SFKs, Ras, and the classic MAPK pathway couple muscarinic receptor activation to increased Na-HCO3 cotransport activity in renal epithelial cells

R. Brooks Robey1,2,4,*, Ofelia S. Ruiz1,3,4,*, Jessica Baniqued1, Dolores Mahmud1,4, Doris Joy D. Espiritu1,4, Angelito A. Bernardo1,4
Jose A. L. Arruda1,2,4
With the Technical Assistance of Yi-Yong Qiu

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 [Rafright-arrowMAPK/ERK kinase (MEK)right-arrowERK]. 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


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ABSTRACT
INTRODUCTION
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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 [Rafright-arrowMAPK/ ERK kinase (MEK)right-arrowextracellular 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.


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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 beta -galactosidase reporter gene construct (pSV · beta -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 beta -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 [gamma -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 beta -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 [gamma -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 beta -glycerol phosphate, 25 MgCl2, 5 EGTA, 1 Na3VO4, 1 dithiothreitol, and 167 µM ATP supplemented with tracer quantities of [gamma -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%.


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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.


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Fig. 1.   Cholinergic stimulation of NBC activity is prevented by pharmacological inhibitors of Src family kinases (SFK). Intracellular pH (pHi) was fluorometrically monitored in 2'7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-loaded cells under nominally Cl-free physiological conditions in the presence of 25 mM NaHCO3 and 1 mM amiloride. Na removal by equimolar substitution with choline resulted in a decrease in pHi that rapidly and completely recovered on the reintroduction of Na. The initial rate of pHi recovery (partial pH/partial t) was then taken as a measure of renal Na-HCO3 cotransporter (NBC) activity. The influence of general tyrosine kinase inhibition (herbimycin; A and B) and selective SFK inhibition {4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1); C and D} on basal and carbachol-stimulated (100 µM × 5 min) NBC activity was then evaluated. Recovery curves from representative experiments are depicted in A and C, and the corresponding rates of pHi recovery are depicted in B and D as the means ± SE for at least 5 separate experiments. pHi recovery was uniformly faster in carbachol-stimulated cells (, A and C) than in unstimulated controls (open circle , A and C). This difference was not apparent in the presence of either 1 µM herbimycin (, A) or 100 nM PP1 (black-triangle, C). Neither herbimycin (, A) nor PP1 (triangle , C) alone altered the rate of pHi recovery. Both herbimycin (B) and PP1 (D) completely prevented the increase in NBC activity by carbachol (filled bars), whereas neither inhibitor altered basal activity (hatched bars; NS).

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.


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Fig. 2.   COOH-terminal Src kinase (Csk) overexpression also prevents cholinergic stimulation of NBC activity. NBC activity was measured in both unstimulated and carbachol-stimulated (100 µM × 5 min) Csk-overexpressing or wild-type control cells. pHi recovery curves from a representative experiment are depicted in A (open circle , unstimulated cells; , carbachol-stimulated cells; diamond , Csk-overexpressing cells; black-lozenge , carbachol-stimulated Csk-overexpressing cells). The corresponding NBC activities for at least 4 such experiments are depicted in B and are given as the means ± SE. Carbachol increased NBC activity normally in cells transfected with the empty parent vector (-). In contrast, Csk overexpression (+) prevented cholinergic stimulation of NBC activity without altering basal cotransport activity.

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.


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Fig. 3.   Carbachol increases Src tyrosine phosphorylation and kinase activity. A: carbachol (100 µM) rapidly increased Src phosphorylation. Increased tryosine phosphorylation was observed within 1 min and was maximal within 3 min, after which phosphotyrosine content declined. Representative paired anti-Src and anti-phosphotyrosine (Src-P) immunoblots are depicted, with an unstimulated A431 cell lysate included as a control. This experiment was repeated at least twice with similar results. B: carbachol also increased Src kinase activity within 5 min as evaluated by an in vitro immunoprecipitated kinase activity assay (*P < 0.01 vs. unstimulated controls). This effect was prevented by pretreatment with 1 µM herbimycin (**P < 0.02 vs. carbachol alone). C: the SFK-selective inhibitor, PP1 (100 nM), mimicked herbimycin's ability to prevent cholinergic stimulation of Src tyrosine phosphorylation, compatible with the ability of both inhibitors to prevent the corresponding increase in NBC activity (Fig. 1).

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 (Rafright-arrowMEKright-arrowERK). 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.


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Fig. 4.   MAPK/ERK kinase (MEK) inhibition prevents cholinergic stimulation of NBC activity. Classic MAPK pathway involvement in carbachol-stimulated (100 µM × 5 min.) NBC activity was evaluated pharmacologically using the MEK1/2-selective inhibitor PD98059. pHi recovery curves from a representative experiment are depicted in A (open circle , unstimulated cells; , +carbachol; diamond , +PD98059; black-lozenge , +carbachol and PD98059). The corresponding NBC activities for at least 4 such experiments are depicted in B and are given as the means ± SE. PD98059 completely prevented the increase in NBC activity by carbachol (filled bars) without altering basal activity (hatched bars; NS).

Carbachol activates the classic MAPK pathway. To directly test the ability of carbachol to activate the classic MAPK pathway (Rafright-arrowMEKright-arrowERK), 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.


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Fig. 5.   Carbachol increases classic MAPK pathway activation. A: 100 µM carbachol increased Raf-1 kinase cascade activity within 5 min. B: extracellular signal-regulated kinases 1 and 2 (ERK1/2) activity was similarly increased within 5 min, and this effect was prevented by the MEK1/2-selective inhibitor PD98059. The ability of carbachol to activate both proximal (Raf-1) and distal (ERK1/2) elements of the classic MAPK module, coupled with the ability of PD98059 to prevent ERK1/2 activation, is consistent with sequential cholinergic activation of all elements of this module (Rafright-arrowMEKright-arrowERK).

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.


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Fig. 6.   Cholinergic stimulation of ERK1/2 activity requires both M1 muscarinic receptor activation and SFK activity. Classic MAPK pathway activation by carbachol (100 µM × 5 min) was inhibited by both the M1 muscarinic antagonist pirenzepine (100 µM) and the SFK inhibitor PP1 (100 nM). Similar results were obtained with the nonselective acetylcholine receptor antagonist atropine (data not shown).

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.


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Fig. 7.   Cholinergic activation of the classic MAPK pathway requires Ras activation. Following transient transfection with either an N17-Ras expression vector (+ N17-Ras) or an empty pUSEamp(+) parent vector (Control), ERK1/2 activity was assayed in both unstimulated (hatched boxes) and carbachol-stimulated (100 µM × 5 min; black boxes) cells. Similar results were obtained in cells transfected with an N17-Ras adenoviral expression vector [kindly provided by B. B. Kahn (11); data not shown].


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 (Rafright-arrowMEKright-arrowERK) 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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