up in a modified Ussing chamber, where transepithelial potential difference and transepithelial resistance could be determined. Microelectrodes were used to measure intracellular Cl− activity (a[Formula: see text]), basolateral or apical membrane potentials ( V mBL or V mC), and the voltage divider ratio. When a basal cell was impaled, V mBL was −73 ± 4.3 mV and a[Formula: see text] was 16.4 ± 2.1 mM, which were similar in presence or absence of bicarbonate. Removal of serosal Cl−caused a transient depolarization of V mBL and a decrease in a[Formula: see text] of 6.5 ± 0.9 mM. The depolarization and the rate of decrease of a[Formula: see text] were inhibited by ∼60% in the presence of the Cl−-channel blocker flufenamate. Serosal bumetanide significantly decreased the rate of change of a[Formula: see text] on removal and readdition of serosal Cl−. When a luminal cell was impaled, V mC was −65 ± 3.6 mV and a[Formula: see text] was 16.3 ± 2.2 mM. Removal of luminal Cl− depolarized V mC and decreased a[Formula: see text] by only 2.5 ± 0.9 mM. Subsequent removal of Cl− from the serosal bath decreased a[Formula: see text]in the luminal cell by an additional 6.4 ± 1.0 mM. A plot of V mBL measurements vs. log a[Formula: see text]/log a[Formula: see text] (a[Formula: see text] is the activity of Cl− in a luminal or serosal bath) yielded a straight line [slope ( S) = 67.8 mV/decade of change in a[Formula: see text]/a[Formula: see text]]. In contrast, V mC correlated very poorly with log a[Formula: see text]/a[Formula: see text] ( S = 18.9 mV/decade of change in a[Formula: see text]/a[Formula: see text]). These results indicate that 1) in rabbit EEC, a[Formula: see text] is higher than equilibrium across apical and basolateral membranes, and this process is independent of bicarbonate; 2) the basolateral cell membrane possesses a conductive Cl− pathway sensitive to flufenamate; and 3) the apical membrane has limited permeability to Cl−, which is consistent with the limited capacity for transepithelial Cl− transport. Transport of Cl− at the basolateral membrane is likely the dominant pathway for regulation of intracellular Cl−.

Selective permeability barrier to urea in shark rectal gland

2005 ◽  
Vol 289 (1) ◽  
pp. F83-F89 ◽  
Author(s):  
Joshua D. Zeidel ◽  
John C. Mathai ◽  
John D. Campbell ◽  
Wily G. Ruiz ◽  
Gerard L. Apodaca ◽  
...  
Keyword(s):  
Activation Energy ◽  
Epithelial Cells ◽  
Water Flow ◽  
Water Permeability ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Water Flux ◽  
Rectal Gland ◽  
Basolateral Membranes ◽  
Urea Permeability

Elasmobranchs such as the dogfish shark Squalus acanthius achieve osmotic homeostasis by maintaining urea concentrations in the 300- to 400-mM range, thus offsetting to some degree ambient marine osmolalities of 900–1,000 mosmol/kgH2O. These creatures also maintain salt balance without losing urea by secreting a NaCl-rich (500 mM) and urea-poor (18 mM) fluid from the rectal gland that is isotonic with the plasma. The composition of the rectal gland fluid suggests that its epithelial cells are permeable to water and not to urea. Because previous work showed that lipid bilayers that permit water flux do not block flux of urea, we reasoned that the plasma membranes of rectal gland epithelial cells must either have aquaporin water channels or must have some selective barrier to urea flux. We therefore isolated apical and basolateral membranes from shark rectal glands and determined their permeabilities to water and urea. Apical membrane fractions were markedly enriched for Na-K-2Cl cotransporter, whereas basolateral membrane fractions were enriched for Na-K-ATPase. Basolateral membrane osmotic water permeability (Pf) averaged 4.3 ± 1.3 × 10−3 cm/s, whereas urea permeability averaged 4.2 ± 0.8 × 10−7 cm/s. The activation energy for water flow averaged 16.4 kcal/mol. Apical membrane Pf averaged 7.5 ± 1.6 × 10−4 cm/s, and urea permeability averaged 2.2 ± 0.4 × 10−7 cm/s, with an average activation energy for water flow of 18.6 kcal/mol. The relatively low water permeabilities and high activation energies argue strongly against water flux via aquaporins. Comparison of membrane water and urea permeabilities with those of artificial liposomes and other isolated biological membranes indicates that the basolateral membrane urea permeability is fivefold lower than would be anticipated for its water permeability. These results indicate that the rectal gland maintains a selective barrier to urea in its basolateral membranes.

Dependence of intracellular Na+ concentration on apical and basolateral membrane Na+ influx in frog skin

1985 ◽  
Vol 249 (5) ◽  
pp. F662-F671
Author(s):  
J. S. Stoddard ◽  
S. I. Helman
Keyword(s):  
Epithelial Cells ◽  
Frog Skin ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Na Transport ◽  
Maximal Inhibition ◽  
Basolateral Membranes ◽  
Isotopic Method ◽  
Rate Of Increase ◽  
Kinetics Of

An isotopic method was developed to measure the intracellular Na+ content of the transepithelial Na+ transport pool of frog skin. Isolated epithelia (no corium) were labeled with 24Na either asymmetrically, from apical (Aa) or basolateral (Ab) solutions, or symmetrically (Aab). Transport pool Na+ could be identified from the kinetics of washout of 24Na carried out in the presence of 1 mM ouabain, 100 microM amiloride, and 1 mM furosemide that served to trap cold Na+ and 24Na within the transport pool. In control epithelia, Aab averaged 64.1 neq/cm2 (13.9 mM), and maximal inhibition of apical membrane Na+ entry with 100 microM amiloride caused Aab to decrease to 24.3 neq/cm2 (5.3 mM). Ouabain caused Aab to increase markedly to 303 neq/cm2 in 30 min, whereas amiloride inhibition of apical membrane Na+ entry reduced markedly the rate of increase of Aab caused by ouabain (7.3 neq X cm-2 X min-1 in control and 1.7 neq X cm-2 X min-1 in the presence of amiloride). These data, in part, confirmed the existence of an important basolateral membrane permeability to Na+ that was measured in separate studies of the bidirectional 24Na fluxes at the basolateral membranes of the cells. Both sets of data were supportive of the idea that a significant Na+ recycling exists at the basolateral membranes of the cells that contributes to the Na+ load on the pump and Na+ recycling participates in the regulation of the Na+ concentration of the Na+ transport pool of these epithelial cells.

Na+ transport pathways in secretory acinar cells: membrane cross talk mediated by [Cl-]i

1994 ◽  
Vol 267 (1) ◽  
pp. C146-C156 ◽  
Author(s):  
M. A. Robertson ◽  
J. K. Foskett
Keyword(s):  
Epithelial Cells ◽  
Cross Talk ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Acinar Cells ◽  
Resting Cells ◽  
Nonselective Cation Channel ◽  
Na Transport ◽  
Cell Shrinkage ◽  
Transport Pathways

Fluid secretion by epithelial cells can be modulated by agents that activate Cl- channels in the apical membrane. To sustain secretion, Cl- influx across the basolateral membrane must also be accelerated. To examine the cellular mechanisms that couple Cl- efflux across the apical membrane to Na(+)-coupled Cl- entry across the basolateral membrane, we employed optical imaging techniques, utilizing single rat salivary acinar cells. Na+ influx was negligible in resting cells but was rapidly increased by carbachol due to activation of a Na(+)-H+ exchanger, a Na(+)-K(+)-2Cl- cotransporter, and, most likely, a nonselective cation channel. Receptor stimulation was not necessary, since elevation of intracellular Ca2+ concentration ([Ca2+]i) by thapsigargin activated the Na+ transporters at equivalent rates. Cell acidification, activation of protein kinase C, cell shrinkage, and other events associated with the rise of [Ca2+]i had little effect on Na+ transport in resting cells. Nevertheless, stimulation of cells in a medium that prevented normal Ca(2+)-induced cell shrinkage prevented activation of all three transport pathways. The block of the activation was not overcome by osmotic shrinkage but was relieved when [Cl-]i was allowed to fall, including conditions in which [Cl-]i fell in the absence of cell shrinkage. Activation of a Na(+)-H+ exchanger, Na(+)-K(+)-2Cl- cotransporter, and nonselective cation channel therefore exhibits a requirement for agonist-induced fall in [Cl-]i. Low [Cl-]i may create a permissive environment for Ca(2+)-dependent activation of multiple Na(+)-transport pathways, providing a mechanism for cross talk that coordinates transport activities of the apical and basolateral membranes in secretory epithelial cells.

Mechanisms of basolateral Na+ transport in rabbit esophageal epithelial cells

1999 ◽  
Vol 276 (2) ◽  
pp. G507-G517 ◽  
Author(s):  
Solange Abdulnour-Nakhoul ◽  
Serhat Bor ◽  
Nese Imeryuz ◽  
Roy C. Orlando
Keyword(s):  
Epithelial Cells ◽  
Ussing Chamber ◽  
Basolateral Membrane ◽  
Potential Difference ◽  
Esophageal Epithelium ◽  
Na Transport ◽  
Ph Measurements ◽  
Dependent Component ◽  
Esophageal Epithelial Cells ◽  
Basolateral Membrane Potential

We examined the mechanisms of cellular Na+ transport, both Cl− dependent and Cl− independent, in the mammalian esophageal epithelium. Rabbit esophageal epithelium was dissected from its muscular layers and mounted in a modified Ussing chamber for impalement with ion-selective microelectrodes. In bicarbonate Ringer, transepithelial potential difference was −14.9 ± 0.9 mV, the transepithelial resistance ( R TE) was 1,879 ± 142 Ω ⋅ cm2, the basolateral membrane potential difference ( V mBL) was −53 ± 1.5 mV, and the intracellular activity of Na+([Formula: see text]) was 24.6 ± 2.1 mM. Removal of Na+ and Cl− from the serosal and luminal baths decreased [Formula: see text] to 6.6 ± 0.6 mM. Readdition of Na+ to the serosal bath in the absence of Cl− increased[Formula: see text] by 21.8 ± 3.0 mM, whereas V mBL and R TE remained unchanged. When serosal Na+ was readded in the presence of amiloride the increase in[Formula: see text] and the rate of Na+ entry were decreased by ∼50%. 5-( N-ethyl- N-isopropyl)amiloride mimicked the effect of amiloride, whereas phenamil did not. Subsequent readdition of Cl− to the serosal bath further increased [Formula: see text] by 4.4 ± 1.9 mM. When the cells were acid loaded by pretreatment with[Formula: see text] in nominally[Formula: see text]-free Ringer, intracellular pH measurements showed a pHi recovery that is dependent on the presence of Na+ in the serosal bath and that can be blocked by amiloride. These data indicate that esophageal epithelial cells possess a Na+-dependent, amiloride-sensitive electroneutral mechanism for Na+entry consistent with the presence of a basolateral Na+/H+ exchanger. The ability of Cl− to further enhance Na+ entry supports the existence of at least one additional Cl−-dependent component of basolateral Na+entry.

scholarly journals Antimicrotubule drugs inhibit the polarized insertion of an intracellular glycoprotein pool into the apical membrane of Madin-Darby canine kidney (MDCK) cells

1992 ◽  
Vol 103 (3) ◽  
pp. 677-687 ◽  
Author(s):  
G.K. Ojakian ◽  
R. Schwimmer
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Immunogold Electron Microscopy ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Transport Pathways ◽  
Intracellular Targeting ◽  
Polarized Delivery ◽  
Membrane Recognition

Previous experiments on MDCK cells have demonstrated that the polarized appearance of a 135 kDa glycoprotein (gp135) on the apical plasma membrane can occur through the insertion of both newly synthesized gp135 as well as a pre-existing gp135 intracellular pool. In this study, anticytoskeletal drugs were utilized to determine the role of the cytoskeleton in the polarized delivery of gp135. Colchicine and nocodazole produced a 15–20% inhibition in the apical surface accumulation of newly synthesized gp135 and inhibited the appearance of the gp135 pool by approximately 33%, while cytochalasin D had no affect on the apical accumulation of either newly synthesized gp135 or the gp135 pool. These results indicate that microtubules, but not microfilaments, are involved in the intracellular targeting of gp135. Quantitative immunogold electron microscopy of nocodazole-treated cells demonstrated that gp135 was not mistargeted to the basolateral membrane, suggesting the possibility that some vesicles containing gp135 did not fuse with the apical membrane and remained in the cells. These experiments demonstrate that microtubules are an important component of gp135 insertion into the apical membrane. They also suggest that gp135 resides within vesicles which have an apical membrane recognition signal and cannot fuse with the basolateral membrane. The possibility that these data, and those of others, could support a hypothesis for the presence of two constitutive apical transport pathways is discussed.

scholarly journals Putative linear motifs mediate the trafficking to apical and basolateral membranes

2020 ◽  
Author(s):  
Laszlo Dobson ◽  
András Zeke ◽  
Levente Szekeres ◽  
Tamás Langó ◽  
Gábor Tusnády
Keyword(s):  
Epithelial Cells ◽  
Drug Transport ◽  
Basolateral Membrane ◽  
Transmembrane Proteins ◽  
Transport Processes ◽  
Membrane Domains ◽  
Protein Distribution ◽  
Basolateral Membranes ◽  
Linear Motifs ◽  
Cellular Components

AbstractCell polarity refers to the asymmetric organisation of cellular components in various cells. Epithelial cells are the best known examples of polarized cells, featuring apical and basolateral membrane domains. Despite huge efforts, the exact rules governing the protein distribution in such domains are still elusive. In this study we examined linear motifs accumulating in these parts and based on the results we prepared ‘Classical’ and Convolutional Neural Networks to classify human transmembrane proteins localizing into apical/basolateral membranes. Asymmetric expression of drug transporters results in vectorial drug transport, governing the pharmacokinetics of numerous substances, yet the data on how proteins are sorted in epithelial cells is very scattered. The provided dataset may offer help to experimentalists to characterize novel molecular targets to regulate transport processes more precisely.

scholarly journals Sorting of Marburg Virus Surface Protein and Virus Release Take Place at Opposite Surfaces of Infected Polarized Epithelial Cells

2001 ◽  
Vol 75 (3) ◽  
pp. 1274-1283 ◽  
Author(s):  
Christian Sänger ◽  
Elke Mühlberger ◽  
Elena Ryabchikova ◽  
Larissa Kolesnikova ◽  
Hans-Dieter Klenk ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Surface Protein ◽  
Hemorrhagic Fever ◽  
Marburg Virus ◽  
Virus Surface ◽  
Mdckii Cells ◽  
Vesicular Stomatitis ◽  
Polarized Epithelial Cells

ABSTRACT Marburg virus, a filovirus, causes severe hemorrhagic fever with hitherto poorly understood molecular pathogenesis. We have investigated here the vectorial transport of the surface protein GP of Marburg virus in polarized epithelial cells. To this end, we established an MDCKII cell line that was able to express GP permanently (MDCK-GP). The functional integrity of GP expressed in these cells was analyzed using vesicular stomatitis virus pseudotypes. Further experiments revealed that GP is transported in MDCK-GP cells mainly to the apical membrane and is released exclusively into the culture medium facing the apical membrane. When MDCKII cells were infected with Marburg virus, the majority of GP was also transported to the apical membrane, suggesting that the protein contains an autonomous apical transport signal. Release of infectious progeny virions, however, took place exclusively at the basolateral membrane of the cells. Thus, vectorial budding of Marburg virus is presumably determined by factors other than the surface protein.

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.

Potassium conductance in rabbit esophageal epithelium

1993 ◽  
Vol 265 (1) ◽  
pp. G28-G34 ◽  
Author(s):  
W. E. Khalbuss ◽  
R. Alkiek ◽  
C. G. Marousis ◽  
R. C. Orlando
Keyword(s):  
Steady State ◽  
Epithelial Cells ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Potassium Conductance ◽  
Short Circuit Current ◽  
Sodium Absorption ◽  
High K ◽  
Esophageal Epithelial Cells ◽  
Apical Membranes

K+ conductance in apical and basolateral cell membranes of rabbit esophageal epithelial cells was investigated within intact epithelium by impalement with conventional microelectrodes from luminal or serosal sides. Under steady-state conditions, K+ conductance was demonstrated in basolateral, but not apical, membranes by showing 1) membrane depolarization upon exposure to either solutions high in K+ (20-65 mM) or containing Ba2+, tetraethylammonium, or quinine, and 2) a resistance ratio that increased on exposure to high K+ solution and decreased on exposure to Ba2+, quinine, and tetraethylammonium. From exposures to high K+, the apparent K+ transference number and electromotive force generated at the basolateral membrane were calculated and found to be 0.42 +/- 0.01 and -83 +/- 3 mV, respectively. Furthermore, basolateral K+ conductance was shown to be important for maintaining resting net transepithelial Na+ absorption in that high K+ or barium inhibited the transepithelial potential difference and short-circuit current of Ussing-chambered epithelia. We conclude that under steady-state conditions the basolateral, but not apical, membranes of esophageal epithelial cells contain a K(+)-conductive pathway and that this pathway is important for active sodium absorption.

ClC-2 in guinea pig colon: mRNA, immunolabeling, and functional evidence for surface epithelium localization

2002 ◽  
Vol 283 (4) ◽  
pp. G1004-G1013 ◽  
Author(s):  
Marcelo Catalán ◽  
Isabel Cornejo ◽  
Carlos D. Figueroa ◽  
María Isabel Niemeyer ◽  
Francisco V. Sepúlveda ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Guinea Pig ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Distal Colon ◽  
Surface Epithelium ◽  
Patch Clamp Technique ◽  
Chloride Currents ◽  
Kinetics Of

The principal function of the colon in fluid homeostasis is the absorption of NaCl and water. Apical membrane Na+ channels, Na+/H+ and Cl−/HCO[Formula: see text] exchangers, have all been postulated to mediate NaCl entry into colonocytes. The identity of the basolateral exit pathway for Cl− is unknown. We have previously demonstrated the presence of the ClC-2 transcript in the guinea pig intestine. Now we explore in more detail, the tissue and cellular distribution of chloride channel ClC-2 in the distal colon by in situ hybridization and immunohistochemistry. The patch-clamp technique was used to characterize Cl− currents in isolated surface epithelial cells from guinea pig distal colon and these were compared with those mediated by recombinant guinea pig (gp)ClC-2. ClC-2 mRNA and protein were found in the surface epithelium of the distal colon. Immunolocalization revealed that, in addition to some intracellular labeling, ClC-2 was present in the basolateral membranes but absent from the apical pole of colonocytes. Isolated surface epithelial cells exhibited hyperpolarization-activated chloride currents showing a Cl− > I− permeability and Cd2+ sensitivity. These characteristics, as well as some details of the kinetics of activation and deactivation, were very similar to those of recombinant gpClC-2 measured in parallel experiments. The presence of active ClC-2 type currents in surface colonic epithelium, coupled to a basolateral location for ClC-2 in the distal colon, suggests a role for ClC-2 channel in mediating basolateral membrane exit of Cl− as an essential step in a NaCl absorption process.

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