scholarly journals Redistribution of immunofluorescence of CFTR anion channel and NKCC cotransporter in chloride cells during adaptation of the killifish Fundulus heteroclitus to sea water

2002 ◽  
Vol 205 (9) ◽  
pp. 1265-1273 ◽  
Author(s):  
W. S. Marshall ◽  
E. M. Lynch ◽  
R. R. F. Cozzi
Keyword(s):  
Fundulus Heteroclitus ◽  
Apical Membrane ◽  
Sea Water ◽  
Basolateral Membrane ◽  
Anion Channel ◽  
Chloride Cells ◽  
Transmembrane Conductance Regulator ◽  
Pavement Cells ◽  
Cell Complexes ◽  
C Terminus

SUMMARY Cellular distribution of cystic fibrosis transmembrane conductance regulator (CFTR) immunofluorescence was detected by monoclonal antibody directed to the C terminus of killifish CFTR (kfCFTR) in chloride cells of fresh water (FW) adapted fish and animals transferred to sea water (SW) for 24h, 48h and 14+ days. Confocal microscopy allowed localization within mitochondria-rich (MR) cells to be determined as superficial (i.e. in the apical membrane) or deeper within the cytoplasm of the cells. In FW, 90 % of MR cells had diffuse kfCFTR immunofluorescence in the central part of the cytosol, with only 8.1 % having apical kfCFTR, which was 6.6±0.54 μm below the microridges of surrounding pavement cells. Curiously, FW but not SW pavement cells also had positive immunofluorescence to kfCFTR. After 24h in SW, a time when kfCFTR expression is elevated, a condensed punctate immunofluorescence appeared among 18.8 % of MR cells, 13.4±0.66 μm(mean ± S.E.M.) below the surface of the cells. By 48h, a majority(76.3 %) of MR cells had punctate kfCFTR distribution and the distance from the surface was less (7.8±0.2 μm), a distribution approaching the SW-acclimated condition (i.e. all MR cells showing kfCFTR immunofluorescence,6.1±0.04 μm below the surface). In contrast, NKCC immunofluorescence was condensed and localized in lateral parts of MR cell complexes in FW animals and then redistributed to the whole basal cytoplasm after acclimation to SW. CFTR, the anion channel responsible for Cl- secretion in marine teleosts, redistributes in MR cells during SW acclimation by condensation of a diffuse distribution below the apical crypt, followed by translocation and insertion in the apical membrane. NKCC, the cotransporter that translocates Cl- across the basolateral membrane, moves from an eccentric cytosolic location in FW to a diffuse basolateral localization in SW chloride cells.

Bumetanide blocks CFTRG Cl in the native sweat duct

1999 ◽  
Vol 276 (1) ◽  
pp. C231-C237 ◽  
Author(s):  
M. M. Reddy ◽  
P. M. Quinton
Keyword(s):  
Basolateral Membrane ◽  
Electrical Conductance ◽  
Anion Channel ◽  
Membrane Resistance ◽  
Activation Process ◽  
Transmembrane Conductance Regulator ◽  
Sweat Duct ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Human Sweat Duct

Bumetanide is well known for its ability to inhibit the nonconductive Na+-K+-2Cl−cotransporter. We were surprised in preliminary studies to find that bumetanide in the contraluminal bath also inhibited NaCl absorption in the human sweat duct, which is apparently poor in cotransporter activity. Inhibition was accompanied by a marked decrease in the transepithelial electrical conductance. Because the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is richly expressed in the sweat duct, we asked whether bumetanide acts by blocking this anion channel. We found that bumetanide 1) significantly increased whole cell input impedance, 2) hyperpolarized transepithelial and basolateral membrane potentials, 3) depolarized apical membrane potential, 4) increased the ratio of apical-to-basolateral membrane resistance, and 5) decreased transepithelial Cl− conductance ( G Cl). These results indicate that bumetanide inhibits CFTR G Clin both cell membranes of this epithelium. We excluded bumetanide interference with the protein kinase A phosphorylation activation process by “irreversibly” phosphorylating CFTR [by using adenosine 5′- O-(3-thiotriphosphate) in the presence of a phosphatase inhibition cocktail] before bumetanide application. We then activated CFTR G Clby adding 5 mM ATP. Bumetanide in the cytoplasmic bath (10−3 M) inhibited ∼71% of this ATP-activated CFTR G Cl, indicating possible direct inhibition of CFTR G Cl. We conclude that bumetanide inhibits CFTR G Clin apical and basolateral membranes independent of phosphorylation. The results also suggest that >10−5 M bumetanide cannot be used to specifically block the Na+-K+-2Cl−cotransporter.

Low-conductance anion channel activated by cAMP in teleost Cl- -secreting cells

1995 ◽  
Vol 268 (4) ◽  
pp. R963-R969 ◽  
Author(s):  
W. S. Marshall ◽  
S. E. Bryson ◽  
A. Midelfart ◽  
W. F. Hamilton
Keyword(s):  
Fundulus Heteroclitus ◽  
Apical Membrane ◽  
Primary Cultures ◽  
Anion Channel ◽  
Disulfonic Acid ◽  
Anion Channels ◽  
Cyclic Monophosphate ◽  
Multiple Copies ◽  
Ion Substitution ◽  
High Conductance

We studied characteristics and modulation of ion channels in primary cultures of opercular epithelium from the euryhaline marine killifish Fundulus heteroclitus. Primary cultures, 17-28 h old, retain mitochondria-rich Cl- cells identifiable by fluorescence microscopy. Cell-attached patches revealed frequent low-conductance 8.1 +/- 0.35 pS channels that usually became inactive on excision; high-conductance anion channels were not apparent. Ion substitution experiments demonstrated selectivity for Cl- over gluconate of 1:0.07. With addition of 1-isobutyl-3-methylxanthine (0.1 mM) and dibutyryladenosine 3',5'-cyclic monophosphate (1.0 mM) to the bath, incidence of the channel increased from 35.3 to 61.9% of total patches (n = 156 and 21, respectively), and incidence of patches with multiple copies of the channel increased markedly from 2.2 to 38.5%. Epithelial Cl- transport was inhibited by mucosally added diphenylamine-2-carboxylic acid (1.0 mM) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.1-1.0 mM). The anion channel was absent from cultured killifish corneal epithelium, a tissue that lacks Cl- cells. We conclude that a low-conductance anion channel of Cl- cells, likely in the apical membrane, may account for adenosine 3',5'-cyclic monophosphate-activated Cl- secretion by marine fish.

Expression of cystic fibrosis transmembrane conductance regulator in a model epithelium

1994 ◽  
Vol 266 (4) ◽  
pp. L405-L413 ◽  
Author(s):  
D. N. Sheppard ◽  
M. R. Carson ◽  
L. S. Ostedgaard ◽  
G. M. Denning ◽  
M. J. Welsh
Keyword(s):  
Cystic Fibrosis ◽  
Apical Membrane ◽  
Single Cells ◽  
Basolateral Membrane ◽  
Recombinant Vaccinia Virus ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Monovalent Ions ◽  
Rat Thyroid ◽  
Cystic Fibrosis Transmembrane

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation and by intracellular nucleotides. The function of CFTR, like other recombinant ion channels, has generally been studied in single cells using voltage-clamp techniques. However, because CFTR is normally located in the apical membrane of epithelia we wanted to develop a system to study the function of recombinant CFTR expressed in an epithelium. We chose Fischer rat thyroid (FRT) epithelia for two reasons. First, when grown on permeable filter supports, FRT cells form polarized epithelia with a high transepithelial resistance. Second, they have no endogenous cAMP-regulated Cl- channels in their apical membrane. We expressed CFTR in FRT epithelia either transiently, using recombinant vaccinia virus, or stably, using a retrovirus. To measure apical membrane Cl- currents, we permeabilized the basolateral membrane to monovalent ions with nystatin and imposed a large transepithelial Cl- concentration gradient. cAMP agonists stimulated apical membrane Cl- currents in FRT epithelia infected with wild-type CFTR (vTF-CFTR) but not in FRT epithelia infected with either control virus (vTF7-3) or CFTR containing the delta F508 mutation (vTF-delta F508). These Cl- currents had properties similar to those of cAMP-activated Cl- currents in cells expressing endogenous or recombinant CFTR.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells

2009 ◽  
Vol 20 (8) ◽  
pp. 2337-2350 ◽  
Author(s):  
Mark R. Silvis ◽  
Carol A. Bertrand ◽  
Nadia Ameen ◽  
Franca Golin-Bisello ◽  
Michael B. Butterworth ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Expression System ◽  
Anion Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
T84 Cells ◽  
Anion Secretion ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.

scholarly journals Structural diversity of occluding junctions in the low-resistance chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus).

1980 ◽  
Vol 87 (2) ◽  
pp. 488-497 ◽  
Author(s):  
S A Ernst ◽  
W C Dodson ◽  
K J Karnaky
Keyword(s):  
Fundulus Heteroclitus ◽  
Short Circuit ◽  
Freeze Fracture ◽  
Short Circuit Current ◽  
Chloride Cells ◽  
Rectal Gland ◽  
Secretory Cells ◽  
Pavement Cells ◽  
Occluding Junctions ◽  
Mucosal Side

The structural features of the chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus) were examined by thin-section and freeze-fracture electron microscopy, with particular emphasis on the morphological appearance of occluding junctions. This epithelium is a flat sheet consisting predominantly of groups of mitochondriarich chloride cells with their apices associated to form apical crypts. These multicellular groups are interspersed in an otherwise continuous pavement cell epithelial lining. The epithelium may be mounted in Ussing-type chambers, which allow ready access to mucosal and serosal solutions and measurement of electrocal properties. The mean short-circuit current, potential difference (mucosal-side negative), and DC resistance for 19 opercular epithelia were, respectively, 120.0 +/- 18.2 microA/cm2, 12.3 +/- 1.7 mV, and 132.5 +/- 26.4 omega cm2. Short-circuit current, a direct measure of Cl- transport, was inhibited by ouabain (5 micron) when introduced on the serosal side, but not when applied to the mucosal side alone. Autoradiographic analysis of [3H]-ouabain-binding sites demonstrated that Na+,K+-ATPase was localized exclusively to basolateral membranes of chloride cells; pavement cells were unlabeled. Occluding junctions between adjacent chloride cells were remarkably shallow (20-25 nm), consisting of two parallel and juxtaposed junctional strands. Junctional interactions between pavement cells or between pavement cells and chloride cells were considerably more elaborate, extending 0.3-0.5 micron in depth and consisting of five or more interlocking junctional strands. Chloride cells at the lateral margins of crypts make simple junctional contacts with neighboring chloride cells and extensive junctions with contiguous pavement cells. Accordingly, in this heterogeneous epithelium, only junctions between Na+,K+-ATPase-rich chloride cells are shallow. Apical crypts may serve, therefore, as focal areas of high cation conductivity across the junctional route. This view is consistent with the electrical data showing that transmural resistance across the opercular eptihelium is low, and with recent studies demonstrating that transepithelial Na+ fluxes are passive. The simplicity of these junctions parallels that described recently for secretory cells of avian salt gland (Riddle and Ernst, 1979, J. Membr. Biol., 45:21-35) and elasmobranch rectal gland (Ernst et al., 1979, J. Cell Biol., 83:(2, Pt. 2):83 a[Abstr.]) and lends morphological support to the concept that paracellular ion permeation plays a central role in ouabain-sensitive transepithelial NaCl secretion.

scholarly journals Mummichog gill and operculum exhibit functionally consistent claudin-10 paralog profiles and Claudin-10c hypersaline response

Biology Open ◽  
2021 ◽  
Author(s):  
Chun Chih Chen ◽  
William S. Marshall ◽  
George N. Robertson ◽  
Regina R.F. Cozzi ◽  
Scott P. Kelly
Keyword(s):  
Fundulus Heteroclitus ◽  
Sea Water ◽  
Expression Profiles ◽  
Protein Abundance ◽  
Transmembrane Conductance Regulator ◽  
The Novel ◽  
Specific Expression ◽  
Secretion Pathway ◽  
Organ Specific ◽  
Organ Specific Expression

Claudin (Cldn) -10 tight junction (TJ) proteins are hypothesized to form the paracellular Na+ secretion pathway of hyposmoregulating mummichog (Fundulus heteroclitus) branchial epithelia. Organ-specific expression profiles showed that only branchial organs (the gill and opercular epithelium, OE) exhibited abundant cldn-10 paralog transcripts, which typically increased following sea water (SW) to hypersaline (2SW) challenge. Post-translational properties, protein abundance, and ionocyte localization of Cldn-10c, were then examined in gill and OE. Western blot analysis revealed two Cldn-10c immunoreactive bands in the mummichog gill and OE at ∼29 kDa and ∼40 kDa. The heavier protein could be eliminated by glycosidase treatment, demonstrating the novel presence of a glycosylated Cldn-10c. Protein abundance of Cldn-10c increased in gill and OE of 2SW-exposed fish. Cldn-10c localized to the sides of gill and OE ionocyte apical crypts and partially colocalized with cystic fibrosis transmembrane conductance regulator and F-actin, consistent with TJ complex localization. Cldn-10c immunofluorescent intensity increased but localization was unaltered by 2SW conditions. In support of our hypothesis, cldn-10/Cldn-10 TJ protein dynamics in gill and OE of mummichogs and TJ localization are functionally consistent with the creation and maintenance of salinity-responsive, cation-selective pores that facilitate Na+ secretion in hyperosmotic environments.

Dynamics of Pavement Cell–Chloride Cell Interactions During Abrupt Salinity Change in FUNDULUS HETEROCLITUS

2001 ◽  
Vol 204 (11) ◽  
pp. 1889-1899 ◽  
Author(s):  
K. Daborn ◽  
R. R. F. Cozzi ◽  
W. S. Marshall
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Fundulus Heteroclitus ◽  
Sea Water ◽  
Osmotic Shock ◽  
Chloride Cells ◽  
Bathing Solution ◽  
Pavement Cell ◽  
Stress Changes ◽  
Scanning Electron

SUMMARY Freshwater-adapted killifish (Fundulus heteroclitus) opercular epithelia were dissected and subjected to blood-side hypertonic bathing solution in Ussing-style chambers to simulate the increase in blood osmolality during migration to sea water. Conversely, seawater-acclimated killifish opercular epithelia were subjected to hypotonic bathing solutions to simulate the initial stages of migration to fresh water. Freshwater-acclimation (hypertonic stress) induced a rapid (approximately 30min) increase in membrane conductance (Gt) from 3.10±0.56 to 7.52±1.15mScm−2 (P<0.01, N=27), whereas seawater-acclimation (hypotonic stress) induced a rapid decrease in Gt from 8.22±1.15 to 4.41±1.00mScm−2 (P<0.01, N=27; means ± s.e.m.). Control seawater-acclimated membranes had a density of apical crypts (where chloride cells are exposed to the environment; detected by scanning electron microscopy) of 1133±96.4cryptsmm−2 (N=12), whereas the hypotonically shocked specimens had a lower crypt density of 870±36.7cryptsmm−2 (P<0.01 N=10; means ± s.e.m.). Hypertonic shock of freshwater membranes increased crypt density from 383.3±73.9 (N=12) to 630±102.9cryptsmm−2 (P<0.05; N=11; means ± s.e.m.). There was no change in density of chloride cells, as detected by fluorescence microscopy; hence, osmotic stress changes the degree of exposure, not the number of chloride cells. Cytochalasin D (5.0μmoll−1) completely blocked the conductance response to hypotonic shock and the reduction in apical crypt density measured by scanning electron microscopy, while phalloidin (33μmoll−1), colchicine (3×10−4moll−1) and griseofulvin (1.0μmoll−1) were ineffective. Actin imaging by phalloidin staining and confocal microscopy revealed extensive actin cords in pavement cell microridges and a ring of actin at the apex of chloride cells. We conclude that the actin cytoskeleton of chloride cells is required to maintain crypt opening and that osmotic shock causes chloride cells to adjust their apical crypt size.

Apical membrane chloride channels in a colonic cell line activated by secretory agonists

1988 ◽  
Vol 254 (4) ◽  
pp. C505-C511 ◽  
Author(s):  
D. R. Halm ◽  
G. R. Rechkemmer ◽  
R. A. Schoumacher ◽  
R. A. Frizzell
Keyword(s):  
Cell Line ◽  
Chloride Channels ◽  
Single Channel ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Anion Channel ◽  
Cell Types ◽  
Cl Channel ◽  
Evoked Activity ◽  
Apical Membranes

We characterized the anion channel responsible for the increase in apical membrane Cl secretion using a model salt-secreting epithelium, the T84 colonic cell line. The adenosine 3',5'-cyclic monophosphate (cAMP)-mediated secretagogues, prostaglandin E2, forskolin, and 8-bromo-cAMP, evoked activity of an outwardly rectifying Cl channel in previously quiet cell-attached membrane patches. The channel remained active in excised, inside-out membranes, where its single-channel conductance was 40-45 pS at 0 mV with 160 mM NaCl in pipette and bath. Selectivities were PCl/PNa = 50 and for halides I(1.8)/Br(1.4)/Cl(1.0)/F(0.4). This halide sequence illustrates that the ability of various anions to undergo transepithelial secretion is determined by the selectivity of the basolateral membrane Cl entry step rather than by the apical Cl channel. Open-channel probability increased with depolarization, an effect that would adjust the rate of Cl exit across secretory cell apical membranes with agonist-induced changes in apical membrane potential. Comparison with the properties of Cl channels detected in other cell types suggests that this cAMP-stimulated Cl channel is uniquely present in the apical membranes of salt-secreting epithelial cells.

Ion-secreting epithelia: chloride cells in the head region of Fundulus heteroclitus

1980 ◽  
Vol 238 (3) ◽  
pp. R185-R198 ◽  
Author(s):  
K. J. Karnaky
Keyword(s):  
Fundulus Heteroclitus ◽  
Chloride Transport ◽  
Basolateral Membrane ◽  
Freeze Fracture ◽  
Chloride Ion ◽  
Salt Gland ◽  
Chloride Cells ◽  
Head Region ◽  
Zonulae Occludentes ◽  
Apical Side

Transporting cells of ion-secreting epithelia are characterized by similar morphological patterns that include rich supplies of mitochondria, exotic patterns of surface amplification, and basolateral, plasma-membrane location of Na-K-ATPase, even though the direction of sodium transport across these epithelia is toward the apical side. Several new models for NaCl secretion propose that sodium, extruded into the intercellular space by Na-K-ATPase, reaches the apical side via the zonulae occludentes. Very recent freeze-fracture electron microscopy of avian salt gland and teleost chloride cells reveals that transporting cells are joined by simple, shallow zonulae occludentes. These observations lend morphological support to the concept that paracellular sodium ion permeation plays a central role in secretion. The chloride ion may traverse the epithelium via a transcellular route, entering the cell at the basolateral membrane by a chloride carrier linked to the cotransport of sodium down its electrochemical gradient into the cell. Finally, the chloride ion may exit the cell across the apical membrane by electrical forces. This review summarizes biochemical, morphological, and electrophysiological aspects of these new secretory models and the important contribution of a half century of research on teleost osmoregulatory mechanisms, including the chloride cell, to our understanding of sodium and chloride transport across secretory epithelia.

Chloride conductance of CFTR facilitates basal Cl−/HCO3−exchange in the villous epithelium of intact murine duodenum

2005 ◽  
Vol 288 (6) ◽  
pp. G1241-G1251 ◽  
Author(s):  
Janet E. Simpson ◽  
Lara R. Gawenis ◽  
Nancy M. Walker ◽  
Kathryn T. Boyle ◽  
Lane L. Clarke
Keyword(s):  
Exchange Rate ◽  
Apical Membrane ◽  
Anion Channel ◽  
Duodenal Mucosa ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Anion Exchanger 1 ◽  
Direct Exposure ◽  
Mucus Barrier ◽  
Cf Transmembrane Conductance Regulator

Villi of the proximal duodenum are situated for direct exposure to gastric acid chyme. However, little is known about active bicarbonate secretion across villi that maintains the protective alkaline mucus barrier, a process that may be compromised in cystic fibrosis (CF), i.e., in the absence of a functional CF transmembrane conductance regulator (CFTR) anion channel. We investigated Cl−/HCO3−exchange activity across the apical membrane of epithelial cells located at the midregion of villi in intact duodenal mucosa from wild-type (WT) and CF mice using the pH-sensitive dye BCECF. Under basal conditions, the Cl−/HCO3−exchange rate was reduced by ∼35% in CF compared with WT villous epithelium. Cl−/HCO3−exchange in WT and CF villi responded similarly to inhibitors of anion exchange, and membrane depolarization enhanced rates of Cl−out/HCO3−inexchange in both epithelia. In anion substitution studies, anionin/HCO3−outexchange rates were greater in WT epithelium using Cl−or NO3−, but decreased to the level of the CF epithelium using the CFTR-impermeant anion, SO42−. Similarly, treatment of WT epithelium with the CFTR-selective blocker glybenclamide decreased the Cl−/HCO3−exchange rate to the level of CF epithelium. The mRNA expression of Slc26a3 (downregulated in adenoma) and Slc26a6 (putative anion exchanger-1) was similar between WT and CF duodena. From these studies of murine duodenum, we conclude 1) characteristics of Cl−/HCO3−exchange in the villous epithelium are most consistent with Slc26a6 activity, and 2) Cl−channel activity of CFTR facilitates apical membrane Cl−in/HCO3−outexchange by providing a Cl−“leak” under basal conditions.

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