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Organ Hypertrophic Signaling within Caveolae Membrane Subdomains Triggered by Ouabain and Antagonized by PST 2238

2004; Elsevier BV; Volume: 279; Issue: 32 Linguagem: Inglês

10.1074/jbc.m402187200

ISSN

1083-351X

Autores

Mara Ferrandi, Isabella Molinari, Paolo Barassi, E. Minotti, Giuseppe Bianchi, Paolo Ferrari,

Tópico(s)

Metabolism, Diabetes, and Cancer

Resumo

In addition to inhibition of the Na-K ATPase, ouabain activates a signal transduction function, triggering growth and proliferation of cultured cells even at nanomolar concentrations. An isomer of ouabain (EO) circulates in mammalians at subnanomolar concentrations, and increased levels are associated with cardiac hypertrophy and hypertension. We present here a study of cardiac and renal hypertrophy induced by ouabain infused into rats for prolonged periods and relate this effect to the recently described ouabain-induced activation of the Src-EGFr-ERK signaling pathway. Ouabain infusion into rats (15 μg/kg/day for 18 weeks) doubled plasma ouabain levels from 0.3 to 0.7 nm and increased blood pressure by 20 mm Hg (p < 0.001), cardiac left ventricle (+11%, p < 0.05), and kidney weight (+9%, p < 0.01). These effects in vivo are associated with a significant enrichment of α1, β1, γa Na-K ATPase subunits together with Src and EGFr in isolated renal caveolae membranes and activation of ERK1/2. In caveolae, direct Na-K ATPase/Src interactions can be demonstrated by co-immunoprecipitation. The interaction is amplified by ouabain, at a high affinity binding site, detectable in caveolae but not in total rat renal membranes. The high affinity site for ouabain is associated with Src-dependent tyrosine phosphorylation of rat α1 Na-K ATPase. The antihypertensive compound, PST 2238, antagonized all ouabain-induced effects at 10 μg/kg/day in vivo or 10-10-10-8min vitro. These findings provide a molecular mechanism for the in vivo pro-hypertrophic and hypertensinogenic activity of ouabain, or by analogy those of EO in humans. They also explain the pharmacological basis for PST 2238 treatment. In addition to inhibition of the Na-K ATPase, ouabain activates a signal transduction function, triggering growth and proliferation of cultured cells even at nanomolar concentrations. An isomer of ouabain (EO) circulates in mammalians at subnanomolar concentrations, and increased levels are associated with cardiac hypertrophy and hypertension. We present here a study of cardiac and renal hypertrophy induced by ouabain infused into rats for prolonged periods and relate this effect to the recently described ouabain-induced activation of the Src-EGFr-ERK signaling pathway. Ouabain infusion into rats (15 μg/kg/day for 18 weeks) doubled plasma ouabain levels from 0.3 to 0.7 nm and increased blood pressure by 20 mm Hg (p < 0.001), cardiac left ventricle (+11%, p < 0.05), and kidney weight (+9%, p < 0.01). These effects in vivo are associated with a significant enrichment of α1, β1, γa Na-K ATPase subunits together with Src and EGFr in isolated renal caveolae membranes and activation of ERK1/2. In caveolae, direct Na-K ATPase/Src interactions can be demonstrated by co-immunoprecipitation. The interaction is amplified by ouabain, at a high affinity binding site, detectable in caveolae but not in total rat renal membranes. The high affinity site for ouabain is associated with Src-dependent tyrosine phosphorylation of rat α1 Na-K ATPase. The antihypertensive compound, PST 2238, antagonized all ouabain-induced effects at 10 μg/kg/day in vivo or 10-10-10-8min vitro. These findings provide a molecular mechanism for the in vivo pro-hypertrophic and hypertensinogenic activity of ouabain, or by analogy those of EO in humans. They also explain the pharmacological basis for PST 2238 treatment. Until recently, the main, if not unique, function ascribed to the integral membrane protein Na-K ATPase is the maintenance and regulation of the electrochemical gradient across the cell membrane in all tissues (1Lingrel J.B. Kuntzweiler T. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar). Ouabain and other steroidal cardenolides (2Schoner W. Eur. J. Biochem. 2002; 269: 2440-2448Crossref PubMed Scopus (306) Google Scholar) or bufadienolides (3Bagrov A.Y. Dmitrieva R.I. Fedorova O. Kazakov G. Roukoyatkina N. Shpen V. Am. J. Hypertension. 1996; 9: 982-990Crossref PubMed Scopus (44) Google Scholar) are considered to be the specific inhibitors of the Na-K ATPase activity. However, in recent years, several studies have indicated that Na-K ATPase can also act as a signal transducer in response to the interaction with its natural ligand ouabain (4Xie Z. Askari A. Eur. J. Biochem. 2002; 269: 2434-2439Crossref PubMed Scopus (511) Google Scholar). This finding originates mainly from studies carried out on cultured rat cardiomyocytes or renal tubular cells based on effects on cell growth and hypertrophy of ouabain in the micromolar range. At these rather high concentrations, which, however, do not seem to affect the bulk intracellular Na+ and Ca2+ concentrations (5Liu J. Tian J. Haas M. Shapiro J.I. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27838-27844Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar), ouabain activates: (a) tyrosine phosphorylation of the epidermal growth factor receptor (EGFr), 1The abbreviations used are: EGFr, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid; CS, control normotensive rats; EO, endogenous ouabain; HR, heart rate; LVFW, left ventricle free-wall; MT, total renal membranes; OS, ouabain-infused rats; SBP, systolic blood pressure; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Src, and p42/44 mitogen-activated protein kinase (MAPKs) in both neonatal rat cardiac myocytes and A7r5 cells (4Xie Z. Askari A. Eur. J. Biochem. 2002; 269: 2434-2439Crossref PubMed Scopus (511) Google Scholar, 6Haas M. Wang H. Tian J. Xie Z. J. Biol. Chem. 2002; 277: 18694-18702Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar); (b) the same signaling pathway within the cellular membrane microdomain of caveolae in isolated perfused rat heart (7Liu L. Mohammadi L. Aynafshar B. Wang H. Li D. Liu J. Ivanov A. Xie Z. Askari A. Am. J. Physiol. Cell. Physiol. 2003; 284: C1550-C1560Crossref PubMed Scopus (159) Google Scholar); and (c) slow intracellular Ca2+ oscillations in rat tubular cells that favor the association of Na-K ATPase with the inositol 1,4,5-trisphosphate receptor (InsP3R) in a signaling microdomain (8Miyakawa-Naito A. Uhlen P. Lal M. Aizman O. Mikoshiba K. Brismar H. Zelenin S. Aperia A. J. Biol. Chem. 2003; 278: 50355-50361Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Recently, it has been shown that even subnanomolar ouabain concentrations stimulate growth of cultured rat tubular cells, via activation of an extracellular signal-regulated kinase (ERK)-dependent pathway and, surprisingly, in view of the fact that these rat cells contain only the α1 isoform of Na-K ATPase, ouabain inhibits a component of active 86Rb uptake with a high affinity (9Dmitrieva R.I. Doris P.A. J. Biol. Chem. 2003; 278: 28160-28166Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The endogenous ouabain (EO), or a close related isomer (10Tymiak A. Norman J.A. Bolgar M. DiDonato G. Lee H. Parker W. Lo L.C. Berova N. Nakanishi K. Haber E. Haupert G. Proc. Natl. Acad. Sci. 1993; 90: 8189-8193Crossref PubMed Scopus (220) Google Scholar), circulates in mammalians at concentrations in the subnanomolar range (11Ferrandi M. Manunta P. Balzan S. Hamlyn J.M. Bianchi G. Ferrari P. Hypertension. 1997; 30: 886-896Crossref PubMed Scopus (119) Google Scholar, 12Manunta P. Messaggio E. Ballabeni C. Sciarrone M.T. Lanzani C. Ferrandi M. Hamlyn J.M. Cusi D. Galletti F. Bianchi G. Hypertension. 2001; 38: 198-203Crossref PubMed Scopus (100) Google Scholar, 13Balzan S. Neglia D. Ghione S. D'Urso G. Baldacchino M.C. Montali U. L'Abbate A. Eur. J. Heart Failure. 2001; 3: 165-171Crossref PubMed Scopus (42) Google Scholar) and has been implicated in the development of hypertension (12Manunta P. Messaggio E. Ballabeni C. Sciarrone M.T. Lanzani C. Ferrandi M. Hamlyn J.M. Cusi D. Galletti F. Bianchi G. Hypertension. 2001; 38: 198-203Crossref PubMed Scopus (100) Google Scholar, 14Hamlyn J.M. Ringel R. Schaeffer J. Levinson P.D. Hamilton B. Kowarski A.A. Blaustein M.P. Nature. 1982; 300: 650-652Crossref PubMed Scopus (520) Google Scholar), alterations of renal sodium reabsorption and cardiac and renal complications (12Manunta P. Messaggio E. Ballabeni C. Sciarrone M.T. Lanzani C. Ferrandi M. Hamlyn J.M. Cusi D. Galletti F. Bianchi G. Hypertension. 2001; 38: 198-203Crossref PubMed Scopus (100) Google Scholar, 13Balzan S. Neglia D. Ghione S. D'Urso G. Baldacchino M.C. Montali U. L'Abbate A. Eur. J. Heart Failure. 2001; 3: 165-171Crossref PubMed Scopus (42) Google Scholar, 14Hamlyn J.M. Ringel R. Schaeffer J. Levinson P.D. Hamilton B. Kowarski A.A. Blaustein M.P. Nature. 1982; 300: 650-652Crossref PubMed Scopus (520) Google Scholar, 15Manunta P. Stella P. Rivera R. Ciurlino D. Cusi D. Ferrandi M. Hamlyn J.M. Bianchi G. Hypertension. 1999; 34: 450-456Crossref PubMed Scopus (161) Google Scholar, 16Pierdomenico S. Bucci A. Manunta P. Rivera R. Ferrandi M. Hamlyn J.M. Lapenna D. Cuccurullo F. Mezzetti A. Am. J. Hypertension. 2001; 14: 44-50Crossref PubMed Scopus (95) Google Scholar). Furthermore, the hypertensinogenic activity of low ouabain concentrations might also be ascribed to a specific vasotonic effect as demonstrated in rodent vessels (17Blaustein M.P. Juhaszova M. Golovina V.A. Clin. Exp. Hypertension. 1998; 20: 691-703Crossref PubMed Scopus (107) Google Scholar, 18Shelly D.A. He S. Moseley A. Weber C. Stegemeyer M. Lynch R. Lingrel J. Paul R.J. Am. J. Physiol. 2004; 286: C813-C820Crossref PubMed Scopus (73) Google Scholar). However, a direct demonstration that the ouabain/Na-K ATPase signaling effects are also relevant to the cardiovascular effects of EO in vivo is still lacking and the following questions have to be elucidated, at least in the experimental rat model. 1) Do in vivo variations of rat plasma ouabain, within the subnanomolar concentrations range, produce cardiac and renal hypertrophy and activate an ERK-dependent transduction pathway mediated by the interaction of α1 Na-K ATPase with signaling proteins in defined membrane microdomains, such as caveolae? 2) Is it possible to reproduce in vitro the in vivo signaling effects by demonstrating that subnanomolar concentrations of ouabain trigger interactions among Na-K ATPase, Src, and EGFr? The new ouabain-antagonist, PST 2238 (19Quadri L. Bianchi G. Cerri A. Fedrizzi G. Ferrari P. Gobbini M. Melloni P. Sputore S. Torri M. J. Med. Chem. 1997; 40: 1561-1564Crossref PubMed Scopus (55) Google Scholar, 20Ferrari P. Torielli L. Ferrandi M. Padoani G. Duzzi L. Florio M. Conti F. Melloni P. Vesci L. Corsico N. Bianchi G. J. Pharmacol. Exp. Ther. 1998; 285: 83-94PubMed Google Scholar) is an important tool in this study. We have demonstrated previously that PST 2238 selectively binds to Na-K ATPase and not to other general or hormonal receptors (20Ferrari P. Torielli L. Ferrandi M. Padoani G. Duzzi L. Florio M. Conti F. Melloni P. Vesci L. Corsico N. Bianchi G. J. Pharmacol. Exp. Ther. 1998; 285: 83-94PubMed Google Scholar) involved in blood pressure regulation or hormonal steroid control, antagonizes the pressor effect of subnanomolar ouabain concentrations and normalizes the ouabain-induced renal Na-K ATPase up-regulation in rats and renal cells (20Ferrari P. Torielli L. Ferrandi M. Padoani G. Duzzi L. Florio M. Conti F. Melloni P. Vesci L. Corsico N. Bianchi G. J. Pharmacol. Exp. Ther. 1998; 285: 83-94PubMed Google Scholar). This pharmacological tool has now been used to substantiate the specificity of the ouabain/Na-K ATPase interaction in caveolae and to provide evidence on its molecular mechanism of action. Materials—Osmotic mini-pumps (Mod. 2002, Mod. 2004, Alzet, Charles River, Calco, Italy). The following chemicals were used: Ouabain (Sigma); Amlodipine besylate (Aapin Chemicals Limited-UK); the ouabain-antagonist PST 2238 (17β-(3-furyl)-5β-androstan-3β,14β,17α-triol) (Fig. 1), a digitoxigenin derivate (Sigma-tau, Pomezia, Rome, Italy) whose synthesis and pharmacological characteristics are described elsewhere (19Quadri L. Bianchi G. Cerri A. Fedrizzi G. Ferrari P. Gobbini M. Melloni P. Sputore S. Torri M. J. Med. Chem. 1997; 40: 1561-1564Crossref PubMed Scopus (55) Google Scholar, 20Ferrari P. Torielli L. Ferrandi M. Padoani G. Duzzi L. Florio M. Conti F. Melloni P. Vesci L. Corsico N. Bianchi G. J. Pharmacol. Exp. Ther. 1998; 285: 83-94PubMed Google Scholar). [γ-32P]ATP (0.5-3 Ci/mmol, 3000 Ci/mmol) and [3H]ouabain (15 Ci/mmol) (Amersham Biosciences). The following antibodies were used: anti-caveolin (BD); anti-clathrin (Cymbus, CBL); anti-α1 and anti-β1 Na-K ATPase (UBI); anti-α2 Na-K ATPase (McB2, from K. Sweadner); anti-α3 Na-K ATPase (Biomol); anti-Src (Santa Cruz Biotechnology); anti-Src, GD11 clone (UBI); Src-Tyr418 and Src-Tyr529 (BIOSOURCE); ERK and dual-phosphorylated (Thr202/Tyr204) ERK (Cell Signaling); anti-γa/γb Na-K ATPase (C33), anti-γa, and anti-γb Na-K ATPase (from S. Karlish) and anti-EGFr (from P. Di Fiore); anti-phosphotyrosine PY99 (Santa Cruz Biotechnology); and recombinant active Src and Src-substrate peptide (UBI); Src inhibitor PP2 (Calbiochem). In Vivo Studies—All procedures were in accordance with Institutional Guidelines for animal care. Three-week-old male Sprague-Dawley rats (Harlan, IN), weighing 100-110 g, were subcutaneously implanted with osmotic mini-pumps, releasing either 15 μg/kg/day of ouabain (OS rats, n = 20) for 18 weeks or sterile saline (CS rats, n = 10) (20Ferrari P. Torielli L. Ferrandi M. Padoani G. Duzzi L. Florio M. Conti F. Melloni P. Vesci L. Corsico N. Bianchi G. J. Pharmacol. Exp. Ther. 1998; 285: 83-94PubMed Google Scholar). At the 6th week of ouabain infusion, OS rats were randomly assigned to two groups (n = 10 each): the first (OS-treated) received PST 2238 orally at 10 μg/kg/day, suspended in 0.5% w/v Methocel, and the second group (OS controls) only vehicle. Systolic blood pressure (SBP) and heart rate (HR) were measured weekly in conscious rats by tail-cuff plethysmography (BP recorder, U. Basile, Italy). At the end of the experiment, rats were sacrificed by cervical dislocation. Blood was collected and plasma separated by centrifugation. Heart and kidney were excised and weights normalized for tibia length. Plasma and Tissue Ouabain Determination—Ouabain was extracted from plasma, left ventricle free-wall (LVFW), and kidney and measured by a radioimmunoassay, as described (11Ferrandi M. Manunta P. Balzan S. Hamlyn J.M. Bianchi G. Ferrari P. Hypertension. 1997; 30: 886-896Crossref PubMed Scopus (119) Google Scholar). Rat Kidney Caveolae Preparation and ERK1/2 Measurement—Rat kidneys were homogenized in: (mm) 200 sodium carbonate, pH 11, 2 sodium orthovanadate, 100 mg/liter Pefabloc and centrifuged at 100,000 × g for 30 min. The cytosol was utilized for total and dual-phosphorylated (Thr202/Tyr204) ERK1/2 quantification by Western blotting. The pellet was used for caveolae isolation following a detergent-free method and fractionation on a 5-45% sucrose gradient (21Song K.S. Shengwen L. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar). Nineteen fractions were automatically collected and protein content determined (Pierce). The distribution along the gradient of the plasma membrane, Golgi, and endoplasmic reticulum was established by measuring alkaline phosphatase, α-mannosidase II, and α-glucosidase II activity, respectively (22van't Hof W. Resh M.D. J. Cell Biol. 1997; 136: 1023-1035Crossref PubMed Scopus (124) Google Scholar). Na-K ATPase Activity Assay—Na-K ATPase activity was measured by [32P]ATP hydrolysis method (23Ferrandi M. Tripodi G. Salardi S. Florio M. Modica R. Barassi P. Parenti P. Shainskaja A. Karlish S. Bianchi G. Ferrari P. Hypertension. 1996; 28: 1018-1025Crossref PubMed Scopus (96) Google Scholar) in sucrose fractions washed in: (mm) 250 sucrose, 30 histidine, pH 7.2, and in purified Na-K ATPase obtained from rat kidney medulla, as described (24Jorgensen P.L. Kidney Int. 1986; 29: 10-20Abstract Full Text PDF PubMed Scopus (179) Google Scholar). To investigate the effect of Src on Na-K ATPase activity, recombinant Src kinase, or its medium, was incubated with purified rat renal α1 Na-K ATPase (protein ratio 1:50) for 30 min at 30 °C in a buffer containing: (mm) 150 NaCl, 3 MgCl2, 3 ATP, and 70 Hepes-Tris, pH 7.4, supplemented with 3 mm MnCl2. The effect of increasing concentrations of ouabain was evaluated. Na-K ATPase activity was measured as above (23Ferrandi M. Tripodi G. Salardi S. Florio M. Modica R. Barassi P. Parenti P. Shainskaja A. Karlish S. Bianchi G. Ferrari P. Hypertension. 1996; 28: 1018-1025Crossref PubMed Scopus (96) Google Scholar). [3H[Ouabain Binding and Scatchard Analysis—Washed renal caveolae were incubated at 37 °C for 1 h with increasing concentrations of [3H]ouabain in a medium containing 3 mm MgCl2, 5 mm phosphoric acid, pH 7.4. Concentrations of labeled ouabain higher than 10-6m were obtained by isotopic dilution with cold ouabain. Nonspecific binding was determined in the presence of 20 mm ouabain. The separation of bound from free-labeled ouabain was achieved by a rapid filtration method on Whatman GF/F filters, which were washed three times and counted for radioactivity. Co-immunoprecipitation Experiments—Washed renal caveolae were incubated for 30 min at 30 °C in the absence or presence of ouabain in: (mm) 10 Tris, 10 MgCl2, 5 MnCl2, 0.25 EGTA, 0.025 sodium orthovanadate, 80 NaCl, 2 ATP, 100 mg/liter Pefabloc, pH 7.4. When specified, PST 2238 or amlodipine were added. The samples were incubated with 0.2% CHAPS for 15 min at 4 °C and then with anti-Src antibody (GD11 clone), conjugated to protein G-Sepharose beads (Sigma) for 4 h. A sample of caveolae, incubated with a non-immune IgG (Sigma), instead of the anti-Src antibody, was used as control. The immunocomplexes were precipitated, washed three times with the immunoprecipitation buffer containing CHAPS, and boiled with Laemmli sample buffer. Supernatants were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting and probed with specific antibodies. In Vitro Src Phosphorylation of Na-K ATPase—Na-K ATPase phosphorylation by Src was measured in vitro by 32Pi incorporation method (A) and Western blotting with an anti-phosphotyrosine antibody (PY99) (B). Method A: 3 μg of purified rat renal α1 Na-K ATPase were incubated for 10 min at 30 °C in the absence, or presence of 50 ng of recombinant Src protein kinase in a medium containing: (mm) 10 Tris, pH 7.4, 10 MgCl2, 5 MnCl2, 0.25 EGTA, 0.025 sodium orthovanadate, 80 NaCl, 0.1 ATP, and [32P]ATP (1500 cpm/nmol, specific activity 3000 Ci/mmol). The reaction was stopped by addition of Laemmli sample buffer. The samples were then subjected to SDS-polyacrylamide gel electrophoresis, followed by autoradiography. Method B: Na-K ATPase was incubated with Src kinase, as in method A, in a medium supplemented with 2 mm cold ATP in the absence of [32P]ATP. When specified, before the addition of Na-K ATPase, Src kinase was preincubated with its specific inhibitor, PP2 (5 μm), for 30 min at 4 °C or with a specific Src substrate peptide (500 μm), instead of Na-K ATPase. The effect of 10-10-10-8m ouabain was tested. The reaction was stopped by addition of Laemmli sample buffer. The samples were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting and probed with specific antibodies to verify the tyrosine phosphorylation of Na-K ATPase (PY99) and Src kinase (Src-Tyr418 and Src-Tyr529). Western Blotting—Samples were separated by SDS-polyacrylamide gel electrophoresis (7-15% acrylamide in glycine or 12% acrylamide in Tricine gels), blotted and overnight incubated at 4 °C with specific primary antibodies, followed by 1 h incubation with horseradish peroxidase-linked secondary antibody and chemiluminescent reaction (Lumi-Glo reagent, Cell Signaling). Autoradiography was performed, bands were scanned with Bio-Rad GS710 densitometer and quantified by Bio-Rad Quantity One Software. Blots were stripped (Re-Blot plus, Chemicon) and reprobed no more than three times. Statistical Analysis—Data are reported as mean ± S.E. The difference among groups was analyzed by one-way analysis of variance, followed by Fisher's least squares difference test. p < 0.05 was considered statistically significant. Ouabain dose-response curves and Scatchard plots were analyzed by a nonlinear regression program (GraphPad Prism Software, version 3). Ouabain Effects on Blood Pressure and Organ Weight—Ouabain infusion doubled plasma ouabain concentration in OS (0.7 ± 0.07 nm, n = 10, p < 0.001) as compared with CS rats (0.3 ± 0.04 nm, n = 10), reaching a value close to that of plasma EO found in hypertensive patients with cardiac hypertrophy (15Manunta P. Stella P. Rivera R. Ciurlino D. Cusi D. Ferrandi M. Hamlyn J.M. Bianchi G. Hypertension. 1999; 34: 450-456Crossref PubMed Scopus (161) Google Scholar, 16Pierdomenico S. Bucci A. Manunta P. Rivera R. Ferrandi M. Hamlyn J.M. Lapenna D. Cuccurullo F. Mezzetti A. Am. J. Hypertension. 2001; 14: 44-50Crossref PubMed Scopus (95) Google Scholar) and in hypertensive rat models (11Ferrandi M. Manunta P. Balzan S. Hamlyn J.M. Bianchi G. Ferrari P. Hypertension. 1997; 30: 886-896Crossref PubMed Scopus (119) Google Scholar). Tissue ouabain content (ng/g tissue) was increased 3-fold in OS LVFW (0.96 ± 0.091, n = 10, p < 0.01) and kidney (10.9 ± 1.4, n = 10, p < 0.01) as compared with CS (LVFW: 0.31 ± 0.033, n = 10; kidney: 3.93 ± 0.3, n = 10). Before starting with ouabain infusion, all rats had a similar SBP (130-135 mmHg). After 18 weeks, SBP was significantly increased in OS as compared with CS controls (+20 mmHg, p < 0.001) (Fig. 2A). The oral treatment with PST 2238 (10 μg/kg/day) significantly reduced SBP in OS rats (-20 mmHg, n = 10, p < 0.001 versus OS, Fig. 2A) within 3 weeks. Neither ouabain nor PST 2238 affected HR (beats/min: CS, 360 ± 8.3; OS, 381 ± 9.5; OS + PST 2238: 358 ± 9) and body weight (g, CS, 459 ± 7; OS, 466 ± 8; OS + PST 2238: 456 ± 6). Ouabain infusion increased cardiac LVFW (+11%, p < 0.05) and kidney weight (+9%, p < 0.01) in OS rats as compared with CS controls (Fig. 2, B and C). PST 2238 treatment prevented the ouabain-induced cardiac and kidney hypertrophy (Fig. 2, B and C). The Ca2+ antagonist amlodipine (oral dose 5 mg/kg/day), taken as an antihypertensive reference compound, significantly reduced SBP in OS rats (-20 mmHg, p < 0.01) but did not affect cardiac and kidney weight (data not shown). These data indicate that in vivo variations of ouabain concentrations within the subnanomolar range, similar to those reported for EO in humans, cause organ hypertrophy in rats besides raising blood pressure. The demonstration that amlodipine prevents the ouabain-induced increase of blood pressure without affecting in vivo organ hypertrophy excludes that the prohypertrophic effect of ouabain might be secondary to pressure overload. Expression of Na-K ATPase and Signaling Molecules in Total Renal Membranes and Caveolae—Preliminary Western blotting analysis of Na-K ATPase, EGFr, and Src in total renal membranes (MT) from CS, OS, and PST 2238-treated OS rats (n = 6) indicated that ouabain infusion significantly increased β1 Na-K ATPase subunit (+16%, p < 0.01) paralleled by an increase, although not statistically significant, of the α1 Na-K ATPase subunit (+11%), EGFr (+14%), and Src (+10%) compared with CS. In the PST 2238-treated OS rats all these differences disappeared. In order to investigate the possibility that the ouabain-induced effects on the protein expression observed in MT are amplified in restricted membrane subdomains, we isolated and characterized renal caveolae from CS, OS, PST 2238-treated OS rats. A typical protein distribution of the renal membranes fractionated on the sucrose density gradient is depicted in Fig. 3. The low density fractions (Fig. 3A, fractions 5-8, containing 5% of the total proteins) contained the specific markers of caveolae (25Razani B. Woodman S.E. Lisanti M.P. Pharmacol. Rev. 2002; 54: 431-467Crossref PubMed Scopus (848) Google Scholar, 26Shaul P. Anderson R.G. Am. J. Physiol. 1998; 276: L843-L851Google Scholar), such as caveolin 1, EGFr, and Src, which were enriched 30-, 40-, and 22-fold, respectively, compared with renal MT (Fig. 3B) and were therefore referred to as caveolae. Interestingly, caveolae were enriched 4-fold in α1, β1, and the γa splice variant of the Na-K ATPase subunits as compared with MT (Fig. 3B). The γb variant was not detected. The absence of clathrin, the marker for coated pits (Fig. 3B), excludes contamination by these vesicles. Mannosidase and glucosidase activities were also absent (not shown) in caveolae, thus excluding possible Golgi or endoplasmic reticulum membrane contaminations. The high buoyant density fractions 11-15 (Fig. 3A), positive for all these markers, including clathrin and γa and γb splice variants of Na-K ATPase (not shown), were referred to as plasma membrane. Effect of Ouabain on the Expression of Na-K ATPase and Signaling Molecules in Renal Caveolae—A representative immunoblotting of the Na-K ATPase subunits and signaling molecules in caveolae isolated from one CS, OS, and PST 2238-treated OS rat is shown in Fig. 4A. The densitometric analysis performed on caveolae from six rats for each group, expressed as percent increase over CS, is shown in Fig. 4B and C. Caveolin 1 content was similar in the three groups of rats. Ouabain significantly increased the recruitment of EGFr, Src, and phosphorylated Src at Tyr418, the Src active form (26Shaul P. Anderson R.G. Am. J. Physiol. 1998; 276: L843-L851Google Scholar) (Fig. 4B) in the OS group. The phospho/non-phospho Src ratio resulted 45% increased in OS over CS control rats (p < 0.01). Moreover, the content of the Na-K ATPase subunits was significantly increased in OS caveolae as compared with CS controls (Fig. 4C). PST 2238 largely reverted all the ouabain-induced effects in caveolae of OS rats (Fig. 4). It reduced the ouabain-induced targeting into caveolae of EGFr, Src, Src-Tyr418 (Fig. 4, A and B), normalized the phospho/non-phospho Src ratio to the level of CS controls and reduced the Na-K ATPase subunit content, particularly the α and β subunits (Fig. 4C). Effect of Ouabain on ERK1/2 Activation—We further investigated whether the effect of ouabain infusion on Na-K ATPase, Src, EGFr expression into caveolae might result in the activation of the ERK pathway and whether PST 2238 might reverse this effect. Fig. 5 shows the results obtained by probing kidney extracts from CS, OS, and PST 2238-treated OS rats (n = 6) with anti-total and anti-dual phosphorylated ERK1/2 antibodies. Ouabain caused a significant increase of total p44 (ERK1) and p42 (ERK2) levels as compared with CS controls and PST 2238 reverted this effect (Fig. 5A). Interestingly, ouabain induced an increase of dual-phosphorylated forms of ERK1/2 versus CS controls that was abolished by PST 2238 (Fig. 5B). These data demonstrate, for the first time, that the in vivo variation of circulating ouabain within the subnanomolar concentration range raises the expression levels of defined Na-K ATPase subunits and signaling molecules into caveolae and the concomitant activation of the ERK1/2 pathway. Na-K ATPase Activity and Ouabain Affinity in Rat Renal Caveolae—Since previous work indicated that ouabain signaling depends upon its interaction with Na-K ATPase (5Liu J. Tian J. Haas M. Shapiro J.I. Askari A. Xie Z. J. Biol. Chem. 2000; 275: 27838-27844Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 6Haas M. Wang H. Tian J. Xie Z. J. Biol. Chem. 2002; 277: 18694-18702Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), we investigated whether rat α1 Na-K ATPase localized in caveolae retains its catalytic activity and affinity for ouabain. This latter aspect is crucial when assessing the physiological relevance of nanomolar concentrations of ouabain in rat kidney where only the ouabain-resistant α1 isoform has been detected (27Lucking K. Nielsen J.M. Pedersen P.A. Jorgensen P. Am. J. Physiol. 1996; 271: F253-F260PubMed Google Scholar). The enzymatic activity of renal MT and caveolae, obtained from CS rats (n = 4), was respectively 3.6 and 2.4 μmol of Pi/min/mg protein as compared with 6 μmol of Pi/min/mg protein for a partially purified rat renal α1 Na-K ATPase. The inhibitory ouabain dose-response curves measured in rat renal caveolae showed the presence of a predominant component with low affinity for ouabain (IC50 = 1.3 × 10-4m), close to that of the purified renal Na-K ATPase (Fig. 6A) and MT (not shown). However, only in caveolae, the ouabain dose-response curve was best fitted according to a two-binding site model (nonlinear regression program, GraphPad Prism Software: one-binding site model, R2 = 0.89; two-binding site model, R2 = 0.99, p < 0.0001, F = 84.09) showing a second minor component (around 25%) of Na-K ATPase having a higher affinity for ouabain (IC50 = 1.2 × 10-7m) (Fig. 6A, inset) and activity of 0.6 μmol of Pi/min/mg protein. Ouabain Binding to Na-K ATPase in Rat Renal Caveolae: Effect of PST 2238—The presence of a ouabain high affinity Na-K ATPase component in rat renal caveolae was further investigated by measuring the [3H]ouabain binding in preparations obtained from CS rats. The ouabain dose-response curve (Scatchard plot, Fig. 6B) was analyzed by a nonlinear regression program comparing one (R2 = 0.76) or two classes (R2 = 0.97) of binding sites (GraphPad Prism Software). Data were best fitted by a two-site model (p < 0.0001, F = 27.02) revealing the presence of the predominant ouabain-resistant Na-K ATPase isoform (Ki = 10-5m; Bmax = 260 pmol of ouabain bound/mg of protein) and a minor component with higher affinity for ouabain (Ki = 2 × 10-7m;

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