Mechanism of activation of ERK and H-K-ATPase byisoproterenol in rat cortical c - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:36 |只看该作者 |倒序浏览 |打印

作者：NicolasLaroche-Joubert, SophieMarsy, StéphanieLuriau, MartineImbert-Teboul,  AlainDoucet作者单位：Laboratoire de Biologie Intégrée des CellulesRénales, Service de Biologie Cellulaire, Commissariat al‘Energie Atomique, Saclay, Unité de Recherche Associée185 Centre National de la Recherche Scientifique, 91191 Gif-sur-Yvette Cedex, France
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      【摘要】
   Isoproterenol stimulates H-K-ATPaseactivity in rat cortical collecting duct -intercalated cells througha PKA-dependent pathway. This study aimed at determining the signalingpathway 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 increasedphosphorylation of ERK in a PKA-dependent manner, and inhibition of theERK phosphorylation prevented the stimulation of H-K-ATPase. Antibodiesagainst the monomeric G protein Ras or the kinase Raf-1 curtailed thestimulation of H-K-ATPase by isoproterenol, whereas antibodies againstthe related proteins Rap-1 and B-Raf had no effect. Pertussis toxin andinhibition of tyrosine kinases with genistein also curtailed isoproterenol-induced stimulation of H-K-ATPase. It is proposed thatactivation of PKA by isoproterenol induces the phosphorylation of -adrenergic receptors and the switch from G s toG i coupling. In turn, -subunits released fromG i would activate a tyrosine kinase-Ras-Raf-1 pathway,leading to the activation of ERK1/2 and of H-K-ATPase.
      【关键词】 Ras extracellular signalregulated kinase
   INTRODUCTION
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H-K-ATPASES ARE P-TYPE ATPASES that exchange intracellular proton against extracellularpotassium at the expense of ATP hydrolysis ( 10 ). GastricH-K-ATPase was the first H-K-ATPase to be discovered and remains thearchetype 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 ATPaseactivity 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 thatcollecting duct H-K-ATPase also participates in the regulation ofacid-base balance ( 10 ). However, regulation of potassiumand acid-base balances in the collecting duct may be accounted for bydifferent H-K-ATPases. As a matter of fact, collecting ducts fromnormal and potassium-deprived rats express at least two distinctSch-28080-sensitive ATPases that are insensitive and sensitive toouabain, respectively ( 4 ).
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Rat cortical collecting ducts (CCDs) consist of at least threecell types characterized by distinct morphological and functional features. Principal cells are involved in sodium, potassium, and watertransport, whereas - and -intercalated cells are the site ofproton and bicarbonate secretion, respectively. Functionally, the threecell types of the rat CCD also differ by the presence of specific Gprotein-coupled hormone receptors activating adenylyl cyclase:principal cells express vasopressin V 2 receptors, whereas - and -intercalated cells express calcitonin and -adrenergic receptors, respectively. This hormone selectivity was used to localizefunctionally H-K-ATPases in the different CCD cell types: Sch-28080-sensitive ATPase is stimulated by calcitonin andisoproterenol in normal rats, demonstrating that it is expressed in - and -intercalated cells, whereas in potassium-deprived rats itis activated by vasopressin and therefore originates from principalcells ( 15 ). These findings further support the hypothesisthat H-K-ATPase-mediated regulation of acid-base balance and potassiumbalance in collecting duct might be accounted for by two distinct formsof H-K-ATPase originating from different cell types. They alsodemonstrate that besides long-term regulation of expression, H-K-ATPaseactivity is modulated in the short term by posttranscriptionalmechanisms. The stimulation of H-K-ATPase by calcitonin andisoproterenol observed in CCDs of normal rats, along with that ofH-ATPase previously reported ( 25 ), likely participates tothe short-term regulation of proton transport by these two hormones( 25, 26 ).; a, h0 |3 x# }- Y  i( J3 ^, R

The signaling pathways underlying calcitonin- and isoproterenol-inducedactivation of H-K-ATPase are different. Although stimulation ofH-K-ATPase by either hormone is mediated by cAMP ( 15 ), the action of isoproterenol relies on the activation of PKA, whereas thatof calcitonin is independent of PKA. The PKA-independent mechanism ofactivation of H-K-ATPase by calcitonin in -intercalated cells hasbeen characterized: the calcitonin-induced increase in cAMP activatesthe cAMP-activated guanine-nucleotide exchange factor Epac I, which inturn activates a cascade that includes the monomeric G protein Rap-1,the B-Raf kinase, the MAP kinase kinase MEK, and the extracellularsignal-regulated kinases ERK1/2 ( 16 ). Activation of ERK1/2leads to the stimulation of H-K-ATPase activity through anontranscriptional mechanism. In contrast, the PKA-dependent signalingmechanism of isoproterenol in -intercalated cells has not beenidentified as yet and was therefore investigated in the present study.Because PKA-dependent activation of ERK has been reported in theliterature ( 5, 11, 32 ), we determined the potentialinvolvement of ERK in isoproterenol-induced activation of H-K-ATPaseand the cascade leading to ERK phosphorylation.
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MATERIALS AND METHODS
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Animal preparation and tubule microdissection. Experiments were carried out in male Sprague-Dawley rats anesthetizedwith pentobarbital sodium (50 mg/kg body wt). CCDs were dissected at4°C from collagenase-treated kidneys as described previously( 12 ). After microdissection, they were photographed todetermine their length, which served for normalization of the results.Unless indicated otherwise, microdissection was carried out in asolution containing (in mM) 120 NaCl, 5 KCl, 1 MgSO 4, 4 NaHCO 3, 0.2 NaH 2 PO 4, 0.15 Na 2 HPO 4, 5 glucose, 0.5 CaCl 2, 0.08 dextran, 2 lactate, 20 HEPES, and 4 essential and nonessential aminoacids, as well as 0.03 vitamins and 1 mg/ml BSA. The pH was adjusted to7.4 and osmotic pressure to 400 mosmol/kgH 2 O 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.* O: W) B, D/ S2 k7 j5 ~+ s1 D

Pretreament with inhibitors and hormones. Microdissected CCDs were preincubated with specific inhibitors ofdifferent intermediates of signaling pathways or with their solventbefore incubation with hormones. Pools of CCDs dissected fromcollagenase-treated kidneys (2- to 4-mm length) were pretreated with orwithout 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 fromCalbiochem (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, exceptU-0126, which was dissolved in DMSO. The corresponding control groupscontained the same concentration of DMSO ( vol/vol).

When specific inhibitors were not commercially available, we evaluatedthe effect of antibodies directed against several signaling proteins.Intracellular entry of antibodies was made possible by transientpermeabilization 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 ina 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. CCDswere then transferred into the usual dissection medium and incubatedwith hormones as described above. All antibodies used were from SantaCruz Biotechnology (Tebu, Le Perray en Yvelines, France):affinity-purified rabbit polyclonal antibody against a peptide mappingat the COOH terminus of human B-Raf (sc-166); rabbit polyclonal IgGagainst an epitope mapping near the COOH terminus of human Rap-1A(sc-65); a monoclonal IgG 1 antibody raised against apeptide mapping at the COOH terminus of Raf-1 p74 of human origin(sc-7267); and an anti-Ras, affinity-purified rat monoclonal antibodyderived by fusion of spleen cells from a rat immunized with Y3Ag 1.2.3. rat myeloma cells (sc-35). All these antibodies were directed againstthe active portion of the proteins ( 2, 7, 30 ) and, exceptfor the anti Raf-1 antibody, were previously reported to displayinhibitory properties ( 16, 27 ).

H-K-ATPase activity assay. H-K-ATPase activity was determined with the radiochemical microassaypreviously described ( 4 ) and adapted for microplate assay.Briefly, to avoid contamination with Na-K-ATPase activity, CCDs wererinsed in a cold Na -free solution containing (in mM) 0.8 MgSO 4, 1 MgCl 2, 0.5 CaCl 2, 100 Tris · HCl, 1 mg/ml BSA, and mannitol up to 400 mosmol/kgH 2 O, at pH 7.4. After transfer within 0.5 µl ofrinsing solution into a 96-well flat-bottom plastic microplate, sampleswere permeabilized by adding 2 µl of hypotonic solution (10 mMNa -free Tris · HCl, pH 7.4, with orwithout 10 4 M Sch-28080) to each sample and freezing ondry-ice. After thawing and addition of 10 µl of assay medium (seecomposition below), the microplate was incubated at 37°C for 15 min.Incubation was stopped by cooling and adding 300 µl of an ice-coldsuspension of 15% (wt/vol) activated charcoal. After centrifugation,50-µl aliquots of each supernatant were transferred to a 96-wellsample microplate for Cerenkov counting (Trilux microbeta 1450, Wallac, Finland).
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H-K-ATPase activity was distinguished from other ATP-hydrolyzingactivities on the basis of its sensitivity to Sch-28080, a specificinhibitor of renal H-K-ATPase activities ( 4 ). Thus, foreach experimental condition, eight samples were divided into twogroups, one for measuring total ATPase activity and the other formeasuring ATPase activity in the presence of Sch-28080. H-K-ATPase activity was calculated as the difference between these two mean ATPase activities.  v; m# O0 P/ y3 U, K
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The ATPase assay medium contained (in mM) 25 Tris · HCl, 10 MgCl 2, 1 EGTA, 2.5 KCl, 50 Tris-ATP, and 200 nCi of [ - 32 P]ATP with orwithout 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 for1 h with or without H-89 and then at 37°C for 10 min with orwithout isoproterenol. Samples were then transferred with 0.5 µl ofincubation medium into 14.5 µl of lysis buffer containing (in mM) 100 NaCl, 1.5 MgCl 2, 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 at15,000 g for 10 min, and the supernatant was removed andstored at 80°C until use." a$ s( `6 r& W. a3 ]5 N

Supernatants were resuspended (vol/vol) in 2× Laemmli, heated at100°C for 5 min, and analyzed by SDS-PAGE. After electrophoresis on10% 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) nonfatdry milk. After washing in TBS-Tween, membranes were incubated for 45 min with an anti-rabbit IgG antibody (dilution 1/5,000) coupled tohorseradish peroxidase (Transduction Laboratories, Lexington, KY) inTBS-Tween. The antigen-antibody complexes were detected bychemiluminescence with the Super Signal Substrate method (Pierce)according to the manufacturer's instructions.

In each experiment, the amounts of proteins loaded onto each lane ofthe electrophoresis gel corresponded to the same initial length ofisolated CCDs (±5%). The chemiluminescence of each lane wasquantified by densitometry (arbitrary units) and expressed in eachexperiment as percentage of the control lane (no isoproterenol treatment). Results are expressed as means ± SE from several animals.2 V- G+ b! A* T  O( C

Measurement of inositol phosphate production. Assays were performed by using the technique developed for proximaltubules ( 20 ) with slight modifications ( 6 ).Briefly, CCDs were microdissected in the dissection solutionsupplemented with ibuprofen (10 µM) and adenosine deaminase (0.5 U/ml). Samples were radiolabeled in 50 µl of this medium containing50 µCi myo-[ 3 H]inositol (1 mCi/ml, Amersham PharmaciaBiotech, Orsay, France) and 2 mM CaCl 2 for 2 h at30°C. Thereafter, tubules were successively rinsed five times in 1 mlof incubation solution (similar to dissection solution except thatCaCl 2 was 0.5 mM), and pools of CCDs (4- to 7-mm length)were incubated in the presence or absence of isoproterenol at 37°Cfor 15 min in the incubation solution supplemented with 20 mM LiCl. Thereaction was stopped, and the phosphoinositides, free inositol,glycerophospho inositol, and inositol phosphates (IPs) were separatedby Dowex chromatography, and their associated radioactivity wascounted. Production of IPs was expressed as the percentage of the totalradioactivity incorporated in tubules. For each condition, productionof IPs was determined in quintuplicate and expressed as the mean value." d$ Q+ a- O5 c* s
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Statistics. Results are given as means ± SE from different animals. Data werecompared according to either paired or unpaired Student's t -test, as appropriate, or, when more than two groups werecompared, according to ANOVA with Fisher's protected least significantdifference test., h  i! l8 T: P( z5 ~8 L. ^5 e. n6 m
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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 ofH-K-ATPase by calcitonin in -intercalated cells of the rat CCD( 16 ), we evaluated whether 1 ) isoproterenolactivates ERK1/2 in CCDs and 2 ) activation of ERK1/2mediates the stimulation of H-K-ATPase.* K' i# C; i" }5 n6 g% p" R8 K) F

Western blot analysis using an antibody against phospho-ERKs p42 andp44 demonstrated that isoproterenol increased the phosphorylation 50% and that this effect was abolished by the PKA inhibitor H-89 (Fig. 1 A ).
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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 theabsence (control; C) or presence of 1 µM H-89 (H89), rat corticalcollecting 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 andp44) was revealed by Western blotting using a specific anti-phospho-ERKantibody. Top : representative gels from the same animal. Ineach experiment, the densitometry of each lane (p42 and p44) wasexpressed as the percentage of the control value (absence of H-89 andof isoproterenol). Bottom : values are means ± SE of resultsfrom 4 animals. Statistical comparison between groups was performed byANOVA with Fisher's protected least significant difference (PLSD)test. * P B : roleof ERK1/2 on hormone-induced stimulation of H-K-ATPase. Afterpreincubation at 30°C for 45 min without (control) or with 5 µMU-0126, CCDs were incubated at 37°C for 10 min without (filled bars)or with 1 µM isoproterenol (shaded bars). Thereafter, H-K-ATPaseactivity was determined. N, no. of experiments. Statisticalcomparison between groups was performed by ANOVA with Fisher's PLSDtest. ** P7 u: ]! T  d9 x1 G4 w
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This finding is compatible with the involvement of ERK activation inthe stimulation of H-K-ATPase not only by calcitonin but also byisoproterenol. Indeed, the preincubation with 5 µM U-0126, aninhibitor of the ERK kinase MEK ( 8 ), abolished the stimulation of H-K-ATPase induced by isoproterenol (Fig. 1 B ).
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Activation pathway of MEK by isoproterenol. MEK may by activated through phosphorylation by a kinase of the Raffamily, Raf-1 or B-Raf. Raf kinases are themselves activated bymonomeric G proteins of the Ras family. We therefore evaluated thepossible 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 theactivity of Ras curtailed the stimulatory effect of isoproterenol (Fig. 2 A ). In contrast, pretreatmentwith a polyclonal antibody directed against the related G proteinB-Raf, previously shown to inhibit calcitonin-induced stimulation ofH-K- ATPase ( 16 ), did not alter the effect ofisoproterenol on H-K-ATPase (Fig. 2 B, left ). Similarly, thepretreatment with a monoclonal antibody mapping a COOH-terminal epitopeof Raf-1 abolished the stimulatory effect of isoproterenol onH-K-ATPase (Fig. 2 A, right ), whereas an antibody againstB-Raf, which inhibits calcitonin-induced activation of H-K-ATPase( 16 ), had no effect (Fig. 2 B, right ). Thespecificity of action of the Ras and Raf-1 antibodies toward theircognate protein is assessed by the fact that these two antibodies hadno effect on the related proteins Rap1 and B-Raf ( 16 ).Together, these findings suggest that isoproterenol-induced activationof ERK1/2 and H-K-ATPase is mediated by the Ras/Raf-1/MEK1/2 cascade." y. [% [2 i$ Z4 V
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Fig. 2. Role of Ras and Raf-1 in isoproterenol-inducedstimulation 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 ) afterstreptolysin-O permeabilization. Thereafter, samples were incubated at37°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 comparisonbetween groups was performed by ANOVA with Fisher's PLSD test.* P P P0 y+ K/ c$ D: q; B) B4 q' M

Because Ras can be activated by PLC, we investigated whetherisoproterenol might stimulate PLC. Results in Fig. 3 indicate that in CCDs, isoproterenoldid not increase the production of IPs, whereas vasopressin andprostaglandin E 2 used as positive controls were efficient.* k, o6 ~" S: k3 d5 y7 K. ?
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Fig. 3. Isoproterenol does not stimulate phospholipase C. Afterrat CCDs were loaded with [myo- 3 H]inositol, they werepreincubated at 37°C for 15 min without (basal) or with 1 µMisoproterenol, 1 µM PGE 2, or 100 nM AVP. Thereafter,inositol phosphates (IPs) were separated and their radioactivity wasdetermined. Values are means ± SE of results from 4 animals andare expressed as the percentage of the total radioactivity incorporatedin CCDs. Statistical comparison between groups was performed by ANOVAwith Fisher's PLSD test. * P P7 ]7 V7 |5 L' e
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Evidence for G s -to-G i switch of the -adrenergic receptor. Results reported above indicate that stimulation of H-K-ATPase byisoproterenol is mediated by activation of PKA and MEK but isindependent of B-Raf, a classic effector of PKA. Several reports haveshown that PKA-mediated phosphorylation of the -adrenergic receptorcan induce a switch from G s to G i coupling( 5, 32 ). Such a switch may lead to the release of -subunits from G i, the stimulation of Ras bynon-receptor tyrosine kinases such as Src, and the activation of Raf-1and MEK ( 5, 11 ). Thus we evaluated whether this pathwaymight account for isoproterenol-induced stimulation of H-K-ATPase inCCD -intercalated cells. For this purpose, we determined the role ofG i and tyrosine kinase activity on the stimulation ofH-K-ATPase by isoproterenol.
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Preincubation with 0.5 µg/ml of the G i inhibitorpertussis toxin (Fig. 4 A ) orwith 100 µM of tyrosine kinase inhibitor genistein (Fig. 4 B ) did not modify basal H-K-ATPase activity but totally blocked its stimulation by isoproterenol.

Fig. 4. Role of G i and tyrosine kinases onisoproterenol-induced stimulation of H-K-ATPase. Rat CCDs werepreincubated at 30°C for 120 min without inhibitor (control) or with0.5 µg/ml pertussis toxin (PTX; A ) or 100 µM genistein( B ). Thereafter, samples were incubated at 37°C for 10 minwithout hormone (filled bars) or with 1 µM isoproterenol (shadedbars) before measurement of H-K-ATPase activity. N, no. ofexperiments. Values are means ± SE. Statistical comparisonbetween groups was performed by ANOVA with Fisher's PLSD test.** P& i( U; @2 {0 \7 i. k- D
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DISCUSSION

In the present study, we determined the signaling pathwaysresponsible for the stimulation of H-K- ATPase byisoproterenol in CCD -intercalated cells (Fig. 5 ). Although the signalization of theeffect of isoproterenol on H-K-ATPase in -intercalated cells isdifferent from that of calcitonin in -intercalated cells, resultsindicate that the initial and terminal steps of H-K-ATPase stimulationare similar in the two cell types: the stimulation is initiated by theproduction of cAMP and finally results from the activation of ERKs.However, the coupling between cAMP production and activation of ERK isdistinct in the two cell types.

Fig. 5. Schematic pathway underlying the stimulation ofH-K- ATPase activity by isoproterenol in -intercalated cells ofCCDs. AC, adenylyl cylcase; R, receptor.3 J9 B/ D- y* o
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As previously shown, ERK activation in -intercalated cells is aPKA-independent process involving the cAMP-activated guanine nucleotideexchange factor Epac I, the monomeric G protein Rap-1 and the B-Rafkinase ( 16 ). In contrast, in -intercalated cells, it isdependent on PKA ( 16 ) and is not related to the activation of Rap-1 and B-Raf (Fig. 2 B, left and right ), aspreviously reported in response to -adrenergic agonists in HEK-293cells ( 23 ). Instead, activation of ERK in -intercalatedcells relies on the stimulation of the classic Ras/Raf-1 pathway (Fig. 2 A, left and right ). Data from the literatureindicate that activation of the monomeric G protein Ras is controlledby the guanine nucleotide exchange factor Sos. Activation of Sos mayresult either from activation of membrane receptor tyrosine kinases andthe recruitment of Grb2 and Sos to the membrane or from a cascade ofnon-receptor tyrosine kinases (in particular, Pyk2 and Src) triggeredby release of calcium in response to PLC activation by proteinG q -coupled receptors. Although activation of the Ras/Raf-1pathway by isoproterenol in CCDs seemed dependent on tyrosine kinaseactivity, because it was abolished by genistein (Fig. 4 B ),it is unlikely to result from the two classic modes of activationof Ras because 1 ) isoproterenol binds to a Gprotein-coupled receptor and not to tyrosine kinase receptors; 2 ) isoproterenol does not activate phospholipase C in -intercalated cells (Fig. 3 ); and 3 ) isoproterenol'seffects on ERKs and H-K-ATPase rely on the activation of PKA( 16 ).) p% V6 N. {" l( ~$ b  h5 P
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An alternate mode of activation of Ras by 2 -adrenergicreceptors has been observed in HEK-293 cells ( 5 ) and incardiomyocytes ( 32 ). This PKA-dependent pathway involvesdirect activation of the non-receptor tyrosine kinase Src, and hence ofSos and Ras, by the -subunits of pertussis toxin-sensitive Gproteins. Activation of this pathway requires phosphorylation of the 2 -adrenergic receptor by PKA, because it is blocked byH-89 or in the presence of a mutant receptor lacking the PKAphosphorylation site. It has been proposed that PKA phosphorylation ofthe receptor, known to induce its heterologous desensitization, alsoallows the receptor to switch its coupling from G s toG i, thereby triggering the release of -subunits. Thatinhibition of PKA ( 16 ), G i (Fig. 4 A ), and of tyrosine kinase activity (Fig. 4 B )blocked the stimulation of H-K-ATPase by isoproterenol is consistentwith the involvement of this signaling pathway in isoproterenol-inducedstimulation of H-K-ATPase in -intercalated cells of rat CCD.! h- E+ \* V* j7 D+ a7 E

It is worth mentioning that PKA has been reported to inhibit theRas/Raf-1-dependent activation of ERKs through either inhibition of Rasbinding ( 31 ) or direct inhibition of Raf-1 kinase activity ( 18, 22 ). The absence of such an inhibition in -intercalated cells may result from the different intracellularcompartmentalization 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, theG s -to-G i switch that promotes the activation ofRas and Raf-1 is accompanied by the desensitization of theisoproterenol receptor and therefore also promotes the deactivation ofthe cAMP-PKA pathway. Thus it is likely that Rap-1 activation may occurat a time when PKA is no longer activated.

Although a causal relationship between activation of ERK andstimulation of H-K-ATPase can be established, on the basis of theinhibition of isoproterenol-induced stimulation of H-K-ATPase by U-0126(Fig. 1 B ), the mechanism of ERK action has not been investigated. Phosphorylation of ERK is known to trigger itstranslocation into the nucleus, where it controls the expression ofspecific genes through activation of transcription factors. However,the time course of isoproterenol-induced activation of H-K-ATPase isnot compatible with de novo protein synthesis but suggests thatphosphorylated ERKs may activate preexisting ATPase units. Severalobservations support the notion that isoproterenol may stimulateH-K-ATPase activity through exocytotic insertion into the plasmamembrane of intracellular H-K-ATPase units: 1 ) in CCDs, H-K-ATPase colocalizes with H-ATPase ( 3 ), another protonpump that is controlled through exocytosis/endocytosis in CCDs( 24 ); 2 ) in gastric mucosa, expression ofH-K-ATPase at the luminal membrane of parietal cells is achievedthrough exocytosis ( 9 ); and 3 ) ERKscontrol exocytosis in several cell types ( 14, 21, 29 ).* `5 Q2 S) c. m; T; I) Y. F6 J+ N' L

In conclusion, the present study provides original data regardingseveral aspects of protein G s -coupled receptorsignalization in native kidney cells because it characterizesH-K-ATPase as a cytosolic effector of ERK signalization and provides anew example of the G s -to-G i switch in -adrenergic signalization in native cells.
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