Departments of 1 Oral Physiology and 2 Biochemistry, Meikai University, School of Dentistry, Sakada-shi, Saitama 350-0283, Japan; and 3 Membrane Biology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda MD 20892
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We
studied the phosphorylation of the secretory
Na+-K+-2Cl cotransporter (NKCC1)
in rat parotid acinar cells. We have previously shown that NKCC1
activity in these cells is dramatically upregulated in response to
-adrenergic stimulation and that this upregulation correlates with
NKCC1 phosphorylation, possibly due to protein kinase A (PKA). We show
here that when ATP is added to purified acinar basolateral membranes
(BLM), NKCC1 is phosphorylated as a result of membrane-associated
protein kinase activity. Additional NKCC1 phosphorylation is seen when
PKA is added to BLMs, but our data indicate that this is due to an
effect of PKA on endogenous membrane kinase or phosphatase activities,
rather than its direct phosphorylation of NKCC1. Also, phosphopeptide
mapping demonstrates that these phosphorylations do not take place at
the site associated with the upregulation of NKCC1 by
-adrenergic
stimulation. However, this upregulatory phosphorylation can be mimicked
by the addition of cAMP to permeabilized acini, and this effect can be
blocked by a specific PKA inhibitor. These latter results provide good evidence that PKA is indeed involved in the upregulatory
phosphorylation of NKCC1 and suggest that an additional factor present
in the acinar cell but absent from isolated membranes is required to bring about the phosphorylation.
exocrine glands; fluid secretion; stimulus-secretion coupling; cation-chloride cotransporter
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE "SECRETORY"
ISOFORM of the Na+-K+-2Cl
cotransporter, NKCC1 (also known as BSC2) plays a central role in the
secretion of salt and water by many exocrine epithelia (5,
8). In these tissues NKCC1 is localized to the basolateral
membrane of the secretory cell, where it concentrates Cl
in the cytoplasm above electrochemical equilibrium. The apical membrane
typically contains a secretagogue-activated Cl
channel.
During stimulation, Cl
thus enters the cell via NKCC1 and
exits via the apical Cl
channel. The resulting
transepithelial Cl
secretion then leads to the secretion
of salt and osmotically obliged water. There is now good evidence from
a number of these tissues that NKCC1 fluxes also are dramatically
increased as a result of secretagogue action and that this effect is
not simply due to the changes in electrochemical driving forces for
Na+, K+, and Cl
that accompany
stimulation (3, 4, 6, 15, 18). Rather, this upregulation
appears to arise directly from the increased activity of individual cotransporters.
In previous studies from our laboratories we have examined this
phenomenon in some detail in rat parotid acinar cells (3, 9,
17-19). In particular, we have characterized the
upregulation of NKCC1 activity induced by both muscarinic and
-adrenergic stimulation (Ca2+-mobilizing and
cAMP-generating stimuli, respectively, in these cells). In the case of
-adrenergic stimulation, which mainly concerns us here, we have
shown that there is a close correlation between isoproterenol-induced
increases in NKCC1 transport activity and NKCC1 phosphorylation
(18, 19). More specifically, we found that the
isoproterenol dose responses for both of these phenomena were
essentially identical, with a half-maximal effect at ~20 nM agonist
in each case, and that isoproterenol stimulation was accompanied by the
increased phosphorylation of a 17-kDa peptide resulting from NKCC1
digestion by V8 protease. In addition, these effects are paralleled by
an increase in the number of high-affinity binding sites for the NKCC1
inhibitor bumetanide in membranes prepared from stimulated acini
(9). These results are consistent with the hypothesis that
-adrenergic stimulation and the accompanying phosphorylation result
in the activation of previously quiescent, or near-quiescent, transporters.
In contrast to many other exocrine epithelia, salivary glands secrete
fluid in response to Ca2+-mobilizing rather than
cAMP-generating agonists (the increased NKCC1 activity resulting from
the latter is thought to account for the increased fluid secretion
observed when -adrenergic stimulation is superimposed on muscarinic
stimulation in these tissues; see, for example, Ref. 18).
Thus
-adrenergic stimulation of salivary acini does not result in
the intracellular Cl
loss and cell shrinkage that
typically accompany fluid secretion by exocrine cells (14,
18), and the involvement of these effects in the
isoproterenol-induced upregulation of the acinar NKCC1 can therefore be
excluded. In addition, we have shown that the effect of isoproterenol
on NKCC1 can be prevented by protein kinase inhibitors and mimicked by
permeant cAMP analogs as well as maneuvers known to increase cytosolic
cAMP levels (18, 19), suggesting the involvement of
protein kinase A (PKA). However, somewhat surprisingly we also have
found that the site of isoproterenol-induced phosphorylation is found
in the NH2-terminal cytosolic tail of NKCC1
(9) which does not contain the sole PKA consensus site in
this cotransporter.
Here we continue our studies of the phosphorylation events associated with the rat parotid NKCC1. Briefly stated, we show that NKCC1 is phosphorylated by endogenous kinases associated with isolated rat parotid acinar basolateral membranes (BLMs). Additional NKCC1 phosphorylation is seen in the presence of added PKA, but our data suggest that this is due to an effect of PKA on endogenous membrane kinase or phosphatase activities rather than its direct phosphorylation of NKCC1. Moreover, these phosphorylations do not take place at the site associated with the upregulation of NKCC1 by isoproterenol stimulation. Finally, we show that the phosphorylation of NKCC1 induced by isoproterenol treatment can be mimicked by the addition of cAMP to permeabilized acini and that this effect can be blocked by a specific peptide inhibitor of PKA. Taken together, these results provide strong evidence that PKA is indeed involved in the phosphorylation of NKCC1 induced by isoproterenol treatment but that an additional, non-membrane associated factor (for example, a cytosolic kinase activated by PKA) is required to effect the phosphorylation event.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
[-32P]ATP (3,000 Ci/mmol) was obtained from NEN.
Molecular weight standards and prepoured SDS-PAGE and tricine gels were
from Novex. Protein G/Sepharose beads were from Pierce.
L-Tosylamido-2-phenylethyl chloromethyl ketone,
phenylmethylsulfonyl fluoride, ovalbumin, and the PKA inhibitor
PKI(5-24) were from Sigma. The catalytic subunit of
PKA (cPKA) also was obtained from Sigma (P8289) and was reconstituted
in deionized water containing 5 mM dithiothreitol (DTT) at a
concentration of 2 U/µl. Pepstatin, leupeptin, and V8 protease were
from Boehringer Mannheim. Calyculin A, K252a, okadaic acid, and
staurosporine were from Calbiochem. Bisindolylmaleimide I HCl,
lavendustin A, microcystin-LR, and H-89 were from LC Laboratories.
Membrane preparation. Rat parotid BLMs were prepared by isopycnic centrifugation as previously described (12). Briefly, rat parotid glands were minced and vigorously homogenized in an isotonic sucrose buffer. The resulting homogenate was centrifuged (2,500 g, 5 min) to remove cellular debris, and the supernatant was recentrifuged (22,000 g, 20 min) to recover a crude membrane pellet. This pellet was spun on a Percoll gradient from which a fraction highly enriched in BLMs was recovered. The basolateral membrane fraction was then washed twice in buffer A (10 mM HEPES buffered with Tris to pH 7.4 plus 100 mM mannitol) containing 100 mM KCl and 1 mM EDTA, resuspended at a protein concentration of 5-10 mg/ml, snap frozen in aliquots, and stored above liquid nitrogen. Relative to the starting tissue homogenate, the activity of the basolateral membrane marker K-stimulated p-nitrophenyl phosphatase is enriched 15-20 times in this final basolateral membrane preparation (12). On the day of an experiment a suitable number of aliquots of BLMs were thawed at room temperature for 30 min, diluted in buffer A containing 1 mM EGTA, centrifuged at 100,000 g for 60 min, and resuspended in buffer A containing 1 mM EGTA at a protein concentration of 1 mg/ml.
Antibody preparation and conjugation.
The antibodies used in these studies (9), -wCT(m) and
-wCT(r), were raised (in mice and rabbits, respectively) against a
6× His fusion protein corresponding to the COOH-terminal 454 amino
acids of rat NKCC1. In Western blots of rat parotid BLMs (not shown),
these antisera recognize a single 170-kDa protein, the molecular mass
we previously established as that of (fully glycosylated) rat NKCC1 in
this tissue. For immunoprecipitation studies, antibodies were
preconjugated to protein G beads as previously described
(9) and suspended in SS containing 1% ovalbumin as a
slurry of 50% beads and 50% buffer. In control experiments (not shown), we established that both
-wCT(m) and
-wCT(r) yield
quantitative immunoprecipitations of rat NKCC1 under the experimental
conditions described here, and we have not detected any differences in
their behavior.
-wCT(m) was used in the experiments shown in Figs. 1-5 and
-wCT(r) was used in the experiment shown in Fig. 6.
|
|
|
|
|
|
Phosphorylation studies using intact rat parotid BLMs.
Phosphorylation reactions were carried out at 30°C in buffer I
(buffer A plus 20 mM sodium acetate, 60 mM potassium
acetate, 60 mM KCl, 10 mM magnesium acetate, and 1 mM EGTA) containing 0.2 mg/ml rat parotid BLMs and other additions as indicated. The reaction was initiated by the addition of 40 µM
[-32P]ATP and stopped by the addition of an
appropriate solution (see below). Unless otherwise stated,
phosphorylation reactions were stopped 30 s after the addition of
[
-32P]ATP. Under these experimental conditions we
found that the half-time of [
-32P]ATP hydrolysis was
~10 s (data not shown; release of 32Pi from
ATP was monitored by using the method described in Ref. 13).
Phosphorylation studies using permeabilized rat parotid acini.
Rat parotid acini were prepared as previously described
(19). Phosphorylation reactions were carried out at 37°C
in buffer I containing a 20% suspension of acini, 0.1 mM
sodium orthovanadate, and other additions as indicated (final volume
100 µl). The reaction was initiated by the addition of 40 µM
[-32P]ATP and 0.1 mM digitonin and was stopped by the
addition of 500 µl of SS. After the addition of SS, insoluble debris
was removed by centrifugation at 10,000 g for 10 min, and
the resulting supernatant was combined with 30 µl (slurry) of
preconjugated protein G beads for immunoprecipitation of NKCC1 as
described in Phosphorylation studies using intact rat parotid
BLMs.
Digestion with V8 protease. Digestion with V8 protease was carried out as previously described (9, 19). Briefly, bands corresponding to immunoprecipitated rat NKCC1 were cut from dried SDS-PAGE gels, rehydrated in 50 mM NH4HCO3 (pH 8.0) plus 1 mM DTT, and incubated with V8 protease (20 U/ml) for 6 h. The digested material recovered from the gel fragments was then dried, taken up in sample buffer, and subjected to tricine-SDS electrophoresis.
Gel electrophoresis and autoradiography. SDS-PAGE, tricine-SDS electrophoresis, autoradiography, and densitometry were carried out as previously described (9). Densitometric results are given as means ± SE for the number of replicates indicated. For SDS-PAGE we employed 4-20% gradient gels. For tricine-SDS electrophoresis we used 16% gels. Unless otherwise noted, the results shown are from SDS-PAGE gels.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The two left-hand lanes of Fig. 1
show the results of an experiment in which rat parotid BLMs were
incubated with [-32P]ATP for 30 s or 30 min, as
described in METHODS, and then analyzed by SDS-PAGE and
autoradiography. After 30 s of incubation, a number of proteins,
particularly in the molecular mass range 50-200 kDa, were labeled
with 32P. Because the BLMs used in these experiments were
extensively washed during their preparation (see METHODS),
this result indicates the presence of one or more protein kinases
tightly associated with the rat parotid BLM. In control experiments
(not shown) we have demonstrated that overall BLM protein
phosphorylation reaches a maximum ~30 s after the addition of
[
-32P]ATP under our experimental conditions (as
indicated in METHODS, the half-time of
[
-32P]ATP hydrolysis was ~10 s under these
conditions). After 30 min of incubation, much of the membrane
phosphorylation is lost (Fig. 1), likewise demonstrating the presence
of significant membrane-associated protein phosphatase activity (as
indicated below, this loss of phosphorylation can be blocked by
inhibition of protein phosphatases, confirming that it is not due to
nonspecific effects such as proteolysis). As illustrated in the two
right-hand lanes of Fig. 1, when NKCC1 was immunoprecipitated from
these membranes, we found that it was one of the phosphoproteins
detected. Analysis by scanning densitometry indicated that
phosphorylation of NKCC1 at 30 s was 3.40 ± 0.08 times that
observed at 30 min (n = 7).
Figure 2 illustrates the effects of various protein kinase inhibitors on the ATP-induced phosphorylation of NKCC1 in rat parotid BLMs. The effects of 10 nM staurosporine and 100 nM K252a (general serine/threonine kinase inhibitors with IC50 values in the range 1-10 nM and 20-200 nM, respectively), 1 µM H-89 (a relatively specific inhibitor of PKA with IC50 ~0.05 µM), 3 µM lavendustin A (a relatively specific inhibitor of tyrosine kinases with IC50 ~0.01 µM), and 2 µM bisindolylmaleimide I (a relatively specific inhibitor of protein kinase C with IC50 <0.1 µM for most isotypes) are shown. Marked inhibition of phosphorylation is seen with staurosporine and K252a, which were used in their usual effective range, but there is little or no effect of the other kinase inhibitors, all of which were used at concentrations at least an order of magnitude above their IC50 values. When we examined the effects of these protein kinase inhibitors on the ATP-induced phosphorylation of all BLM proteins (cf., 2 left-hand lanes of Fig. 1), qualitatively similar results were found (not shown).
Figure 3 shows the effects of various protein phosphatase inhibitors on the dephosphorylation of NKCC1 by membrane-associated protein phosphatase(s). The effects of calyculin A, okadaic acid, and microcystin-LR, all used at 100 nM, are shown in Fig. 3, left. Both calyculin A and microcystin-LR are known to inhibit serine/threonine protein phosphatase type 1 at concentrations of ~1 nM, and all three compounds inhibit serine/threonine protein phosphatase type 2A at concentrations of ~1 nM. However, even at 100 nM concentration these compounds have only modest effects on the dephosphorylation of NKCC1 by membrane-associated phosphatases; we suspect that this is because there are relatively small quantities of these predominantly cytosolic phosphatases that copurify with the BLM. In contrast, the more general phosphatase inhibitor, sodium orthovanadate, whose effects are shown in Fig. 3, right, markedly inhibits the dephosphorylation of NKCC1 at 0.1 mM concentration. Qualitatively similar results were found for the effects of these protein phosphatase inhibitors on overall dephosphorylation of all BLM proteins by membrane-associated phosphatases (not shown).
As already discussed, our previous studies demonstrate that the
activity of NKCC1 is dramatically upregulated in response to
-adrenergic stimulation of rat parotid acini and indicate that this
effect is the result of NKCC1 phosphorylation. Our results also suggest
that PKA may be involved in this effect. To explore this possibility
further, we tested the effects of adding cAMP or cPKA to rat parotid
BLMs in the presence of [
-32P]ATP. The results of such
an experiment are shown in Fig. 4. The
top, left-hand panel of Fig. 4 shows an autoradiograph of immunoprecipitated NKCC1 showing apparent increases in phosphorylation by both treatments relative to an untreated control. The top, right-hand panel of Fig. 4 shows an autoradiograph of V8 protease digests of these samples run on a tricine-SDS gel; in these digests we
see a single phosphopeptide with molecular mass ~7 kDa. The bottom
panel of Fig. 4 presents a densitometric analysis confirming that the
phosphorylation of this 7-kDa peptide is increased by the presence of
cAMP or cPKA. The effect of cAMP in this experiment is presumably due
to the presence of PKA bound to A-kinase anchoring proteins (AKAPs)
(1, 16) in the BLM and thereby copurified with them. We
have, in fact, previously presented evidence for the presence of
endogenous PKA and its cAMP-dependent phosphorylation of
Na+-K+-ATPase in this BLM preparation
(10).
However, as also stated earlier, the phosphorylation of NKCC1 that
accompanies isoproterenol stimulation of intact acini and correlates
with functional NKCC1 upregulation is associated with a 17-kDa peptide
resulting from V8 protease digestion. Although an ~7-kDa
phosphopeptide also was seen in these previous V8 protease digests
(9, 19), its phosphorylation was not significantly affected by isoproterenol treatment. To better understand the effect of
cPKA on the 7-kDa phosphopeptide shown in Fig. 4, we carried out the
experiment shown in Fig. 5. Here BLMs
were preincubated with unlabeled ATP in the presence or absence of
cPKA, and then additions were made to both reactions to bring them to
the same levels of cPKA and the PKA inhibitor H-89 (final concentration 1 µM), before adding [-32P]ATP and measuring NKCC1
phosphorylation. As can be readily appreciated from Fig. 5,
significantly more incorporation of 32P into NKCC1 is seen
in the membranes preincubated with cPKA. However, because cPKA was
present in both reactions with [
-32P]ATP, this
increase in phosphorylation could not be due to a direct
phosphorylation of NKCC1 by cPKA. Instead, the results of this
experiment are consistent with the hypothesis that cPKA treatment of
acinar BLMs results in the activation of endogenous membrane-associated
kinase(s) or the inhibition of membrane phosphatases, which in turn
leads to increased phosphorylation of NKCC1.
To be able to evaluate this result in the context of our earlier
experiments with intact acini, we carried out the experiment illustrated in Fig. 6. Here
digitonin-permeabilized acini were incubated with
[-32P]ATP alone, with [
-32P]ATP in
the presence of cAMP, or with [
-32P]ATP plus cAMP and
PKI(5-24), a specific peptide inhibitor of PKA. After
incubation, the phosphorylation of NKCC1 was analyzed by V8 protease
digestion. In this digest, three phosphopeptides are shown (Fig. 6,
top), the 17-kDa and 7-kDa phosphopeptides previously
observed in NKCC1 digests from intact acini (9, 19) and a
10-kDa phosphopeptide only seen with permeabilized acini (a 5.7-kDa
phosphopeptide reported in Ref. 19 was not seen in our
more recent experiments described here and in Ref. 9,
where NKCC1 was immunopurified before gel purification and V8 protease digestion; in the experiments reported in Ref.
19 we digested gel slices containing all BLM proteins with
Mr ~170 kDa, so the 5.7-kDa phosphopeptide is
presumably from another ~170-kDa membrane protein). As shown in Fig.
6, the addition of cAMP to permeabilized acini results in the
characteristic increase in phosphorylation of the 17-kDa peptide
observed in our previous studies with intact acini stimulated with
isoproterenol (9, 19). This result is consistent with our
earlier observations that the effects of isoproterenol on NKCC1 can be
mimicked by maneuvers that increase intracellular cAMP levels
(18, 19). Moreover, as also shown in Fig. 6, the effect of
cAMP on phosphorylation of the 17-kDa peptide can be blocked by
PKI(5-24), providing strong evidence that PKA itself
is indeed involved in this phosphorylation event in permeabilized
acini, and thus presumably in intact acini as well. The fact that cPKA
is unable to phosphorylate the 17-kDa peptide when it is added to
isolated acinar BLM (Fig. 4) suggests that there is a participant in
this phosphorylation reaction that is present in the acinar cell but
absent from isolated membranes.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have demonstrated here that isolated rat parotid BLMs possess significant membrane-associated protein kinase and protein phosphatase activities and that one of the proteins phosphorylated and dephosphorylated by these enzymes is NKCC1 (Figs. 1-3). The membrane-associated protein kinase activity appears to be of the serine/threonine type, because its effect on NKCC1 was blocked by the general serine/threonine kinase inhibitors staurosporine and K252a; however, specific inhibitors of PKA, tyrosine kinase, and protein kinase C had little or no effect on this kinase activity even at high concentrations (Fig. 2). The membrane-associated phosphatase activity was inhibited by the nonspecific phosphatase inhibitor sodium orthovanadate, but more specific inhibitors of protein phosphatases 1 and 2A were relatively ineffective (Fig. 3). Moreover, because our experiments were carried out in essentially Ca2+-free medium (1 mM EGTA), the involvement of protein phosphatase 2B (calcineurin) also can be excluded. Further experiments designed to identify the specific protein kinases and phosphatases involved in these effects were not attempted.
The phosphorylation of NKCC1 produced by the membrane-associated
protein kinase activity was on a 7-kDa peptide resulting from V8
protease digestion (Fig. 4). Additional phosphorylation of this peptide
was seen when isolated BLMs were incubated with ATP in the presence of
cAMP (Fig. 4). This effect is presumably due to the presence of
endogenous PKA in parotid BLMs. It has been shown that membrane-bound
AKAPs bind PKA via its regulatory subunit RII and thereby anchor it
near specific target proteins (1, 16). These target
proteins are then rapidly phosphorylated in response to increased
cellular cAMP, which releases cPKA from RII. Phosphorylation of
Na+-K+-ATPase in response to cAMP addition has,
in fact, previously been demonstrated in this same parotid BLM
preparation and the involvement of AKAPs confirmed (10).
As already emphasized, however, the upregulation of NKCC1 function
associated with isoproterenol treatment and increased acinar cAMP
levels correlates with the phosphorylation of a 17-kDa peptide
resulting from V8 protease digestion. Initially we wondered if PKA was
actually anchored near NKCC1 via AKAPs but had been stripped off during
the BLM preparation, thus accounting for the absence of phosphorylation of the 17-kDa peptide with cAMP treatment. However, attempts to reload
membrane-bound AKAPs with PKA, followed by centrifugation and cAMP plus
[-32P]ATP treatment, failed to yield phosphorylation
of the 17-kDa peptide (not shown). Likewise, direct addition of cPKA
plus [
-32P]ATP to BLM did not result in
phosphorylation of the 17-kDa peptide, although NKCC1 phosphorylation
of the 7-kDa peptide was increased (Fig. 4).
We did find, however, that preincubation of BLMs with cPKA before the
addition of [-32P]ATP resulted in markedly higher
levels of NKCC1 phosphorylation than observed in untreated controls
(Fig. 5). This result suggests that the effects of cAMP and cPKA on
phosphorylation of NKCC1 are due to activation of the endogenous
membrane-associated kinase activity or inhibition of membrane
phosphatases, rather than a direct phosphorylation of NKCC1 by cPKA.
Thus it appears as if PKA may be unable to directly phosphorylate NKCC1
at any site in isolated BLMs.
We previously observed a 7-kDa phosphopeptide in V8 protease digests of immunoprecipitated NKCC1 from intact rat parotid acini (9, 19). Because the endogenous protein kinase activity of the rat parotid BLM appears to be quite high and is clearly able to overpower the endogenous protein phosphatase activity (Fig. 1), we speculate that phosphorylation of NKCC1 on the 7-kDa peptide site is maintained near saturation levels in the intact acinar cell. This could account for the observation that no increase in phosphorylation at this site is observed with isoproterenol stimulation despite the resulting activation of PKA. The significance of this phosphorylation of NKCC1 in rat parotid acinar cells remains to be determined.
The fact that the phosphorylation of NKCC1 induced by isoproterenol
treatment can be mimicked by the addition of cAMP to permeabilized acini and that this effect can blocked by a specific peptide inhibitor of PKA (Fig. 6) provides strong evidence for the involvement of PKA in
this effect. Because this phosphorylation cannot be produced by the
addition of PKA to BLM, we conclude that some factor present in intact
cells but missing in isolated BLMs may be involved. Further experiments
are required to identify this (putative) participant in the
phosphorylation reaction; however, an obvious candidate would be a
cytosolic kinase that is activated by PKA. In this regard, Forbush and
colleagues have presented evidence that NKCC1 can be upregulated as a
result of phosphorylation by an as yet unidentified
Cl-dependent kinase (7, 11) and that this
phosphorylation can be reversed by protein phosphatase type 1, which is
directly targeted to NKCC1 (2). Whether these same enzymes
are involved in
-adrenergic stimulation-dependent NKCC1
phosphorylation in salivary glands remains to be determined. If this is
the case, however, then this kinase must be activated by a
non-Cl
-dependent mechanism in these tissues because, as
already stressed, intracellular Cl
levels in salivary
acinar cells are not affected by
-adrenergic stimulation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Bruce J. Baum for helpful discussions and encouragement during the course of this work.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: R. J. Turner, Bldg. 10, Rm. 1A06, 10 Center Dr. MSC 1190, National Institutes of Health, Bethesda MD 20892-1190 (E-mail: rjturner{at}nih.gov).
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.
10.1152/ajpcell.00352.2001
Received 26 July 2001; accepted in final form 27 November 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Coghlan, VM,
Bergeson SE,
Langeberg L,
Nilaver G,
and
Scott JD.
A-kinase anchoring proteins: a key to selective activation of cAMP-responsive elements.
Mol Cell Biochem
127-128::
309-319,
1993.
2.
Darman, RB,
Flemmer A,
and
Forbush B.
Modulation of ion transport by direct targeting of protein phosphatase type 1 to the Na-K-Cl cotransporter.
J Biol Chem
276:
34359-34362,
2001
3.
Evans, RL,
and
Turner RJ.
Upregulation of Na+-K+-2Cl cotransporter activity in rat parotid acinar cells by muscarinic stimulation.
J Physiol
499:
351-359,
1997[Abstract].
4.
Forbush, B, III,
Haas M,
and
Lytle C.
Na-K-Cl cotransport in the shark rectal gland. I. Regulation in the intact perfused gland.
Am J Physiol Cell Physiol
262:
C1000-C1008,
1992
5.
Haas, M,
and
Forbush B, III.
The Na-K-Cl cotransporter of secretory epithelia.
Annu Rev Physiol
62:
515-534,
2000[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
Lytle C.
[Cl]i-dependent phosphorylation of the Na-K-Cl cotransport protein of dog tracheal epithelial cells.
J Biol Chem
270:
28955-28961,
1995
8.
Kaplan, MR,
Mount DB,
and
Delpire E.
Molecular Mechanisms of NaCl Cotransport.
Annu Rev Physiol
58:
649-668,
1996[ISI][Medline].
9.
Kurihara, K,
Moore-Hoon ML,
Saitoh M,
and
Turner RJ.
Characterization of the phosphorylation event resulting in upregulation of the salivary Na+-K+-2Cl cotransporter by beta-adrenergic stimulation.
Am J Physiol Cell Physiol
277:
C1184-C1193,
1999
10.
Kurihara, K,
Nakanishi N,
and
Ueha T.
Regulation of Na+-K+-ATPase by cAMP-dependent protein kinase anchored on membrane via its anchoring protein.
Am J Physiol Cell Physiol
279:
C1516-C1527,
2000
11.
Lytle, C,
and
Forbush B, III.
The Na-K-Cl cotransport protein of shark rectal gland. II. Regulation by direct phosphorylation.
J Biol Chem
267:
25438-25443,
1992
12.
Manganel, M,
and
Turner RJ.
Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles.
J Membr Biol
102:
247-254,
1988[ISI][Medline].
13.
Martin, JB,
and
Doty DM.
Determination of inorganic phosphate, modification of isobutyl alcohol procedure.
Anal Chem
21:
965-967,
1949[ISI].
14.
Martinez, JR,
Cassity N,
and
Reed P.
Effects of isoproterenol on Cl transport in rat submandibular salivary gland acini.
Arch Oral Biol
33:
505-509,
1988[ISI][Medline].
15.
Matthews, JB,
Smith JA,
Tally KJ,
Awtrey CS,
Nguyen H,
Rich J,
and
Madara JL.
Na-K-2Cl cotransport in intestinal epithelial cells.
J Biol Chem
269:
15703-15709,
1994
16.
Mochly-Rosen, D.
Localization of protein kinases by anchoring proteins: a theme in signal transduction.
Science
268:
247-251,
1995[ISI][Medline].
17.
Paulais, M,
and
Turner RJ.
Activation of the Na+-K+-2Cl cotransporter in rat parotid acinar cells by aluminum fluoride and phosphatase inhibitors.
J Biol Chem
267:
21558-21563,
1992
18.
Paulais, M,
and
Turner RJ.
-Adrenergic upregulation of the Na+-K+-2Cl
cotransporter in rat parotid acinar cells.
J Clin Invest
89:
1142-1147,
1992[ISI][Medline].
19.
Tanimura, A,
Kurihara K,
Reshkin SJ,
and
Turner RJ.
Involvement of direct phosphorylation in the regulation of the rat parotid Na+-K+-2Cl cotransporter.
J Biol Chem
270:
25252-25258,
1995