(Received for publication, July 17, 1995)
From the
We identify a 175-kDa membrane phosphoprotein (pp175) in rat
parotid acini whose properties correlate well with the
Na-K
-2Cl
cotransporter previously characterized functionally and biochemically
in this tissue. pp175 was the only phosphoprotein immunoprecipitated by
an anti-Na
-K
-2Cl
cotransporter antibody and the only membrane protein whose
phosphorylation state was conspicuously altered after a brief (45-s)
exposure of acini to the
-adrenergic agonist isoproterenol.
Phosphopeptide mapping provided evidence for three phosphorylation
sites on pp175, only one of which was labeled in response to
isoproterenol treatment. The half-maximal effect of isoproterenol on
phosphorylation of pp175 (
20 nM) was in excellent
agreement with its previously demonstrated up-regulatory effect on
cotransport activity. Increased phosphorylation of pp175 was also seen
following acinar treatment with a permeant cAMP analogue and with
forskolin, conditions that have likewise been shown to up-regulate the
cotransporter. Combined with earlier results from our laboratory, these
data provide strong evidence that the up-regulation of the
cotransporter by these agents is due to direct phosphorylation mediated
by protein kinase A. AlF
treatment, which
results in an up-regulation of cotransport activity comparable with
that observed with isoproterenol (
6-fold), caused a similar
increase in phosphorylation of pp175. However, hypertonic shrinkage and
treatment with the protein phosphatase inhibitor calyculin A, which
also up-regulate the cotransporter (
3-fold and
6-fold,
respectively) caused no change in the phosphorylation level.
Furthermore, although acinar treatment with the muscarinic agonist
carbachol results in a dramatic up-regulation of cotransport activity
and a concomitant phosphorylation of pp175, no phosphorylation of pp175
was seen with the Ca
-mobilizing agent thapsigargin,
which is able to fully mimic the up-regulatory effect of carbachol on
transport activity. Taken together, these results indicate that direct
phosphorylation is only one of the mechanisms involved in
secretagogue-induced regulation of the rat parotid
Na
-K
-2Cl
cotransporter.
Because of its experimental accessibility, relative homogeneity
and rich hormonal responsiveness, the rat parotid gland is rapidly
becoming one of the more popular mammalian experimental models for the
study of the mechanism(s) and regulation of epithelial fluid and
electrolyte secretion(1, 2) . Work from a number of
laboratories has established that salt and water secretion by the
acinar cells, which comprise the bulk of this gland, is due to
transepithelial Cl movement(1, 2, 3) . The active step in
this process is Cl
entry across the acinar
basolateral membrane, a large component of which has been shown to be
due to Na
-K
-2Cl
cotransport(4, 5) .
Consistent with its important
role in secretion, we have shown that the activity of the rat parotid
Na-K
-2Cl
cotransporter is regulated by a number of physiological and other
potentially physiologically relevant stimuli. We first demonstrated a
substantial (
6-fold) up-regulation of cotransporter activity
following
-adrenergic stimulation and provided good evidence that
this was due to a phosphorylation event mediated by cyclic
AMP-dependent protein kinase(6) . This up-regulation is
paralleled in vivo by an increase in salivary flow seen when
sympathetic (adrenergic) stimulation, arising, for example, from
mastication, is superimposed on parasympathetic (muscarinic)
stimulation(7) , the main fluid secretory stimulus for the
gland. In a later publication (8) we demonstrated that the rat
parotid Na
-K
-2Cl
cotransporter is up-regulated (again
6-fold) by aluminum fluoride
(AlF
), an activator of G-proteins, and by
calyculin A, a protein phosphatase inhibitor. Based on several factors,
including diverse sensitivity to blockade of up-regulation by protein
kinase inhibitors and the observation that AlF
does not induce cAMP generation in the rat parotid, we have
argued that the mechanisms of action of AlF
and calyculin A on the cotransporter are different from that of
-adrenergic stimulation and from one another(8) .
More
recently (9) ()we have shown that
Na
-K
-2Cl
cotransport activity in these cells is also increased by muscarinic
stimulation (>15-fold) and by hypertonic shrinkage (
3-fold).
Our data suggest that these latter effects are also unrelated to one
another and unrelated to the effect of
-adrenergic stimulation
(see ``Discussion''). At this time our understanding of these
up-regulatory events is still incomplete, and the physiological
significance of some of these stimuli remains to be determined.
However, our results clearly demonstrate that the rat parotid
Na
-K
-2Cl
cotransporter is under tight regulatory control, in all likelihood by
multiple intracellular signaling pathways, and thus that it provides a
particularly rich experimental system for the study of transport
regulation by hormonal and other stimuli.
In the present paper we
explore these phenomena further by studying the effects of these
various up-regulatory stimuli on the phosphorylation state of the
Na-K
-2Cl
cotransport protein itself. Although it is generally accepted that
phosphorylation events play an important role in cellular signaling,
relatively few studies have actually directly explored their possible
involvement in the regulation of facilitative membrane transport
proteins. We show here that there is a good correlation between
increased transport activity and increased transporter phosphorylation
following
-adrenergic stimulation and AlF
treatment of rat parotid acini, suggesting that the regulation of
the cotransporter by these stimuli is due to direct phosphorylation.
Somewhat surprisingly, however, this was not the case for the other
stimuli studied, in spite of the fact that some of these agents have
been shown to increase both the transport activity and the
phosphorylation state of
Na
-K
-2Cl
cotransporters in lower species(11, 12) . These
observations indicate that the
Na
-K
-2Cl
cotransporter in the rat parotid is regulated both via direct
phosphorylation and via other, as yet unidentified, mechanisms.
The
digestion medium was Earle's minimum essential medium (Biofluids,
Rockville, MD) containing 0.22 units/ml collagenase P, 2 mM glutamine, and 1% bovine serum albumin. The physiological salt
solution (PSS) contained 135 mM NaCl, 5.8 mM KCl, 1.8
mM CaCl, 0.8 mM MgSO
, 0.73
mM NaH
PO
, 11 mM glucose, 20
mM HEPES (pH 7.4 with NaOH), 2 mM glutamine, and 1%
bovine serum albumin. The digestion medium and PSS were continuously
gassed with 95% O
, 5% CO
and 100%
O
, respectively. The stop solution for the
P
labeling studies contained 100 mM NaCl, 20 mM HEPES (pH 7.4 with NaOH), 10 mM Na
ATP, 50 mM NaF, 15 mM sodium
pyrophosphate, 100 µM sodium orthovanadate, 5 mM EDTA, 300 µM phenylmethylsulfonyl fluoride, 100
µML-tosylamido-2-phenylethyl chloromethyl
ketone, 1.5 µM pepstatin, and 1.5 µM leupeptin.
Protein concentration was measured with the BCA protein assay system (Pierce).
Aliquots (200 µl) of labeled cells were incubated with the agents indicated at 37 °C in siliconized glass tubes (Sigmacote number SL-2; Sigma). The incubation was terminated by the addition of 800 µl of ice-cold stop solution and disruption by immersion in a Branson B-12 sonicator bath (Shelton, CT) as follows. Each sample was first sonicated for 30 s and then placed on ice. After disruption of all samples in this way, each sample was subsequently sonicated to clarity.
A 400-µl aliquot of Triton extract was incubated overnight at 4 °C with immune or non-immune sheep serum (7 µl/100 µg of extract protein). Prewashed protein G-Sepharose beads (10 µl of beads/µl of serum) were then added. After 30 min of additional incubation, the beads were collected by centrifugation and washed six times with washing buffer. The tube was changed for the last spin. Protein retained by the washed beads was then eluted with 100 µl of electrophoresis sample buffer.
Immunoprecipitation
of [H]bumetanide binding activity from the above
lipid-stabilized Triton extracts was carried out using immune and
nonimmune IgG preabsorbed onto protein G-Sepharose beads. This was done
in order to avoid any possible interference of serum with the
[
H]bumetanide binding assay and to allow
quantitation of protein remaining after immunoprecipitation (see
``Results''). Protein G beads were washed twice with Buffer K
containing 0.3% Triton X-100 and 0.15% phosphatidylserine (sonicated to
clarity as above) and then resuspended in the same buffer containing 1%
ovalbumin and incubated for 40 min with immune or nonimmune sheep serum
(10 µl of beads/µl of serum; total volume
300 µl). The
beads were then washed three times in Buffer K plus 0.3% Triton X-100
and 0.15% phosphatidylserine, added to the lipid-stabilized Triton
extract (70 µl of beads/100 µg of extract protein), and
incubated for 2 h at 4 °C. After removal of the beads by
centrifugation, [
H]bumetanide binding activity
remaining in the resulting supernatant was determined by the method
given below.
Figure 1:
Effects of isoproterenol on protein
phosphorylation in rat parotid acinar cells. Rat parotid acini were
labeled with P
(see ``Experimental
Procedures'') and then incubated with (+) or without
(-) 1 µM isoproterenol for 45 s at 37 °C. After
disruption of the cells by sonication, the resulting homogenates were
centrifuged to produce cytosolic and particulate fractions, and the
particulate fractions were further separated into Triton extracts and
Triton-insoluble fractions (see ``Experimental Procedures''
for details). Aliquots of the cytosolic fractions (Cy), Triton
extracts (TE) and Triton-insoluble (TI) fractions
were then analyzed by SDS-PAGE (4-20% gradient gel) and
autoradiography. The total volumes of the cytosolic fractions, Triton
extracts, and Triton-insoluble fractions were 1 ml, 0.7 ml, and 0.7 ml,
respectively, and the respective volumes of each fraction run on the
gel were in the ratio 1.0:0.7:0.7 (protein loaded 39, 4.1, and 2.4
µg, respectively). The positions of the molecular weight markers
are indicated on the left of the autoradiograph. The
bands indicated by the arrows are discussed under
``Results.''
A number of factors discussed in the remainder of
the paper provide strong evidence that the 175-kDa phosphoprotein
(pp175) identified above is (a major part or all of) the rat parotid
Na-K
-2Cl
cotransporter.
Figure 2:
Effects of isoproterenol concentration and
cAMP on the phosphorylation of pp175. P
-Labeled acini were exposed to the
concentrations of stimuli indicated for 45 s at 37 °C and then
Triton-extracts were isolated and analyzed by SDS-PAGE and
autoradiography as in Fig. 1. The phosphorylation of the 175-kDa
phosphoprotein (pp175) identified in Fig. 1was quantified by
scanning densitometry of the resulting autoradiographs. The pp175
phosphorylation determined in this way for each experimental condition
has been normalized to the pp175 phosphorylation determined from a
control (untreated) sample from the same preparation run on the same
gel. The results shown are the means ± S.E. of three or more
independent experiments. A, phosphorylation of pp175 versus isoproterenol (ISO) concentration. B,
phosphorylation of pp175 after acinar treatment with isoproterenol, the
permeant cAMP analogue dibutyryl cAMP (DBcAMP), and the
activator of adenylate cyclase forskolin (FOR).
In parotid acinar cells,
cAMP is thought to be the major intracellular messenger mediating the
effects of -adrenoreceptor stimulation. In our earlier work (6) we also demonstrated that significant up-regulation of
cotransport activity was seen following acinar treatment with permeant
analogues of cAMP and with forskolin, which increases intracellular
cAMP by direct activation of the catalytic subunit of adenylate
cyclase. Consistent with the effects of these agents on transport, in Fig. 2B we show that increased phosphorylation of pp175
is likewise seen when acini are treated with the permeant cAMP analogue
dibutyryl cAMP and with forskolin.
The strong correlation
established in Fig. 2between the effects of isoproterenol and
cAMP on phosphorylation of pp175 and their previously documented
effects on Na-K
-2Cl
cotransport activity (6) supports the hypotheses that pp175 is
the rat parotid
Na
-K
-2Cl
cotransporter and that its up-regulation by isoproterenol is due to
direct phosphorylation.
Figure 3:
Immunoprecipitation of
Na-dependent bumetanide binding activity by antiserum
against the parotid bumetanide binding protein. A lipid-stabilized
Triton extract of the particulate fraction of rat parotid acini was
prepared and subjected to immunoprecipitation using protein G-Sepharose
beads preincubated with nonimmune sheep serum or immune serum raised
against the rabbit parotid bumetanide binding protein (see
``Experimental Procedures''). The
Na
-dependent component of
[
H]bumetanide binding (see ``Experimental
Procedures'') in the lipid-stabilized Triton extract before
immunoprecipitation (Control) and in the supernatant remaining
after immunoprecipitation with immune (I) or nonimmune (N) serum are shown. The results are the means ± S.E.
of three independent experiments. The data from each experiment were
normalized to the binding observed in the lipid-stabilized extract
before immunoprecipitation (5.28 ± 0.75 pmol/mg protein, n = 3); the data were also corrected for the dilution arising
from addition of protein G-Sepharose beads.
When this
polyclonal antiserum was used in immunoprecipitation studies with
Triton extracts from P
-labeled cells it
specifically precipitated pp175. This result is illustrated in Fig. 4. Here we compare autoradiographs of SDS-PAGE gels of
material immunoprecipitated with nonimmune (N) or immune (I) serum from extracts of
P
-labeled
acini pretreated with (+) or without(-) 1 µM
isoproterenol. A single phosphoprotein appearing as a diffuse band
centered at M
175,000 whose phosphorylation
is markedly increased by isoproterenol treatment is seen in the
precipitate from immune serum. No phosphoproteins were
immunoprecipitated by nonimmune serum. In addition, when the
supernatants remaining after immunoprecipitation with immune serum were
examined by SDS-PAGE and autoradiography, no
P
-labeled band at 175 kDa was detectable (not
shown), indicating that all of the labeled protein at 175 kDa is
recognized by the
anti-Na
-K
-2Cl
cotransporter antibody.
Figure 4:
Immunoprecipitation of pp175 by
antiserum against the parotid bumetanide binding protein. Rat parotid
acini labeled with P
were incubated with
(+) or without(-) 1 µM isoproterenol for 45 s
at 37 °C. The Triton extract of the particulate fraction was then
immunoprecipitated with immune (I) or nonimmune (N)
sheep serum as described under ``Experimental Procedures.''
The figure shows autoradiographs of SDS-PAGE gels of the
initial Triton extracts on the left, the immunoprecipitates
from untreated cells in the center, and the immunoprecipitates
from isoproterenol-treated cells on the right, as
indicated.
Figure 5:
Phosphopeptide mapping of pp175 using V8
protease. Rat parotid acini were labeled with P
and then incubated with (+) or without(-) 1 µM isoproterenol for 45 s at 37 °C as usual. Digestion of pp175
with V8 protease (for 6 or 12 h, as indicated) and Tricine-SDS
electrophoresis were then carried out as described under
``Experimental Procedures.'' The figure shows an
autoradiograph of a representative Tricine-SDS gel. The positions of
the molecular weight markers are indicated on the left of the autoradiograph.
On average little difference was found between the
phosphorylation patterns observed after 6 and 12 h of V8 protease
digestion. The density of labeling of the 7.5-kDa band did, however,
decrease significantly between 6 and 12 h of protease treatment (20
± 5% and 31 ± 9% decreases were found in digests from
control and isoproterenol-treated cells, respectively), presumably
indicating continued slow digestion of this peptide by V8 protease. No
other significant increase or decrease in labeling with time of
protease treatment was observed. In particular, paired t tests
provided no evidence for a systematic shift of P
from the 7.5-kDa to the 5.7-kDa peptide with time. This
observation argues against the possibility that the latter peptide may
be a digestion product of the former. Since all of the labeled protein
at 175 kDa is recognized by our
anti-Na
-K
-2Cl
cotransporter antibody (see above), all three of these labeled peptides
are presumably associated with the transporter. Thus the results
illustrated in Fig. 5are consistent with the presence of at
least three phosphorylation sites on pp175, only one of which is
phosphorylated in response to isoproterenol treatment.
Figure 6:
Effects of
AlF, calyculin A, hypertonic shrinkage,
and low Cl
medium on the phosphorylation of pp175.
P
-labeled acini were exposed to the stimuli
indicated below, and the resulting phosphorylation of pp175 was
quantitated and normalized as described in the caption to Fig. 2. The stimuli were 1 µM isoproterenol for 45
s (ISO), 15 mM NaF plus 10 µM AlCl
for 260 s (AlF
), 1 µM calyculin A for 300 s (CA), PSS plus 80 mM sucrose for 40 s (SUC), and low chloride medium for 120 s (low Cl). Cells were switched to low chloride medium by
diluting acini in PSS 1:1 with PSS in which NaCl was replaced with
NaNO
. Previous work from our laboratory has shown that the t
for
Cl
loss
from rat parotid acinar cells switched to Cl
-free
medium is
2 min at 26 °C (S.I. Lee and R.J. Turner, unpublished
results). Thus this treatment (at 37 °C) is expected to lower
intracellular Cl
levels by at least 25%. The results
shown are the means ± S.E. of three or more independent
experiments.
In the shark rectal gland
experimental maneuvers that reduce intracellular chloride concentration
have been shown to result in up-regulation of
Na-K
-2Cl
cotransport activity and phosphorylation of the
Na
-K
-2Cl
cotransport protein(11) . However, as also illustrated in Fig. 6, switching rat parotid acini to low chloride medium has
no effect on the phosphorylation of pp175.
Recent work in our
laboratory (9) has demonstrated a dramatic up-regulation of rat
parotid Na-K
-2Cl
cotransport activity by muscarinic stimulation (
15-fold and
25-fold after 30 s of stimulation with 1 µM and 10
µM of the muscarinic agonist carbachol, respectively). In Fig. 7we show that this up-regulation is paralleled by increased
phosphorylation of pp175. However, in additional experiments (9) we have shown that the up-regulatory effect of carbachol on
the cotransporter can be duplicated by the microsomal
Ca
-ATPase inhibitor thapsigargin, which raises
intracellular calcium concentration to levels comparable with that seen
with carbachol, but without interacting with plasma membrane receptors,
and without activating protein kinase C. We illustrate in Fig. 7that treatment of acini with 1 µM thapsigargin under conditions that yield an up-regulation of
cotransport activity comparable with that produced by 1 µM carbachol ( (9) and data not shown), results in no
significant phosphorylation of pp175. In addition, treatment of acini
with the active phorbol ester PMA to activate protein kinase C yields
no phosphorylation of pp175 in the presence or absence of thapsigargin (Fig. 7).
Figure 7:
Effects of carbachol, thapsigargin, and
PMA on the phosphorylation of pp175. P
-labeled
acini were exposed to the stimuli indicated, and the resulting
phosphorylation of pp175 was quantitated and normalized as described in
the caption to Fig. 2. Acini were exposed to carbachol (CCh) for 40 s and to thapsigargin (ThG) and/or PMA
for 120 s. The results shown are the means ± S.E. of three or
more independent experiments.
In this paper we identify a 175-kDa membrane phosphoprotein
(pp175) in the rat parotid whose properties correlate well with the
Na-K
-2Cl
cotransporter characterized functionally in this gland (6) and
with Na
-K
-2Cl
cotransporters previously identified biochemically in salivary glands (15) and other
tissues(11, 20, 21, 22, 23) .
pp175 was the only membrane protein whose phosphorylation state was
conspicuously altered after a brief (45-s) exposure to the
cAMP-mobilizing secretagogue isoproterenol (Fig. 1). We have
previously demonstrated that isoproterenol treatment results in a
substantial up-regulation of
Na
-K
-2Cl
cotransport activity in the rat parotid and provided strong evidence
that this was due to a phosphorylation event mediated by protein kinase
A(6) . Consistent with the identification of pp175 as the
Na
-K
-2Cl
cotransporter, the half-maximal effect of isoproterenol on
phosphorylation of pp175 (
20 nM; Fig. 2A)
was in excellent agreement with its half-maximal effect on cotransport
activity(6) . Phosphopeptide mapping provided evidence for
three phosphorylation sites on pp175 (Fig. 5), only one of which
was labeled in response to isoproterenol treatment.
Increased
phosphorylation of pp175 was also seen following acinar treatment with
a permeant cAMP analogue and with forskolin (Fig. 2B),
conditions that have also been shown to up-regulate the cotransporter,
presumably by the same mechanism as isoproterenol(6) . In
addition, pp175 was the only phosphoprotein immunoprecipitated by an
antibody raised against the rabbit parotid
Na-K
-2Cl
cotransporter (Fig. 4). This antibody also quantitatively
immunoprecipitated sodium-dependent bumetanide binding activity from a
detergent extract of the rat parotid (Fig. 3) consistent with
the expected properties of an anti-cotransporter antibody. Taken
together with its molecular weight, which is in the expected range
(150,000-195,000) of previously identified
Na
-K
-2Cl
cotransporters(15, 20, 21, 22, 23) ,
the above results provide convincing evidence that pp175 is the
Na
-K
-2Cl
cotransporter of the rat parotid.
The effects of cAMP-dependent
secretagogues on the phosphorylation state of
Na-K
-2Cl
cotransporters recently identified in the shark rectal gland ((11) ; a 195-kDa phosphoprotein) and the avian salt gland ((23) ; a 170-kDa phosphoprotein) have also been studied.
Consistent with the results presented here, in both these tissues a
strong correlation between apparent up-regulation of transport activity
and cotransporter phosphorylation was
observed(11, 23) .
In their study of the
phosphorylation of the shark rectal gland
Na-K
-2Cl
cotransporter, Lytle and Forbush (11) also showed that
transport up-regulation by osmotic shrinkage is accompanied by parallel
increases in transporter phosphorylation. In addition, they showed that
maneuvers that decrease intracellular chloride concentration in this
tissue result in increased cotransporter phosphorylation. This latter
observation is consistent with previous suggestions from this group
that the decreased intracellular chloride concentration that
accompanies secretion by the gland may itself play a role in the
activation of the cotransporter(10, 11, 22) .
In the avian salt gland Torchia et al.(12, 23) have demonstrated that
Ca
-mobilizing secretagogues also result in
cotransporter phosphorylation. This is apparently due to the combined
effect of increased intracellular calcium concentration and activation
of protein kinase C, since it can be mimicked by the application of a
Ca
ionophore plus an active phorbol ester but not by
either of these treatments alone(12) . Treatment with the
protein phosphatase inhibitor okadaic acid also resulted in
cotransporter phosphorylation in the avian salt gland(12) .
The above results from the shark rectal gland and avian salt gland
are consistent with the hypothesis that direct phosphorylation,
possibly at different sites by different stimuli, plays a central role
in the regulation of
Na-K
-2Cl
cotransport activity in these tissues. However, the situation is
clearly more complex in the rat parotid. Although the up-regulation of
transport activity seen with isoproterenol and
AlF
(both
6-fold) correlates well
with the phosphorylation of pp175 induced by these agents ( Fig. 2and Fig. 6), this is not the case for treatment
with the protein phosphatase inhibitor calyculin A or hypertonic
shrinkage (Fig. 6). Despite the fact that both these latter
treatments result in significant up-regulation of the cotransporter
(
6-fold and
3-fold, respectively; (8) and Footnote
1), neither causes significant phosphorylation of pp175. Furthermore,
although acinar treatment with the muscarinic agonist carbachol results
in a dose-dependent phosphorylation of pp175 (Fig. 7), no
phosphorylation is produced by the Ca
-mobilizing
agent thapsigargin, which is able to fully mimic the dramatic
up-regulatory effect of carbachol on transport activity (
15-fold
for treatment with either 1 µM carbachol or 1
µM thapsigargin; (9) ). Moreover, treatment of
acini with the active phorbol ester PMA to activate protein kinase C
yielded no phosphorylation of pp175 in the presence or absence of
thapsigargin (Fig. 7). Taken together, these latter results
indicate that the phosphorylation of pp175 seen with muscarinic
stimulation is not required for up-regulation of cotransport activity
in the rat parotid and, in addition, suggest that this phosphorylation
is not due to protein kinase C.
Finally, two observations made here indicate that the increase in cotransporter phosphorylation associated with decreased intracellular chloride concentration in the shark rectal gland (see above) is not seen in the rat parotid: (i) increased phosphorylation of pp175 is not observed after suspension of acini in low chloride medium (Fig. 6), and (ii) increased phosphorylation of pp175 is not observed after thapsigargin treatment (Fig. 6), which is expected to lead to a secretion-induced decrease in intracellular chloride concentration similar to that observed with muscarinic stimulation(4) .
As already indicated, the
experimental evidence available to date suggests that the
up-regulations of the parotid
Na-K
-2Cl
cotransporter by treatment with
-adrenergic agonists, muscarinic
agonists, hypertonic shrinkage, AlF
, and
calyculin A, all occur via different intracellular mechanisms. Briefly
stated, this conclusion is based on the observations that the effects
of
-adrenergic and muscarinic stimulation are secondary to
increased intracellular levels of cAMP (6) and
Ca
(9) , respectively, while the effects of
hypertonic shrinkage, AlF
, and calyculin
A are apparently independent of both of these intracellular messengers (8) .
The effects of these latter three stimuli on
the cotransporter can, however, be distinguished by their sensitivities
to inhibition by the compound K252a (K
0.6
µM,
20 µM, and 20 µM,
respectively; (8) and Footnote 1). It is nevertheless always
possible that the effects of some of these stimuli may be related. For
example, two stimuli may act at different steps in the same
up-regulatory pathway, resulting in the apparent differences discussed
above.
We also considered the possibility that agents which resulted in an up-regulation of cotransport activity without a concomitant increase in the phosphorylation of pp175 might be acting via phosphorylation of another membrane-associated protein. However, close examination of autoradiographs of Triton extracts from cells treated with thapsigargin, hypertonic shrinkage, or calyculin A did not reveal changes in the phosphorylation pattern of proteins at any molecular weight.
The results presented here support the hypothesis that the
up-regulations of the rat parotid
Na-K
-2Cl
cotransporter by
-adrenergic stimulation and
AlF
treatment are due to direct
phosphorylation of the transporter itself, whereas other mechanisms are
clearly involved in the up-regulatory effects of muscarinic
stimulation, hypertonic shrinkage, and calyculin A treatment.