1 Section of Nephrology, Department of Medicine, 2 Department of Physiology and Biophysics, and 3 Department of Pathology, University of Illinois at Chicago College of Medicine, and 4 Veterans Affairs Chicago Health Care System, West Side Division, Chicago, Illinois 60612
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cholinergic agents are
known to affect the epithelial transport of H2O and
electrolytes in the kidney. In proximal tubule cells, cholinergic
agonists increase basolateral Na-HCO3 cotransport activity
via M1 muscarinic receptor activation. The signaling intermediates that couple these G protein-coupled receptors to cotransporter activation, however, are not well defined. We therefore sought to identify distal effectors of muscarinic receptor activation that contribute to increased NBC activity in cultured proximal tubule
cells. As demonstrated previously for acute CO2-regulated cotransport activity, we found that inhibitors of Src family kinases (SFKs) or the classic mitogen-activated protein kinase (MAPK) pathway
prevented the stimulation of NBC activity by carbachol. The ability of
carbachol to activate Src, as well as the proximal (Raf) and distal
[extracellular signal-regulated kinases 1 and 2 (ERK1/2)] elements of
the classic MAPK module, was compatible with these findings.
Cholinergic stimulation of ERK1/2 activity was also completely
prevented by overexpression of a dominant negative mutant of Ras
(N17-Ras). Taken together, these findings suggest a requirement for the
sequential activation of SFKs, Ras, and the classic MAPK pathway
[RafMAPK/ERK kinase (MEK)
ERK]. These findings provide
important insights into the molecular mechanisms underlying cholinergic
regulation of NBC activity in renal epithelial cells. They also
suggest a specific mechanism whereby cholinergic stimulation of the
kidney can contribute to pH homeostasis.
sodium-bicarbonate cotransport; proximal tubule; epithelial cell; mitogen-activated protein kinase; Src family kinases; carboxyterminal Src kinase; Ras; intracellular pH
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CHOLINERGIC STIMULATION OF renal epithelial muscarinic
receptors is known to modulate the tubular handling of salt and water (19, 30, 35, 36), but the specific mechanisms underlying cholinergic regulation of epithelial ion transport are not well understood. In proximal tubule cells, cholinergic agonists have been
shown to rapidly increase basolateral Na-HCO3 cotransport (NBC) activity (25, 26). NBC activity is a major
determinant of intracellular pH (pHi) in these cells and
may therefore serve as a prototype for the characterization of
cholinergic regulation of ion transport and pHi in renal
epithelial cells. We have previously shown that carbachol stimulates
basolateral NBC activity in primary cultures of rabbit renal proximal
tubule cells via M1 muscarinic acetylcholine receptor
activation (26). We have also demonstrated the general
involvement of tyrosine kinase activity in cholinergic stimulation of
NBC activity (25), but specific signaling effectors have
not yet been identified. We have recently reported such an effector
function for the Src family of nonreceptor tyrosine kinases (SFKs),
including Src, in acute CO2-stimulated NBC activity
(28). We therefore sought to examine whether SFKs, and Src
in particular, play a role in the cholinergic stimulation of NBC
activity in this cell type. To this end, we examined selective
inhibitors of SFKs for the ability to attenuate the effects of
carbachol in the opossum kidney (OK) proximal tubule cell line. In
parallel, we investigated the ability of carbachol to increase both Src phosphorylation and activity. Because both Ras and the classic mitogen-activated protein kinase (MAPK) pathway
[RafMAPK/ ERK kinase (MEK)
extracellular signal-regulated
kinases 1 and 2 (ERK1/2)] are known distal effectors of SFK signaling,
we also examined their individual roles in the modulation of NBC
activity by carbachol.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. The pH-sensitive fluorophore 2'7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) was obtained from Molecular Probes (Eugene, OR) as the membrane-permeable acetoxymethyl ester (BCECF-AM). Amiloride was purchased from Research Biochemicals (Natick, MA). Herbimycin A (herbimycin), 2'-amino-3'-methoxyflavone (PD98059), 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1), 4-amino-7-phenylpyrazolo[3,4-d]-pyrimidine (PP3), and hygromycin B were obtained from Calbiochem (San Diego, CA), and recombinant phosphotyrosine-specific RC20 antibodies were purchased from Transduction Laboratories (Lexington, KY). ERK1/2- and Src-specific antibodies were obtained from Upstate Biotechnology (Lake Placid, NY), as were the SFK, ERK1/2, and Raf-1 kinase assay kits employed herein. The ECL enhanced chemiluminescence detection system from Amersham Pharmacia (Arlington Heights, IL) was used to analyze all immunoblots. All other immunoblotting reagents, including nitrocellulose membranes, were obtained from Bio-Rad (Hercules, CA). Cell culture reagents, including LipofectAMINE lipofection reagent, were obtained from GIBCO-BRL (Grand Island, NY). Unless otherwise noted, all other reagents, including carbachol and pirenzepine, were obtained from Sigma (St. Louis, MO) and were the finest quality available.
Cell culture. Mycoplasma-free American OK cells were obtained from the American Type Culture Collection (Rockville, MD) at passage 37. Cells were routinely maintained in a humidified 37°C-5% CO2 incubator in Eagle's minimum essential medium containing Earle's salts and supplemented with 10% fetal bovine serum. All testing was performed between passages 38 and 48 to minimize the effects of phenotypic variation in continuous culture, and cells were routinely serum deprived for 24 h before and during testing.
Fluorometric assays of pHi and NBC activity. Confluent cell monolayers cultured on uncoated plastic coverslips were loaded with BCECF and continuously monitored for pH-dependent changes in fluorescence as described previously (23, 24). In brief, cells were perfused at 37°C with a Cl-free physiologic solution consisting of (in mM) 25 NaHCO3, 110 sodium gluconate, 5 potassium gluconate, 2 CaSO4, 0.5 MgSO4, 1 KH2PO4, 10 glucose, and 9 HEPES, pH 7.40 and supplemented with 1 mM amiloride to minimize the contributions of cellular Cl/HCO3 and Na/H exchange activities. Extracellular pH was maintained constant at 7.40 throughout. After a stable basal fluorescence signal was obtained, Na was removed by equimolar substitution with choline, resulting in an immediate decrease in pHi and pH-sensitive BCECF fluorescence. On the reintroduction of Na, pHi and fluorescence rapidly and fully recovered, and NBC activity was taken as the initial rate of this recovery as described previously (1, 29). pH-sensitive BCECF fluorescence at 500 nm was routinely calibrated at the completion of each experiment in the presence of elevated extracellular potassium and the ionophore nigericin (to equilibrate intracellular and extracellular pH). All measurements were performed by dual-wavelength monitoring and ratiometric analysis at pH-sensitive (500 nm) and -insensitive (450 nm) excitation wavelengths (F500/F450).
Heterologous transgene overexpression.
We have previously characterized OK cells stably overexpressing a
wild-type rat COOH-terminal Src kinase (Csk) transgene under the
control of a retroviral long-terminal repeat sequence
(28). These cells were grown to confluence in normal
growth medium supplemented with 100 µg/ml hygromycin B before
testing, and Csk overexpression was routinely confirmed by immunoblot
analysis as described previously (28). The dominant
interfering S17N mutant of H-Ras (N17-Ras) was also transiently
overexpressed in OK cells by using a commercially available N17-Ras
expression vector [pUSEamp(+)/N17-Ras; Upstate Biotechnology]. The
S17N mutation in N17-Ras markedly attenuates the affinity of Ras for
GTP without substantially affecting its affinity for GDP, and
overexpression of this mutant has been shown to disrupt endogenous Ras
signaling, presumably via competition for endogenous GTP/GDP exchange
factors (5, 11). Transient gene transfer was achieved by
using LipofectAMINE lipofection reagent according to the
manufacturer's recommendations and as described previously
(23). Transfection efficiency was assessed by using a
-galactosidase reporter gene construct (pSV ·
-gal; Promega) as reported previously (21) and was typically at
least 20-30%. N17-Ras overexpression was also routinely confirmed
by immunoblot analysis (data not shown). OK cells transiently
transfected with the empty pUSEamp(+) parent vector were routinely
analyzed in parallel as transfection controls. Major findings were
confirmed in cells overexpressing N17-Ras or
-galactosidase
transgenes following adenoviral transfer as described previously
(11).
Src phosphorylation and kinase activity assays.
Src phosphorylation was assessed by quantitative immunoblot analysis of
whole cell lysates using both Src-specific polyclonal antisera and
recombinant monoclonal anti-phosphotyrosine antibodies (RC20) as
described previously (28). Src kinase activity was evaluated in parallel using a commercially available immunoprecipitated kinase activity assay (Upstate Biotechnology) according to the manufacturer's recommendations. In brief, we tested the in vitro ability of Src immunoprecipiates to phosphorylate a synthetic oligopeptide substrate (KVEKIGEGTYGVVYK) corresponding to residues 6-20 of p34cdc2 (7). Samples
were incubated in (in mM, unless noted otherwise) 25 Tris · HCl
(pH 7.2), 31.3 MgCl2, 25 MnCl2, 0.5 EGTA, 0.5 dithiothreitol, 62.5 µM Na3VO4, and 112.5 µM ATP containing 10 µCi [-32P]ATP at 30°C for
10 min before the reaction was stopped by the addition of
trichloroacetic acid. After application to P81 phosphocellulose paper,
unincorporated radionuclides were eluted with 7.5% (vol/vol) phosphoric acid. Phosphotranferase activity was then assayed as specific 32P incorporation into the substrate by liquid
scintillation counting.
ERK1/2 activity assays.
Total ERK1/2 kinase activity was measured using a
commercially-available in vitro immunoprecipitated kinase activity
assay (Upstate Biotechnology) according to the manufacturer's
recommendations. In brief, ERK1/2 immunoprecipitates prepared from
whole cell lysates were tested for the ability to phosphorylate myelin
basic protein in the presence of inhibitors of protein kinase A (PKI),
PKC (PKC inhibitor peptide), and calmodulin kinase II (compound
R24571). The final reaction mixture consisted of (in mM, unless noted
otherwise) 20 MOPS (pH 7.2), 25 -glycerol phosphate, 16.9 MgCl2, 5 EGTA, 1 Na3VO4, 1 dithiothreitol, 5 µM PKC inhibitor peptide, 0.5 µM PKI, 5 µM
compound R24571, and 112.5 µM ATP containing 10-µCi [
-32P]ATP for 10 min at 30°C before application to
P81 phosphocellulose paper squares. Unincorporated 32P was
eluted with 7.5% (vol/vol) phosphoric acid, and the remaining incorporated radioactivity was assayed by liquid scintillation counting. Major findings were also confirmed using a nonradioactive immunoprecipitated ERK1/2 activity assay as described previously (20, 22).
Raf-1 kinase cascade activity assays.
Raf-1 kinase activity was measured by using a commercially available
kit (Upstate Biotechnology) according to the manufacturer's recommendations. In brief, Raf-1 immunoprecipitates were prepared from
total cell lysates and were examined for the ability to activate an in
vitro phosphorylation cascade involving inactive MEK1- and ERK2-GST
fusion proteins. Activation of this cascade was monitored as specific
32P incorporation into the substrate myelin basic protein
in (in mM, unless noted otherwise) 20 MOPS, pH 7.2, 25 -glycerol
phosphate, 25 MgCl2, 5 EGTA, 1 Na3VO4, 1 dithiothreitol, and 167 µM ATP
supplemented with tracer quantities of [
-32P]ATP for
10 min at 30°C. Aliquots were spotted onto P81 phosphocellulose paper, and incorporated radioactivity was assayed by liquid
scintillation counting following the elution of unincorporated
32P with 7.5% (vol/vol) phosphoric acid. Positive control
samples containing activated recombinant ERK2 were routinely assayed in parallel.
Statistical analysis. All results were expressed as means ± SE for at least three, and typically five or more, independent experiments, unless noted otherwise. Statistical comparisons were routinely performed by t-testing for paired or unpaired data where appropriate using a significance level of 95%.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cholinergic stimulation of NBC activity is prevented by both
general and SFK-selective tyrosine kinase inhibitors.
We have previously shown that the cholinergic agonist carbachol
increases proximal tubule cell NBC activity in a time- and dose-dependent manner (26). NBC activity was maximally
increased within 1-5 min of stimulation by 10 µM carbachol
(apparent ED50 ~0.1 µM), and this effect was completely
prevented by general inhibitors of tyrosine kinase activity
(25). In the present study, we found that pretreatment of
OK cells with the tyrosine kinase inhibitor herbimycin, at a
concentration (1 µM) known to inhibit Src kinase activity
(34), prevented cholinergic stimulation of NBC activity by
100 µM carbachol without altering basal transport activity (Fig.
1, A and B). To
assess the specificity of this effect for SFKs, we also tested the
tyrphostin PP1, a SFK-selective tyrosine kinase inhibitor
(14), for the ability to mimic the effects of more general
inhibitors of tyrosine kinases. As shown in Fig. 1, C and
D, 100 nM PP1 completely prevented carbachol's effect on
NBC activity without altering basal transport activity, a finding that
is compatible with SFK involvement in this effect.
|
Cholinergic stimulation of NBC activity is also inhibited by Csk
overexpression.
Csk is an endogenous inhibitor of SFKs that is expressed by proximal
tubule cells (6, 28). To verify the involvement of SFKs,
we tested the ability of Csk overexpression to inhibit cholinergic
stimulation of NBC activity. OK cells stably overexpressing Csk have
been described previously (28). As shown in Fig.
2, Csk-overexpressing cells exhibited
normal basal NBC activity, consistent with our previous report.
Cholinergic stimulation, however, failed to increase NBC activity in
Csk-overexpressing cells, but not in cells transfected with the empty
parent vector (Fig. 2) or in untransfected control cells (data not
shown), further suggesting the involvement of SFKs.
|
Carbachol increases both Src kinase activity and phosphorylation.
As shown in Fig. 3A,
immunodetectable Src tyrosine phosphorylation was rapidly increased by
100 µM carbachol, and this increase was prevented by pretreatment
with 1 µM herbimycin (data not shown). As shown in Fig.
3B, carbachol similarly increased immunoprecipitatable Src
kinase activity ~70% above basal levels within 5 min, and this
effect was also inhibited by herbimycin. As depicted in Fig. 3C, 100 nM PP1 mimicked the effect of herbimycin on
carbachol-stimulated Src phosphorylation. This finding is consistent
with the ability of 100 nM PP1 to prevent cholinergic stimulation of
NBC activity (Fig. 1, C and D). When tested in
parallel, the negative control analog, PP3, was unable to mimic these
effects (data not shown), further suggesting specificity for SFKs such
as Src.
|
Cholinergic stimulation of NBC activity is prevented by MEK1/2
inhibition.
A variety of agents that signal through SFKs also signal through the
classic MAPK pathway (RafMEK
ERK). We have previously shown that
activation of this signaling pathway plays an important role in the
stimulation of NBC activity by CO2 (28).
Figure 4 shows the effect of the specific
MEK1/2 inhibitor, PD98059, on carbachol stimulation of NBC activity.
This inhibitor did not alter basal cotransport activity but completely
prevented the stimulatory effect of carbachol. These results are
compatible with a role for the classic MAPK pathway in the cholinergic
stimulation of NBC activity.
|
Carbachol activates the classic MAPK pathway.
To directly test the ability of carbachol to activate the classic MAPK
pathway (RafMEK
ERK), we assayed both Raf-1 and ERK1/2 activity,
representing the proximal and distal components of this signaling
module, respectively. In OK cells, cholinergic stimulation by carbachol
increased Raf-1 cascade signaling over 80% above control levels within
5 min. (P < 0.02; Fig.
5A). As shown in Fig.
5B, carbachol also increased ERK1/2 kinase activity ~60% above control levels in the same timeframe (P < 0.001), and this increase was completely prevented by pretreatment with
PD98059 (P < 0.001). ERK1/2 phosphorylation increased
in parallel and was similarly prevented by PD98059 (data not shown).
These findings are in agreement with those depicted in Fig. 4 above.
|
Cholinergic activation of the classic MAPK pathway requires
M1 muscarinic receptor activation and SFK activation.
To integrate our present findings with those reported previously
(26) and to further compare the specific cholinergic
signaling requirements for classic MAPK pathway activation with those
already identified for stimulation of NBC activity, we also examined
the ability of pirenzepine and PP1 to attenuate activation of ERK1/2 by
carbachol. In separate experiments, we found that the cholinergic stimulation of ERK1/2 activity was blocked by 100 µM pirenzepine (P < 0.001; Fig.
6A) or 100 nM PP1
(P < 0.001; Fig. 6B), suggesting selectivity for both the M1 muscarinic receptor and SFKs.
Neither inhibitor had a significant effect on basal ERK1/2 activity.
|
N17-Ras overexpression abrogates cholinergic stimulation of
ERK1/2 activity.
To test the Ras dependence of Raf-1 activation by carbachol, we
transfected OK cells with the dominant interfering S17N mutant of Ras
(N-17 Ras). In control cells transfected with pUSEamp(+) alone,
carbachol increased ERK1/2 activity over 150% above control levels
(P < 0.05; Fig. 7). In
contrast, N17-Ras overexpression completely abrogated cholinergic
stimulation of ERK1/2 activity (Fig. 7), suggesting a requirement for
Ras in this effect.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cholinergic stimulation of the kidney has profound effects on both renal hemodynamics and the epithelial transport of water and electrolytes (10, 15-19, 30, 35, 36). Effects on transport are largely mediated by muscarinic receptors located on renal epithelial cells, but the mechanisms coupling muscarinic receptor activation to epithelial ion transport are poorly understood. In the present study, we address at least one specific mechanism whereby cholinergic stimulation influences epithelial cell NBC activity and pHi. We further suggest that this may serve as a prototype for the study of direct cholinergic regulation of epithelial ion transport.
Although there is considerable heterogeneity in G protein-coupled receptor (GPCR) signaling, there is also significant overlap in the number and types of distal signaling effectors involved. SFKs are well known to couple to both hepta-spanning GPCRs and receptor tyrosine kinases (13). We demonstrate herein that cholinergic stimulation of NBC activity in OK cells involves SFK activation. A similar role for SFKs has been reported for the stimulatory effect of acute acidosis on both the apical Na/H exchanger (NHE3) and the basolateral NBC (28, 37, 38). As in the case of CO2 (28), the effect of cholinergic stimulation was blocked by the general tyrosine kinase inhibitor herbimycin and the SFK-selective tyrphostin PP1. Stable overexpression of Csk, an endogenous negative regulator of SFKs, similarly prevented cholinergic stimulation of NBC activity. Carbachol also increased both Src phosphorylation and activity in a manner that is temporally consistent with a role in coupling muscarinic receptor activation to cholinergic stimulation of NBC activity, effects that were uniformly inhibited by herbimycin, PP1, and Csk. Taken together, our findings provide strong support for the contention that SFKs play an important role in the stimulation of NBC activity by a variety of stimuli, including CO2 and carbachol. They also suggest a specific role for Src in the cholinergic stimulation of NBC activity, but they do not exclude contributions by other SFKs expressed in the kidney and this cell type, particularly Fyn and Yes (2, 12, 31).
The components of the classic MAPK pathway (RafMEK
ERK) are
well-described distal effectors of Src signaling in a number of cell
types (8, 9, 32), including proximal tubule cells (6, 28). In the present work, the ability of selective
MEK1/2 inhibition by PD98059 to prevent cholinergic stimulation of NBC activity suggests the involvement of classic MAPK pathway activation in
this process. The ability of carbachol to stimulate both ERK1/2 phosphorylation and activity is compatible with this interpretation, as
is the ability of PD98059 to inhibit these effects.
Finally, GTP-activated Ras commonly couples both receptor and nonreceptor tyrosine kinase signaling to classic MAPK pathway activation (3, 5). Although Ras-independent mechanisms have been described (4). The ability of N17-Ras overexpression to completely inhibit cholinergic-stimulated ERK1/2 activity suggests a requirement for endogenous Ras activation in the present context. Carbachol's ability to activate Raf-1, the proximal element of the classic MAPK pathway commonly activated by Ras (3, 4), is also compatible with a signaling effector function for Ras in coupling cholinergic stimulation to classic MAPK pathway activation.
The experiments reported herein, which were performed on confluent OK cell monolayers, serve to both confirm and extend our previous observations in primary cultures of rabbit proximal tubule cells (25, 26). Taken together, our findings suggest a direct effect of carbachol on NBC activity. Both the timecourse of activation and our previous demonstration of increased NBC activity in basolateral membrane vesicles prepared from carbachol-stimulated cells (25) are compatible with this interpretation. Although not directly examined, these findings could also be taken to suggest that confluent OK cell monolayers exhibit incomplete cell polarity or incomplete tight junction formation, thus permitting unrestricted access to both "apical" and "basolateral" cell surfaces under the experimental conditions employed. We cannot, however, presently exclude contributions by carbachol-stimulated increases in paracellular permeability that might serve to unmask otherwise "hidden" basolateral receptors and/or NBC.
We previously demonstrated that cholinergic stimulation of proximal tubule cell NBC activity involves M1 muscarinic receptor activation (26). In the present work, we have shown that muscarinic stimulation is coupled to increased NBC activity via SFKs, Ras, and the classic MAPK pathway. Cholinergic stimulation of NBC activity thus shares a common requirement for signaling elements implicated in acute acidosis-stimulated basolateral NBC (28) and apical Na/H exchange (37, 38) activities in this cell type. Elements of this signaling cascade have also been reported to play an important role in angiotensin II stimulation of these coordinately regulated transport activities (27, 33). It is reasonable, therefore, to speculate that these proximate signaling intermediates play a central role in the regulation of epithelial cell pHi by a variety of stimuli, and in pH homeostasis in general. Although it is not presently known how classic MAPK pathway activation is coupled to increased NBC activity, the identification of signaling effectors distal to ERK1/2 will provide important clues to the molecular mechanisms underlying this coupling, as well as the coordinated regulation of ion transport activities localized to different cellular interfaces in polarized epithelia.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported, in part, by US Department of Veterans Affairs Merit Review Awards (to R. B. Robey, A. A. Bernardo, and J. A. L. Arruda). Portions of this work were presented in preliminary form at the 31st and 32nd Annual Meetings of the American Society of Nephrology in Philadelphia, PA (October 26, 1998) and Miami, FL (November 5, 1999), respectively.
![]() |
FOOTNOTES |
---|
* R. B. Robey and O. S. Ruiz contributed equally to this work.
Address for reprint requests and other correspondence: J. A. L. Arruda, Dept. of Medicine, Section of Nephrology, Univ. of Illinois at Chicago College of Medicine, 820 South Wood St., Rm. 418W CSN (M/C 793), Chicago, IL 60612-7315 (E-mail: JAArruda{at}uic.edu).
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.
Received 20 September 2000; accepted in final form 17 January 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alpern, RJ.
Mechanism of basolateral membrane H+/OH/HCO3
transport in the rat proximal convoluted tubule.
J Gen Physiol
86:
613-636,
1985[Abstract].
2.
Arreaza, G,
Melkonian KA,
LaFevre-Bernt M,
and
Brown DA.
Triton X-100-resistant membrane complexes from cultured kidney epithelial cells contain the Src family protein tyrosine kinase p62yes.
J Biol Chem
269:
19123-19127,
1994
3.
Avruch, J,
Zhang XF,
and
Kyriakis JM.
Raf meets Ras: completing the framework of a signal transduction pathway.
Trends Biochem Sci
19:
279-283,
1994[ISI][Medline].
4.
Barnard, D,
Diaz B,
Clawson D,
and
Marshall M.
Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms.
Oncogene
17:
1539-1547,
1998[ISI][Medline].
5.
Boguski, MS,
and
McCormick F.
Proteins regulating Ras and its relatives.
Nature
366:
643-654,
1993[ISI][Medline].
6.
Chen, JK,
Capdevila J,
and
Harris RC.
Overexpression of C-terminal Src kinase blocks 14,15-epoxyeicosatrienoic acid-induced tyrosine kinase phosphorylation and mitogenesis.
J Biol Chem
275:
13789-13792,
2000
7.
Cheng, HC,
Nishio H,
Hatase O,
Ralph S,
and
Wang JH.
A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases.
J Biol Chem
267:
9248-9256,
1992
8.
Della Rocca, GJ,
van Biesen T,
Daaka Y,
Luttrell DK,
Luttrell LM,
and
Lefkowitz RJ.
Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors: convergence of Gi- and Gq-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase.
J Biol Chem
272:
19125-19132,
1997
9.
Dikic, I,
Tokiwa G,
Lev S,
Courtneidge SA,
and
Schlessinger J.
A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation.
Nature
383:
547-550,
1996[ISI][Medline].
10.
Earley, LE,
and
Friedler RM.
The effects of combined renal vasodilatation and pressor agents on renal hemodynamics and the tubular reabsorption of sodium.
J Clin Invest
45:
542-551,
1966[ISI][Medline].
11.
Gnudi, L,
Frevert EU,
Houseknecht KL,
Erhardt P,
and
Kahn BB.
Adenovirus-mediated gene transfer of dominant negative RasAsn17 in 3T3L1 adipocytes does not alter insulin-stimulated PI3-kinase activity or glucose transport.
Mol Endocrinol
11:
67-76,
1997
12.
Grandaliano, G,
Monno R,
Ranieri E,
Gesualdo L,
and
Schena FP.
Regenerative and proinflammatory effects of thrombin on human proximal tubular cells.
J Am Soc Nephrol
11:
1016-1025,
2000
13.
Gutkind, JS.
The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades.
J Biol Chem
273:
1839-1842,
1998
14.
Hanke, JH,
Gardner JP,
Dow RL,
Changelian PS,
Brissette WH,
Weringer EJ,
Pollok BA,
and
Connelly PA.
Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor: study of Lck- and FynT-dependent T cell activation.
J Biol Chem
271:
695-701,
1996
15.
Harvey, RB.
Effects of acetylcholine infused into renal artery of dogs.
Am J Physiol
211:
487-492,
1966[ISI][Medline].
16.
Hunt, R.
Vasodilator reactions.
Am J Physiol
45:
197-230,
1918.
17.
Nahmod, VE,
and
Lanari A.
Abolition of autoregulation of renal blood flow by acetylcholine.
Am J Physiol
207:
123-127,
1964[ISI].
18.
Pinter, GG,
O'Morchoe CCC,
and
Sikand RS.
Effect of acetylcholine on urinary electrolyte excretion.
Am J Physiol
207:
979-982,
1964[ISI].
19.
Pirola, CJ,
Alvarez AL,
Balda MS,
Finkielman S,
and
Nahmod VE.
Evidence for cholinergic innervation in dog renal tissue.
Am J Physiol Renal Fluid Electrolyte Physiol
257:
F746-F754,
1989
20.
Robey, RB,
Ma J,
and
Santos AVP
Regulation of mesangial cell hexokinase activity by PKC and the classic MAPK pathway.
Am J Physiol Renal Physiol
277:
F742-F749,
1999
21.
Robey, RB,
Osawa H,
Printz RL,
and
Granner DK.
Transient gene transfer into myotubes following differentiation in culture.
BioTechniques
20:
40-42,
1996[ISI][Medline].
22.
Robey, RB,
Raval BJ,
Ma J,
and
Santos AVP
Thrombin is a novel regulator of hexokinase activity in mesangial cells.
Kidney Int
57:
2308-2318,
2000[ISI][Medline].
23.
Robey, RB,
Ruiz O,
Santos AVP,
Ma J,
Kear F,
Wang LJ,
Li CJ,
Bernardo AA,
and
Arruda JAL
pH-dependent fluorescenceof a heterologously expressed Aequorea green fluorescent protein mutant: in situ spectral characteristics and applicability to intracellular pH estimation.
Biochemistry
37:
9894-9901,
1998[ISI][Medline].
24.
Roos, A,
and
Boron WF.
Intracellular pH.
Physiol Rev
61:
296-434,
1991.
25.
Ruiz, OS,
Qiu YY,
Cardoso LR,
and
Arruda JAL
Regulation of the renal Na-HCO3 cotransporter: IX. Modulation by insulin, epidermal growth factor and carbachol.
Regul Pept
77:
155-161,
1998[ISI][Medline].
26.
Ruiz, OS,
Qiu YY,
Cardoso LR,
and
Arruda JAL
Regulation of the renal Na-HCO3 cotransporter: VII. Mechanism of the cholinergic stimulation.
Kidney Int
51:
1069-1077,
1997[ISI][Medline].
27.
Ruiz, OS,
Qiu YY,
Wang LJ,
Robey RB,
and
Arruda JAL
The classic MAPK pathway: a common mediator of Na-HCO3 cotransporter (NBC) activation by CO2, angiotensin II and carbachol (Abstract).
J Am Soc Nephrol
9:
12A,
1998.
28.
Ruiz, OS,
Robey RB,
Qiu YY,
Wang LJ,
Li CJ,
Ma J,
and
Arruda JAL
Regulation of the renal Na-HCO3 cotransporter. XI. Signal transduction underlying CO2 stimulation.
Am J Physiol Renal Physiol
277:
F580-F586,
1999
29.
Ruiz, OS,
Wang LJ,
Pahlavan P,
and
Arruda JAL
Regulation of renal Na-HCO3 cotransporter. III. Presence and modulation by glucocorticoids in primary cultures of the proximal tubule.
Kidney Int
47:
1669-1676,
1995[ISI][Medline].
30.
Stein, JH,
Reineck JH,
Osgood RW,
and
Ferris TF.
Effect of acetylcholine on proximal tubular sodium reabsorption in the dog.
Am J Physiol
220:
227-232,
1971[ISI][Medline].
31.
Sugawara, K,
Sugawara I,
Sukegawa J,
Akatsuka T,
Yamamoto T,
Morita M,
Mori S,
and
Toyoshima K.
Distribution of c-yes-1 gene product in various cells and tissues.
Br J Cancer
63:
508-513,
1991[ISI][Medline].
32.
Thomas, SM,
and
Brugge JS.
Cellular functions regulated by Src family kinases.
Annu Rev Cell Dev Biol
13:
513-609,
1997[ISI][Medline].
33.
Tsuganezawa, H,
Preisig PA,
and
Alpern RJ.
Dominant negative c-Src inhibits angiotensin II induced activation of NHE3 in OKP cells.
Kidney Int
54:
394-398,
1998[ISI][Medline].
34.
Uehara, Y,
and
Fukazawa H.
Use and selectivity of herbimycin A as inhibitor of protein-tyrosine kinases.
Methods Enzymol
201:
370-379,
1991[ISI][Medline].
35.
Vander, AJ.
Effects of acetylcholine, atropine and physostigmine on renal function in the dog.
Am J Physiol
206:
492-498,
1964[ISI].
36.
Wang, T,
and
Chan YL.
Cholinergic effect on rat proximal convoluted tubule.
Pflügers Arch
415:
533-539,
1990[ISI][Medline].
37.
Yamaji, Y,
Amemiya M,
Cano A,
Preisig PA,
Miller RT,
Moe OW,
and
Alpern RJ.
Overexpression of Csk inhibits acid-induced activation of NHE-3.
Proc Natl Acad Sci USA
92:
6274-6278,
1995[Abstract].
38.
Yamaji, Y,
Tsuganezawa H,
Moe OW,
and
Alpern RJ.
Intracellular acidosis activates c-Src.
Am J Physiol Cell Physiol
272:
C886-C893,
1997