Mechanism of activation of ERK and H-K-ATPase by isoproterenol in rat cortical collecting duct

Nicolas Laroche-Joubert, Sophie Marsy, Stéphanie Luriau, Martine Imbert-Teboul, and Alain Doucet

Laboratoire de Biologie Intégrée des Cellules Rénales, Service de Biologie Cellulaire, Commissariat a l'Energie Atomique, Saclay, Unité de Recherche Associée 1859, Centre National de la Recherche Scientifique, 91191 Gif-sur-Yvette Cedex, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isoproterenol stimulates H-K-ATPase activity in rat cortical collecting duct beta -intercalated cells through a PKA-dependent pathway. This study aimed at determining the signaling pathway underlying this effect. H-K-ATPase activity was determined in microdissected collecting ducts preincubated with or without specific inhibitors or antibodies against intracellular signaling proteins. Transient cell membrane permeabilization with streptolysin-O allowed intracellular access to antibodies. Isoproterenol increased phosphorylation of ERK in a PKA-dependent manner, and inhibition of the ERK phosphorylation prevented the stimulation of H-K-ATPase. Antibodies against the monomeric G protein Ras or the kinase Raf-1 curtailed the stimulation of H-K-ATPase by isoproterenol, whereas antibodies against the related proteins Rap-1 and B-Raf had no effect. Pertussis toxin and inhibition of tyrosine kinases with genistein also curtailed isoproterenol-induced stimulation of H-K-ATPase. It is proposed that activation of PKA by isoproterenol induces the phosphorylation of beta -adrenergic receptors and the switch from Gs to Gi coupling. In turn, beta gamma -subunits released from Gi would activate a tyrosine kinase-Ras-Raf-1 pathway, leading to the activation of ERK1/2 and of H-K-ATPase.

Ras; extracellular signal-regulated kinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

H-K-ATPASES ARE P-TYPE ATPASES that exchange intracellular proton against extracellular potassium at the expense of ATP hydrolysis (10). Gastric H-K-ATPase was the first H-K-ATPase to be discovered and remains the archetype of this family of ATPases. It is a ouabain-insensitive, omeprazole- and Sch-28080-sensitive ATPase located in the apical membrane of gastric parietal cells where it energizes HCl secretion (9). Subsequently, other H-K-ATPases were characterized in the distal colon and in the kidney collecting duct (13, 28). Based on the finding that Sch-28080-sensitive ATPase activity is increased in the collecting duct of potassium-deprived rats compared with normal ones, it was initially thought that renal H-K-ATPase was mainly involved in potassium transport (19). Later, functional studies demonstrated that collecting duct H-K-ATPase also participates in the regulation of acid-base balance (10). However, regulation of potassium and acid-base balances in the collecting duct may be accounted for by different H-K-ATPases. As a matter of fact, collecting ducts from normal and potassium-deprived rats express at least two distinct Sch-28080-sensitive ATPases that are insensitive and sensitive to ouabain, respectively (4).

Rat cortical collecting ducts (CCDs) consist of at least three cell types characterized by distinct morphological and functional features. Principal cells are involved in sodium, potassium, and water transport, whereas alpha - and beta -intercalated cells are the site of proton and bicarbonate secretion, respectively. Functionally, the three cell types of the rat CCD also differ by the presence of specific G protein-coupled hormone receptors activating adenylyl cyclase: principal cells express vasopressin V2 receptors, whereas alpha - and beta -intercalated cells express calcitonin and beta -adrenergic receptors, respectively. This hormone selectivity was used to localize functionally H-K-ATPases in the different CCD cell types: Sch-28080-sensitive ATPase is stimulated by calcitonin and isoproterenol in normal rats, demonstrating that it is expressed in alpha - and beta -intercalated cells, whereas in potassium-deprived rats it is activated by vasopressin and therefore originates from principal cells (15). These findings further support the hypothesis that H-K-ATPase-mediated regulation of acid-base balance and potassium balance in collecting duct might be accounted for by two distinct forms of H-K-ATPase originating from different cell types. They also demonstrate that besides long-term regulation of expression, H-K-ATPase activity is modulated in the short term by posttranscriptional mechanisms. The stimulation of H-K-ATPase by calcitonin and isoproterenol observed in CCDs of normal rats, along with that of H-ATPase previously reported (25), likely participates to the short-term regulation of proton transport by these two hormones (25, 26).

The signaling pathways underlying calcitonin- and isoproterenol-induced activation of H-K-ATPase are different. Although stimulation of H-K-ATPase by either hormone is mediated by cAMP (15), the action of isoproterenol relies on the activation of PKA, whereas that of calcitonin is independent of PKA. The PKA-independent mechanism of activation of H-K-ATPase by calcitonin in alpha -intercalated cells has been characterized: the calcitonin-induced increase in cAMP activates the cAMP-activated guanine-nucleotide exchange factor Epac I, which in turn activates a cascade that includes the monomeric G protein Rap-1, the B-Raf kinase, the MAP kinase kinase MEK, and the extracellular signal-regulated kinases ERK1/2 (16). Activation of ERK1/2 leads to the stimulation of H-K-ATPase activity through a nontranscriptional mechanism. In contrast, the PKA-dependent signaling mechanism of isoproterenol in beta -intercalated cells has not been identified as yet and was therefore investigated in the present study. Because PKA-dependent activation of ERK has been reported in the literature (5, 11, 32), we determined the potential involvement of ERK in isoproterenol-induced activation of H-K-ATPase and the cascade leading to ERK phosphorylation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Animal preparation and tubule microdissection. Experiments were carried out in male Sprague-Dawley rats anesthetized with pentobarbital sodium (50 mg/kg body wt). CCDs were dissected at 4°C from collagenase-treated kidneys as described previously (12). After microdissection, they were photographed to determine their length, which served for normalization of the results. Unless indicated otherwise, microdissection was carried out in a solution containing (in mM) 120 NaCl, 5 KCl, 1 MgSO4, 4 NaHCO3, 0.2 NaH2PO4, 0.15 Na2HPO4, 5 glucose, 0.5 CaCl2, 0.08 dextran, 2 lactate, 20 HEPES, and 4 essential and nonessential amino acids, as well as 0.03 vitamins and 1 mg/ml BSA. The pH was adjusted to 7.4 and osmotic pressure to 400 mosmol/kgH2O with mannitol. For Western blotting analysis, antiproteases [1 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml aminoethylbenzenesulfonyl fluoride (AEBSF), and 10 µg/ml antipain] were added to the dissection solution.

Pretreament with inhibitors and hormones. Microdissected CCDs were preincubated with specific inhibitors of different intermediates of signaling pathways or with their solvent before incubation with hormones. Pools of CCDs dissected from collagenase-treated kidneys (2- to 4-mm length) were pretreated with or without the inhibitors at 30°C for 45-120 min before treatment with isoproterenol (10 min at 37°C). Isoproterenol treatment was stopped by cooling the samples at 4°C. Bisindolylmaleimide I was from Calbiochem (Merck Eurolab, Fontenay sous bois, France), and 1,4-diamino-2,3-dicyano-1,4-bis-(o-aminophenylmercapto)butadiene (U-0126) was from Sigma RBI (Sigma-Aldrich, St. Quentin Fallavier, France). All inhibitors were prepared from aqueous solutions, except U-0126, which was dissolved in DMSO. The corresponding control groups contained the same concentration of DMSO (<0.1% vol/vol).

When specific inhibitors were not commercially available, we evaluated the effect of antibodies directed against several signaling proteins. Intracellular entry of antibodies was made possible by transient permeabilization of CCD cells by streptolysin-O (1, 17). For this purpose, pools of CCDs were first preincubated at 37°C for 8 min and thereafter for 90 min at 4°C with or without the antibody in a medium containing (in mM) 137 NaCl, 3 KCl, 5 glucose, 20 PIPES, 1 mg/ml BSA, and 0.2 IU/ml streptolysin-O (Sigma-Aldrich) at pH 6.8. CCDs were then transferred into the usual dissection medium and incubated with hormones as described above. All antibodies used were from Santa Cruz Biotechnology (Tebu, Le Perray en Yvelines, France): affinity-purified rabbit polyclonal antibody against a peptide mapping at the COOH terminus of human B-Raf (sc-166); rabbit polyclonal IgG against an epitope mapping near the COOH terminus of human Rap-1A (sc-65); a monoclonal IgG1 antibody raised against a peptide mapping at the COOH terminus of Raf-1 p74 of human origin (sc-7267); and an anti-Ras, affinity-purified rat monoclonal antibody derived by fusion of spleen cells from a rat immunized with Y3Ag 1.2.3. rat myeloma cells (sc-35). All these antibodies were directed against the active portion of the proteins (2, 7, 30) and, except for the anti Raf-1 antibody, were previously reported to display inhibitory properties (16, 27).

H-K-ATPase activity assay. H-K-ATPase activity was determined with the radiochemical microassay previously described (4) and adapted for microplate assay. Briefly, to avoid contamination with Na-K-ATPase activity, CCDs were rinsed in a cold Na+-free solution containing (in mM) 0.8 MgSO4, 1 MgCl2, 0.5 CaCl2, 100 Tris · HCl, 1 mg/ml BSA, and mannitol up to 400 mosmol/kgH2O, at pH 7.4. After transfer within 0.5 µl of rinsing solution into a 96-well flat-bottom plastic microplate, samples were permeabilized by adding 2 µl of hypotonic solution (10 mM Na+-free Tris · HCl, pH 7.4, with or without 10-4 M Sch-28080) to each sample and freezing on dry-ice. After thawing and addition of 10 µl of assay medium (see composition below), the microplate was incubated at 37°C for 15 min. Incubation was stopped by cooling and adding 300 µl of an ice-cold suspension of 15% (wt/vol) activated charcoal. After centrifugation, 50-µl aliquots of each supernatant were transferred to a 96-well sample microplate for Cerenkov counting (Trilux microbeta 1450, Wallac, Finland).

H-K-ATPase activity was distinguished from other ATP-hydrolyzing activities on the basis of its sensitivity to Sch-28080, a specific inhibitor of renal H-K-ATPase activities (4). Thus, for each experimental condition, eight samples were divided into two groups, one for measuring total ATPase activity and the other for measuring ATPase activity in the presence of Sch-28080. H-K-ATPase activity was calculated as the difference between these two mean ATPase activities.

The ATPase assay medium contained (in mM) 25 Tris · HCl, 10 MgCl2, 1 EGTA, 2.5 KCl, 50 Tris-ATP, and 200 nCi of [gamma -32P]ATP with or without 300 µM Sch-28080.

Western blot analysis of phospho-ERKs. Pools of 30 CCDs dissected in medium supplemented with leupeptin (25 µg/ml) and aprotinin (25 µg/ml) were preincubated at 37°C for 1 h with or without H-89 and then at 37°C for 10 min with or without isoproterenol. Samples were then transferred with 0.5 µl of incubation medium into 14.5 µl of lysis buffer containing (in mM) 100 NaCl, 1.5 MgCl2, 2 sodium pyrophosphate, 2.5 glycerophosphate, 30 NaF, 1 EGTA, 20 HEPES, 1 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml AEBSF, and 10 µg/ml antipain at pH 7.4, vortexed, and kept on ice for 1 h. Cell lysate was centrifuged at 15,000 g for 10 min, and the supernatant was removed and stored at -80°C until use.

Supernatants were resuspended (vol/vol) in 2× Laemmli, heated at 100°C for 5 min, and analyzed by SDS-PAGE. After electrophoresis on 10% polyacrylamide gels, proteins were electrotransferred onto polyvinylidine difluoride membranes (Immobilon-P, Millipore) and incubated for 1 h with anti-phospho-ERK1/2 antibody (dilution 1/2,000) in PBS with 0.1% Tween (vol/vol) and with 5% (wt/vol) nonfat dry milk. After washing in TBS-Tween, membranes were incubated for 45 min with an anti-rabbit IgG antibody (dilution 1/5,000) coupled to horseradish peroxidase (Transduction Laboratories, Lexington, KY) in TBS-Tween. The antigen-antibody complexes were detected by chemiluminescence with the Super Signal Substrate method (Pierce) according to the manufacturer's instructions.

In each experiment, the amounts of proteins loaded onto each lane of the electrophoresis gel corresponded to the same initial length of isolated CCDs (±5%). The chemiluminescence of each lane was quantified by densitometry (arbitrary units) and expressed in each experiment as percentage of the control lane (no isoproterenol treatment). Results are expressed as means ± SE from several animals.

Measurement of inositol phosphate production. Assays were performed by using the technique developed for proximal tubules (20) with slight modifications (6). Briefly, CCDs were microdissected in the dissection solution supplemented with ibuprofen (10 µM) and adenosine deaminase (0.5 U/ml). Samples were radiolabeled in 50 µl of this medium containing 50 µCi myo-[3H]inositol (1 mCi/ml, Amersham Pharmacia Biotech, Orsay, France) and 2 mM CaCl2 for 2 h at 30°C. Thereafter, tubules were successively rinsed five times in 1 ml of incubation solution (similar to dissection solution except that CaCl2 was 0.5 mM), and pools of CCDs (4- to 7-mm length) were incubated in the presence or absence of isoproterenol at 37°C for 15 min in the incubation solution supplemented with 20 mM LiCl. The reaction was stopped, and the phosphoinositides, free inositol, glycerophospho inositol, and inositol phosphates (IPs) were separated by Dowex chromatography, and their associated radioactivity was counted. Production of IPs was expressed as the percentage of the total radioactivity incorporated in tubules. For each condition, production of IPs was determined in quintuplicate and expressed as the mean value.

Statistics. Results are given as means ± SE from different animals. Data were compared according to either paired or unpaired Student's t-test, as appropriate, or, when more than two groups were compared, according to ANOVA with Fisher's protected least significant difference test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Role of ERKs in isoproterenol-induced stimulation of H-K-ATPase. Because activation of the ERK pathway mediates the stimulation of H-K-ATPase by calcitonin in alpha -intercalated cells of the rat CCD (16), we evaluated whether 1) isoproterenol activates ERK1/2 in CCDs and 2) activation of ERK1/2 mediates the stimulation of H-K-ATPase.

Western blot analysis using an antibody against phospho-ERKs p42 and p44 demonstrated that isoproterenol increased the phosphorylation of ERK1/2 by >50% and that this effect was abolished by the PKA inhibitor H-89 (Fig. 1A).


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Fig. 1.   Activation of ERK mediates isoproterenol action. A: isoproterenol (Iso)-induced phosphorylation of ERK1/2: effect of H-89. After preincubation at 30°C for 1 h in the absence (control; C) or presence of 1 µM H-89 (H89), rat cortical collecting ducts (CCDs) were incubated at 37°C for 10 min without (filled bars) or with 1 µM isoproterenol (shaded bars). Thereafter, samples were solubilized in Triton X-100, and phospho-ERK1/2 (p42 and p44) was revealed by Western blotting using a specific anti-phospho-ERK antibody. Top: representative gels from the same animal. In each experiment, the densitometry of each lane (p42 and p44) was expressed as the percentage of the control value (absence of H-89 and of isoproterenol). Bottom: values are means ± SE of results from 4 animals. Statistical comparison between groups was performed by ANOVA with Fisher's protected least significant difference (PLSD) test. *P < 0.05 vs. control values. B: role of ERK1/2 on hormone-induced stimulation of H-K-ATPase. After preincubation at 30°C for 45 min without (control) or with 5 µM U-0126, CCDs were incubated at 37°C for 10 min without (filled bars) or with 1 µM isoproterenol (shaded bars). Thereafter, H-K-ATPase activity was determined. N, no. of experiments. Statistical comparison between groups was performed by ANOVA with Fisher's PLSD test. **P < 0.001 vs. control values (no isoproterenol).

This finding is compatible with the involvement of ERK activation in the stimulation of H-K-ATPase not only by calcitonin but also by isoproterenol. Indeed, the preincubation with 5 µM U-0126, an inhibitor of the ERK kinase MEK (8), abolished the stimulation of H-K-ATPase induced by isoproterenol (Fig. 1B).

Activation pathway of MEK by isoproterenol. MEK may by activated through phosphorylation by a kinase of the Raf family, Raf-1 or B-Raf. Raf kinases are themselves activated by monomeric G proteins of the Ras family. We therefore evaluated the possible role of Raf kinases and Ras G protein in isoproterenol-induced stimulation of H-K-ATPase.

The pretreatment of CCDs with a monoclonal antibody that inhibits the activity of Ras curtailed the stimulatory effect of isoproterenol (Fig. 2A). In contrast, pretreatment with a polyclonal antibody directed against the related G protein B-Raf, previously shown to inhibit calcitonin-induced stimulation of H-K- ATPase (16), did not alter the effect of isoproterenol on H-K-ATPase (Fig. 2B, left). Similarly, the pretreatment with a monoclonal antibody mapping a COOH-terminal epitope of Raf-1 abolished the stimulatory effect of isoproterenol on H-K-ATPase (Fig. 2A, right), whereas an antibody against B-Raf, which inhibits calcitonin-induced activation of H-K-ATPase (16), had no effect (Fig. 2B, right). The specificity of action of the Ras and Raf-1 antibodies toward their cognate protein is assessed by the fact that these two antibodies had no effect on the related proteins Rap1 and B-Raf (16). Together, these findings suggest that isoproterenol-induced activation of ERK1/2 and H-K-ATPase is mediated by the Ras/Raf-1/MEK1/2 cascade.


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Fig. 2.   Role of Ras and Raf-1 in isoproterenol-induced stimulation of H-K-ATPase. Rat CCDs were preincubated at 4°C for 90 min in the absence (control) or presence of antibodies (dilution 1:100) directed against Ras (A, left), Raf-1 (A, right), Rap-1 (B, left), or B-Raf (B, right) after streptolysin-O permeabilization. Thereafter, samples were incubated at 37°C for 10 min without hormone (filled bars) or 1 µM isoproterenol (shaded bars) before measurement of H-K-ATPase activity. N, no. of experiments. Values are means ± SE. Statistical comparison between groups was performed by ANOVA with Fisher's PLSD test. *P < 0.05, **P < 0.005, and ***P < 0.001 vs. control groups (no isoproterenol).

Because Ras can be activated by PLC, we investigated whether isoproterenol might stimulate PLC. Results in Fig. 3 indicate that in CCDs, isoproterenol did not increase the production of IPs, whereas vasopressin and prostaglandin E2 used as positive controls were efficient.


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Fig. 3.   Isoproterenol does not stimulate phospholipase C. After rat CCDs were loaded with [myo-3H]inositol, they were preincubated at 37°C for 15 min without (basal) or with 1 µM isoproterenol, 1 µM PGE2, or 100 nM AVP. Thereafter, inositol phosphates (IPs) were separated and their radioactivity was determined. Values are means ± SE of results from 4 animals and are expressed as the percentage of the total radioactivity incorporated in CCDs. Statistical comparison between groups was performed by ANOVA with Fisher's PLSD test. *P < 0.01 and **P < 0.001 vs. basal.

Evidence for Gs-to-Gi switch of the beta -adrenergic receptor. Results reported above indicate that stimulation of H-K-ATPase by isoproterenol is mediated by activation of PKA and MEK but is independent of B-Raf, a classic effector of PKA. Several reports have shown that PKA-mediated phosphorylation of the beta -adrenergic receptor can induce a switch from Gs to Gi coupling (5, 32). Such a switch may lead to the release of beta gamma -subunits from Galpha i, the stimulation of Ras by non-receptor tyrosine kinases such as Src, and the activation of Raf-1 and MEK (5, 11). Thus we evaluated whether this pathway might account for isoproterenol-induced stimulation of H-K-ATPase in CCD beta -intercalated cells. For this purpose, we determined the role of Gi and tyrosine kinase activity on the stimulation of H-K-ATPase by isoproterenol.

Preincubation with 0.5 µg/ml of the Gi inhibitor pertussis toxin (Fig. 4A) or with 100 µM of tyrosine kinase inhibitor genistein (Fig. 4B) did not modify basal H-K-ATPase activity but totally blocked its stimulation by isoproterenol.


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Fig. 4.   Role of Galpha i and tyrosine kinases on isoproterenol-induced stimulation of H-K-ATPase. Rat CCDs were preincubated at 30°C for 120 min without inhibitor (control) or with 0.5 µg/ml pertussis toxin (PTX; A) or 100 µM genistein (B). Thereafter, samples were incubated at 37°C for 10 min without hormone (filled bars) or with 1 µM isoproterenol (shaded bars) before measurement of H-K-ATPase activity. N, no. of experiments. Values are means ± SE. Statistical comparison between groups was performed by ANOVA with Fisher's PLSD test. **P < 0.005 vs. control groups (no isoproterenol).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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In the present study, we determined the signaling pathways responsible for the stimulation of H-K- ATPase by isoproterenol in CCD beta -intercalated cells (Fig. 5). Although the signalization of the effect of isoproterenol on H-K-ATPase in beta -intercalated cells is different from that of calcitonin in alpha -intercalated cells, results indicate that the initial and terminal steps of H-K-ATPase stimulation are similar in the two cell types: the stimulation is initiated by the production of cAMP and finally results from the activation of ERKs. However, the coupling between cAMP production and activation of ERK is distinct in the two cell types.


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Fig. 5.   Schematic pathway underlying the stimulation of H-K- ATPase activity by isoproterenol in beta -intercalated cells of CCDs. AC, adenylyl cylcase; R, receptor.

As previously shown, ERK activation in alpha -intercalated cells is a PKA-independent process involving the cAMP-activated guanine nucleotide exchange factor Epac I, the monomeric G protein Rap-1 and the B-Raf kinase (16). In contrast, in beta -intercalated cells, it is dependent on PKA (16) and is not related to the activation of Rap-1 and B-Raf (Fig. 2B, left and right), as previously reported in response to beta -adrenergic agonists in HEK-293 cells (23). Instead, activation of ERK in beta -intercalated cells relies on the stimulation of the classic Ras/Raf-1 pathway (Fig. 2A, left and right). Data from the literature indicate that activation of the monomeric G protein Ras is controlled by the guanine nucleotide exchange factor Sos. Activation of Sos may result either from activation of membrane receptor tyrosine kinases and the recruitment of Grb2 and Sos to the membrane or from a cascade of non-receptor tyrosine kinases (in particular, Pyk2 and Src) triggered by release of calcium in response to PLC activation by protein Gq-coupled receptors. Although activation of the Ras/Raf-1 pathway by isoproterenol in CCDs seemed dependent on tyrosine kinase activity, because it was abolished by genistein (Fig. 4B), it is unlikely to result from the two classic modes of activation of Ras because 1) isoproterenol binds to a G protein-coupled receptor and not to tyrosine kinase receptors; 2) isoproterenol does not activate phospholipase C in beta -intercalated cells (Fig. 3); and 3) isoproterenol's effects on ERKs and H-K-ATPase rely on the activation of PKA (16).

An alternate mode of activation of Ras by beta 2-adrenergic receptors has been observed in HEK-293 cells (5) and in cardiomyocytes (32). This PKA-dependent pathway involves direct activation of the non-receptor tyrosine kinase Src, and hence of Sos and Ras, by the beta gamma -subunits of pertussis toxin-sensitive G proteins. Activation of this pathway requires phosphorylation of the beta 2-adrenergic receptor by PKA, because it is blocked by H-89 or in the presence of a mutant receptor lacking the PKA phosphorylation site. It has been proposed that PKA phosphorylation of the receptor, known to induce its heterologous desensitization, also allows the receptor to switch its coupling from Gs to Gi, thereby triggering the release of beta gamma -subunits. That inhibition of PKA (16), Gi (Fig. 4A), and of tyrosine kinase activity (Fig. 4B) blocked the stimulation of H-K-ATPase by isoproterenol is consistent with the involvement of this signaling pathway in isoproterenol-induced stimulation of H-K-ATPase in beta -intercalated cells of rat CCD.

It is worth mentioning that PKA has been reported to inhibit the Ras/Raf-1-dependent activation of ERKs through either inhibition of Ras binding (31) or direct inhibition of Raf-1 kinase activity (18, 22). The absence of such an inhibition in beta -intercalated cells may result from the different intracellular compartmentalization of PKA on one hand and of Ras/Raf-1 on the other, or from a difference in the time course of activation of PKA and Raf-1. According to the above-mentioned hypothesis, the Gs-to-Gi switch that promotes the activation of Ras and Raf-1 is accompanied by the desensitization of the isoproterenol receptor and therefore also promotes the deactivation of the cAMP-PKA pathway. Thus it is likely that Rap-1 activation may occur at a time when PKA is no longer activated.

Although a causal relationship between activation of ERK and stimulation of H-K-ATPase can be established, on the basis of the inhibition of isoproterenol-induced stimulation of H-K-ATPase by U-0126 (Fig. 1B), the mechanism of ERK action has not been investigated. Phosphorylation of ERK is known to trigger its translocation into the nucleus, where it controls the expression of specific genes through activation of transcription factors. However, the time course of isoproterenol-induced activation of H-K-ATPase is not compatible with de novo protein synthesis but suggests that phosphorylated ERKs may activate preexisting ATPase units. Several observations support the notion that isoproterenol may stimulate H-K-ATPase activity through exocytotic insertion into the plasma membrane of intracellular H-K-ATPase units: 1) in CCDs, H-K-ATPase colocalizes with H-ATPase (3), another proton pump that is controlled through exocytosis/endocytosis in CCDs (24); 2) in gastric mucosa, expression of H-K-ATPase at the luminal membrane of parietal cells is achieved through exocytosis (9); and 3) ERKs control exocytosis in several cell types (14, 21, 29).

In conclusion, the present study provides original data regarding several aspects of protein Gs-coupled receptor signalization in native kidney cells because it characterizes H-K-ATPase as a cytosolic effector of ERK signalization and provides a new example of the Gs-to-Gi switch in beta -adrenergic signalization in native cells.


    FOOTNOTES

Address for reprint requests and other correspondence: A. Doucet, CNRS FRE 2468 and IFR 58, Institut des Cordeliers, 15 rue de l'Ecole de Médecine, 75270 Paris Cedex 6, France (E-mail: alain.doucet{at}bhdc.jussieu.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajprenal.00394.2002

Received 6 November 2002; accepted in final form 6 January 2003.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 284(5):F948-F954
0363-6127/03 $5.00 Copyright © 2003 the American Physiological Society




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