A self-referencing Cl--selective microelectrode (Cl- SrE) was developed and used to detect changes in the direction and magnitude of the Cl- flux (J(Cl)) from the apical region of cultured foetal distal lung epithelial cells (FDLEs) as a function of external Cl- concentration ([Cl-]e) and in response to pharmacological challenges. The technique, which is similar to that developed for other ion-selective microelectrodes, centres on the oscillation of a Cl--selective microelectrode between known points, micrometres apart, orthogonal to the plasma membrane. Application of the Fick principle to the differential voltage obtained per excursion amplitude (the referenced signal) yields the Cl- flux (pmol × cm(−2) × s(−1)). A Cl- effusion gradient was used to confirm that empirical measurements of J(Cl) using the Cl- SrE were statistically similar to predicted flux values calculated from the fall in [Cl-] with distance from the tip of the effusion source. Apical J(Cl) was then measured as a function of [Cl-]e from polarised FDLE cultures grown on permeable supports. At [Cl-]e<50 mmol × l(−1), an apical-to-basolateral (inward) flux, maximal at 400 pmol × cm(−2) × s(−1), was observed; this reverted to a continuous basolateral-to-apical (outward) flux of 203 pmol × cm(−2) × s(−1) at [Cl]e>100 mmol × l(−1). At [Cl-]e>100 mmol × l(−1), isoproterenol (basolaterally applied, 10 micromol × l(−1)) activated a Cl- influx of 561 pmol × cm(−2) × s(−1), whereas UTP (apically applied, 100 micromol × l(−1)) stimulated a Cl- efflux of 300 pmol × cm(−2) × s(−1). In all cases, 50–70 % of J(Cl) was abolished by Cl- channel blockade using 10 micromol × l(−1) diphenylamine-2-carboxylic acid (DPC) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that the Cl- SrE resolves a Cl- gradient in the microenvironment of the apical region of lung epithelia that varies in both direction and magnitude as a function of external [Cl-]e and in response to Cl- channel blockade and to beta2 adrenoreceptor and P2Y receptor agonists.

REFERENCES

Boucher
R. C.
(
1994
).
Human airway ion transport.
I. Am. J. Respir. Crit. Care Med
150
,
271
–.
Clarke
L. L.
,
Boucher
R. C.
(
1992
).
Chloride secretory response to extracellular ATP in human normal and cystic fibrosis nasal epithelia.
Am. J. Physiol
265
,
348
–.
Clunes
M. T.
,
Collett
A.
,
Baines
D. L.
,
Bovell
D. L.
,
Murphie
H.
,
Inglis
S. K.
,
McAlroy
H. L.
,
Olver
R. E.
,
Wilson
S. M.
(
1998
).
Culture substrate-specific expression of P2Y(2) receptors in distal lung epithelial cells isolated from foetal rats.
Br. J. Pharmac
124
,
845
–.
Hull
J.
,
Skinner
W.
,
Robertson
C.
,
Phelan
P.
(
1998
).
Elemental content of airway surface liquid from infants with cystic fibrosis.
Am. J. Respir. Crit. Care Med
157
,
10
–.
Illek
B.
,
Tam
A.W.
,
Fischer
H.
,
Machen
T. E.
(
1999
).
Anion selectivity of apical membrane conductance of Calu 3 human airway epithelium.
Pflugers Arch
437
,
812
–.
Illek
B.
,
Yankaskas
J. R.
,
Machen
T. E.
(
1997
).
cAMP and genistein stimulate HCO3conductance through CFTR in human airway epithelia.
Am. J. Physiol
272
,
752
–.
Inglis
S. K.
,
Collett
A.
,
McAlroy
H. L.
,
Wilson
S. M.
,
Olver
R. E.
(
1999
).
Effect of luminal nucleotides on Clsecretion and Na+absorption in distal bronchi.
Pflugers Arch
438
,
621
–.
Jaffe
L. F.
,
Nuccitelli
R.
(
1974
).
An ultrasensitive vibrating probe for measuring steady extracellular currents.
J. Cell Biol
63
,
614
–.
Jiang
X.
,
Ingbar
D.H.
,
O'Grady
S. M.
(
1998
).
Adrenergic stimulation of Na+transport across alveolar epithelial cells involves actibation of apical Clchannels.
Am. J. Physiol
275
,
1610
–.
Kaji
D. M.
(
1993
).
Na+/K+/2Clcotransport in medullary thick ascending limb cells: Kinetics and bumetanide binding.
Biochim. Biophys. Acta
1152
,
289
–.
Kemp
P. J.
,
Olver
R. E.
(
1996
).
The cellular basis of G protein regulation of alveolar ion channels.
Exp. Physiol
81
,
493
–.
Knowles
M. R.
,
Robinson
J. M.
,
Wood
R. E.
,
Pue
C. A.
,
Mentz
W. M.
,
Wager
G. C.
,
Gatzy
J. T.
,
Boucher
R. C.
(
1997
).
Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects.
J. Clin. Invest
100
,
2588
–.
Kondo
Y.
,
Buhrer
T.
,
Seiler
K.
,
Fromter
E.
,
Simon
W.
(
1989
).
A new double-barrelled, ionophore-based microelectrode for chloride ions.
Pflugers Arch
414
,
663
–.
Kuhtreiber
W. M.
,
Jaffe
L. F.
(
1990
).
Detection of extracellular calcium gradients with a calcium-specific vibrating electrode.
J. Cell Biol
110
,
1565
–.
Land
S. C.
,
Porterfield
D. M.
,
Sanger
R. H.
,
Smith
P. J. S.
(
1999
).
The self-referencingoxygen-selective microelectrode:detection of transmembrane O2flux from single cells.
J. Exp. Biol
202
,
211
–.
Land
S. C.
,
Sanger
R. H.
,
Smith
P. J. S.
(
1997
).
O2-availability modulates transmembrane Ca2+-flux via second-messenger pathways in anoxia-tolerant hepatocytes.
J. Appl. Physiol
82
,
776
–.
Mason
S. J.
,
Paradiso
A. M.
,
Boucher
R. C.
(
1991
).
Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human and cystic fibrosis airway epithelium.
Br. J. Pharmac
103
,
1649
–.
Matsui
H.
,
Grubb
B. R.
,
Tarran
R.
,
Randell
S. H.
,
Gatzy
J. T.
,
Davis
C. W.
,
Boucher
R. C.
(
1998
).
Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease.
Cell
95
,
1005
–.
Muller-Berger
S.
,
Samarzija
I.
,
Kunimi
M.
,
Yamada
H.
,
Fromter
E.
,
Seki.
G.
(
1999
).
A stop-flow microperfusion technique for rapid determination of HCO3absorption/H+-secretion by isolated renal tubules.
Pflugers Arch
439
,
208
–.
Ramminger
S.
,
Collett
A.
,
Baines
D. L.
,
Murphie
H.
,
McAlroy
H. L.
,
Olver
R. E.
,
Inglis
S. K.
,
Wilson
S. M.
(
1999
).
P2Y2receptor-mediated inhibition of ion transport in distal lung epithelial cells.
Br. J. Pharmac
128
,
293
–.
Ruckes
C.
,
Blank
U.
,
Moller
K.
,
Rieboldt
J.
,
Lindemann
H.
,
Munker
G.
,
Clauss
W.
,
Weber
W.-M.
(
1997
).
Amiloride-sensitive Na+channels in human nasal epithelium are different from classical epithelial Na+channels.
Biochem. Biophys. Res. Commun
237
,
488
–.
Schellhase
D. E.
,
Emrie
P. A.
,
Fisher
J. H.
,
Shannon
J. M.
(
1989
).
Ontogeny of surfactant apoproteins in the rat.
Ped. Res
26
,
167
–.
Shirihai
O.
,
Smith
P. J. S.
,
Hammar
K.
,
Dagan
D.
(
1998
).
H+and K+gradient generated by microglia H/K ATPase.
Glia
23
,
339
–.
Trimarchi
J. R.
,
Liu
L.
,
Porterfield
D. M.
,
Smith
P. J. S.
,
Keefe
D. L.
(
2000
).
Oxidative phosphorylation-dependent and- independent oxygen consumption by individual preimplantation mouse embryos.
Biol. Reprod
62
,
1866
–.
Tsuruoka
S.
,
Schwartz
G. J.
(
1999
).
Mechanisms of HCO3secretion in the rabbit connecting segment.
Am. J. Physiol
277
,
567
–.
Uyekubo
S. N.
,
Fischer
H.
,
Maminishkis
A.
,
Illek
B.
,
Miller
S. S.
,
Widdicombe
J. H.
(
1998
).
cAMP-dependent absorption of chloride across airway epithelium.
Am. J. Physiol
275
,
1219
–.
Whisenant
N.
,
Khademazad
M.
,
Muallem
S.
(
1993
).
Regulatory interaction of ATP, Na+and Clin the turnover cycle of the Na+/K+/2Cl cotransporter.
J. Gen. Physiol
101
,
889
–.
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