(Received for publication, August 2, 1995; and in revised form, September 22, 1995)
From the
Basolateral Na-K-Cl cotransport activity in primary cultures of
dog tracheal epithelial cells is stimulated by -adrenergic agents,
such as isoproterenol, and by apical UTP, which acts through an apical
P
-purinergic receptor. While at least part of the
stimulatory effect of isoproterenol appears to involve direct
activation of the cotransporter via cAMP-dependent protein kinase,
cotransport stimulation by apical UTP is entirely secondary to apical
Cl
efflux and a resultant decrease in intracellular
[Cl
]
([Cl
]
) and/or cell
shrinkage (Haas, M., and McBrayer, D. G.(1994) Am. J. Physiol. 266, C1440-C1452). In the secretory epithelia of the shark
rectal gland and avian salt gland, Na-K-Cl cotransport activation by
both cAMP-dependent and cAMP-independent secretagogues has been shown
to be accompanied by phosphorylation of the cotransport protein itself
(Lytle, C., and Forbush, B., III(1992) J. Biol. Chem. 267,
25438-25443; Torchia, J., Lytle, C., Pon, D. J., Forbush, B.,
III, and Sen, A. K.(1992) J. Biol. Chem. 267,
25444-25450). In the present study, we immunoprecipitate the
170-kDa Na-K-Cl cotransport protein of dog tracheal epithelial
cells with a monoclonal antibody against the cotransporter of the
intestinal cell line T84. Incubation of confluent primary cultures of
tracheal cells with isoproterenol and apical UTP increases
basolateral-to-apical
Cl
flux 3.4- and
2.6-fold, respectively, and produces similar increases (3.2- and
2.8-fold, respectively) in
P incorporation into the
170-kDa cotransport protein. Decreasing
[Cl
]
(without
concomitant cell shrinkage) by incubating cultures with apical nystatin
and reduced apical [Cl
]
([Cl
]
) likewise
increases both cotransport activity and cotransport protein
phosphorylation. These effects become more pronounced with greater
reductions in [Cl
]
;
after 20 min of incubation with nystatin and 32 mM
[Cl
]
, cotransport
activity and
P incorporation into the cotransport protein
are increased 2.8- and 2.7-fold, respectively, similar to increases
seen with apical UTP. 2-3-fold increases in cotransporter
activity and phosphorylation are also seen in nystatin-treated cells
under hypertonic conditions (50 mM sucrose added apically and
basolaterally). These findings suggest a close correlation between
Na-K-Cl cotransport activity and phosphorylation of the
170-kDa
cotransport protein. The latter is phosphorylated in response to both
reduced [Cl
]
and cell
shrinkage, either or both of which are likely to be involved in
secondary cotransport activation in response to apical UTP.
In secretory epithelia, both cAMP-dependent and cAMP-independent
secretagogues augment ion transport through multiple transport pathways
to produce net salt and fluid secretion; these pathways include apical
Cl channels and basolateral K
channels as well as the basolateral Na-K-Cl cotransporter and
other basolateral salt influx
pathways(1, 2, 3, 4, 5, 6) .
Na-K-Cl cotransport is the primary basolateral influx pathway for
Na
and Cl
in dog tracheal
epithelium(1, 2, 3, 7) , and
cotransport activity is increased both by isoproterenol and cAMP
analogues, as well as by apical triphosphate nucleotides such as UTP,
which binds to an apical P
-purinergic receptor and
subsequently stimulates net salt and fluid secretion in a
cAMP-independent manner(8, 9, 10) . Previous
studies employing primary cultures of dog tracheal epithelial cells
have provided evidence that cAMP-dependent cotransport activation
involves, at least in part, direct cotransport activation via
cAMP-dependent protein kinase. By contrast, cotransport activation in
response to apical UTP is entirely secondary to apical Cl
efflux via activated channels(8, 9) . The
cellular signal for this secondary cotransport activation in dog
tracheal cells is likely to be a decrease in intracellular
[Cl
]
([Cl
]
) and/or in cell
volume; both reduced [Cl
]
and cell shrinkage can produce a level of cotransport
activation similar to that seen with apical UTP(9) .
Furthermore, both reduced [Cl
]
and cell shrinkage are known to activate Na-K-Cl cotransport
in a variety of tissues(11, 12, 13) ,
including several secretory
epithelia(14, 15, 16, 17) . In rat
parotid acini, it was recently shown that reduced
[Cl
]
, and not cell
shrinkage, is required for basolateral cotransport activation in
response to carbachol(4) , though this has not yet been
resolved for other secretory epithelia including those of mammalian
airways.
Studies of Lytle and Forbush (14, 15) in
shark rectal gland epithelium and of Torchia and co-workers (18, 19) in avian salt gland epithelium have
demonstrated phosphorylation of the Na-K-Cl cotransport protein itself
in response to cAMP-dependent and cAMP-independent secretagogues.
Increased phosphorylation of the cotransport protein was also observed
following incubation of intact shark rectal gland tubules in hypertonic
and in Cl-free media to reduce cell volume and
[Cl
]
, respectively (14, 15) . Furthermore, in the shark rectal gland it
was shown that increasing degrees of hypertonic cell shrinkage produce
increasing and approximately proportional stimulation of cotransport
protein phosphorylation and [
H]benzmetanide
binding (14) , the latter having previously been shown to
correlate closely with Na-K-Cl cotransport
activity(16, 20) . Thus, in these secretory epithelia
it appears that Na-K-Cl cotransport activation involves phosphorylation
of the cotransport protein itself. These results are consistent with
findings in a variety of epithelial and nonepithelial tissues
demonstrating that stimulation of Na-K-Cl cotransport by hormones,
second messengers, and cell shrinkage can be blocked by protein kinase
inhibitors and that protein phosphatase inhibitors augment cotransport
activity both in the presence and absence of cotransport
stimuli(14, 21, 22, 23, 24, 25) .
In this study, we examine the phosphorylation of the Na-K-Cl
cotransport protein of dog tracheal epithelial cells in response to
isoproterenol, apical UTP, cell shrinkage, and reduced
[Cl]
. Confluent
primary cell cultures incubated under each of these conditions known to
stimulate Na-K-Cl cotransport exhibit increased phosphorylation of the
170-kDa Na-K-Cl cotransport protein, which is subsequently
isolated by immunoprecipitation using a monoclonal antibody directed
against the Na-K-Cl cotransport protein of T84
cells(26, 27) . Of particular note, the degrees of
cotransporter activation and phosphorylation in response to graded
decreases in [Cl
]
(without concomitant cell shrinkage) are found to be closely
correlated, suggesting the possibility of a
Cl
-sensitive protein kinase that phosphorylates, and
thus activates, the Na-K-Cl cotransporter. A preliminary report has
been presented in abstract form (28) .
As in previous studies(8, 9, 29) , we measured the potential difference and resistance across each Transwell culture daily following optical detection of confluence, using an epithelial volt-ohmmeter with ``chopstick'' electrodes (World Precision Instruments, Sarasota, FL). Maximal potential difference and resistance values were attained 2 days after optical detection of confluence. All experiments were thus performed on day 2 following detection of confluence (day 5-6 in culture).
In experiments where
[Cl]
or medium tonicity was
varied, nystatin (Sigma) was added to the apical media to increase
apical membrane Cl
permeability(9) . As in
our previous studies(9) , cultures were preincubated for 10 min
with apical nystatin (final concentration, 350 units/ml, added from a
200,000-unit/ml stock solution in Me
SO), and nystatin was
present in the apical medium throughout the flux period. Apical media
used in these experiments were synthetic solutions containing 124
mM (NaCl + NaNO
), 4 mM KCl, 10
mM sucrose, 10 mM glucose, 1.5 mM CaCl
, 0.5 mM MgCl
, 14 mM NaHCO
, 1 mM Na
HPO
,
and 10 mM Na-HEPES (Research Organics, Cleveland, OH), pH 7.4,
at 37 °C. The basolateral medium in these experiments was
F12-4x + 1 mM CaCl
. During the initial
10 min of the flux period, the apical medium was isotonic and contained
124 mM Cl
. For experiments in which
[Cl
]
was varied, following
removal of the 10-min sample the apical medium was changed to one
containing 66, 49, or 32 mM [Cl
]
(equimolar substitution with NO
), and this apical medium
was then used for the remainder of the flux period. Where tonicity was
varied, after removal of the 10-min sample the Transwell was
transferred to a well containing F12-4x + 1 mM CaCl
+ 50 mM sucrose, with the same
concentration of
Cl
as present during
the initial 10 min. The apical medium then used for the remainder of
the flux period was the 124 mM Cl
synthetic
medium described above, to which 50 mM sucrose was added.
Flux data are calculated as ratios of stimulated flux rate/control
flux rate. The control rate is determined as the average of the counts
in the four samples of apical medium collected during the first 10 min
of each incubation, over which time flux rates remain
linear(9) . For incubations to which isoproterenol or UTP is
added, the incubation proceeds for 10 additional minutes after addition
of agonist, and the stimulated flux rate is determined as the average
of the counts in the final two samples of the incubation (i.e. 17.5- and 20-min samples). For incubations in which
[Cl]
or medium tonicity is
varied, the incubation proceeds for 20 min after the medium change, and
the stimulated flux rate is again taken to be the average of the counts
in the final two samples of the incubation (i.e. 27.5- and
30-min samples).
Following the incubations, the cultures were
washed rapidly by immerson in ice-cold, isotonic HEPES-buffered saline
(pH 7.4). The filters were then cut from the Transwell supports and
added to microcentrifuge tubes containing 0.4 ml of ice-cold lysing
buffer containing 140 mM NaCl, 20 mM Na-HEPES (pH 7.4
at 2 °C), and the following mixture of phosphatase and protease
inhibitors: 5 mM Na-EDTA, 5 mM NaF, 5 mM
Na pyrophosphate, 0.1 mM Na
orthovanadate, 300 µM phenylmethylsulfonyl fluoride,
100 µMN-tosyl-L-phenylalanine
chloromethyl ketone, 1.5 µM pepstatin, and 1.5 µM leupeptin (Sigma or Boehringer Mannheim). To ensure an adequate
P signal, 4-6 similarly incubated cultures comprised
a single sample, and 2-3 filters from a given sample were added
to a single microcentrifuge tube. The contents of each tube were then
sonicated for 20 s with a probe sonifier (model 50 Sonic Dismembrator,
Fisher, setting 2) with the tube immersed in ice. Each tube was then
tightly capped and vortexed vigorously. The contents of tubes
containing filters from the same sample were then combined in 30-ml
Corex tubes, 3 ml of additional lysing buffer was added to each tube,
and the contents of each tube were sonicated for an additional 15 s
with the tube immersed in ice. The contents of each tube were then
transferred to 50-ml centrifuge tubes to which 20 ml of ice-cold lysing
buffer was added; the tubes were then vortexed, and the filters were
removed with forceps. The samples were then centrifuged successively
for 5 min at 200
g and for 15 min at 2,500
g, both at 0 °C. The pellets were discarded, and the
supernatant was centrifuged for 45 min at 50,000
g and
0 °C. The pellet from this latter centrifugation, containing crude
plasma membranes, was then suspended in lysing buffer containing 1% SDS
(Sigma). These SDS-containing samples were then vortexed and incubated
on ice for 10 min. To each sample, an equal volume of lysing buffer
containing 9% Triton X-100 (Pierce) and a second, equal volume of
lysing buffer without added detergent were then added. This mixture was
incubated on ice for 50 min and then centrifuged in a microcentrifuge
to remove insoluble debris. The supernatant was then mixed with a mouse
monoclonal antibody developed by Lytle et al.(26) against a
38-kDa peptide encoding the
carboxyl-terminal 310 residues of the Na-K-Cl cotransporter of the T84
intestinal epithelial cell line (antibody T4, (26) and (27) ) (35 µg of T4 added per Transwell in the sample).
Antibody T4, which preferentially reacts with the SDS-denatured form of
the Na-K-Cl cotransport protein(26, 27) , was purified
from hybridoma supernatants by affinity chromatography using protein
G-Sepharose (Sigma) prior to use in these experiments. Immediately
following T4 addition, sufficient lysing buffer was then added to each
sample to reduce its concentration of Triton X-100 to 2%, and the
samples were incubated overnight at 4 °C on a rotating shaker. The
next morning, protein G-Sepharose (1 µl of packed beads per µg
of T4) was added to each sample, and the samples were incubated an
additional 2 h at 4 °C on the rotating shaker. The beads were then
washed five times with ice-cold lysing buffer containing 1% Triton
X-100, once with HEPES-buffered saline without added detergent, mixed
with SDS-containing Laemmli sample buffer(31) , and incubated
at 50 °C for 1 h. The samples were then centrifuged; the
supernatants as well as prestained protein standards (molecular mass
range 27-180 kDa, Sigma) were loaded onto a 6% Laemmli
SDS-polyacrylamide gel(31) .
The proteins separated on the
gel were then transferred electrophoretically to polyvinylidene
fluoride (PVDF) membranes (Immobilon-P, Millipore, Bedford, MA), which
were subjected to autoradiography to determine P
incorporation. Following this, the PVDF transfers were subjected to
Western blotting with T4 (1.65 µg/ml) to determine the amount of
170 kDa protein in each sample. Western blotting was done using
the ECL method and reagents (Amersham, Arlington Heights, IL), except
for the peroxidase-conjugated secondary antibody (anti-mouse IgG) which
was from Sigma. Films from autoradiography and Western blots were then
analyzed by densitometry to determine the relative amounts
(stimulated/control) of
P and protein present in the
170 kDa region; from these data ratios (stimulated/control) of
P incorporated/unit protein were calculated for each
sample. Densitometry peaks were corrected for background which was
determined from regions of the gel lane in which there were no distinct
band(s) identified; this was typically the top of the gel and/or the
region between the 36.5 kDa standard and the tracking dye. For studies
with isoproterenol and apical UTP, control levels of
P
incorporation were determined in the absence of added agonist; for
studies of effects of reduced [Cl
]
and cell shrinkage control levels were determined from isotonic
incubations with 124 mM apical
[Cl
]. At least one appropriate control
incubation was included in all experiments.
The Western blot in Fig. 1shows that the monoclonal
antibody T4, raised against the carboxyl-terminal domain of the T84
cell Na-K-Cl cotransport protein(26, 27) , reacts
primarily with one protein from SDS-solubilized dog tracheal epithelial
cell membranes of molecular mass 170 kDa (lane 1). Our
immunoprecipitation procedure using T4 (see ``Materials and
Methods'') effectively removes all of this
170-kDa protein
from a supernatant containing SDS-solubilized dog tracheal epithelial
membrane proteins (lane 2). The immunoprecipitated material (lane 3) contains primarily the
170-kDa protein, though a
45-kDa band is also noted on this and most Western blots of T4
immunoprecipitates. The molecular mass of the major immunoprecipitated
protein is in good agreement with that of a single peak of specific
labeling identified by photolabeling of dog tracheal epithelial cells
with the photosensitive bumetanide analogue,
[
H]BSTBA (Fig. 2). We have previously used
[
H]BSTBA photolabeling to identify Na-K-Cl
cotransport proteins of other tissues, including dog and mouse kidney,
duck red blood cells, and shark rectal gland (32, 33, 34) . In each case, the molecular
mass of the protein identified by [
H]BSTBA
labeling was subsequently found to be in excellent agreement with that
determined by Western blotting using anti-cotransporter
antibodies(14, 34) . (
)Thus, the
170-kDa protein immunoprecipitated with T4 appears to represent
the Na-K-Cl cotransporter of dog tracheal epithelial cells.
Figure 1:
Immunoprecipitation of
dog tracheal epithelial Na-K-Cl cotransport protein with antibody T4.
Crude plasma membranes were isolated from four Transwell cultures of
dog tracheal cells by sonication followed by centrifugation. The
membranes were solubilized in 1% SDS and immunoprecipitated with T4 as
described under ``Materials and Methods.'' Shown are Western
blots using antibody T4; lane 1 contains a sample of
solubilized membranes (12.5% of the total sample) before
immunoprecipitation; lane 2 contains an equivalent-sized
sample of the solubilized membranes after incubation with T4 and
protein G-Sepharose; lane 3 contains 50% of the total
immunoprecipitated material. Locations of molecular weight markers are
indicated (TD, tracking dye). The 170-kDa Na-K-Cl
cotransport protein is completely immunoprecipitated using T4. We do
not observe a band corresponding to antibody T4 on this blot of the
immunoprecipitated material (lane 3), which employed a
secondary antibody (peroxidase-conjugated anti-mouse IgG) from Sigma.
However, we do observe a broad band in the
65-kDa region on
similar Western blots of immunoprecipitates employing a secondary
antibody from Amersham, in good agreement with the observed mobility of
T4 heavy chain on Coomassie Blue-stained SDS-polyacrylamide
gels.
Figure 2:
Photoaffinity labeling of primary cultures
of dog tracheal epithelial cells with [H]BSTBA.
Transwell cultures (six per sample) were incubated with 0.3
µM [
H]BSTBA (basolateral), with or
without 20 µM unlabeled bumetanide. The cultures were then
washed and sonicated, and after removal of the filters the remaining
suspension was photolyzed as described under ``Materials and
Methods.'' Plasma membranes were then isolated, solubilized with
SDS, and run on a 7% Laemmli (31) SDS-polyacrylamide gel. The
gel was then stained and cut into 4-mm slices, which were digested with
30% H
O
and counted. Data shown are counts/min
in each slice, minus background of 40 counts/min. Locations of
molecular weight markers on the gel are indicated (TD,
tracking dye). Representative data are from one of three separate
experiments.
Fig. 3shows that when primary cultures of dog tracheal
epithelial cells are incubated with isoproterenol (lane 2) or
apical UTP (lane 3), phosphorylation of the 170-kDa
Na-K-Cl cotransport protein is stimulated. The upper portion of Fig. 3shows the autoradiogram, while the lower portion depicts
the
170-kDa region of a Western blot performed on the same
polyvinylidene difluoride transfer. In four separate experiments, we
find that isoproterenol increases
P incorporation into
this protein band by an average of 3.2-fold and that UTP increases such
phosphorylation by an average of 2.8-fold (Table 1). The degree
of stimulation of cotransport protein phosphorylation in response to
each of these secretagogues correlates well with the stimulation of
basolateral-to-apical
Cl
flux by each
compound, which over a large number of experiments averages 3.4-fold
for isoproterenol and 2.6-fold for apical UTP (Table 1). In Fig. 3we also note a second band in the
45-kDa region, the
phosphorylation of which is increased in response to isoproterenol and
UTP. As noted above (lane 3 of Fig. 1), we also observe
this band on most Western blots of membrane protein immunoprecipitated
using T4. These findings suggest that the
45-kDa band may
represent a proteolytic fragment of the
170-kDa cotransport
protein, which contains a regulatory phosphorylation site, although we
have not studied this further.
Figure 3:
Phosphorylation of the dog tracheal
epithelial Na-K-Cl cotransport protein in response to secretagogues. P-Loaded Transwell cultures (four per sample) were
incubated for 10 min without added agonist (lane 1), with
isoproterenol (5 µM, basolateral, lane 2), or
with apical UTP (10 µM, lane 3). Plasma membranes
were then isolated from each sample by sonication followed by
centrifugation before SDS solubilization of proteins. The
SDS-solubilized membrane proteins were then subjected to
immunoprecipitation using antibody T4 as described under
``Materials and Methods.'' Immunoprecipitated material was
run on a SDS-polyacrylamide gel, and proteins were transferred to
Immobilon for autoradiography and subsequent Western blotting with
antibody T4. The upper portion of the figure shows the
autoradiogram; the lower portion shows the
170-kDa region
of the Western blot subsequently performed on the same transfer. In
this experiment, relative amounts of
P incorporation (per
unit of protein) into the
170-kDa protein are as follows: lane
1, 1.0; lane 2, 2.3; lane 3,
3.2.
Fig. 4shows that
phosphorylation of the 170-kDa Na-K-Cl cotransport protein is also
stimulated by reduced [Cl
]
(lane 2) and by hypertonic cell shrinkage (lane
3) in the absence of secretagogues. In this experiment,
[Cl
]
was reduced by incubating
the cells with apical nystatin and an apical medium containing reduced
(32 mM) [Cl
]. Nitrate substitutes
for Cl
to maintain isotonicity. We have previously
shown (9) that this substitution does not produce cell
shrinkage; thus, reducing [Cl
]
in the absence of concomitant cell shrinkage or hormonal
stimulation stimulates cotransport protein phosphorylation. In eight
separate experiments, we found that reducing apical
[Cl
] to 32 mM in the presence of
apical nystatin stimulated
P incorporation into the
170-kDa cotransport protein by an average of 2.7-fold (Table 1) compared with the level of incorporation seen with 124
mM apical [Cl
] under otherwise
identical conditions. We have done a large number of similar
experiments (including those presented in (9) ) determining the
effect of reducing [Cl
]
on
basolateral-to-apical
Cl
flux. In the
presence of apical nystatin, this flux is limited by Cl
transport across the basolateral membrane and is
90%
bumetanide-sensitive(9) , indicating that it represents
primarily Na-K-Cl cotransport. After a similar period of exposure to
apical nystatin and 32 mM apical
[Cl
] as used for the experiment in Fig. 4(lane 2), we find an average stimulation of
Cl
flux of 2.8-fold, similar to the
2.7-fold increase seen in cotransport protein phosphorylation. Addition
of 50 mM sucrose to both apical and basolateral media (with
124 mM apical [Cl
] in the presence
of apical nystatin), which we find to decrease initial cell water
content by
20%(9) , also stimulates cotransport protein
phosphorylation by an average of 3-fold over four experiments (Table 1, Fig. 4). This degree of stimulation is similar
to the degree of
Cl
flux stimulation
seen in response to hypertonic shrinkage under similar conditions
(2.4-fold; Table 1).
Figure 4:
Phosphorylation of the dog tracheal
epithelial Na-K-Cl cotransport protein in response to reduced
[Cl]
and hypertonic
cell shrinkage in the absence of secretagogues.
P-Loaded
Transwell cultures (four per sample) were incubated for 20 min with 350
units/ml apical nystatin and 124 mM apical
[Cl
]. Following this, the cells were
incubated an additional 20 min in the continued presence of apical
nystatin and 124 mM apical [Cl
]
(control, lane 1), 32 mM apical
[Cl
] (nitrate substitution, lane
2) or 124 mM apical [Cl
] plus
50 mM sucrose apically and basolaterally (lane 3).
Plasma membranes were then isolated from each sample by sonication
followed by centrifugation before SDS solubilization of proteins. The
SDS-solubilized membrane proteins were then subjected to
immunoprecipitation using antibody T4 as described under
``Materials and Methods.'' Immunoprecipitated material was
run on a SDS-polyacrylamide gel, and proteins were transferred to
Immobilon for autoradiography and subsequent Western blotting with
antibody T4. The upper portion of the figure shows the
autoradiogram; the lower portion shows the
170-kDa region
of the Western blot subsequently performed on the same transfer. In
this experiment, relative amounts of
P incorporation (per
unit protein) into the
170-kDa protein are as follows: lane
1, 1.0; lane 2, 3.7; lane 3,
5.2.
The dependence of cotransport protein
phosphorylation on the degree of [Cl]
reduction is examined in Fig. 5. When cells are exposed
for 20 min to apical nystatin and 66 mM apical
[Cl
], a relatively modest increase in
phosphorylation of the
170-kDa protein is observed compared with
the level seen in cells incubated with nystatin and 124 mM apical [Cl
] (lanes 1 and 2 of Fig. 5; also see Table 1). Progressively
greater increases in phosphorylation are seen when apical
[Cl
] is further reduced to 49 mM (lane 3 of Fig. 5) and 32 mM (lane
4). Table 1again shows a strong correlation between
cotransport protein phosphorylation and basolateral-to-apical
Cl
flux stimulated by different degrees
of [Cl
]
reduction.
Figure 5:
Phosphorylation of the dog tracheal
epithelial Na-K-Cl cotransport protein in response to increasing
degrees of reduced [Cl]
in the absence of secretagogues.
P-Loaded
Transwell cultures (six per sample) were incubated for 20 min with 350
units/ml apical nystatin and 124 mM apical
[Cl
], followed by an additional 20 min of
incubation in the continued presence of apical nystatin and 124 mM apical [Cl
] (control, lane
1), 66 mM apical [Cl
] (lane 2), 49 mM apical
[Cl
] (lane 3), or 32 mM apical [Cl
] (lane 4). Nitrate
substituted isosmotically for Cl
; under these
conditions, apical [Cl
] can be reduced from
124 to 32 mM without concurrent cell shrinkage (9) .
After the incubations, plasma membranes were isolated from each sample
by sonication followed by centrifugation before SDS solubilization of
proteins. The SDS-solubilized membrane proteins were then subjected to
immunoprecipitation using antibody T4 as described under
``Materials and Methods.'' Immunoprecipitated material was
run on a SDS-polyacrylamide gel, and proteins were transferred to
Immobilon for autoradiography and subsequent Western blotting with
antibody T4. The upper portion of the figure shows the
autoradiogram; the lower portion shows the
170-kDa region
of the Western blot subsequently performed on the same transfer. In
this experiment, relative amounts of
P incorporation (per
unit of protein) into the
170-kDa protein are as follows: lane
1, 1.0; lane 2, 1.4; lane 3, 2.3; lane
4, 3.0.
The results of this study demonstrate that the stimulation of
basolateral Na-K-Cl cotransport in dog tracheal epithelial cells by
cAMP-dependent and cAMP-independent secretagogues, as well as by
hypertonic cell shrinkage, is accompanied by an increase in
phosphorylation of the Na-K-Cl cotransport protein itself. Furthermore,
the fractional increase in phosphorylation of the 170-kDa
cotransport protein seen in response to each of these cotransport
stimuli is similar to the fractional increase in basolateral-to-apical
Cl
flux promoted by each stimulus. These
results are consistent with the hypothesis that activation of Na-K-Cl
cotransport is achieved primarily through phosphorylation of the
cotransport protein and are similar to results obtained by Lytle and
Forbush (14, 15) in intact tubules from shark rectal
gland.
We also find that reducing
[Cl]
, in the absence of
secretagogue and without concomitant cell shrinkage, increases
phosphorylation of the
170-kDa Na-K-Cl cotransport protein.
Furthermore, an excellent correlation is observed between this
phosphorylation and the stimulation of cotransport activity in response
to graded decreases in [Cl
]
.
Thus, it appears that dog tracheal epithelial cells may contain a
protein kinase and/or phosphatase that is sensitive to the level of
[Cl
]
, with phosphorylation and
activation of the Na-K-Cl cotransport protein resulting when
[Cl
]
becomes sufficiently
reduced. Alternatively, levels of
[Cl
]
may somehow modulate the
activity of other protein kinase(s) that phosphorylate the
cotransporter, such as the putative volume-sensitive ``V
kinase'' activated in response to cell
shrinkage(14, 22, 23) . Treharne et al.(35) recently described two protein kinases in human nasal
epithelial plasma membranes that show peak activity at 40-50
mM [Cl
] and progressive, marked
inhibition when [Cl
] is raised in the range
from 50 to 150 mM. However, the only substrates of these
kinases identified were proteins of 37 and 45 kDa. Furthermore, one of
these kinases was found to be inhibited by nitrate as well as by
Cl
and was also inhibited by DIDS, which we find to
have no significant effect on Na-K-Cl cotransport or its activation in
dog tracheal epithelial cells or other cells (36) . (
)
The stimulatory effect of reducing
[Cl]
on Na-K-Cl cotransport
activation appears to be primarily a regulatory effect, as opposed to a
thermodynamic one. We previously noted that changes in the
thermodynamic driving force for basolateral cotransport produced by
varying apical [Na
] and
[K
] in the presence of apical nystatin had
only small effects on basolateral-to-apical
Cl
fluxes in dog tracheal cell cultures(9) . Furthermore,
reducing [Cl
]
augments
saturable [
H]bumetanide and
[
H]benzmetanide binding in dog tracheal and shark
rectal gland epithelial cells, respectively(9, 15) ;
such binding reflects the activation state of the cotransporter rather
than ion gradients(37) . While we do not know the level of
[Cl
]
present at each apical
[Cl
] with nystatin present,
[Cl
]
in the presence of 124
mM apical [Cl
] is most likely
close to, but not greater than, [Cl
]
in resting cells. In five separate experiments, we found the cell
water content (9) of cultures incubated for 40 min with apical
nystatin, and the 124 mM [Cl
]
apical medium was slightly (mean of 7%) but not significantly lower
than that of cultures incubated under identical conditions without
nystatin. The presence of intracellular anions that do not permeate
nystatin pores thus appears to maintain
[Cl
]
in nystatin-treated cells
well below the level of apical
[Cl
](38) . Notably, reducing apical
[Cl
] to 66 mM appears to markedly
reduce [Cl
]
; cultures exposed
for 40 min to an apical medium with nystatin, 66 mM [Cl
], and gluconate (which, unlike
nitrate, does not permeate nystatin pores) as the substitute anion
undergo a
30% loss of cell water(9) .
Na-K-Cl
cotransport activation in response to apical UTP is entirely secondary
to apical Cl efflux; inhibition of such efflux blocks
cotransport activation by UTP(8, 9) . The cellular
signal for this secondary cotransport activation is not yet known,
though two prime candidates would appear to be cell shrinkage and
reduced [Cl
]
, both of which
occur during secretagogue stimulation in dog tracheal and other
secretory epithelia (3, 4, 39) . In rat
parotid acini, Robertson and Foskett (4) recently demonstrated
that reduced [Cl
]
but not cell
shrinkage is required for carbachol stimulation of basolateral
Na
entry, which in this tissue is mediated both by
Na-K-Cl cotransport and by Na/H exchange. In shark rectal gland
tubules, activation and phosphorylation of the Na-K-Cl cotransporter by
secretagogues can be mimicked by a variety of maneuvers that decrease
[Cl
]
(15) . Reduced
[Cl
]
is also necessary for
agonist activation of Na
influx via
a nonselective cation channel in rat fetal distal lung epithelium (40) and for antidiuretic hormone and cAMP activation of
nonselective cation channels in the distal renal tubule cell line
A6(41) . Our findings in dog tracheal epithelial cells are also
consistent with reduced [Cl
]
as
an intracellular signal for secondary activation of Na-K-Cl
cotransport. The degrees of stimulation of both basolateral-to-apical
Cl
flux and of cotransport protein
phosphorylation seen when [Cl
]
is reduced by incubation with apical nystatin and 32 mM apical [Cl
] are approximately
equivalent to those produced by a dose of UTP (10 µM) that
maximally stimulates transepithelial
Cl
transport and basolateral [
H]bumetanide
binding in primary cultures of tracheal epithelial
cells(8, 9) . In addition, we observe a very strong
correlation between cotransporter activation and phosphorylation in
response to graded decreases in
[Cl
]
. However, our findings
also cannot exlude cell shrinkage as either a primary or adjunct signal
for secondary cotransport activation in dog tracheal cells. A degree of
hypertonicity that produces an initial decline in cell water content of
20% (9) was found to stimulate both cotransporter
activation and phosphorylation to approximately the same extent as that
observed with 10 µM UTP. Like reduced
[Cl
]
, cell shrinkage appears to
stimulate Na-K-Cl cotransport via activation of a protein kinase
(and/or inhibition of a protein phosphatase). This appears not only to
be the case in epithelial cells from dog trachea and shark rectal gland (14, 15) in which shrinkage has been shown to cause
phosphorylation of the cotransporter itself, but also in several other
cell types in which cotransport activation by cell shrinkage has been
found to be blocked by protein kinase
inhibitors(22, 23) . The identity of the putative
``V kinase'' activated by cell shrinkage is also not known,
though it does not appear to be cAMP-dependent protein kinase or
protein kinase C(14, 22, 23) .
Studies aimed at the characterization and possible
differentiation of putative [Cl
]-sensitive
and volume-sensitive kinase(s) responsible for Na-K-Cl cotransport
activation in tracheal epithelial cells are presently being initiated.