Characterization of cyclic AMP-regulated chloride conductance in the pigmented rabbit conjunctival epithelial cells

2002 ◽  
Vol 80 (6) ◽  
pp. 533-540 ◽  
Author(s):  
Michael H.I Shiue ◽  
Hovhannes J Gukasyan ◽  
Kwang-Jin Kim ◽  
Donald D.F Loo ◽  
Vincent H.L Lee
Keyword(s):  
Epithelial Cells ◽  
Cyclic Amp ◽  
Patch Clamp ◽  
Reversal Potential ◽  
Partial Substitution ◽  
Bathing Solution ◽  
Transmembrane Conductance Regulator ◽  
Whole Cell ◽  
C Terminus ◽  
Conjunctival Epithelial Cells

We have previously reported that the pigmented rabbit conjunctiva is a Cl– secreting tissue, subject to cAMP, Ca2+, and PKC modulation. The present study was conducted to characterize, at the cellular and molecular levels, cAMP-regulated Cl– channels in rabbit conjunctival epithelial cells. cAMP-inducible Cl– channel properties were evaluated by monitoring the whole-cell currents using patch clamp techniques. Results showed that 10 μM forskolin significantly stimulated a glibenclamide-inhibitable whole-cell conductance by approximately five-fold. Furthermore, reduction of the Cl– concentration in the bathing solution through partial substitution of NaCl with Na-isethionate resulted in a rightward shift of the reversal potential for both baseline and forskolin-stimulated whole-cell currents from 0 to values close to the theoretical Cl– reversal potential predicted by the Nernst equation. Western blot analysis with a monoclonal antibody recognizing the epitope in the C-terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) showed a positive band at its molecular weight, approximately 170 kD. Immunostaining under confocal microscopy revealed a CFTR specific signal in the apical sections of primary conjunctival epithelial cells. In addition, RT-PCR detection amplified a cDNA fragment 100% identical to the predicted portion of the cloned rabbit CFTR message. The stage is thus set for determining the extent of CFTR contribution to cAMP-regulated Cl– conductance in pigmented rabbit conjunctival epithelial cells.Key words: conjunctiva, chloride current, CFTR, chloride channel, patch clamp, cyclic AMP, whole-cell.

Activation of Cl− currents by intracellular chloride in fibroblasts stably expressing the human cystic fibrosis transmembrane conductance regulator

1993 ◽  
Vol 71 (9) ◽  
pp. 645-649 ◽  
Author(s):  
Xiaodong Wang ◽  
Yoshinori Marunaka ◽  
Ludwik Fedorko ◽  
Sascha Dho ◽  
J. Kevin Foskett ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cyclic Amp ◽  
Patch Clamp ◽  
Single Channel ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Whole Cell ◽  
Cystic Fibrosis Transmembrane

The Cl− conductance of a mouse fibroblast cell line (LTK− cells) that was stably transfected with the human CFTR (cystic fibrosis transmembrane conductance regulator) complementary DNA was studied. Single Cl− channel activity was observed only after treatment of the cells with forskolin, the single-channel conductance being 6.2 ± 0.2 pS with a linear current–voltage relationship. In CFTR+ cells, the whole-cell current at +90 mV increased from 7.3 ± 2.7 pA/pF (n = 12) to 46.1 ± 11.2 pA/pF (n = 5) after addition of dibutyryl-cyclic AMP (10−4 M) to the bath. Increasing the intracellular Cl− concentration to 150 mM activated linear Cl− currents in the absence of cyclic AMP in CFTR+ (n = 42) but not in CFTR− cells (n = 4). Similar Cl− current was also activated by high intracellular I− concentration. These results indicate that the CFTR-induced Cl− conductance in LTK− cells can be activated by either cyclic AMP or high intracellular halide concentrations.Key words: cystic fibrosis transmembrane conductance regulator (CFTR), chloride channel, cyclic AMP, whole-cell patch clamp, single-channel patch clamp.

A patch-clamp study: secretagogue-induced currents in rat peritoneal mast cells

1989 ◽  
Vol 256 (3) ◽  
pp. C560-C568 ◽  
Author(s):  
M. Kuno ◽  
T. Okada ◽  
T. Shibata
Keyword(s):  
Mast Cells ◽  
Patch Clamp ◽  
Reversal Potential ◽  
Whole Cell ◽  
Current Voltage ◽  
Peritoneal Mast Cells ◽  
Induced Currents ◽  
Rat Peritoneal Mast Cells ◽  
Whole Cell Recordings ◽  
Current Voltage Relation

Ca2+ entry through plasma membrane has been considered to play a significant role in elevating cytosolic free Ca2+ concentrations during stimulus-secretion coupling in mast cells, but electrophysiological evidence of the Ca2+ channels is lacking. We examined the properties of secretagogue (compound 48/80)-induced currents in rat peritoneal mast cells, using the patch-clamp technique. In the whole cell recordings, the addition of compound 48/80 induced transient currents that were suppressed by Cd or reduced by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). In Ringer solution containing 2 mM Ca2+, the current-voltage relation was fairly linear from -100 to 50 mV and the reversal potential was 14 +/- 10.1 mV (n = 9). When the external Ca2+ was approximately 1 microM, the compound 48/80-induced currents were marginal, but readmission of Ca2+ or Ba2+ to the bath solution led to an appearance of the currents. In the cell-attached patches, the stimulation enhanced the activity of inward current mediated by Ba2+. The unitary inward Ba2+ current was characterized by the unitary conductance of 10.5 +/- 2.0 pS (n = 10) with isotonic BaCl2 pipette solution, the extrapolated reversal potential of 60.7 +/- 16.0 mV (n = 10) positive to the resting membrane potentials. The percent open time of the inward Ba2+ current channel was not appreciably changed by voltage. In some whole cell recordings, an increase in openings of the cation-selective channel (20-45 pS) was identified in the stimulated cells. When the external Na+ was completely replaced by choline+, the compound 48/80-induced currents had a fairly linear current-voltage relation.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional coupling in bovine ciliary epithelial cells is modulated by carbachol

1997 ◽  
Vol 273 (6) ◽  
pp. C1876-C1881 ◽  
Author(s):  
J. W. Stelling ◽  
T. J. C. Jacob
Keyword(s):  
Epithelial Cells ◽  
Patch Clamp ◽  
Lucifer Yellow ◽  
Electrical Coupling ◽  
Ciliary Epithelium ◽  
Posterior Chamber ◽  
Functional Coupling ◽  
Half Time ◽  
Whole Cell ◽  
Junctional Communication

The functional coupling of the ciliary epithelium was studied in isolated pairs (couplets) of pigmented ciliary epithelial (PCE) and nonpigmented ciliary epithelial (NPCE) cells using the whole cell patch clamp and the fluorescent dye lucifer yellow. One cell of the pair (usually the NPCE cell of a NPCE-PCE cell couplet) was accessed with a 2–5 MΩ electrode, containing 1–2 mM lucifer yellow, in the whole cell configuration of the patch clamp. After voltage-clamp experiments were completed, cells were viewed under a fluorescent microscope to confirm that the cells were coupled. The electrical coupling of the cells was also studied by calculating the capacitance (using the time-domain technique), assuming a “supercell” model for coupled cells. The mean capacitance of coupled pairs was 79.8 ± 4.3 (SE) pF ( n = 47) compared with single cell capacitances of 36.8 ± 3.4 pF ( n = 10) for PCE cells and 38.1 ± 3.1 pF ( n = 15) for NPCE cells. Octanol, carbachol (CCh), and raised extracellular Ca2+ concentration ([Ca2+]o) all caused uncoupling in pairs (couplets) of coupled NPCE and PCE cells. At room temperature (22–24°C), the capacitance of the couplets decreased from 70.5 ± 8.0 to 48.0 ± 5.2 pF ( n = 5) when exposed to octanol (1 mM), from 73.8 ± 9.2 to 43.2 ± 9.5 pF ( n = 4) when exposed to CCh (100 μM), and from 80.5 ± 6.7 to 49.9 ± 7.8 pF ( n = 4) when exposed to 10 mM [Ca2+]o. The response to CCh was dose dependent; at higher temperatures of 34–37°C, 10 μM CCh caused a 38% reduction in capacitance, from 53.7 ± 9.7 to 33.5 ± 3.3 pF ( n = 7) with a half-time of 249 s, and 100 μM CCh caused a 49% reduction in capacitance, from 51.3 ± 5.6 to 26.0 ± 2.4 pF ( n = 7) with a half-time of 124 s. After pairs uncoupled and the uncoupling agent was washed out, the cell pairs often exhibited an increase in capacitance that we interpreted as “recoupling” or a reopening of the gap junctional communication pathway; the half-time for this process was 729 s after uncoupling with 100 μM CCh and 211 s after uncoupling with 10 μM CCh. This interpretation was confirmed optically by the spread of lucifer yellow into both cells of an uncoupled pair with a time course corresponding to the increase in electrical coupling. The controllable coupling of ciliary epithelial cells extends the idea of a functional syncytium involved in active transport. PCE cells take up solute and water from the blood, which then cross to NPCE cells via gap junctions and from there are secreted into the posterior chamber of the eye. Modulation of the coupling between NPCE and PCE cells may provide a mechanism to control secretion.

scholarly journals Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

10.3791/55700 ◽  
2017 ◽  
Author(s):  
Bao Li Zhang ◽  
Da Yuan Gao ◽  
Xiao Xu Zhang ◽  
Shuo Shi ◽  
Winnie Shum
Keyword(s):  
Epithelial Cells ◽  
Patch Clamp ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

Chloride channels in the apical membrane of thyroid epithelial cells are regulated by cyclic AMP

1995 ◽  
Vol 147 (3) ◽  
pp. 441-448 ◽  
Author(s):  
J R Bourke ◽  
O Sand ◽  
K C Abel ◽  
G J Huxham ◽  
S W Manley
Keyword(s):  
Epithelial Cells ◽  
Cyclic Amp ◽  
Chloride Channels ◽  
Apical Membrane ◽  
Reversal Potential ◽  
Channel Structure ◽  
Resting Membrane Potential ◽  
Prostaglandin E ◽  
Excised Patches ◽  
Apical Membranes

Abstract Porcine thyroid epithelial cells cultured as a monolayer with their apical membranes facing the medium are known to absorb Na+ and to secrete the anions Cl− and HCO3−. Chloride channels were found in the apical membrane, and displayed a reversal potential close to the resting membrane potential, linear current–voltage relationships, a conductance at physiological temperature of 6·5 pS, and a small but significant permeability to HCO3−. Stimulation of ion transport with prostaglandin E2 or 8-(4-chlorophenylthio) adenosine 3′:5′-cyclic monophosphate promoted activation of Cl− channels in cell-attached patches, and excised patches were reactivated by exposure of their cytoplasmic surface to protein kinase A and ATP. Physiological temperatures were necessary for activation of Cl− channels in cell-attached patches. The channels exhibited sub-states with a conductance exactly half that of the full unit conductance, suggesting a dual-barrelled channel structure. It is concluded that the apical membrane of thyroid epithelial cells contains cyclic AMP-activated Cl− channels controlling anion transport. Journal of Endocrinology (1995) 147, 441–448

scholarly journals Generation and functional characterization of epithelial cells with stable expression of SLC26A9 Cl− channels

2016 ◽  
Vol 310 (7) ◽  
pp. L593-L602 ◽  
Author(s):  
Johanna J. Salomon ◽  
Stephan Spahn ◽  
Xiaohui Wang ◽  
Joachim Füllekrug ◽  
Carol A. Bertrand ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Patch Clamp ◽  
Western Blotting ◽  
Therapeutic Target ◽  
Stable Expression ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Rat Thyroid

Recent studies identified the SLC26A9 Cl− channel as a modifier and potential therapeutic target in cystic fibrosis (CF). However, understanding of the regulation of SLC26A9 in epithelia remains limited and cellular models with stable expression for biochemical and functional studies are missing. We, therefore, generated Fisher rat thyroid (FRT) epithelial cells with stable expression of HA-tagged SLC26A9 via retroviral transfection and characterized SLC26A9 expression and function using Western blotting, immunolocalization, whole cell patch-clamp, and transepithelial bioelectric studies in Ussing chambers. We demonstrate stable expression of SLC26A9 in transfected FRT (SLC26A9-FRT) cells on the mRNA and protein level. Immunolocalization and Western blotting detected SLC26A9 in different intracellular compartments and to a lesser extent at the cell surface. Whole cell patch-clamp recordings demonstrated significantly increased constitutive Cl− currents in SLC26A9-FRT compared with control-transduced FRT (Control-FRT) cells ( P < 0.01). Similar, transepithelial measurements showed that the basal short circuit current was significantly increased in SLC26A9-FRT vs. Control-FRT cell monolayers ( P < 0.01). SLC26A9-mediated Cl− currents were increased by cAMP-dependent stimulation (IBMX and forskolin) and inhibited by GlyH-101, niflumic acid, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), as well as RNAi knockdown of WNK1 implicated in epithelial osmoregulation. Our results support that these novel epithelial cells with stable expression of SLC26A9 will be a useful model for studies of pharmacological regulation including the identification of activators of SLC26A9 Cl− channels that may compensate deficient cystic fibrosis transmembrane regulator (CFTR)-mediated Cl− secretion and serve as an alternative therapeutic target in patients with CF and potentially other muco-obstructive lung diseases.

scholarly journals The Effect of Hypertonic Media on Water Permeability of Frog Urinary Bladder

1973 ◽  
Vol 61 (1) ◽  
pp. 110-124 ◽  
Author(s):  
P. Ripoche ◽  
J. Bourguet ◽  
M. Parisi
Keyword(s):  
Epithelial Cells ◽  
Urinary Bladder ◽  
Cyclic Amp ◽  
Antidiuretic Hormone ◽  
Water Permeability ◽  
Prostaglandin E1 ◽  
Physiological State ◽  
Solution Temperature ◽  
Bathing Solution ◽  
Frog Urinary Bladder

The frog urinary bladder undergoes, in some conditions, a marked increase of its water permeability when incubated in hypertonic media. This increase was observed with various nonpermeant solutes. It seems to result from the shrinkage of an osmo-sensitive compartment of the tissue, probably the epithelial cells. Many similarities were found between this effect and the physiological increase in water permeability (hydrosmotic response) elicited by antidiuretic hormone (ADH): both were dependent on the physiological state of the animals, and although the response was slower after hyperosmolar than after hormonal challenge, the patterns of response were similar, and in both cases markedly dependent on bathing solution temperature. Norepinephrine and prostaglandin E1, which in this tissue reduce the hydrosmotic action of ADH, presumably by inhibiting the adenyl cylase also reduced the effect of hyperosmolarity. Conversely this effect was potentiated by incubation in the presence of oxytocin, exogenous cyclic AMP, and theophylline, conditions in which the intracellular concentration of cyclic AMP is increased. These data demonstrate that the response to hyperosmolarity is elicited, at least partly, by mechanisms also involved in the physiological hydrosmotic response to ADH.

Adult alveolar epithelial cells express multiple subtypes of voltage-gated K+ channels that are located in apical membrane

2003 ◽  
Vol 284 (6) ◽  
pp. C1614-C1624 ◽  
Author(s):  
So Yeong Lee ◽  
Peter J. Maniak ◽  
David H. Ingbar ◽  
Scott M. O'Grady
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Reversal Potential ◽  
Alveolar Epithelial Cells ◽  
Α Subunit ◽  
Whole Cell ◽  
Voltage Gated ◽  
Alveolar Epithelial ◽  
Kv Channels ◽  
Α Subunits

Whole cell perforated patch-clamp experiments were performed with adult rat alveolar epithelial cells. The holding potential was −60 mV, and depolarizing voltage steps activated voltage-gated K+ (Kv) channels. The voltage-activated currents exhibited a mean reversal potential of −32 mV. Complete activation was achieved at −10 mV. The currents exhibited slow inactivation, with significant variability in the time course between cells. Tail current analysis revealed cell-to-cell variability in K+ selectivity, suggesting contributions of multiple Kv α-subunits to the whole cell current. The Kv channels also displayed steady-state inactivation when the membrane potential was held at depolarized voltages with a window current between −30 and 5 mV. Analysis of RNA isolated from these cells by RT-PCR revealed the presence of eight Kv α-subunits (Kv1.1, Kv1.3, Kv1.4, Kv2.2, Kv4.1, Kv4.2, Kv4.3, and Kv9.3), three β-subunits (Kvβ1.1, Kvβ2.1, and Kvβ3.1), and two K+ channel interacting protein (KChIP) isoforms (KChIP2 and KChIP3). Western blot analysis with available Kv α-subunit antibodies (Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3) showed labeling of 50-kDa proteins from alveolar epithelial cells grown in monolayer culture. Immunocytochemical analysis of cells from monolayers showed that Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3 were localized to the apical membrane. We conclude that expression of multiple Kv α-, β-, and KChIP subunits explains the variability in inactivation gating and K+ selectivity observed between cells and that Kv channels in the apical membrane may contribute to basal K+ secretion across the alveolar epithelium.

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