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
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ABSTRACT |
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Isoproterenol stimulates H-K-ATPase
activity in rat cortical collecting duct -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
-adrenergic receptors and the switch from Gs to
Gi coupling. In turn,
-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
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INTRODUCTION |
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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 - and
-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
- and
-intercalated cells express calcitonin and
-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
- and
-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 -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
-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.
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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 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 104 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).
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.
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.
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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 of
H-K-ATPase by calcitonin in -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.
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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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Evidence for Gs-to-Gi switch of the
-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
-adrenergic receptor
can induce a switch from Gs to Gi coupling
(5, 32). Such a switch may lead to the release of
-subunits from G
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
-intercalated cells. For this purpose, we determined the role of
Gi and tyrosine kinase activity on the stimulation of
H-K-ATPase by isoproterenol.
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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 -intercalated cells (Fig.
5). Although the signalization of the
effect of isoproterenol on H-K-ATPase in
-intercalated cells is
different from that of calcitonin in
-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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As previously shown, ERK activation in -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
-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
-adrenergic agonists in HEK-293
cells (23). Instead, activation of ERK in
-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
-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 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
-subunits of pertussis toxin-sensitive G
proteins. Activation of this pathway requires phosphorylation of the
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
-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
-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
-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
-adrenergic signalization in native cells.
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FOOTNOTES |
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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.
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