Regulation of the renal
Na-HCO3 cotransporter. XI.
Signal transduction underlying CO2
stimulation
Ofelia S.
Ruiz,
R. Brooks
Robey,
Yi-Yong
Qiu,
Long Jiang
Wang,
Cheng Jin
Li,
Jianfei
Ma, and
Jose A. L.
Arruda
University of Illinois at Chicago, College of Medicine, and Chicago
Veterans Affairs Health Care System, West Side Division, Chicago,
Illinois 60612-7315
 |
ABSTRACT |
We have previously shown that
CO2 stimulation of the renal
Na-HCO3 cotransporter (NBC)
activity is abrogated by general inhibitors of protein tyrosine
kinases. The more selective inhibitor herbimycin also blocked this
effect at concentrations known to preferentially inhibit Src family
kinases (SFKs). We therefore examined a role for SFKs in
CO2-stimulated NBC activity. To
this end, we engineered OK cells to express the COOH-terminal Src
kinase (Csk), a negative regulator of SFKs.
CO2 stimulated NBC activity
normally in
-galactosidase-expressing and untransfected control
cells. In contrast, Csk-expressing cells had normal baseline NBC
activity that was not stimulated by
CO2. CO2 stimulation increased both
total SFK activity and specific tyrosine phosphorylation of Src. The
specific MEK1/2 inhibitor PD-98059 completely inhibited
the CO2 stimulation of NBC
activity as well as the accompanying phosphorylation and activation of ERK1/2. Our data suggest the involvement of both SFKs, probably Src,
and the "classic" MAPK pathway in mediating
CO2-stimulated NBC activity in
renal epithelial cells.
Src family kinase; ERK1/2 kinase; phosphorylation
 |
INTRODUCTION |
VECTORIAL HCO3
transport from the renal proximal tubule cell into the blood is
mediated by the basolateral
Na-HCO3 cotransporter, NBC (2). We
have shown that NBC is regulated by ambient pH (27), by glucocorticoids
(23), and by activators of both G protein-coupled receptors, e.g.,
carbachol, angiotensin II, parathyroid hormone (21, 22, 24), and
receptor protein tyrosine kinases, e.g., insulin and epidermal growth
factor (EGF) (19). A number of pH-dependent and pH-independent signal
transduction mechanisms, involving G proteins (25), protein kinase A
(PKA), PKC, and calcium-calmodulin kinase (26), have been
implicated as mediators of these effects. Of relevance to the present
work, we have previously shown that general inhibitors of protein
tyrosine kinases prevent the adaptive increase in NBC activity in
response to acute CO2 elevation,
suggesting that tyrosine phosphorylation is involved in NBC regulation
(20). The nonreceptor protein tyrosine kinase c-Src plays a role in the
analogous adaptive increase in renal brush border Na/H exchanger
activity (NHE3) that accompanies metabolic acidosis (32). Moreover, Src
kinase activity is increased by both metabolic acidosis and decreased
intracellular pH
(pHi) in a murine
proximal tubule cell line (34). We therefore sought to examine whether
Src family kinase (SFK), and Src in particular, may play a
similar role in the regulation of NBC by
CO2. To this end, we tested the
ability of COOH-terminal Src kinase (Csk), a negative regulator of
SFKs, to influence NBC activity when expressed in cultured rabbit
proximal tubule cells and OK (American opossum kidney) cells. Since the
"classic" MAPK pathway mediates a number of downstream effects of
Src activation, we also examined its role in the stimulation of NBC by
CO2.
 |
MATERIALS AND METHODS |
Materials. Mycoplasma-free OK cells
were obtained from the American Type Culture Collection (Rockville, MD)
at passage 37. Mammalian expression vectors containing full-length
cDNAs for rat (pLXSH/Csk) and chicken (pcDNA1/Csk) Csk
were provided by Drs. Jonathan Cooper (University of Washington,
Seattle, WA) and Hidesaburo Hanafusa (Rockefeller University, New York,
NY), respectively. Standard DMEM-F12 cell culture medium was purchased
from Atlanta Biologicals (Norcross, GA), and all other cell culture
reagents, including LipofectAMINE. were obtained from GIBCO-BRL (Life
Technologies, Gaithersburg, MD). The acetoxymethyl ester
of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) was purchased from Molecular Probes (Eugene,
OR), amiloride was from Research Biochemicals (Natick, MA), hygromycin
was from Calbiochem Novabiochem (San Diego, CA), c-Src
and Csk specific antibodies were from Santa Cruz Biotechnologies (Santa
Cruz, CA), and RC20 anti-phosphotyrosine was from Transduction
Laboratories (Lexington, KY), ERK1/2-specific antibodies and protein
tyrosine kinase assay kits specific for SFKs and ERK1/2 were obtained
from Upstate Biotechnology (Lake Placid, NY). The enhanced
chemiluminescent detection kit (ECL) used to analyze immunoblots was
obtained from Amersham (Arlington Heights, IL). All other chemicals
were purchased from Sigma Chemicals (St. Louis, MO).
Isolation and culture of rabbit proximal tubule
segments and culture of OK cells. Proximal tubules were
isolated from New Zealand White rabbits as previously described (4,
27). Electron microscopy confirmed that these preparations were
homogenous, with over 98% of the isolated tubules exhibiting
morphological characteristics of proximal tubules. We have previously
shown that explant cultures from these tubules retain morphological, enzymatic, and transport functions of the original epithelium (19-26). Isolated proximal tubules were routinely suspended and cultured in defined serum-free growth medium: a mixture (50:50) of
DMEM-F12 medium containing 24 mM
NaHCO3, 192 IU/ml penicillin, and
200 µg/ml streptomycin, 5 µg/ml bovine insulin, 5 µg/ml human transferrin, and 5 × 10
8 M hydrocortisone.
Cultures were maintained at 37°C in a 5%
CO2 incubator at pH 7.40. Growth
medium was changed regularly every 3-4 days. Confluence was
typically achieved within 8-10 days, after which cells were
detached by trypsinization and replated on clear plastic coverslips.
Confluence was typically achieved 2-3 days later, and cells were
then used for fluorometric assays of NBC activity. Cultured proximal
tubule cells were rendered quiescent by removal of hormones 48 h prior
to experimental procedures.
OK cells were cultured in standard Eagle's MEM medium supplemented
with 10% fetal calf serum and were maintained in 5%
CO2 at 37°C. To minimize the
effects of phenotypic variation in culture, we routinely tested cells
between passages 38 and 43. OK cells were typically serum deprived for
24 h prior to and during any experimental procedures.
Fluorometric assay of
pHi and NBC activity in cultured renal
epithelial cells.
pHi was continuously monitored
using the pH-sensitive fluorescent probe, BCECF as previously described
(4, 23). In brief, cells grown on coverslips were perfused with a
Cl-free solution to minimize contributions by both the Na-dependent
Cl/HCO3 exchanger and the
HCO3 /Cl
exchanger. Amiloride, 1 mM, was also included to inhibit
Na/H exchanger activity. The cell chamber was perfused prior to assay
with a Cl-free solution containing (in mM) 25 NaHCO3, 110 sodium gluconate, 5 potassium gluconate, 9 HEPES (pH 7.40), 2 CaSO4, 1 KH2PO4,
0.5 MgSO4, 10 glucose, and 1 amiloride at a rate of 20 ml/min at 37°C. Extracellular pH was
maintained constant at 7.40. Once a stable fluorescence signal was
achieved, Na was removed by equimolar substitution of choline for Na.
Na removal resulted in a decrease in
pHi that rapidly and fully
recovered upon Na readdition. NBC activity was assayed as the initial
rate of pHi recovery following the
addition of NaHCO3 to cells
perfused in the absence of Na. pHi
recovery was inhibitable by DIDS and, in the absence of chloride, was
primarily attributable to NBC activity (2). pH-sensitive BCECF
fluorescence was routinely calibrated in the presence of elevated
extracellular K+ and the ionophore
nigericin (to equilibrate intracellular and extracellular pH). All
measurements were performed by dual-wavelength monitoring and
ratiometric analysis (18) at pH-sensitive and pH-insensitive excitation
wavelengths
(F500 /F450)
at the completion of each experiment. Buffer capacity of the cells was
determined from the pHi change as
described (18).
Lipofection-mediated gene transfer.
Transfection with mammalian expression vectors encoding rat (pLXSH/Csk)
or chicken (pcDNA1/Csk) Csk was accomplished by lipofection using
LipofectAMINE (GIBCO-BRL) according to the manufacturer's
recommendations. Rat Csk was expressed in OK cells under the control of
a retroviral long terminal repeat (LTR) promoter, and
stable transfectants were selected by hygromycin resistance conferred
by the parent pLXSH vector (11). Individual clones were isolated and
propagated in complete growth medium supplemented with 100 µg/ml
hygromycin. The cells were incubated until they reached confluence
before testing. Overexpression of the Csk protein was tested in
10-12 clones by Western blot analysis. Transient transfection of
rabbit proximal tubule cells was achieved in a similar manner but using
pcDNA1/Csk, which expresses chicken Csk under the control of a
cytomegalovirus promoter (28).
Cell lysate preparation and
immunoblotting. Confluent cell monolayers (75 cm2) were rinsed with ice-cold
phosphate-buffered saline and harvested in 1 ml of modified RIPA buffer
[50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5%
sodium deoxycholate, and 0.1% SDS] supplemented with 100 µg/ml
phenylmethylsulfonyl fluoride. After incubation on ice for 30 min and
centrifugation at 15,000 g for 20 min
at 4°C, the resulting supernatant was mixed with SDS gel-loading buffer [50 mM Tris-Cl (pH 6.8), 100 mM dithiotreitol, 2% SDS, and 0.1% bromophenol blue], boiled for 3 min, and separated by 10% SDS-PAGE. Resolved proteins were transferred onto a nitrocellulose membrane, which was then blocked by 2.5% fraction V bovine serum albumin in TBST (20 mM Tris-Cl, pH 8.0, 150 mM NaCl, and 0.5% Tween
20) for 2 h at room temperature. Following incubation with rabbit
polyclonal anti-Csk antibodies for 2 h at 4°C, specific bands
corresponding to Csk were detected and quantitated by incubation with
horseradish peroxidase-labeled secondary antibodies using the ECL
detection kit (Amersham).
Detection of specific Src and ERK1/2
phosphorylation. To examine specific phosphorylation,
Src immunoprecipitates were analyzed by parallel immunoblot analyses
using both rabbit polyclonal anti-Src (Santa Cruz Biotechnologies) and
mouse monoclonal anti-phosphotyrosine (RC20) antibodies (Transduction
Laboratories). Specific ERK1/2 phosphorylation was similarly examined
in ERK1/2 immunoprecipitates using both mouse polyclonal anti-ERK1/2
(Upstate Biotechnology) and mouse monoclonal RC20 antibodies. Detection
and quantitation of individual species were uniformly performed using a
commercially available chemiluminescent detection system (ECL, Amersham).
Measurement of SFK activity. The SFK
activity was evaluated using a commercially available kinase assay kit
(Upstate Biotechnology). In brief, we tested the ability of cell
lysates to specifically phosphorylate a synthetic peptide substrate of
SFKs corresponding to residues 6-20 of cdc2 (9). Samples were
incubated in a solution containing 10 mM Tris · HCl
(pH 7.2), 12.5 mM MgCl2, 10 mM
MnCl2, 0.2 mM EGTA, 25 µM
Na3VO4,
0.2 mM dithiothreitol, 50 µM ATP, and 10 µCi
[
-32P] ATP at
30°C for 10 min before stopping the reaction by the addition of
trichloroacetic acid. An aliquot was applied to a P81 phosphocellulose
paper. Unincorporated 32P was
eluted with 7.5% phosphoric acid, and the remaining incorporated radioactivity was determined by liquid scintillation counting.
Measurement of ERK1/2 kinase activity.
Total ERK1/2 kinase activity was measured as previously described (30)
using a commercially available kit (Upstate Biotechnology). In brief,
cell lysates were tested for the ability to specifically phosphorylate
myelin basic protein in the presence of inhibitors of PKC, PKA, and
calmodulin kinase II. Samples were incubated in 100 µM ATP, 15 mM
MgCl2, containing 10 µCi
[
-32P]ATP for 10 min at 30°C before application to P81 phosphocellulose paper.
Unincorporated 32P was eluted with
7.5% phosphoric acid, and the remaining incorporated radioactivity was
determined by liquid scintillation counting.
Statistical analysis. Results are
expressed as means ± SE, and statistical comparisons were performed
by t-testing for both paired or
unpaired data where appropriate.
 |
RESULTS |
Effects of herbimycin on
CO2 stimulation of NBC
activity.
We have previously shown that 10%
CO2-stimulated NBC activity in
cultured rabbit proximal tubule cells can be blocked by general tyrosine kinase inhibitors. Pretreatment of OK cells with herbimycin at
concentrations known to preferentially inhibit SFKs,
<10
6 M (7), had no effect
on basal NBC activity, but completely blocked the increase following
exposure to 10% CO2 (Fig.
1). These findings are in agreement with
our previous observations (20) and are compatible with a postulated
role for tyrosine kinases, and SFKs in particular, in regulating NBC
activity in proximal tubule cells.


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Fig. 1.
Summary of effect of herbimycin on
CO2 stimulation of NBC activity.
Intracellular pH (pHi) was
measured as previously described (Ref. 23). Cells were continuously
perfused with physiological solution containing 25 mM
NaHCO3 and 1 mM amiloride at pH
7.40. Once a stable fluorescence signal was achieved, Na was removed by
equimolar substitution of choline for Na. Na removal resulted in a
decrease in pHi that rapidly and
fully recovered upon Na readdition. NBC activity was assayed as the
initial rate of pHi recovery
following addition of NaHCO3 to
cells perfused in absence of Na. A:
representative experiment showing effect of herbimycin on
CO2-stimulated activity. Upon
removal of NaHCO3, decrease in
pHi was the same in all groups
(not shown). Addition of NaHCO3
resulted in recovery of pHi (NBC
activity) that was greater in 10%
CO2-treated than in control cells
or in cells pretreated with herbimycin.
B: OK cells were preincubated with
vehicle (control) or herbimycin,
10 6 M, and then exposed to
either 5% CO2 or 10%
CO2 for 5 min at 37°C.
pHi was measured using the
pH-sensitive fluorescent probe, BCECF-AM. Each bar represents mean ± SE; n = 6. Buffer capacity was
not different between control and
CO2-treated cells (26.6 ± 0.8 vs. 27.6 ± 0.9 mmol/pH unit). NS, not significant.
|
|
Overexpression of Csk in OK cells and rabbit proximal
tubule cells. To better address the role of SFKs on NBC
activation, we tested the functional consequences of impaired SFK
activation in both OK cells and cultured rabbit proximal tubule cells
(see below). To this end, we expressed a Csk transgene and examined the
effects on both basal and
CO2-stimulated NBC activity.
Untransfected cells and cells stably transfected with a
-galactosidase reporter gene were employed as controls. Cultured
proximal tubule cells were transiently transfected with the same
expression vector. Overexpression of Csk protein was confirmed by
Western blot analysis (Fig. 2).

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Fig. 2.
Overexpression of COOH-terminal Src kinase (Csk) in OK cells. Western
blot analysis of untransfected control (lane
1) and stably transfected (lane
2) OK cells with Csk. Cell lysates were resolved on a
10% SDS-PAGE, transferred onto a nitrocellulose membrane, and probed
with anti-Csk polyclonal antibody.
|
|
Effect of Csk overexpression on
CO2-stimulated NBC
activity.
Figure 3A
shows the effect of 10% CO2 on
NBC activity in cultured rabbit proximal tubule cells transfected with
pLXSH/Csk or with an empty control vector. In cells transfected with
vector alone, 10% CO2 stimulated
NBC activity normally from 1.49 ± 0.07 to 2.02 ± 0.19 pH
units/min (P < 0.001). In contrast,
10% CO2 failed to stimulate NBC
activity in cells expressing Csk [Csk, 1.39 ± 0.09; Csk + 10% CO2, 1.20 ± 0.11 pH
units/min; not significant (NS)]. Baseline NBC activity was not
different between cells transfected with an empty control vector and
pLXSH/Csk. In proximal tubule cells transiently expressing the chicken
homolog of Csk, similar results were obtained. The 10%
CO2 failed to stimulate NBC (Csk, 1.49 ± 0.10 vs. Csk + 10%
CO2, 1.25 ± 0.1 pH units/min;
NS, n = 8), suggesting that the effect
is not species specific.

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Fig. 3.
Effect of Csk overexpression on
CO2-stimulated NBC activity.
A: rabbit proximal tubule cells were
transiently transfected with an empty control vector or with pLXSH/Csk
cDNA, and NBC activity was measured in cells exposed either to 5% or
10% CO2 for 5 min. Each bar
represents mean ± SE; n = 8. B: NBC activity was measured in
untransfected (control) and stably transfected OK cells exposed to
either 5% or 10% CO2 for 5 min.
Each bar represents mean ± SE; n = 6.
|
|
Figure 3B shows the results obtained
in OK cells stably expressing rat Csk. In cells transfected with
control vector alone, 10% CO2
increased NBC activity significantly from 1.0 ± 0.07 to 2.02 ± 0.19 pH units/min (P < 0.01),
whereas in cells expressing Csk, 10%
CO2 failed to stimulate NBC
completely (Csk, 0.7 ± 0.08 vs. Csk + 10%
CO2, 0.82 ± 0.08 pH
units/min). Baseline NBC activity was not different between control
cells and cells stably expressing Csk. Additional experiment showed
that Csk-transfected cells had baseline NBC activity not different from
controls (1.06 ± 0.05 vs. 1.10 ± 0.07 pH units/min;
n = 6, NS).
Effect of Csk overexpression on dexamethasone
stimulation of NBC activity. Figure
4 shows that stable Csk expression did not prevent the stimulatory effect of
10
8 M dexamethasone on NBC
activity [control, 1.30 ± 0.02; dexamethasone, 1.78 ± 0.05 (P < 0.001); Csk, 1.33 ± 0.10; dexamethasone + Csk, 1.80 ± 0.06 pH units/min
(P < 0.01)]. The increase in
NBC activity by dexamethasone was of the same magnitude in both control
and Csk-expressing cells, indicating that the effect of Csk is specific for CO2.

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Fig. 4.
Effect of Csk overexpression on dexamethasone stimulation of NBC
activity. NBC activity was assayed in untransfected (control) and
stable pLXSH/Csk-transfected OK cells pretreated with either vehicle or
dexamethasone, 10 8 M, for
48 h as previously described (24). Each bar represents mean ± SE;
n = 6.
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|
Effect of CO2 on
SFK activity in OK cells.
Figure 5A shows the effect of 10%
CO2 on SFK activity.
CO2 increased SFK activity by over
50% above control levels within 3 min and then decreased toward
baseline levels at 30 min. Activation of NBC and SFK activity by
CO2 was also correlated with
increased tyrosine phosphorylation of endogenous Src, which was
similarly blocked by herbimycin, suggesting a causal relationship.
Figure 5B
shows that 10% CO2 increased
phosphorylation of Src by twofold, and herbimycin decreased
phosphorylation to control levels (expressed as percentage of control,
100%; CO2, 212.8 ± 28.8%,
P < 0.001; CO2 + herbimycin, 78.3 ± 13.4%, n = 4).

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Fig. 5.
Effect of CO2 on Src family kinase
(SFK) activity. A: SFK activity was
measured in OK cell lysates previously exposed to 10%
CO2 for the indicated time periods
using a commercially available kit. Each time point represents mean ± SE (n = 4) and is expressed as
the CO2-stimulated increase in SFK
activity. * P < 0.05 and
P < 0.001 vs. controls.
B: OK cell lysates exposed to 5%
CO2 (lanes
1 and 2) or to 10%
CO2 (lanes
3 and 4) for 5 min
in absence (lane 3) or in presence
of herbimycin (lane 4) were
immunoprecipitated with IgG (lane 1)
or with Src-specific polyclonal antibodies (lanes
2-4). Immunoprecipitates were probed with a
monoclonal anti-phosphotyrosine kinase antibody. Phosphoproteins were
detected by enhanced chemiluminescence. Result of a representative
experiment is shown.
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|
Effect of MEK1/2 inhibition on
CO2-stimulated NBC
activity.
Because a number of signaling pathways involving Src activation proceed
through the classic MAPK pathway (Ras
Raf1
MEK1/2
ERK1/2), we utilized the specific MEK1/2 inhibitor PD-98059 to
examine the role of this pathway. Figure 6
shows that the inhibitor failed to alter baseline activity of the
cotransporter (control, 1.58 ± 0.17; inhibitor, 1.53 ± 0.13 pH
units/min), but completely abolished the effect of 10%
CO2
(CO2, 2.17 ± 0.14;
CO2 + inhibitor, 1.57 ± 0.15 pH units/min; P < 0.02).

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Fig. 6.
Effect of MEK1/2 inhibition on
CO2-stimulated NBC activity. NBC
activity was assayed in OK cells pretreated with vehicle or PD-98059,
10 µM, and then exposed to 5% or 10%
CO2. Each bar represents mean ± SE; n = 6.
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Effect of CO2 on
ERK1/2 kinase activity.
The finding that PD-98059 inhibits
CO2 stimulation of NBC activity
suggests involvement of the classic MAPK pathway. Consistent with this
interpretation, 10% CO2 increased
ERK1/2 activity by over 50% (Fig.
7A), and
this effect was also blocked by PD-98059. Tyrosine phosphorylation of
ERK1/2 correlated with activation. Figure 7,
B and
C, shows that
CO2 increased phosphorylation of ERK1/2, which was likewise blocked by PD-98059 (expressed as percentage of control, 100%; CO2, 231.6 ± 28.2%, P < 0.001;
CO2 + PD-98059, 90.1 ± 31.6%,
P < 0.001, n = 4).


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Fig. 7.
Effect of CO2 on ERK1/2 kinase
activity. A: ERK1/2 kinase activity
was measured in lysates from OK cells exposed to 5% or 10%
CO2 in absence or in presence of
PD-98059. Each bar represents mean ± SE;
n = 6. B: lysates from OK cells exposed to
5% (lane 2) or 10%
CO2 (lane
3) or pretreated with angiotensin II,
10 7 M
(lane 4) or
10 11 M
(lane 5), were immunoprecipitated
with either IgG (lane 2) or with
ERK1/2-specific polyclonal antibodies (lanes
3-5). Lane 1 was a 3T3 lysate, a positive control. Immunoblotting was done using
anti-phosphotyrosine monoclonal antibodies as a probe. Result of a
representative experiment, performed in triplicate, is shown.
Angiotensin II was used as positive control, and results are consistent
with published results (31). C: OK
cells lysates exposed to 5% CO2
(lanes 1 and
2) or 10%
CO2 (lanes
3 and 4) in absence
(lane 3) or presence of PD-98059
(lane 4) were immunoprecipitated
with IgG (lane 1) or with
ERK1/2-specific polyclonal antibody (lanes
2-4). Immunoprecipitates were probed with a
monoclonal anti-phosphotyrosine kinase antibody. Phosphoproteins were
detected by enhanced chemiluminescence. Result of a representative
experiment is shown.
|
|
 |
DISCUSSION |
The role of SFKs in modulating NBC and NHE3 activities has only
recently been examined, but SFKs are clearly involved (10, 32, 34).
This closely related family of tyrosine kinases is phylogenetically
conserved and in the mouse is composed of at least nine members (Src,
Yes, Fyn, Lyn, Lck, Hck, Fgn, Plk, and Yrk) (7, 12, 29). Src, Yes, and
Fyn are ubiquitously expressed and have been identified in renal
proximal tubule cells (3, 29). Heterologous expression of Csk
(COOH-terminal Src kinase) specifically inhibits the activation of all
known Src kinase family members (3, 11, 14, 17) and prevents the
acidosis-induced increase in NHE3 activity in MCT medullary collecting
duct cells (34). A decrease in
pHi, both in the presence or
absence of extracellular acidosis, increases both Src kinase activity
and specific phosphorylation of endogenous 60- to 70-kDa and 120-kDa proteins in these cells (32). We have previously shown that activation
of tyrosine kinases by a variety of extracellular stimuli, including
insulin, EGF, and carbachol, can increase NBC activity in cultured
proximal tubule cells (19). The finding that tyrosine kinase inhibitors
prevent the effect of metabolic acidosis on NHE3 and
CO2 on NBC activity is consistent
with the hypothesis that SFKs may be involved in the regulation of both
NHE3 and NBC.
OK cells are derived from proximal tubule cells and retain
morphological and functional characteristics of normal proximal tubule,
including regulated apical NHE3 and basolateral NBC activities. They
are thus ideally suited for the simultaneous study of their regulation
(8). Heterologous expression of Csk completely blocked the effects of
CO2 on NBC activity in both OK
cells and cultured rabbit proximal tubule cells.
CO2 stimulation also increased
endogenous SFK activity and phosphorylation. Taken together, these
findings suggest that Src plays a role in the stimulation of NBC
activity by CO2.
Activation of Src results in signaling through the classic MAPK cascade
(Ras
Raf
MEK1/2
ERK1/2) (1, 3, 5, 6, 8,
13-17, 30, 31). The "classic" MAPK pathway signaling through
ERK1/2 is activated by a variety of extracellular stimuli, including
growth factors. The upstream activator of ERK1/2, MEK1/2, is
specifically inhibited by PD-98059. It is therefore of great interest
that PD-98059-inhibitable MEK1/2 and ERK1/2 activation plays a major
role in the stimulation of NHE1 by growth factors and arginine
vasopressin (AVP) (1, 5). The finding that PD-98059 also
blocks the effect of CO2 on NBC
activity, coupled with the observation that
CO2 increases both specific ERK1/2
phosphorylation and kinase activity, strongly suggests that the classic
MAPK pathway is involved in NBC regulation. The major components of
this pathway are ubiquitously expressed along the axial nephron, and
both MEK1/2 and ERK1/2 are stimulated by EGF-1 and angiotensin II in
the kidney, with the latter acting mainly in the proximal tubule (30).
The specific mechanisms whereby ERK1/2 augments a membrane transporter activity have not been elucidated. ERK1/2 kinase activation by AVP in
platelets and by growth factors in other tissues can increase NHE1
activity independent of specific Tyr phosphorylation (5, 6). In the
brain, there is substantial evidence that angiotensin II affects
norepinephrine transporters via classic MAPK pathway activation (15,
33). Activation of ERK1/2 also results in phosphorylation of the
membrane AT1 receptor, presumably
through direct physical association with ERK1/2 (33). The above
evidence clearly suggests that ERK1/2 activation can alter membrane
transport activity by unknown mechanisms.
In conclusion, we have demonstrated that
CO2 stimulation of NBC activity
involves both SFK and ERK1/2 activation.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Jonathan Cooper (University of Washington, Seattle)
and Hidesaburo Hanafusa (Rockefeller University, NY) for providing us
with rat pLXSH/Csk and chicken pcDNA1/Csk cDNAs, respectively.
 |
FOOTNOTES |
Portions of this study were presented at the 30th Annual Meeting of the
American Society of Nephrology in San Antonio, TX, on November 3, 1997.
This work was supported by funds from the Veterans Affairs Central
Office Merit Review Program (to J. A. L. Arruda).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: O. S. Ruiz,
Univ. of Illinois at Chicago, Section of Nephrology, M/C 793, 820 South
Wood St., Chicago, IL 60612-7315 (E-mail:
osruiz{at}uic.edu).
Received 28 January 1999; accepted in final form 8 June 1999.
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