W. A. Bernbaum Center for Cystic Fibrosis Research, Departments of Pediatrics and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4948
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ABSTRACT |
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In this study, we
tested the hypothesis that intracellular Cl
(Cl
and thus the activation of Na-K-Cl cotransport (NKCC1) in a
Calu-3 cell line. The
1-adrenergic agonist methoxamine (MOX) and hypertonic sucrose increased Cl
]i). Titration of
[Cl
]i from 20-140 mM in
nystatin-permeabilized cell monolayers did not affect the baseline
activity of PKC-
, PKC-
, or rottlerin-sensitive NKCC1. At 200 mM
Cl
, rottlerin-sensitive NKCC1 was activated, and PKC
isotypes were localized predominantly to a particulate fraction. MOX
induced a biphasic increase in NKCC1 activity and PKC-
in activity
and particulate localization of PKC-
and -
. Activity of NKCC1 and PKC-
decreased with increasing Cl
]i levels. Rottlerin inhibited the
effects of MOX, which indicates that PKC-
was required for
activation of NKCC1. The results indicate that, in airway epithelial
cells, a Cl
is necessary. Further, high Cl
, which results in increased
enzyme activity.
-adrenergic; methoxamine; volume; hyperosmotic stress; bumetanide; shrinkage; cystic fibrosis; rottlerin
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INTRODUCTION |
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NA-K-2CL
COTRANSPORTERS (NKCCS) comprise a family of
Cl-dependent cation cotransporters that are expressed in
many animal cells (24). This widespread distribution
suggests a fundamental role for NKCCs in physiological functions. In
most cells that have been well studied, NKCCs are activated by cell
shrinkage and thus play a potentially critical role in cell-volume
regulation. Epithelia NKCCs serve secretory and absorptive functions
depending on the localization of the NKCC to the basolateral or apical
plasma membrane, respectively. Absorptive functions of NKCCs are best observed in the absorptive epithelium of the thick ascending limb of
Henle's loop of mammalian kidney. Secretory functions are well studied
in a number of epithelia including those of the colon, trachea, small
intestine, and salivary gland (24).
Bumetanide-sensitive NKCC1 is localized to the basolateral plasma
membrane of epithelia and participates in homeostatic control of cell
volume and secretion of fluids and electrolytes. In epithelia of the
colon and trachea, for example, Cl secretion is rapidly
elicited by agents that act on cAMP. cAMP-mediated activation of apical
Cl
channels is generally modeled as the major regulatory
event to elicit secretion. However, basolateral NKCC1 is necessary for secretion because it supplies Cl
for secretion. The
intracellular Cl
concentration
([Cl
]i) itself has been linked to
regulation of NKCC1 activity in secretory epithelia and avian
erythrocytes (22, 24) as well as in dialyzed squid axon
(1). In general, a reduction in intracellular Cl
(Cl
secretion as a
coordinated control of Cl
exit at the apical membrane
through Cl
channels and entry at the basolateral membrane
through NKCC1. However, the mechanism by which
[Cl
]i regulates NKCC1 activity is not well understood.
One mechanism that is thought to control activity of NKCC1 is NKCC1
phosphorylation through the action of serine and threonine protein
kinases. NKCC1 isoforms from mammalian epithelia share similar putative
sites for phosphorylation by protein kinases A and C (PKA and
PKC; Ref. 24). Yet elevations in PKA alone do not fully
explain the activation of NKCC1 in shark rectal gland cells and human
tracheal epithelial cells (9). Rather, we have shown that
activation of NKCC1 in human tracheal epithelial cells and in a Calu-3
cell line can be isolated from activation of Cl channels
through stimulation with an
1-adrenergic agonist
(9, 10, 12). Our studies also demonstrate a signaling
mechanism in which PKC-
is required for
1-adrenergic
activation of NKCC1 (11). An earlier study
(8) on rabbit tracheal epithelial cells showed that NKCC1
is activated by hyperosmotic stress, which causes a rapid loss of water
and leads to cell shrinkage and subsequent compensatory uptake of ions
via NaCl or NKCC.
In this study, we investigated the sensitivity of human airway
epithelial NKCC1 activity and PKC- activity to
[Cl
]i in Calu-3 cells. A Calu-3 epithelial
cell line has been shown by others and by us to express NKCC1 mRNA and
hormone-mediated activation of NKCC1 (4, 10). We first
identified experimental conditions that allow alterations in
Cl
1-adrenergic
agonist) and by hyperosmolarity induced using sucrose. Next, we studied
the effects of varying [Cl
]i on the
activity of NKCC1 and on the activity and subcellular distribution of
PKC-
and -
. The results indicate that NKCC1 is quiescent over a
range of [Cl
]i until stimulated by hormone.
Peak net activity of NKCC1 and PKC-
occurs at 20 mM
Cl
to a particulate fraction.
Surprisingly, at very high Cl
1-adrenergic agonist. Agonist
stimulation of NKCC1 is sensitive to rottlerin (an inhibitor of
PKC-
). Therefore, PKC-
is necessary for activation of NKCC1 even
at very high [Cl
]i. Thus, in human airway
epithelial cells, a Cl
activity is necessary.
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MATERIALS AND METHODS |
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Cell culture. Calu-3 cells were grown in cell culture in Earle's balanced salt solution supplemented with 2.4 mM L-glutamine and 10% fetal bovine serum on 0.4-µm pore size Transwell-Clear polyester filter inserts (Corning Costar, Cambridge, MA). For measurement of cell volume and transepithelial flux, cells were seeded at a density of 2.0 × 106 cells onto filters with a growth area of 4.52 cm2. Cells were incubated in a humidified CO2 incubator at 37°C. Culture medium was changed at 48-h intervals until confluence was reached. Confluence was assessed by microscopic examination of the cell monolayer and by measurement of electrical resistance across the cell monolayer. Transepithelial resistance was quantitated using chopstick electrodes and an epithelial voltohmmeter (EVOM, World Precision Instruments, New Haven, CT). Values were corrected for background resistance of the filter alone bathed in medium. Cell monolayers were serum deprived for 18 h before the experiments.
Measurement of Cl (in nmol) in an
aliquot of incubation medium; and m is the protein content in the cell
lysate. [Cl
]i (in mM) was calculated as
Cl
Measurement of NKCC1 activity.
NKCC1 activity was measured as bumetanide-sensitive,
basolateral-to-apical, unidirectional flux of 86Rb, a
congener of K, as previously described (13). Cell
monolayers were preincubated for 10 min at 37°C with HPSS or HPSS
supplemented with 10 µM bumetanide or 10 µM rottlerin in a
basolateral bathing solution. Cells were permeabilized at the apical
membrane using 175 U/ml nystatin in an apical cytosolic medium
containing (in mM) 20 Na+, 110 K+, 0.4 Mg2+, 4.2 HCO at concentrations <132 mM.
For concentrations >132 mM, sufficient N-methyl-D-glucamine was added to achieve the
desired concentration. Basolateral [Cl
] was held
constant at 136 mM. To assure that treatment with nystatin altered
Cl
]. Treatment with nystatin did not
significantly alter Vi measurements (Table
1). [Cl
]i
(expressed in mM) was calculated from values for Cl
]i was directly proportional to
[Cl
] in the apical bathing medium, which indicates that
treatment with nystatin allowed movement of Cl
between
the cytosol and extracellular medium.
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Immunoprecipitation, PKC activity assay, and Western blot
analysis of PKC isotypes.
Calu-3 cells were grown on filter inserts before being serum deprived
and preequilibrated with an apical perfusate containing 225 U of
nystatin/ml, 20 mM Na+, 110 mM K+, 0.4 mM
Mg2+, 4.2 mM HCO] as described (see
Measurement of NKCC1 activity). Cells were then stimulated
with methoxamine or, as a control, with vehicle of HPSS. Cells were
rapidly immersed in ice-cold PBS to halt the stimulation and were
harvested in 1 ml of lysis buffer consisting of 100 mM NaCl, 50 mM NaF,
50 mM Tris · HCl, pH 7.55, 1% Nonidet P-40, 0.25% sodium
deoxycholate, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, and the
protease inhibitors as described. Lysates were clarified by
microcentrifugation at 4°C for 20 min at 12,000 g and
incubated with antiserum against a specific PKC isotype as previously
described (13). Immune complexes were recovered using
protein G agarose beads that were prewashed and resuspended in lysis
buffer. Western blot analysis of immunoprecipitated protein was used to
titrate antiserum and select an optimal antiserum concentration
(13). Kinase activity of PKC isotypes was measured by
taking immunoprecipitates to a final volume of 50 µl in assay mixture
{50 mM Tris · HCl, pH 7.5, 10 mM
-mercaptoethanol, 10 mM
MgSO4, 40 µg/ml phosphatidylserine, 0.1 µM phorbol
12-myristate 13-acetate (PMA), 50 µM ATP, 10 µg/ml histone III, 3 µCi of [
-32P]ATP, 1 mM sodium orthovanadate, and
protease inhibitors} and incubating at 30°C for 15 min. The
reaction was terminated by addition of 30 µl of glacial acetic acid.
A 40-µl aliquot was spotted on P-81 phosphocellulose paper, washed,
and counted for radioactivity using Cerenkov counting.
Data analysis.
Data are reported as means ± SE. Cl
Materials. 86Rb (sp act 154 Bq/g of Rb; 4,200 Ci/g of Rb) and an enhanced chemiluminescence kit were purchased from Amersham Life Science, 36Cl (sp act 260 MBq/g of Cl, 7.5 mCi/g of Cl) was purchased from ICN Radiochemical, and [14C]urea (sp act 37 GBq/mmol) was purchased from NEN. Transwell-Clear filter inserts were purchased from Fisher Scientific. Polyclonal anti-PKC isotype-specific antibodies were obtained from Santa Cruz Biotechnology, and recombinant PKC isotypes were from Calbiochem (La Jolla, CA). T4 monoclonal antibody was obtained from the Developmental Studies Hybridoma Bank. Methoxamine-HCl, bumetanide, and nystatin were purchased from Sigma Chemical and rottlerin was from Research Products International (Natick, MA). All other chemicals were reagent grade.
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RESULTS |
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Modulation of Cl-adrenergic stimulation.
To measure changes in Cl
1-adrenergic agonist
methoxamine is presented in Fig. 1. We
also calculated means for cumulative data, which are presented in Table
2. Stimulation of Calu-3 cells with
methoxamine resulted in a time-dependent increase in the ratios of
Cl
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Modulation of Cl
|
Modulation of NKCC1 activity by [Cl]i.
To evaluate the effects of [Cl
]i on cell
function in the absence of cell volume changes, we measured NKCC1
activity as bumetanide-sensitive, 86Rb-unidirectional,
basolateral-to-apical flux in nystatin-permeabilized Calu-3 cell
monolayers grown on filter inserts. The apical perfusion medium was
adjusted to match intracellular Na+, K+, and
Mg2+ concentrations, and the [Cl
] in a
basolateral perfusion medium was held constant at 136 mM. The data of
Fig. 3 demonstrate that NKCC1 activity
was detected when [Cl
]i was as low as 10 mM
and as high as 200 mM in cells that were treated with vehicle of HPSS.
NKCC1 activity levels measured at [Cl
]i
from 10 to 140 mM were not significantly different, which indicates that an electrochemical gradient across the basolateral membrane is not
sufficient for stimulation of NKCC1 function. Surprisingly, at
[Cl
]i of 200 mM, bumetanide-sensitive
K+ flux increased significantly to 281 nmol · mg
protein
1 · 10 min
1
(P < 0.001; ANOVA), which accounts for 35.8% of total
K+ flux. Bumetanide-insensitive K+ flux did not
significantly change with increasing [Cl
]i,
which indicates that elevated K+ flux at 200 mM could be
attributed to NKCC1 activity. A comparison of NKCC1 activity at 200 and
40 mM Cl
]i of 45 mM (Table 2), revealed a
difference in bumetanide-sensitive baseline K+ flux. At 40 mM Cl
1 · 10 min
1 (n = 4) and comprised 9.1% of total
K+ flux. In comparison, baseline NKCC1 activity at 200 mM
Cl
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Modulation of in vivo PKC activity by
[Cl]i.
We have previously shown (11-13) that hormone
stimulation rapidly increases activity of PKC-
and -
and that the
increased activity of PKC-
is necessary for activation of NKCC1. Our
results from this study indicated that methoxamine also increases
Cl
might be sensitive to Cl
]i was varied
in permeabilized cell monolayers using nystatin as described (see
Measurement of NKCC1 activity). Increasing apical [Cl
]i from 10 to 200 mM did not
significantly affect baseline activity of PKC-
in cells that were
treated with vehicle (Fig. 4).
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Subcellular distribution of PKC isotypes in Calu-3 cells.
Our previous studies (11-13) of PKC- distribution
in human tracheal epithelial cells localized PKC-
to both cytosol
and a Triton X-100 soluble particulate fraction. We examined the
subcellular distribution of PKC-
and, because its activity also
increases after
1-adrenergic stimulation
(10), we also investigated the distribution of PKC-
in
Calu-3 cells. The results are illustrated in Fig.
5. At the endogenous
[Cl
]i of 47 mM, 54.5% of PKC-
and
37.6% of PKC-
were localized to a particulate fraction. Decreasing
Cl
and -
levels.
Methoxamine increased particulate PKC-
at 20 mM
Cl
to 70.9%. Similarly, at 47 mM Cl
and -
levels increased
after methoxamine stimulation to 81.4 and 62.1%, respectively. At 200 mM Cl
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DISCUSSION |
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[Cl]i has been linked to regulation of
NKCC1 in a number of cells and tissues including secretory epithelia,
avian erythrocytes, and squid axon (1, 6, 24). This study
of Calu-3 airway epithelial cells demonstrates for the first time that
a Cl
is necessary. Increasing [Cl
]i,
independent of changes in cell volume, correlates with shifts in the
subcellular localization of PKC-
and -
(see Fig. 5) and in the
activity and regulation of PKC-
and NKCC1 by the
1-adrenergic agonist methoxamine (see Figs. 3 and 4).
Titration of [Cl
]i from 10 to 140 mM in
permeabilized Calu-3 cells does not alter the baseline activity of
NKCC1 except at [Cl
]i of 200 mM. At this
high [Cl
]i level, baseline NKCC1 activity
increased significantly. This response differs from other reports,
which describe cotransport as strongly dependent on
[Cl
]i (6, 21). In these other
systems, the dependence on [Cl
]i is
biphasic and shows stimulation with increasing
[Cl
]i from 0 to 40 mM followed by
inhibition. The response of Calu-3 cells cannot be explained by a
change in cell volume (see Table 1) or by an increase in PKC-
activity (see Fig. 4). One explanation could be the sensitivity of
NKCC1-regulated K+ flux to intracellular electrolyte
concentration, or conversely water content, which has been postulated
to reflect the dependence of thermodynamic activity of intracellular
proteins on total protein concentration or macromolecular crowding
(18). Macromolecular crowding decreases the dissociation
rate constants of protein complexes leading to prolonged formation of
complexes (23) such as F actin (14) and
kinases/phosphatases (18) that regulate electrolyte
exchange and cotransport pathways. This could explain, for example, an
increase in rottlerin-sensitive baseline NKCC1 activity of almost
sixfold at a [Cl
]i of 200 mM. Rottlerin
blocks methoxamine-stimulated NKCC1 regardless of
[Cl
]i, which indicates that PKC-
is
required for activation of NKCC1 and that hormonal activation is
additive to the effects of high [Cl
]i alone.
Elevating [Cl]i to 200 mM also enhanced
translocation of PKC-
and -
from the cytosol to the cell
periphery (see Fig. 5). Translocation did not, however, lead to a
significant increase in enzyme activity, nor did it prevent a
methoxamine-induced increase in the activity of PKC-
(see Fig. 4).
These results indicate that Calu-3 cells resemble human tracheal
epithelial cells (13) and a CF/T43 airway epithelial cell
line (12), which exhibit an approximately even
distribution of PKC-
between the cytosol and a particulate fraction
and a predominantly cytosolic localization of PKC-
. With Calu-3
cells, we speculate that macromolecular crowding at high
[Cl
]i promotes protein-protein
interactions, which lead to the association of PKC with a particulate
subcellular fraction with slight or no increase in PKC activity. One
indication of a protein-protein interaction is the
coimmunoprecipitation of PKC-
with NKCC1 and vice versa (see Fig.
6). Hormonal stimulation at varying [Cl
]i
produced distinct effects on PKC-
activity. At
[Cl
]i of <140 mM, methoxamine increased
the PKC association with a particulate fraction and rottlerin-sensitive
NKCC1 activity and, in addition, increased the activity of PKC-
.
However, at [Cl
]i of >140 mM, the response
to methoxamine is additive to the effects of high
[Cl
]i and retains a sensitivity to
rottlerin, which suggests a PKC-
-dependent activation of
membrane-associated NKCC1 or a separate pool of NKCC1. Aspects of this
model have yet to be tested in airway epithelial cells.
The [Cl]i in Calu-3 cells was 47.4 mM, as
measured by radioisotopic equilibrium distribution of 36Cl
(see Table 2). This is comparable to the
[Cl
]i values reported for other secretory
epithelia, including shark rectal gland (49 mM, Ref. 5),
dog tracheal epithelial cells (47.2 mM, Ref. 25), and
submandibular acinar cells (56 mM, Ref. 28). In these
cells, stimulation with cAMP-generating agents increased
transepithelial flux, which resulted in secretion. cAMP treatment of
shark rectal gland decreased [Cl
]i from 49 to ~40 mM (5) and reduced dog tracheal epithelial cell
[Cl
]i from 47.2 to 32.2 mM
(25). However, when activity of Cl
]i
is much less than needed to activate NKCC1. A similar finding by
Robertson and Foskett (22) using a rat salivary acinar
cell preparation was consistent with the conclusion that a decrease in
Cl
1-adrenergic activation of NKCC1 is sensitive to a fall in [Cl
]i, but requires stimulation of
PKC-
activity. The results have important implications for diseases
such as cystic fibrosis in which the apparent secretory capacity of
airway epithelium is defective. Studies by Slotki and colleagues
(27) using CFPAC cells, which are derived from pancreatic
adenocarcinoma of a cystic fibrosis patient, demonstrate that secretory
capacity can be limited by low cotransporter activity. This implies
that genetic approaches to correct cystic fibrosis transmembrane
conductance regulator levels to improve secretory capacity will depend,
for success, on optimal functioning of NKCC1. Understanding
intracellular mechanisms leading to activation of NKCC1 is thus
critical for successful development of new therapeutics to correct
secretion in cystic fibrosis.
From previous studies (9, 10), we predicted
agonist-induced increases in Vi and Cl]i appears to be necessary for recovery
from hyperosmotic stress. In cystic fibrosis and other pulmonary
diseases, an increased osmolarity of a mucociliary fluid layer, due to
lack of sufficient fluid secretion, hypersecretion of proteins or
mucus, bacterial infection, or an enhanced inflammatory response,
might induce alterations in epithelial
[Cl
]i that limit the level of NKCC1
activity with consequences for the ability of epithelial cells to
mount a defense through increased fluid secretion. Successful
therapeutic approaches to restore or stimulate secretory capacity must
take into account any sensitivity toward
Cl
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Calvin Cotton for helpful discussions.
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FOOTNOTES |
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This research was supported by National Institutes of Health Grant HL-58598.
Address for reprint requests and other correspondence: C. M. Liedtke, Pediatric Pulmonology, Case Western Reserve Univ., BRB, Rm. 824, 2109 Adelbert Rd., Cleveland, OH, 44106-4948 (E-mail: cxl7{at}po.cwru.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 14, 2001;10.1152/ajplung.00143.2001
Received 25 April 2001; accepted in final form 11 December 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Breitwieser, GE,
Altamirano AA,
and
Russell JM.
Osmotic regulation of Na+-K+-Cl cotransport in squid giant axon is [Cl
]i dependent.
Am J Physiol Cell Physiol
258:
C749-C753,
1990
2.
Clerici, C,
Couette S,
Loiseau A,
Herman P,
and
Amiel C.
Evidence for Na-K-Cl cotransport in alveolar epithelial cells: effect of phorbol ester and osmotic stress.
J Membr Biol
147:
295-304,
1995[ISI][Medline].
3.
D'Andrea, LC,
Lytle C,
Matthews JB,
Hofman P,
Forbush B, III,
and
Madara JL.
Na/K/2Cl cotransporter protein of intestinal epithelial cells: surface distribution, immunoprecipitation as a protein complex, and surface expression in response to cAMP.
J Biol Chem
271:
28969-28976,
1996
4.
Devor, DC,
Singh AK,
Lambert LC,
DeLuca A,
Frizzell RA,
and
Bridges RJ.
Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells.
J Gen Physiol
113:
743-760,
1999
5.
Greger, R,
Schlatter E,
Wang F,
and
Forrest JN, Jr.
Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias). III. Effects of simulation of secretion by cyclic AMP.
Pflügers Arch
402:
376-384,
1984[ISI][Medline].
6.
Haas, M,
and
McBrayer DG.
Na-K-Cl cotransport in nystatin-treated tracheal cells: regulation by isoproterenol, apical UTP, and [Cl]i.
Am J Physiol Cell Physiol
266:
C1440-C1452,
1994
7.
Haas, M,
McBrayer D,
and
Yankaskas J.
Dual mechanisms for Na-K-Cl cotransport regulation in airway epithelial cells.
Am J Physiol Cell Physiol
264:
C189-C200,
1993
8.
Liedtke, CM.
Bumetanide-sensitive Na+ and Cl uptake in rabbit tracheal epithelial cells is stimulated by neurohormones and hypertonicity.
Am J Physiol Lung Cell Mol Physiol
262:
L621-L627,
1992
9.
Liedtke, CM.
Role of protein kinase C in -adrenergic regulation of NaCl(K) cotransport in human airway epithelial cells.
Am J Physiol Lung Cell Mol Physiol
268:
L414-L423,
1995
10.
Liedtke, CM,
Cody D,
and
Cole TS.
Differential regulation of Cl transport proteins by PKC in Calu-3 cells.
Am J Physiol Lung Cell Mol Physiol
280:
L739-L747,
2001
11.
Liedtke, CM,
and
Cole T.
Antisense oligodeoxynucleotide to PKC- blocks
1-adrenergic activation of Na-K-2Cl cotransport.
Am J Physiol Cell Physiol
273:
C1632-C1640,
1997
12.
Liedtke, CM,
and
Cole T.
PKC signalling in CF/T43 cell line: regulation of NKCC1 by PKC-.
Biochim Biophys Acta
1495:
24-33,
2000[ISI][Medline].
13.
Liedtke, CM,
Cole T,
and
Ikebe M.
Differential activation of PKC- and PKC-
by
1-adrenergic stimulation in human airway epithelial cells.
Am J Physiol Cell Physiol
273:
C937-C943,
1997
14.
Lindner, RA,
and
Ralston GB.
Macromolecular crowding: effects on actin polymerisation.
Biophys Chem
66:
57-66,
1997[ISI][Medline].
15.
Lyttle, C,
Xu JC,
Biemesderfer D,
and
Forbush B, III.
Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies.
Am J Physiol Cell Physiol
269:
C1496-C1505,
1995
16.
Matthews, JB,
Smith JA,
Mun EC,
and
Sicklick JK.
Osmotic regulation of intestinal epithelial Na+-K+-Cl cotransport: role of Cl
and F-actin.
Am J Physiol Cell Physiol
274:
C697-C706,
1998
17.
Miley, HE,
Holden D,
Grint R,
Best L,
and
Brown PD.
Regulatory volume increase in rat pancreatic -cells.
Eur J Physiol
435:
227-230,
1998[ISI][Medline].
18.
Parker, JC,
Dunham PB,
and
Minton AP.
Effects of ionic strength on the regulation of Na/H exchange and K-Cl cotransport in dog red blood cells.
J Gen Physiol
105:
677-699,
1995[Abstract].
19.
Payne, JA,
and
Forbush B, III.
Molecular characterization of the epithelial Na-K-Cl cotransporter isoforms.
Curr Opin Cell Biol
7:
493-503,
1995[ISI][Medline].
20.
Payne, JA,
Xu JC,
Haas M,
Lytle CY,
Ward D,
and
Forbush B.
Primary structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na-K-Cl cotransporter in human colon.
J Biol Chem
270:
17977-17985,
1995
21.
Putney, LK,
Vibat CRT,
and
O'Donnell ME.
Intracellular Cl regulates Na-K-Cl cotransport activity in human trabecular meshwork cells.
Am J Physiol Cell Physiol
277:
C373-C383,
1999
22.
Robertson, MA,
and
Foskett JK.
Na+ transport pathways in secretory acinar cells: membrane cross talk mediated by [Cl]i.
Am J Physiol Cell Physiol
267:
C146-C156,
1994
23.
Rohwer, JM,
Postma PW,
Kholodenko BN,
and
Westerhoff HV.
Implications of macromolecular crowding for signal transduction and metabolite channeling.
Proc Natl Acad Sci USA
95:
10547-10552,
1998
24.
Russell, JM.
Sodium-potassium-chloride cotransport.
Physiol Rev
80:
211-276,
2000
25.
Shorofsky, SR,
Field M,
and
Fozzard HA.
Mechanism of Cl secretion in canine trachea: changes in intracellular chloride activity with secretion.
J Membr Biol
81:
1-8,
1984[ISI][Medline].
26.
Simmons, NL,
and
Tivey DR.
The effect of hyperosmotic challenge upon ion transport in cultured renal epithelial layers (MDCK).
Pflügers Arch
421:
503-509,
1992[ISI][Medline].
27.
Slotki, IN,
Breuer WV,
Greger R,
and
Cabantchik ZI.
Long-term cAMP activation of Na+-K+-Cl cotransporter activity in HT-29 human adenocarcinoma cells.
Am J Physiol Cell Physiol
264:
C857-C865,
1993
28.
Zeng, W,
Lee MG,
and
Muallem S.
Membrane-specific regulation of Cl channels by purinergic receptors in rat submandibular gland acinar and duct cells.
J Biol Chem
272:
32956-32965,
1997