Chloride conductive and cotransport mechanisms in cultures of canine tracheal epithelial cells measured by an entrapped fluorescent indicator

A. C. Chao, Jonathan Widdicombe, A. S. Verkman

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

To study Cl conductive and cotransport mechanisms, primary cultures of canine tracheal cells were grown to confluency on thin glass cover slips and on porous filters. Transepithelial resistance was >100 ω·cm2, and short circuit current (Isc=2-20 μA/cm2), representing active secretion of Cl, increased >threefold with addition of 10 μm isoproterenol to the serosal solution. Cells made transiently permeable in hypotonic solution were loaded with the Cl-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (5 mm, 4 min, 150 mOsm). The electrical properties of the cell monolayers were not altered by the loading procedure. Intracellular SPQ fluorescence was monitored continuously by epifluorescence microscopy (excitation 360±5 nm, emission>410 nm). SPQ leakage from the cells was <10% in 60 min at 37°C. Intracellular calibration of SPQ fluorescence vs. [Cl] (0-90 mm) was carried out using high-K buffers containing the ionophores nigericin (5 μm) and tributyltin (10 μm); SPQ fluorescence was quenched with a Stern-Volmer constant of 13 m-1. Intracellular Cl activity was 43±4 mm. Cl flux was measured in response to addition and removal of 114 mm Cl from the bathing solution. Addition of 10 μm isoproterenol increased Cl efflux from 0.10 to 0.27 mm/sec. The increase was inhibited by the Cl-channel blocker diphenylamine-2-carboxylic acid (1 mm). In the absence of isoproterenol, removal of external Na or addition of 0.5 mm furosemide, reduced Cl influx by >fourfold. In ouabain-treated monolayers, removal of external K in the presence of 5 mm barium diminished Cl influx by >twofold, suggesting that Cl entry is in part K dependent. These results establish an accurate optical method for the realtime measurement of intracellular Cl activity in tracheal cells that does not require an electrically tight cell monolayer. The data demonstrate the presence of an isoproterenol-regulated Cl channel and a furosemide-sensitive cation-coupled transport mechanism.

Original languageEnglish (US)
Pages (from-to)193-202
Number of pages10
JournalThe Journal of Membrane Biology
Volume113
Issue number3
DOIs
StatePublished - Feb 1990
Externally publishedYes

Fingerprint

Canidae
Chlorides
Epithelial Cells
Isoproterenol
Hypotonic Solutions
Furosemide
Ouabain
Barium
Glass
Cations
Microscopy
Fluorescence
6-methoxy-N-(3-sulfopropyl)quinolinium

Keywords

  • cell culture
  • chloride transport
  • fluorescence
  • furosemide
  • isoproterenol
  • Na-K-Cl transport
  • SPQ
  • trachea

ASJC Scopus subject areas

  • Physiology
  • Cell Biology
  • Biophysics

Cite this

@article{01869569f97e467ea95c6c9868eaeee0,
title = "Chloride conductive and cotransport mechanisms in cultures of canine tracheal epithelial cells measured by an entrapped fluorescent indicator",
abstract = "To study Cl conductive and cotransport mechanisms, primary cultures of canine tracheal cells were grown to confluency on thin glass cover slips and on porous filters. Transepithelial resistance was >100 ω·cm2, and short circuit current (Isc=2-20 μA/cm2), representing active secretion of Cl, increased >threefold with addition of 10 μm isoproterenol to the serosal solution. Cells made transiently permeable in hypotonic solution were loaded with the Cl-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (5 mm, 4 min, 150 mOsm). The electrical properties of the cell monolayers were not altered by the loading procedure. Intracellular SPQ fluorescence was monitored continuously by epifluorescence microscopy (excitation 360±5 nm, emission>410 nm). SPQ leakage from the cells was <10{\%} in 60 min at 37°C. Intracellular calibration of SPQ fluorescence vs. [Cl] (0-90 mm) was carried out using high-K buffers containing the ionophores nigericin (5 μm) and tributyltin (10 μm); SPQ fluorescence was quenched with a Stern-Volmer constant of 13 m-1. Intracellular Cl activity was 43±4 mm. Cl flux was measured in response to addition and removal of 114 mm Cl from the bathing solution. Addition of 10 μm isoproterenol increased Cl efflux from 0.10 to 0.27 mm/sec. The increase was inhibited by the Cl-channel blocker diphenylamine-2-carboxylic acid (1 mm). In the absence of isoproterenol, removal of external Na or addition of 0.5 mm furosemide, reduced Cl influx by >fourfold. In ouabain-treated monolayers, removal of external K in the presence of 5 mm barium diminished Cl influx by >twofold, suggesting that Cl entry is in part K dependent. These results establish an accurate optical method for the realtime measurement of intracellular Cl activity in tracheal cells that does not require an electrically tight cell monolayer. The data demonstrate the presence of an isoproterenol-regulated Cl channel and a furosemide-sensitive cation-coupled transport mechanism.",
keywords = "cell culture, chloride transport, fluorescence, furosemide, isoproterenol, Na-K-Cl transport, SPQ, trachea",
author = "Chao, {A. C.} and Jonathan Widdicombe and Verkman, {A. S.}",
year = "1990",
month = "2",
doi = "10.1007/BF01870071",
language = "English (US)",
volume = "113",
pages = "193--202",
journal = "Journal of Membrane Biology",
issn = "0022-2631",
publisher = "Springer New York",
number = "3",

}

TY - JOUR

T1 - Chloride conductive and cotransport mechanisms in cultures of canine tracheal epithelial cells measured by an entrapped fluorescent indicator

AU - Chao, A. C.

AU - Widdicombe, Jonathan

AU - Verkman, A. S.

PY - 1990/2

Y1 - 1990/2

N2 - To study Cl conductive and cotransport mechanisms, primary cultures of canine tracheal cells were grown to confluency on thin glass cover slips and on porous filters. Transepithelial resistance was >100 ω·cm2, and short circuit current (Isc=2-20 μA/cm2), representing active secretion of Cl, increased >threefold with addition of 10 μm isoproterenol to the serosal solution. Cells made transiently permeable in hypotonic solution were loaded with the Cl-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (5 mm, 4 min, 150 mOsm). The electrical properties of the cell monolayers were not altered by the loading procedure. Intracellular SPQ fluorescence was monitored continuously by epifluorescence microscopy (excitation 360±5 nm, emission>410 nm). SPQ leakage from the cells was <10% in 60 min at 37°C. Intracellular calibration of SPQ fluorescence vs. [Cl] (0-90 mm) was carried out using high-K buffers containing the ionophores nigericin (5 μm) and tributyltin (10 μm); SPQ fluorescence was quenched with a Stern-Volmer constant of 13 m-1. Intracellular Cl activity was 43±4 mm. Cl flux was measured in response to addition and removal of 114 mm Cl from the bathing solution. Addition of 10 μm isoproterenol increased Cl efflux from 0.10 to 0.27 mm/sec. The increase was inhibited by the Cl-channel blocker diphenylamine-2-carboxylic acid (1 mm). In the absence of isoproterenol, removal of external Na or addition of 0.5 mm furosemide, reduced Cl influx by >fourfold. In ouabain-treated monolayers, removal of external K in the presence of 5 mm barium diminished Cl influx by >twofold, suggesting that Cl entry is in part K dependent. These results establish an accurate optical method for the realtime measurement of intracellular Cl activity in tracheal cells that does not require an electrically tight cell monolayer. The data demonstrate the presence of an isoproterenol-regulated Cl channel and a furosemide-sensitive cation-coupled transport mechanism.

AB - To study Cl conductive and cotransport mechanisms, primary cultures of canine tracheal cells were grown to confluency on thin glass cover slips and on porous filters. Transepithelial resistance was >100 ω·cm2, and short circuit current (Isc=2-20 μA/cm2), representing active secretion of Cl, increased >threefold with addition of 10 μm isoproterenol to the serosal solution. Cells made transiently permeable in hypotonic solution were loaded with the Cl-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (5 mm, 4 min, 150 mOsm). The electrical properties of the cell monolayers were not altered by the loading procedure. Intracellular SPQ fluorescence was monitored continuously by epifluorescence microscopy (excitation 360±5 nm, emission>410 nm). SPQ leakage from the cells was <10% in 60 min at 37°C. Intracellular calibration of SPQ fluorescence vs. [Cl] (0-90 mm) was carried out using high-K buffers containing the ionophores nigericin (5 μm) and tributyltin (10 μm); SPQ fluorescence was quenched with a Stern-Volmer constant of 13 m-1. Intracellular Cl activity was 43±4 mm. Cl flux was measured in response to addition and removal of 114 mm Cl from the bathing solution. Addition of 10 μm isoproterenol increased Cl efflux from 0.10 to 0.27 mm/sec. The increase was inhibited by the Cl-channel blocker diphenylamine-2-carboxylic acid (1 mm). In the absence of isoproterenol, removal of external Na or addition of 0.5 mm furosemide, reduced Cl influx by >fourfold. In ouabain-treated monolayers, removal of external K in the presence of 5 mm barium diminished Cl influx by >twofold, suggesting that Cl entry is in part K dependent. These results establish an accurate optical method for the realtime measurement of intracellular Cl activity in tracheal cells that does not require an electrically tight cell monolayer. The data demonstrate the presence of an isoproterenol-regulated Cl channel and a furosemide-sensitive cation-coupled transport mechanism.

KW - cell culture

KW - chloride transport

KW - fluorescence

KW - furosemide

KW - isoproterenol

KW - Na-K-Cl transport

KW - SPQ

KW - trachea

UR - http://www.scopus.com/inward/record.url?scp=0025020683&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025020683&partnerID=8YFLogxK

U2 - 10.1007/BF01870071

DO - 10.1007/BF01870071

M3 - Article

C2 - 2335807

AN - SCOPUS:0025020683

VL - 113

SP - 193

EP - 202

JO - Journal of Membrane Biology

JF - Journal of Membrane Biology

SN - 0022-2631

IS - 3

ER -