Department of Biochemistry, The August Krogh Institute, University of Copenhagen, DK-2100 Copenhagen, Denmark
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ABSTRACT |
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To identify protein kinases (PK) and phosphatases (PP) involved
in regulation of the
Na+-K+-2Cl
cotransporter in Ehrlich cells, the effect of various PK and PP
inhibitors was examined. The PP-1, PP-2A, and PP-3 inhibitor calyculin
A (Cal-A) was a potent activator of
Na+-K+-2Cl
cotransport (EC50 = 35 nM).
Activation by Cal-A was rapid (<1 min) but transient. Inactivation is
probably due to a 10% cell swelling and/or the concurrent
increase in intracellular
Cl
concentration. Cell
shrinkage also activates the
Na+-K+-2Cl
cotransport system. Combining cell shrinkage with Cal-A treatment prolonged the cotransport activation compared with stimulation with
Cal-A alone, suggesting PK stimulation by cell shrinkage. Shrinkage-induced cotransport activation was pH and
Ca2+/calmodulin dependent.
Inhibition of myosin light chain kinase by ML-7 and ML-9 or of PKA by
H-89 and KT-5720 inhibited cotransport activity induced by Cal-A and by
cell shrinkage, with IC50 values similar to reported inhibition constants of the respective kinases in
vitro. Cell shrinkage increased the ML-7-sensitive cotransport activity, whereas the H-89-sensitive activity was unchanged, suggesting that myosin light chain kinase is a modulator of the
Na+-K+-2Cl
cotransport activity during regulatory volume increase.
dephosphorylation; phosphorylation; bumetanide; myosin light chain kinase; protein kinase A; volume regulation
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INTRODUCTION |
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THE ELECTRONEUTRAL
Na+-K+-2Cl
cotransporter consists of at least two isoforms (reviewed in Refs. 7,
10, and 26).
Na+-K+-2Cl
cotransport plays an important role in transepithelial transport of
salt and water, and in symmetrical cells the cotransporter maintains
cellular Cl
concentration
above electrochemical equilibrium and restores cell volume after cell
shrinkage (7, 10). Activation of
Na+-K+-2Cl
cotransport is one of the first mitogenic signals (1), providing another important function of the cotransporter in symmetrical cells.
A variety of signal transduction pathways appear to be involved in
regulation of
Na+-K+-2Cl
cotransport (7, 10). Activation of cotransport by some agonists has
been ascribed to the concomitant cell shrinkage or to the reduction in
intracellular Cl
concentration
([Cl
]i)
(10, 28), and shrinkage-induced activation of the
Na+-K+-2Cl
cotransporter has been suggested to depend on
[Cl
]i
(10). Agonist-induced activation and cell shrinkage-induced activation
of the cotransporter involve several protein kinases (27). The protein
kinases involved in phosphorylation of the Na+-K+-2Cl
cotransport protein after cell shrinkage (8, 17, 24) remain unidentified.
Dephosphorylation of the
Na+-K+-2Cl
cotransporter in avian erythrocytes is due to Ser/Thr protein
phosphatase type 1 (PP-1) and/or type 2A (PP-2A), as estimated
by the use of the protein phosphatase inhibitors okadaic acid (27) and
calyculin A (Cal-A) (25). Cal-A equipotently inhibits PP-1, PP-2A, and
PP-3 (11, 13). Cal-A activates
Na+-K+-2Cl
cotransport, probably a consequence of increased net phosphorylation of
the cotransporter (24), suggesting that
Na+-K+-2Cl
cotransport is regulated by Ser/Thr kinases.
In aortic endothelial cells a 19-kDa component of the cytoskeleton,
identified as myosin light chain (MLC), is phosphorylated in response
to cell shrinkage (17); the phosphorylation is inhibited by the MLC
kinase (MLCK) inhibitor ML-7
[1-(5-iodonaphthalene-1-sulfonyl)-1-hexahydro-1,4-diazepine], which at micromolar concentrations also inhibits shrinkage-activated Na+-K+-2Cl
cotransport. However, ML-7 is unable to block shrinkage-induced phosphorylation of the
Na+-K+-2Cl
cotransporter in endothelial cells, suggesting that MLCK exerts its
effect indirectly (17).
In Ehrlich cells the
Na+-K+-2Cl
cotransporter mediates regulatory volume increase (RVI) (10) via a
Ca2+/calmodulin (CaM)- and protein
kinase C (PKC)-dependent mechanism (14). Inhibition of PKC, however,
reduced cotransport activity only 20% during RVI (19), suggesting the
involvement of other protein kinases.
We present pharmacological data suggesting continued protein
phosphorylation/dephosphorylation of Ser/Thr residues in the Na+-K+-2Cl
cotransporter (or a regulatory protein) at steady state. We also show
that MLCK and protein kinase A (PKA), directly or indirectly, participate in the isotonic steady-state phosphorylation of the Na+-K+-2Cl
cotransporter and that MLCK appears to modulate the
Na+-K+-2Cl
cotransport activity during RVI.
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MATERIALS AND METHODS |
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Cells
Ehrlich ascites tumor cells (hyperdiploid strain) were maintained and harvested as described previously (14). The cytocrit was adjusted to 4 or 8%, and cells were kept at 37°C.Incubation Media
Isotonic standard medium A was composed of (in mM) 150 Na+, 150 ClUnidirectional K+ Influx
Na+-K+-2ClBefore K+ influx measurements, cells (8% cytocrit) were preincubated in standard isotonic medium with one or a combination of protein phosphatase and kinase inhibitors before addition of experimental medium containing 86Rb+. To measure initial rates of K+ uptake in experiments examining the duration of the cotransport activity, 86Rb+ was added at different times after cellular stimulation (1, 5, 10, and 14 min). In the remaining experiments, 86Rb+ was present in the experimental medium.
Initial rates of K+ uptake were calculated by linear regression from four samples taken 0.5-3 min after addition of the isotope (14). Correlation coefficients between cellular counts per minute and time were >0.99.
Na+-K+-2Cl
cotransport activity during RVI is subject to considerable variation.
We discarded results obtained from Ehrlich cells with a
shrinkage-induced cotransport activity of <10
µmol · g dry
wt
1 · min
1,
since, because of the low stimulation of
Na+-K+-2Cl
cotransport compared with isotonic controls (4 ± 0.6 µmol · g dry
wt
1 · min
1,
n = 18), the results on effects of
protein kinase and phosphatase inhibitors would not be reliable.
Statistical significance was evaluated using Student's paired or independent t-test. Values are means ± SE.
Measurements of Cell Volume
Cell volume was measured using a Coulter counter (15).Reagents
Reagents were of analytic grade; they were purchased from Sigma Chemical (St. Louis, MO) with these exceptions: 86RbCl was from Risø (Roskilde, Denmark), pimozide was a gift from Lundbeck (Copenhagen, Denmark), bumetanide was a gift from Leo Pharmaceuticals (Ballerup, Denmark), BAPTA-AM was from Molecular Probes (Eugene, OR), H-89, KT-5720, ML-7, ML-9, and staurosporine were from Calbiochem (Bad Soden, Germany), and KN-62, KN-04, Cal-A, deltamethrin, permethrin, and chelerythrine were from Alamone Labs (Jerusalem, Israel).Stock solutions of chelerythrine (125 µM), 8-bromo-cAMP (5 mM),
dibutyryl cAMP (100 mM), 8-bromo-cGMP (5 mM), and bradykinin (1 mM)
were prepared in water. Pimozide (10 mM), bumetanide (10 mM),
deltamethrin (100 µM), permethrin (500 µM), ML-7 (2 mM), and Cal-A
(20 µM) were prepared in 96% ethanol. BAPTA-AM (10 mM), H-89
(1-2 mM), KN-62 (10 mM), KN-04 (10 mM), KT-5720 (200 µM), ML-9
(2 mM), and staurosporine (1-2 mM) were dissolved in desiccated DMSO. Stock solutions were stored at 20°C, except H-89,
KN-04, KN-62, ML-7, and staurosporine, which were stored at 4°C.
Cell suspensions received a maximum of 0.7% ethanol or 0.1%
DMSO-0.3% ethanol (vol/vol). Controls received an appropriate volume
of carrier(s).
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RESULTS |
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Transient Activation of the
Na+-K+-2Cl
Cotransporter
Maximal activation of the
Na+-K+-2Cl
cotransporter appeared within the 1st min after addition
of bradykinin, and the activity returned to the resting level ~5 min
after stimulation (Fig. 1). Maximal
activity of the
Na+-K+-2Cl
cotransporter appeared rapidly after stimulation of the cells with a
hypertonic medium with final osmolarity of 400 or 500 mosM (Fig. 1). In
the 400 mosM medium the cotransport activity returned to the resting
level within 8 min, whereas it declined more slowly toward the resting
level in a strong hypertonic medium (500 mosM; Fig. 1), where it was
still significantly higher than the basal level of activity
(P
0.05, independent
t-test) 10 min after the exposure. In
contrast, cotransport activity 5 and 10 min after stimulation with
bradykinin was not significantly different from the basal level
(P
0.20, independent
t-test; Fig. 1). Thus
Na+-K+-2Cl
cotransport activity induced by strong hypertonic treatment outlasts the activity induced by bradykinin or by milder hypertonic challenge probably because of a longer-lasting cell shrinkage.
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Effect of Protein Phosphatase Inhibitors on the
Na+-K+-2Cl
Cotransporter Under Isotonic Conditions
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Cal-A (100 nM) induced maximal activity of the
Na+-K+-2Cl
cotransport activity within the 1st min (Fig.
3) at 43 ± 5 µmol · g dry
wt
1 · min
1
(n = 3). In two independent
experiments the bumetanide-sensitive K+ uptake was measured 15-60
s after addition of Cal-A (data not shown). The uptake was linear,
indicating that maximal cotransport activity is achieved after 15 s of
incubation with Cal-A. After 14 min of incubation with Cal-A the
cotransport activity was not significantly different from the resting
level (P
0.30, independent t-test; Fig. 3). Thus Cal-A-induced
activation of the cotransporter is transient. This shows that some
phosphatase activity remains after addition of Cal-A.
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To examine whether the inactivation of the
Na+-K+-2Cl
cotransporter is secondary to cell swelling induced by Cal-A, cell
volume was monitored using a Coulter counter. Cell volume increased as a result of the Cal-A-induced cotransport activity, promoting a net
uptake of salt and water (Fig. 4).
Inactivation of the
Na+-K+-2Cl
cotransporter after Cal-A treatment, therefore, could be due to the
increase in cell volume or, alternatively, to the concurrent increase
in
[Cl
]i.
To investigate these possibilities, cells were preincubated in a
hypotonic medium (225 mosM) for 5 min. One minute before addition of
experimental medium containing
86Rb+,
some of the cells also received 100 nM Cal-A, which stimulated the
bumetanide-sensitive K+ influx
from 11.1 to 21.5 µmol · g dry
wt
1 · min
1
(n = 1). Thus Cal-A can activate the
cotransporter even in cells swollen to ~1.1 times their normal
volume. The activation was, however, smaller than when cells at normal
volume are activated by Cal-A (Fig. 3).
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Whether inactivation of
Na+-K+-2Cl
cotransport after prolonged exposure to Cal-A is due to cell lysis (32)
was examined by light microscopy of cells exposed to Cal-A for 14 min. No changes in cell morphology were observed; i.e., cotransport
inactivation 14 min after Cal-A exposure is not due to membrane
deformation or cell lysis. The results with Cal-A suggest that PP-1,
PP-2A, and/or PP-3 is involved in maintaining low
Na+-K+-2Cl
cotransport activity under isotonic conditions at steady state.
Ca2+/CaM is involved in the
regulation of the
Na+-K+-2Cl
cotransporter (14). A possible role for the
Ca2+/CaM-dependent PP-2B in the
regulation of the cotransporter was therefore investigated using the
potent PP-2B inhibitor deltamethrin (Ki = 30 pM in in
vitro experiments) (5). The structurally related but inactive compound
permethrin was used as a control. No significant difference in
cotransport activity in cells preincubated with deltamethrin (100 nM,
15 min) or permethrin was observed (Table
1).The same concentration and preincubation
time for deltamethrin were used in intact brain synaptosomes (5). Thus,
although a positive control for the effect of deltamethrin in Ehrlich
cells was lacking, a role for PP-2B in the inactivation of the
cotransporter seems unlikely.
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Effect of Protein Phosphatase Inhibitors on the
Na+-K+-2Cl
Cotransporter After Stimulation With Bradykinin or Hypertonic
Medium
Neither bradykinin nor hypertonicity could increase
Na+-K+-2Cl
cotransport activity further in cells exposed to Cal-A
(P
0.30, independent
t-tests; Table 1). This indicates that
Cal-A induces maximal activation of the cotransporter in Ehrlich cells.
Maximal activation of the cotransporter occurred within 1 min after
stimulation with Cal-A plus bradykinin, with a decline toward the
resting level during the next 10 min (Fig.
5A).
After 5 min the cotransport activity was still significantly higher than the basal activity (P 0.05, independent t-test). Thus cotransport activity induced by Cal-A plus bradykinin was greater and lasted longer
than that induced by bradykinin alone. In contrast, no significant
difference was found between cells treated with Cal-A plus bradykinin
and those treated with Cal-A alone. Therefore, the effects of Cal-A
plus bradykinin on the
Na+-K+-2Cl
cotransporter are not additive. Exposure to Cal-A, hypertonic medium,
or Cal-A plus hypertonic medium activated cotransport maximally within
1 min, and the activity declined toward the resting level during the
next 14 min (Fig. 5B). In cells
exposed to Cal-A plus hypertonic medium, cotransport activity was
significantly higher than at the resting level even after 14 min
(P
0.05, independent
t-tests), whereas neither cells
treated with Cal-A nor those treated with hypertonic medium showed
significantly increased activity after 10 min. Thus inactivation of
cotransport is delayed when cells are exposed to Cal-A plus hypertonic
medium compared with the inactivation in cells in hypertonic medium or after addition of Cal-A, indicating that PP-1, PP-2A, and/or
PP-3 is involved in the inactivation of the
Na+-K+-2Cl
cotransporter in agreement with the results in Table 1. However, other
mechanisms must also be involved, since the process was delayed by
Cal-A but not prevented. Alternatively, Cal-A may not inhibit the
involved phosphatases completely.
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Role of Ca2+ and pH in
Activation of the
Na+-K+-2Cl
Cotransporter
Preliminary experiments at pHo of
7.4 showed that BAPTA-induced acidification caused a marked decrease in
cotransport activity. The pH dependence of the cotransport activity was
thus further investigated. At pHo
of 6.5, cotransport activity during RVI was negligible but increased
with increasing pHo to 24 ± 3.9 µmol · g dry
wt1 · min
1
at pHo of 8.3 (Fig.
6). The decrease in cotransport activity caused by BAPTA-induced acidification suggests that the inhibitory effect of H+ is internal. The data
in Fig. 6 exclude that the inhibitory effect of BAPTA in Table
2 is due to the exposure of the cells to a pHo of 8.3.
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Ca2+/CaM and PKC Are
Involved in Activation of the
Na+-K+-2Cl
Cotransporter
Bradykinin-induced cotransport activity was inhibited by 56% in cells
pretreated with 100 µM pimozide
(P 0.0, paired
t-test), slightly, but not
significantly, inhibited (P
0.40, independent t-test) after
preincubation with chelerythrine (2.5 µM), and inhibited by 75% when
the two inhibitors were combined (Table 2). In the presence of both
inhibitors the cotransport activity was significantly lower than after
separate preincubation with pimozide or chelerythrine (P
0.0, paired
t-tests) and not significantly
different from the activity in unstimulated cells. This pharmacological
evidence indicates the involvement of a
Ca2+/CaM and PKC in the
cotransport activation.
CaM Kinase II Is Not Involved in Activation of the
Na+-K+-2Cl
Cotransporter
MLCK and PKA Are Involved in Activation of
Na+-K+-2Cl
Cotransport After Hypertonic Stimulation
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Because neither ML-7 nor H-89 is entirely specific for MLCK and PKA, respectively (Calbiochem specifications), dose-response experiments were performed for all four inhibitors (Fig. 8). The results support those obtained using ML-7 and H-89. The observed IC50 values are summarized in Table 3.
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Because inhibition of PKA by H-89 decreases
Na+-K+-2Cl
cotransport activity during RVI (Table 2), the effect of stimulation of
PKA with forskolin (300 µM) was tested on the cotransport activity during RVI. Forskolin had no effect, indicating that cAMP may not be
rate limiting (Table 4).
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Cal-A-Activated
Na+-K+-2Cl
Cotransport Is Regulated by MLCK, PKA, and Other Ser/Thr Kinases
Under Isotonic Conditions
Cells were preincubated with Cal-A (control) or Cal-A plus one or two
Ser/Thr protein kinase inhibitors. Relative to the controls (no protein
kinase inhibitor), K+ influx via
the
Na+-K+-2Cl
cotransporter in four paired experiments was reduced to 0.71 ± 0.02 (P
0.001) after addition of ML-7,
0.76 ± 0.05 (P
0.006, paired
t-test) after addition of H-89, and
0.65 ± 0.03 (n = 3, P
0.009, paired
t-test) after addition of ML-7 plus
H-89. In five other paired experiments, staurosporine reduced
Cal-A-activated Na+-K+-2Cl
cotransport activity to 0.50 ± 0.05 (P
0.002, paired
t-test; for absolute values see Table
2). Inhibition of PP-1, PP-2A, and/or PP-3 by Cal-A thus
results in a net phosphorylation caused by MLCK, PKA, and other Ser/Thr
kinases during RVI and at steady state.
MLCK-Sensitive Cotransport Increases After Cell Shrinkage
Figure 9 shows the ML-7-sensitive part of Na+-K+-2Cl
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DISCUSSION |
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Activation of the
Na+-K+-2Cl
Cotransporter in Ehrlich Cells
In cells exposed to hypertonic treatment the cotransport activity
rapidly increases to a maximal level and then declines to the basal
level after ~15 min in strong hypertonic medium (500 mosM) or after
~9 min in a milder hypertonic medium (400 mosM; Fig. 1). This finding
suggests that the duration of the increased cotransport activity after
hypertonic treatment depends on the osmolarity and, hence, the degree
of cell shrinkage.
Na+-K+-2Cl
cotransport activity induced by the 500 mosM hypertonic medium also
lasts longer than the cotransport activity induced by bradykinin, reflecting that the cells are shrunken for a longer period after addition of hypertonic medium than after addition of bradykinin.
Pharmacological Evidence for Involvement of Different Protein
Phosphatases in Regulation of the
Na+-K+-2Cl
Cotransporter Under Isotonic Conditions
Deltamethrin had no effect on the
Na+-K+-2Cl
cotransport activity (Table 1). Provided that deltamethrin has access
to PP-2B in Ehrlich cells (see
RESULTS), we propose that PP-2B is
not involved in regulation of
Na+-K+-2Cl
cotransport. To our knowledge, a role for PP-2B in the regulation of
the
Na+-K+-2Cl
cotransporter has not been investigated in other cell types.
Involvement of Different Protein Phosphatases in Regulation of
the
Na+-K+-2Cl
Cotransporter After Stimulation With Bradykinin or Addition of
Hypertonic Medium
Involvement of Different Protein Kinases in Regulation of the
Na+-K+-2Cl
Cotransporter Under Isotonic Conditions
Involvement of Different Protein Kinases in Activation of the
Na+-K+-2Cl
Cotransporter During RVI and After Agonist Stimulation
Ca2+/CaM-dependent
mechanisms.
Previously we found that activation of
Na+-K+-2Cl
cotransport during RVI is Ca2+/CaM
dependent (14). Here we demonstrate that cotransport activity induced
by bradykinin or cell shrinkage is markedly reduced in cells loaded
with BAPTA, an intracellular chelator of
Ca2+ (Table 2). BAPTA loading of
Ehrlich cells decreases intracellular Ca2+ concentration
([Ca2+]i)
by ~50% compared with control cells but also causes an immediate intracellular acidification (15), which itself inhibits cotransport (Fig. 6). Preincubation of cells in a medium of pH 8.3 prevents the intracellular acidification (15) without preventing
inhibition of the cotransport activity by BAPTA. Cell shrinkage due to
a loss of K+ and
Cl
presumably via the
otherwise silent
K+-Cl
cotransporter is another effect of BAPTA loading (15). However, it is
unlikely that this cell shrinkage results in an inhibition of the
Na+-K+-2Cl
cotransporter. Therefore, activation of
Na+-K+-2Cl
cotransport depends on
[Ca2+]i,
as indicated by the absence of cotransport in BAPTA-loaded cells (Table
2). A substantial decrease in agonist-induced cotransport after
preincubation with BAPTA-AM is also found in endothelial cells (23) and
nasal gland acinar cells (12). In Ehrlich cells no increase in
[Ca2+]i
during RVI could be demonstrated (14), although minor spatially localized increases in
[Ca2+]i
cannot be excluded by the method used.
PKC.
In Ehrlich cells, PKC is maximally activated 1 min after exposure to a
hypertonic medium, and chelerythrine, a specific PKC inhibitor,
inhibits cotransport by 20% during RVI (19). In contrast, no
significant decrease in bradykinin-induced cotransport activity could
be demonstrated here (Table 2). However, the bradykinin-induced Na+-K+-2Cl
cotransport activity is significantly lower in cells treated with
pimozide plus chelerythrine than in cells treated with pimozide alone
(Table 2). This suggests that PKC plays a minor role in activation of the
Na+-K+-2Cl
cotransporter in Ehrlich cells. In general, the role of PKC in the
regulation of the cotransporter is not clear, since, depending on cell
type, activation of PKC can be stimulatory or inhibitory (10).
MLCK.
It has been suggested that MLCK is important for
Na+-K+-2Cl
cotransport during RVI in endothelial cells (17, 24): the cotransport activity on cell shrinkage was inhibited ~30 and 50% at 25 and 100 µM ML-7, respectively, but was not inhibited at 1 µM ML-7. At 100 µM, ML-7 blocks MLC phosphorylation and
Na+-K+-2Cl
cotransport activity, but not phosphorylation of the cotransporter itself (17). However, according to Calbiochem specifications, ML-7 also
inhibits PKA and PKC, with
Ki values of 21 and 42 µM, respectively; thus it is difficult to make definite
conclusions as to which kinase is involved in regulation of cotransport
activation at 100 µM ML-7.
PKA.
PKA inhibitors H-89 and KT-5720 reduced
Na+-K+-2Cl
cotransport during RVI, with IC50
values of 37 and 34 nM, respectively, for the inhibitor-sensitive part
of the influx (Table 3). The IC50 values are close to the reported
Ki values at 48 and 60 nM for H-89 (2) and KT-5720 (6), respectively. On the basis of these pharmacological data, we propose that PKA is involved in the
regulation of the cotransporter in Ehrlich cells. Interestingly, activation of PKA during isotonic steady state or RVI does not result
in enhanced cotransport activity (Table 4). In duck erythrocytes, PKA
has likewise been assigned a role as modulator of
Na+-K+-2Cl
cotransport on the basis of effects of the inhibitors H-9
[N-(2-aminoethyl)-5-isoquinolinesulfonamide] and K-252a (27). The role of cAMP and, therefore, PKA is ambiguous: cAMP stimulates cotransport in some avian cells, whereas in other cell
types, such as fibroblasts, cotransport is inhibited by cAMP (7, 10).
PKA is not likely to be the protein kinase controlling the RVI response
(27). As shown in the present report, the H-89-sensitive part of the
cotransport activity does not increase significantly after cell
shrinkage, suggesting that PKA is involved in the maintenance of
steady-state phosphorylation without being further activated during
RVI.
Activation of MLCK on Cell Shrinkage
In contrast to the H-89-sensitive part of cotransport, the ML-7-sensitive part increases nearly twofold during RVI compared with the activity at steady state (Fig. 9). This suggests that MLCK is further activated during RVI; i.e., cell shrinkage seems to activate MLCK.MLCK is a Ca2+/CaM-dependent
kinase; hence, the fact that pimozide and BAPTA strongly inhibit
cotransport activity during RVI (Table 2) supports the suggested role
for MLCK. Thus the present pharmacological data suggest that the
regulatory pathway involved in shrinkage activation of the
cotransporter involves activation of a volume-sensitive
Ca2+/CaM-dependent MLCK and
concomitant phosphorylation of MLC. MLC phosphorylation, however, may
not be due to MLCK only: PKC is also active during RVI (19) and has MLC
among its substrates. The PKC inhibitor chelerythrine and the
Ca2+/CaM antagonist pimozide seem
to have additive effects (Table 2), suggesting that the activity of PKC
is unrelated to MLCK. Whether the effects of ML-7 and chelerythrine are
additive has not been investigated. Further investigations, based on
molecular and biochemical evidence, are needed to characterize the role of MLCK in the regulation of the
Na+-K+-2Cl
cotransporter.
How Is MLCK Activated?
Two major theories for explaining how cells sense volume alterations and activate ion transport systems have been put forward. The first theory involves the macromolecular crowding of cytosolic solutes (10): on the basis of the inhibitory effect of urea, Lim et al. (21) suggested that Na+-K+-2ClIn conclusion, using various pharmacological tools, we have
demonstrated that the protein kinases MLCK and PKA participate in the
continuous phosphorylation and the protein phosphatases PP-1, PP-2A,
and/or PP-3 in the dephosphorylation of the
Na+-K+-2Cl
cotransporter (or a regulatory protein) during isotonic steady state in
Ehrlich cells. In addition, we found that the ML-7-sensitive cotransport is further activated after cell shrinkage, indicating that
an ML-7-sensitive process, most probably MLCK, plays a regulatory role
during RVI in Ehrlich cells.
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ACKNOWLEDGEMENTS |
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The expert technical assistance of Birgit B. Jørgensen is acknowledged.
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FOOTNOTES |
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This research was supported by Carlsberg Foundation Grants 960305/20-1299 and 960344/40-1099, The Novo Foundation, and Danish Natural Science Research Council Grants 110702-2 and 111228-1.
Address reprint requests to E. K. Hoffmann.
Received 28 October 1996; accepted in final form 30 March 1998.
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