This Article Abstract Full Text (PDF) All Versions of this Article: 145/3/1151    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Isenovic, E. R. Articles by Sowers, J. R. Search for Related Content PubMed PubMed Citation Articles by Isenovic, E. R. Articles by Sowers, J. R.

Angiotensin II Regulation of the Na⁺ Pump Involves the Phosphatidylinositol-3 Kinase and p42/44 Mitogen-Activated Protein Kinase Signaling Pathways in Vascular Smooth Muscle Cells

Department of Internal Medicine (J.R.S.), University of Missouri-Columbia and the H. S. Truman Veterans Affairs Medical Center, Columbia, Missouri 65212; Departments of Cell Biology, Biochemistry, and Medicine (E.R.I., D.B.J., M.H.K., Q.S., N.M., G.G.), State University of New York-Health Science Center, Brooklyn, New York 11201; and Department of Biology (K.K.), Jichi Medical School, Tochigi, Japan

Address all correspondence and requests for reprints to: James R. Sowers, M.D., F.A.C.P., Professor of Medicine, Physiology, and Pharmacology, Thomas W. and Joan F. Burns Missouri Chair in Diabetology, Associate Dean for Clinical Research, Director of the M.U. Diabetes and Cardiovascular Center, Department of Internal Medicine, MA 410, Health Sciences Center, One Hospital Drive, DCO43.00, Columbia, Missouri 65212. E-mail: sowersj{at}health.missouri.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
This investigation used primary cultured rat vascular smooth muscle cells to examine angiotensin II (Ang II) regulation of Na⁺, K⁺-ATPase (Na⁺ pump) activity, and Na⁺ pump ₁- and ß₁-subunit gene transcription. This regulation was mediated through both phosphatidylinositol-3 kinase (PI3K) and p42/44 mitogen-activated protein kinase (p42/44^MAPK) signaling pathways. Both acute (10 min) and prolonged (24 h) treatment with Ang II stimulated Na⁺ pump activity. Also, prolonged exposure to Ang II (24 h) increased promoter transcription of the Na⁺ pump ₁- and ß₁-subunits. Furthermore, PI3K activities because well because p42/44^MAPK phosphorylation were increased within 10 min after Ang II treatment. To determine whether these stimulatory activities of Ang II are acting through Ang II receptors 1 and/or 2 (AT₁, AT₂), cells were pretreated with either AT₁ receptor blocker losartan or the AT₂ receptor blocker PD 123,319. Indeed, these treatments prevented the stimulatory effect of Ang II on Na⁺ pump activity at both acute and 24-h time points. Furthermore, the Ang II-stimulated ₁-subunit promoter transcription was inhibited by losartan but not by the AT₂ receptor blocker. These results indicate that Ang II acts through both the AT₁ and AT₂ receptor to up-regulate Na⁺ pump activity; however, Ang II regulates ₁-gene transcription through AT₁ but not AT₂ receptors. It was also observed that the Ang II-stimulated ß₁-subunit gene transcription is not mediated through either AT₁ or AT₂ receptors. To examine whether the Na⁺/H⁺ exchanger is involved in Ang II-stimulated Na⁺ pump activity, cells were pretreated with amiloride, a specific inhibitor of the Na⁺/H⁺ exchanger. This pretreatment prevented 24 h, but not acute, Ang II-stimulated Na⁺ pump activity. The 24-h Ang II-stimulated ₁-subunit promoter transcription was also inhibited by amiloride. This suggests that the prolonged effect of Ang II on Na⁺ pump activity is dependent on increased Na⁺/H⁺ exchange. Because Ang II treatment for 10 min increased PI3K activity because well because p42/44^MAPK phosphorylation, studies were performed to determine the involvement of PI3K and p42/44^MAPK signaling pathways in both Ang II-stimulated Na⁺ pump activity and ₁- and ß₁-gene transcription. Cells were pretreated with either the PI3K inhibitor wortmannin or the p42/44^MAPK inhibitor PD 98059. Ang II-stimulated PI3K or p42/44^MAPK activity was inhibited by these pretreatments. Furthermore, pretreatment of cells with the PI3K inhibitors wortmannin and LY29404 or the MAPK inhibitors U0126 and PD 98059 were all observed to inhibit Ang II-stimulated Na⁺ pump activity. To more specifically determine the role of PI3K in Ang II-regulation of ₁-and ß₁-gene transcription, cells were cotransfected with a dominant-negative p85 construct. Cotransfection with dominant-negative p85 reduced Ang II-stimulated ₁-but not ß₁-gene transcription in vascular smooth muscle cells. These results indicate that Ang II acts through PI3K/p42/44^MAPK signaling pathways to up-regulate Na⁺ pump activity and ₁-gene transcription and that Ang II-regulated ß₁-gene transcription is not mediated through either PI3K or p42/44 ^MAPK signaling pathways.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
ANGIOTENSIN II (ANG II), the biologically active component of the renin-angiotensin system (RAS), acts through two Ang II receptor subtypes, the AT₁ and the AT₂ receptors, which mediate cardiovascular and other actions of Ang II. The two receptors are comprised of seven transmembrane glycoproteins with a 30% sequence homology (1, 2). The receptor binding sites for agonist and nonpeptide antagonist ligands have been defined, and AT₁ antagonists are effective because antihypertensive agents that are well tolerated (1, 2). The development of specific nonpeptide receptor antagonists has led to major advances in the physiology and therapy of the RAS. All classic physiological effects of Ang II, such as vasoconstriction, cell proliferation, aldosterone and vasopressin release, sodium and water retention, and sympathetic facilitation, are mediated by the AT₁ receptor (1, 2). The AT₂ receptor, in contrast, is up-regulated in pathological conditions, and it counteracts several of the growth responses initiated by the AT₁ and growth factor receptors.

In the vascular smooth muscle cell (VSMC), Ang II regulates via VSMC AT₁ receptors, many processes including the state of contractility/relaxation (3, 4, 5). AT₁ receptors are predominantly coupled to G proteins and signal through phospholipases, inositol phosphates, calcium channels, and a variety of serine/threonine and tyrosine kinases (1, 2, 4). In this regard, Ang II can activate phosphatidylinositol 3-kinase (PI3K), the AT₁ receptor. After Ang II-induced phosphorylation of PI3K’s regulatory p85 subunit, p85 forms complexes with specific phosphotyrosines of either growth factors or adapter proteins such as insulin receptor substrate (IRS)-1, thereby influencing vascular tone and numerous VSMC functions (5, 6). This protein-protein interaction allows PI3K’s catalytic p110 subunit to phosphorylate phosphoinositides at the 3' position of the inositol ring to generate 3-phosphoinositides. These lipids then serve as intermediates for specific downstream signal transduction events, leading to a multitude of biological responses (5, 6, 7, 8). Another important serine/threonine protein kinase activated by Ang II is p42/44 MAPK (p42/44^MAPK) (9, 10). The p42/44^MAPK signaling pathway is a distinct serine-threonine kinase cascade consisting of three enzymes: MAPK kinase kinase, MAPK kinase (MAPKK, MEK, MKK), and MAPK. Upstream activators of the MAPK pathways include small GTPases of the Ras family, and downstream effectors include transcription factors and other kinases (11, 12).

The Na⁺, K⁺-ATPase (Na⁺ pump) is a plasma membrane enzyme that plays a crucial role in VSMC homeostasis, and it maintains Na⁺ and K⁺ gradients between the intra- and extracellular milieu that are important for the maintenance of cell volume and tone (13). Structurally, the minimal units of the Na⁺ pump are two major polypeptides, the - and the ß-subunits having different isoforms, 4 (_1-4) and 3ß (ß_1-ß₃) (13, 14). The subunits are responsible for the catalytic and transport properties of the enzyme because it contains binding sites for cations and ATP, and it also includes a phosphorylation site (14, 15). The ß-subunits are involved in the docking of the Na⁺ pump to the plasma membrane (13, 14).

Despite the physiological and pathophysiological importance of Ang II, possible signal transduction pathways involved in its regulation of the Na⁺ pump remains poorly understood (15, 16). Thus, the aim of this investigation was to further elucidate the signaling mechanisms employed by Ang II in modulating the regulation of Na⁺ pump activity in VSMCs with particular emphasis on both PI3K and MAPK signaling pathways.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
VSMC preparation and culture
VSMCs were isolated from Sprague Dawley rat thoracic aorta by enzymatic dissociation as described previously (17, 18). Primary cultured cells were grown (37 C in 95% humidified air and 5% CO₂) in glucose-free DMEM (without addition of insulin or growth factors) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Serial passages (through passage 7) of VSMCs were obtained by treating confluent cultures with 0.2% trypsin-EGTA in Ca²⁺- and Mg²⁺-free Hanks’ balanced salt solution (Sigma Chemical, St. Louis, MO). Cells were characterized as VSMCs by a hill-and-valley pattern displayed at confluence and by positive immunostaining with a monoclonal antibody to VSMC-specific -actin (Sigma). The Animal Research Committee of the State University of New York Health Science Center at Brooklyn approved the animal experimentation described within this report.

Ouabain-sensitive ⁸⁶Rb uptake
Na⁺ pump activity was determined by measuring ouabain (1 mM)-sensitive ⁸⁶Rb flux based on the principle that ⁸⁶Rb transport displays identical kinetics to K⁺ (19). VSMCs were seeded onto 24-well tissue culture plates at a density of 10,000 cells/well in DMEM/9% fetal bovine serum. Medium was changed every 2–3 d until the cells were 100% confluent (5–7 d). On the day of the experiment, cells were washed three times with RPMI 1640 [containing 102 mM NaCl, 5.6 mM Na₂HPO₄, 5.4 mM KCl, 0.4 mM CaCl₂/2H₂O, 0.4 mM MgSO₄/7H₂O, 10 mM glucose, and 24 HEPES (pH 7.4)] and acclimated in the final wash for 20 min. The treatment paradigm consisted of Ang II for 10 min or 24 h followed by the incubation in the presence or absence of 1 mM ouabain for 10 min in RPMI 1640. Then 1 µCi ⁸⁶Rb was added to all wells, and ⁸⁶Rb flux was allowed to proceed for 10 min. ⁸⁶Rb flux was terminated by addition of ice-cold 100 mM MgCl₂ followed by three washes with the same solution. Cells were solubilized with NaOH and neutralized with HCl, and ⁸⁶Rb uptake was quantified by liquid scintillation counting. Na⁺ pump activity was calculated as a percentage of ouabain-sensitive uptakes vs. total ⁸⁶Rb uptake and expressed as percent of control. All experiments were completed using three to six replicates per treatment per experiment.

Preparation of cell lysates
Quiescent VSMCs in 100-mm culture dishes were incubated with 100 nM Ang II, and Ang II-induced responses were terminated by addition of ice-cold PBS. This dose of Ang II was been previously shown to maximally stimulate Na⁺ pump activity in VSMCs (16). Cells were rinsed again with PBS and lysed by the addition of 1ml lysis buffer [20 mM Tris HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM Na₄P₂O₇, 2 mM Na₃O₄, 1 mM ß-glycerolphosphate, 1% Triton X-100] and the following protease inhibitors: 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 5 µg/ml aprotinin (Sigma). Cells were scraped and lysates were subjected to centrifugation at 13,000 rpm. Clarified supernatants were used fresh or stored at -70 C (20).

Immunoblot analysis
Equal amounts of protein (50 µg) were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane in Tris-glycine transfer buffer containing 20% methanol in a trans-blot cell (Bio-Rad, Hercules, CA). Membranes were blocked in 5% instant nonfat dry milk in Tris-buffered saline [20 mM Tris, 137 mM NaCl (pH 7.6) containing 0.3% Tween 20], washed in Tris-buffered saline, and probed with primary antibody (dilute 1:1000) raised against the p42/44^MAPK kinase (Cell Signaling, Beverly, MA). The immunoblots were subsequently washed and incubated in (1: 5000) horseradish peroxidase-coupled antirabbit IgG antibody (Cell Signaling) for 1 h. The bound antibodies were visualized by enhanced chemiluminescence using the ECL system (Amersham, Piscataway, NJ) and exposure to X-OMAT film (Kodak, Rochester, NY). Signals were quantitated by a densitometry by using NIH 1.60 Software (National Institutes of Health, Bethesda, MD). Multiple exposure of each blot was performed to ensure that signals were within the linear range of the film.

Immunoprecipitation and assay for PI3K activity
Cell lysates prepared, as described above, were probed with an antiphosphotyrosine antibody (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 C with gentle rocking. Protein A/G (20 µl; 50% slurry, Santa Cruz) was added and samples were further incubated for an additional 2 h. The immune complexes were then recovered by centrifugation and used for measuring PI3K activity as previously described (17, 21). The reaction products were separated by thin-layer chromatography on oxalate-pretreated Silica Gel 60 plates in a solvent of chloroform/methanol/4 N ammonia (60:47:17). Cold propidium iodide, phosphatidylinositol phosphate, and phosphatidylinositol diphosphate were run as a standard and visualized by primulin staining. ³²P-labeled phosphatidylinositol 3-P products were measured using a STORM PhosphorImager and calculated by the IMAGEQUANT software (Molecular Dynamics, Sunnyvale, CA).

Transfection and luciferase assay
Cells (40–50% confluent) were transiently transfected using the cytofectene reagent (Bio-Rad) according to the manufacturer’s protocol and a standardized method (17, 20, 22). Briefly, cells in 30-mm plates were transfected with 1 µg of a luciferase reporter plasmid contain a portion of Na⁺ pump ₁-subunit promoter (-1537 to +261) (17, 20) or 1 µg of a luciferase reporter plasmid contains a portion of the Na⁺ pump ß₁-subunit promoter (-871 to +151) (17, 22). The cells were cotransfected with 20 ng luciferase-pRL-SV40 (Promega, Madison, WI) as an internal control. Four hours after transfection, the transfection mix was removed and cells were washed twice with serum-free media. In some experiments where indicated, VSMCs were pretreated for 15–30 min with the stated concentrations of inhibitors (either wortmannin, PD 98059, losartan, or amiloride) before addition of fresh serum-free media in the absence or presence of Ang II (100 nM; 24 h). Cells were washed twice with ice-cold PB and lysed with passive lysis buffer (Promega). Lysates were analyzed for both firefly and Renilla luciferase activity using Promega dual-luciferase reporter assay kit. In some cases in which cells were not cotransfected with pRL-SV40 vector, only firefly luciferase was measured in aliquots containing equal amount of protein. Negative controls of mock-transfected cells and empty vector (pUSEamp, Promega) were incorporated in all experiments. To control for transfection efficiency, pGL3 control plasmid (Promega) containing the firefly luciferase was used in all experiments. In cotransfection experiments with dominant-negative ()p85 (lacking a binding site for the p110 catalytic subunit of PI3K), the cells were transfected with an additional 1 µg of the plasmid and the total amount of DNA in transfected cells was kept constant by addition of the empty vector. Cleared cell-lysates were assayed for luciferase in an Optocomp1 single-sample luminometer. Activity of the Na⁺ pump-promoter reporter constructs was normalized to the activity of Renilla reporter.

Statistical analysis
Values are expressed as mean ± SEM. Statistical significance was evaluated with nonparametric test (Mann-Whitney rank sum test) or ANOVA with the appropriate correction for multiple comparisons (Newman-Keuls method). P < 0.05 was considered significant. All comparisons are vs. control values, unless otherwise specified.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Involvement of PI3K and p42/44^MAPK and role of Na⁺/H⁺ exchanger and Ang II-stimulated Na⁺ pump activity
It has been reported that Ang II (100 nM) stimulates Na⁺ pump activity in VSMCs with maximum effects within 10 min (17). Thus, this concentration of Ang II was used in our experiments to study both the acute (10 min) and prolonged (24 h) effects of Ang II on Na⁺ pump activity. Na⁺ pump activity was determined by measuring ouabain (1 mM)-sensitive ⁸⁶Rb influx (19). Both the acute and prolonged treatment significantly stimulated Na⁺ pump activity above control values by 72% (Fig. 1A) and 63% (Fig. 1B), respectively. To clarify the signaling pathway involved in Ang II regulation of Na⁺ pump activity, we examined the role of both PI3K and p42/44^MAPK signal transduction pathways. To determine whether PI3K is involved, VSMCs were first pretreated with the PI3K blockers wortmannin (50 nM; 30 min) or LY29404 (50 µM; 15 min) before both acute and prolonged Ang II treatment. Ang II-stimulated Na⁺ pump activity was significantly decreased by wortmannin as well as LY 29404 treatments at both 10 min (Fig. 1A) and 24 h (Fig. 1B) without any significant effect on basal Na⁺ pump activity.

View larger version (28K): [in this window] [in a new window]   FIG. 1. Role of PI3K, p42/44MAPK, AT1 and AT2 receptors, and Na+/H+ exchanger in Ang II-stimulated Na+ pump activity. Cells were treated with Ang II (100 nM; 10 min) (A) or 24-h (B) pretreatment with inhibitors: wortmannin (50 nM; 15 min), U0126 (10 µM; 15 min), PD 12339 (10 µM; 15 min), or losartan (1 µM; 15 min) before Ang II treatment. The results are expressed as percent of control (arbitrarily set at 100%). Results are mean SEM, n = 3–6 experiments performed in triplicate. ***, P < 0.001, indicates Ang vs. cont; the other P values shown are inhibitor-treated groups vs. Ang II-treated cells.

The role of the Na⁺/H⁺ exchanger in Ang II stimulation of Na⁺ pump activity was also studied. In this regard, cells were pretreated with 5-(N-ethyl-N-isopropyl amiloride (10 µM; 10 min), a specific inhibitor of the Na⁺/H⁺ exchanger (15, 24). Inhibition of Na⁺/H⁺ exchange did not affect acute Ang II-stimulated Na⁺ pump activity (Fig. 1A) but did significantly diminish prolonged Ang II-stimulated Na⁺ pump activity (Fig. 1B), implicating a role for the Na⁺/H⁺ exchanger in Ang II’s actions.

Role of AT₁ and AT₂ receptors in Ang II-stimulated Na⁺ pump activity
Because Ang II is known to mediate many of its biological effects in VSMCs through activation of the AT₁ receptor (1, 2, 3, 4, 5), the effect of the specific AT₁ receptor antagonist losartan (25) on Na⁺ pump activity was examined. For these studies VSMCs were pretreated with losartan (1 µM; 15 min) before both acute and prolonged Ang II treatment. The Ang II-induced Na⁺ pump activity was completely suppressed both acutely (Fig. 1A) and at 24 h (Fig. 1B) by pretreatment with this AT₁ receptor inhibitor.

We also examined whether the AT₂ receptors were involved in the action of Ang II. For these studies VSMCs were pretreated with the AT₂ receptor antagonist PD 123.319 (1 µM; 15 min) (25) before acute and prolonged Ang II treatment. AT₂ receptor inhibition decreased both acute (Fig. 1A) and prolonged (Fig. 1B) Ang II-stimulated pump activity. Taken together, the results with losartan and PD123319 indicated that Ang II regulates the Na⁺ pump in VSMCs via both AT₁ and AT₂ receptors.

Effects of Ang II on PI3K and p42/p44^MAPK activation
Because our results (Fig. 1, A and B) demonstrate that PI3K/p42/44^MAPK pathways are involved in Ang II-stimulated Na⁺ pump activity, we further investigated the effect of Ang II on stimulation of PI3K activity as well as p42/44^MAPK phosphorylation. Abundance of phosphorylated PI3K in antiphosphotyrosine precipitates was measured using an in vitro assay that quantitates phosphatidylinositol 3 phosphate (Fig. 2) A low level of PI3K activity was present in the basal state, whereas Ang II-stimulated PI3K activity (Fig. 2). However, pretreatment of cells with the PI3K inhibitor wortmannin, significantly reduced the Ang II-stimulated PI3K activity (Fig. 2). In addition, VSMCs were stimulated with Ang II (100 nM; 10 min) and the lysates were directly subjected to immunoblotting with antibody against phosphorylated p42/44^MAPK (Fig. 3). Ang II significantly increased the phosphorylation of p42/44^MAPK after 10 min (Fig. 3). However, when cells were preincubated with PD 98059, Ang II-stimulated phosphorylation of p42/p44^MAPK at 10 min was completely inhibited (Fig. 3). Additionally, another p42/44^MAPK inhibitor U0126 also decreased Ang II-stimulated phosphorylation of p42/44^MAPK (Fig. 3). To determine whether p42/44^MAPK is downstream or upstream from PI3K, VSMCs were treated with wortmannin, and p42/44^MAPK phosphorylation was measured. Wortmannin inhibition of PI3K did not attenuate MAPK phosphorylation (wortmannin = 0.82, 0.22-fold, control = 1-fold, n = 5). These results suggest that PI3K and p42/44^MAPK activation are both involved in Ang II effects on the Na⁺, K⁺-ATPase pump.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Effect of Ang II on PI3K activity in VSMCs. Cells were treated with Ang II for 10 min or pretreated with wortmannin (100 nM; 30 min) and then treated with Ang II for an additional 10 min. A representative autoradiogram from TLC performed on PI3K product (phosphatidylinositol 3 phosphate) is shown at the bottom of the figure. Results are mean SEM; n = 4 experiments. ***, P < 0.001, indicates Ang vs. control; the other P value shown is the inhibitor-treated group vs. Ang II-treated cells.

View larger version (28K): [in this window] [in a new window]   FIG. 3. Effect of Ang II on p42/44MAPK phosphorylation in VSMCs. Cells were treated with Ang II for 10 min or pretreated with PD 98059 (50 µM; 1 h) or U0126 (10 µM; 15 min) and then treated with Ang II for an additional 10 min. A representative immunoblot is shown at the bottom of the figure. Results are mean ± SEM; n = 3–4 experiments; **, P < 0.01, indicates Ang vs. control; the other P values shown are inhibitor-treated groups vs. Ang II-treated cells.

Role of PI3Kp42/44/^MAPK pathways in Ang II regulation of ₁ gene transcription

View larger version (15K): [in this window] [in a new window]   FIG. 4. Role of PI3K and p42/44MAPK in Ang II-stimulated Na+ pump 1-promoter transcription. A luciferase reporter plasmid containing a portion of the Na+ pump 1-subunit promoter between -1537 to +261 bp was transiently transfected into VSMCs, and control values were assessed a relative value of 100%. Cells were treated with Ang II (100 nM; 24 h) or pretreated with wortmannin (100 nM; 30 min), PD 98059 (50 µM; 1 h), or U0126 (10 µM; 15 min) before Ang II treatment. Results are mean ± SEM; n = 3–11 experiments. ***, P < 0.001, indicates Ang vs. control; the other P values shown are inhibitor-treated groups vs. Ang II-treated cells.

View larger version (20K): [in this window] [in a new window]   FIG. 5. Role of PI3K in Ang II-stimulated Na+ pump 1-promoter transcription. Before Ang II treatment, a luciferase reporter plasmid containing a portion of the Na+ pump 1-subunit promoter between -1537 and +261 bp was transiently transfected into VSMCs or VSMCs cotransfected with p85. Control values were assessed a relative value of 100%. After transfection, cells were treated with Ang II (100 nM; 24 h). Results are mean ± SEM; n = 3–11 experiments. ***, P < 0.001, indicates Ang vs. control; the other P value shown is cotransfected vs. Ang II-treated cells.

Ang II-stimulated ₁-gene transcription is blocked with amiloride and losartan in VSMCs

View larger version (14K): [in this window] [in a new window]   FIG. 6. Effect of Na+/H+ exchanger inhibitor (amiloride) or AT1 receptor (losartan) blocker on Ang II-induced Na+ pump 1-promoter transcription. A luciferase reporter plasmid containing a portion of the rat Na+ pump 1-subunit promoter between -1537 and +261 bp was transiently transfected into VSMCs, and control values were assessed a relative value of 100%. Cells were treated with Ang II (100 nM; 24 h) or pretreated with either amiloride (10 µM min) or losartan (1 nM; 15 min) before Ang II treatment. Results are mean ± SEM; n = 3–11 experiments. ***, P < 0.001, indicates Ang vs. control; the other P values shown are inhibitor-treated groups vs. Ang II-treated cells.

View larger version (14K): [in this window] [in a new window]   FIG. 7. Effect of Ang II treatment on Na+ pump ß1-promoter transcription. A luciferase reporter plasmid containing a portion of the rat Na+ pump ß1-subunit promoter between -817 and +151 bp was transiently transfected into VSMCs. Control values were assessed a relative value of 100%. Cells were treated with Ang II (100 nM; 24 h). Results are mean ± SEM; n = 3 experiments. *, P < 0.05, indicates Ang vs. control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Another important signal transduction pathway, MAPK, was also studied because it was previously shown that activation of MAPK via Ras/MEK contributes to Ang II-stimulated vascular contraction, injury, hypertrophy, and hypertension in rodents (10, 30). In the current study, Ang II-induced activation of p42/44^MAPK in VSMCs within 10 min. This activation is transient because incubation with Ang II for 24 h was no longer associated with activation of this kinase pathway, possibly reflecting a tachyphylaxis for this effect of Ang II.

Although there is no previously reported evidence of cross-talk between the p42/44^MAPK and PI3K pathways in VSMCs, in the current investigation inhibition of either p42/p44^MAPK or PI3K activity diminished the induction of Na⁺ pump activity, suggesting that both pathways contribute to the action(s) of Ang II. Thus, the ability of Ang II to induce activation of PI3K (Fig. 2) and p42/44^MAPK (Fig. 3) is consistent with the notion that Ang II activates the Na⁺ pump in VSMCs through both PI3K and p42/44^MAPK signaling pathways (9, 29).

The role of p42/44^MAPK activation in Ang II-stimulated Na⁺ pump activity was assessed, using p42/44^MAPK inhibitor PD 98059. This inhibitor suppressed phosphorylation of p42/44^MAPK in VSMCs treated with Ang II (Fig. 3). Furthermore, pretreatment of cells with PD 98059 also abolished both acute and long-term Ang II-stimulated Na⁺ pump activity. Thus, these data demonstrate that p42/44^MAPK signaling is necessary for the both acute and 24-h Ang II stimulation of the Na⁺ pump in VSMCs.

Ang II receptor 1 (AT₁) stimulation causes rapid but transient p42/44^MAPK activation via multiple signaling pathways that include protein kinase C/Ras/Raf, Pyk-2, and growth factor receptors such as epithelial growth factor (EGF) and platelet-derived growth factor (1, 31). In the rat kidney proximal convoluted tubule, EGF has been reported to stimulate Na⁺ reabsorption mediated by tyrosine phosphorylation. Activation of receptor tyrosine kinases by EGF acts on the Na⁺ pump to stimulate ouabain-sensitive ⁸⁶Rb⁺ uptake (32), suggesting that the AT₁ receptor has a role in Na⁺ pump regulation. In this study we observed that an AT₁ as well as AT₂ receptor antagonist blocked both acute and chronic stimulation of VSMC Na⁺ pump activity by Ang II. As shown in Fig. 1, both the AT₁ receptor inhibitor losartan and the AT₂ blocker PD 123.319 inhibits VSMC Na⁺ pump activity. Therefore, it can be concluded that Ang II-stimulated Na⁺ pump activity is mediated via both AT₁ and AT₂ receptors. In this regard, it is also of interest to note that previous studies looking at other cells types (32, 33) indicate that stimulation of AT₂ receptors may offset the AT₁ receptor-mediated actions of Ang II on Na⁺ pump activity (34, 35). When AT1 is blocked, increased Ang II may act on AT₂ receptors (36).

The effect of the Na⁺/H⁺ exchanger on Ang II action was investigated because it has been previously suggested that an increase in Na⁺ influx is required for Ang II-stimulated Na⁺ pump activity in VSMCs (15, 24, 27, 37). A variety of growth factors and vasoconstrictors activate Na⁺/H⁺ exchange, leading to increases in intracellular Na⁺ and intracellular alkalinization, with secondary activation of the Na⁺ pump to restore homeostasis (27, 37). In the current study, however, acute Ang II stimulation of ⁸⁶Rb flux in VSMCs was not inhibited by amiloride, a relatively specific inhibitor of Na⁺/H⁺ exchange (37, 38), suggesting that this acute stimulation was not dependent on Na⁺/H⁺ activation. By contrast, amiloride inhibits the longer-term (24 h) effect of Ang II on Na⁺ pump activity, demonstrating the importance of increased Na⁺/H⁺ exchange activity and the associated increase in intracellular Na⁺. In this regard, an increase in Na⁺ influx may have a major role in the mechanism of Ang II-stimulated up-regulation of Na⁺ pump 1 promoter transcription. Increased Na⁺ influx may serve as a signal either directly, or indirectly, to up-regulate 1-subunit gene transcription. Consistent with this notion, increased intracellular Na⁺ has been shown to stimulate gene transcription of the Na⁺ pump ₁-isoform in VSMCs (39). Furthermore, it has been reported that treatment of VSMCs with monensin can increase intracellular Na⁺, inducing a dose-dependent up-regulation of Na⁺ pump ₁-, ₂-, and ß₁-subunit mRNA levels (40). Investigators have characterized a transcriptional Na⁺-response mechanism, defining a positive Na⁺-response regulatory region in the ₁- and ₂-genes of the Na⁺ pump, and they detected a Na⁺-response nuclear DNA binding protein (40). It remains to be resolved how increased intracellular Na⁺ stimulates Na⁺ pump ₁-, -, and ß₁-subunit gene transcription.

Transcriptional/translational regulation of Na⁺ pump ₁- and ß₁-subunit isoforms has been reported to be altered in several disease states and also altered in a number of tissues in response to various agonists (15, 39, 40, 41). Although it has been previously shown that Ang II increases ₁- and ß₁-mRNA content in VSMCs (15, 16, 38), the molecular basis of the increase of mRNA levels had not been delineated. In the present investigation, an increase in ₁- and ß₁-gene transcription was detected 24 h after Ang II treatment of the VSMCs. To our knowledge, this is the first report that Ang II stimulates both ₁- and ß₁-gene transcription in VSMCs. The Ang II-stimulated increase in ₁-gene transcription was inhibited by wortmannin, U0126, and PD 98059 as well as in cotransfection with p85, suggesting that this Ang II effect is mediated through both PI3K and p42/44^MAPK signaling. Furthermore, Ang II stimulation of ₁-gene transcription was inhibited by losartan and amiloride, implicating the AT₁ receptor and Na⁺/H⁺ exchange in this process. Our results also show that Ang II-stimulated ß₁-gene transcription is not mediated via PI3K/p42/44^MAPK. Thus, the evaluation of potential signaling pathways having a role in Ang II regulation of ß₁-subunit should be a fruitful area of future investigation.

In summary, results of this investigation indicate that Ang II-stimulated Na⁺ pump activity in VSMCs via binding to AT₁/AT₂ receptors initiates both PI3K and p42/44^MAPK signal transduction pathways, leading to up-regulation of gene transcription of the ₁-catalytic subunit. This effect is observed rapidly and lasts for at least 24 h. Na⁺/H⁺ exchange is also involved in the sustained effects of Ang II on Na⁺ pump activity. The Na⁺ pump is an important mediator of vascular volume, tone intracellular [Ca²⁺], intracellular pH, and growth (42), and abnormal regulation of the Na⁺ pump has been implicated in a number of disease states such as hypertension, diabetes, and arteriosclerosis (1, 2, 24). Because Ang II is a tissue-produced autocrine/paracrine factor, local regulation of the Na⁺ pump by Ang II is of potential physiological significance. In conclusion, our results suggest that regulation of Na⁺ pump activity and gene transcription is mediated via both PI3K and p42/44^MAPK pathways.

	Footnotes

 
This work was supported by grants from the NIH (RO1-HL-63904-01), the Veterans Affairs Merit System (0018), and the American Diabetes Association (RA0095).

Abbreviations: , Dominant-negative; Ang II, angiotensin II; AT₁, AT₂, Ang II receptors 1 and 2; EGF, epithelial growth factor; IRS, insulin receptor substrate; PI3K, phosphatidylinositol-3 kinase; RAS, renin-angiotensin system; VSMC, vascular smooth muscle cell.

Received January 21, 2003.

Accepted for publication November 11, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. De Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T 2001 International Union of Pharmacology. XXIII. The angiotensin II receptors. Pharmacol Rev 52:415–472
2. Kaschina E, Unger T 2003 Angiotensin AT1/AT2 receptors: regulation, signaling and function. Blood Press 12:70–88[CrossRef][Medline]
3. Berk B, Duff J, Marrero Ml 1996 Angiotensin II signal transduction in vascular smooth muscle. In: Sowers JR, ed. Endocrinology of the vasculature. Totowa, NJ: Humana Press; 187–204
4. Regitz-Zagrosek V, Neub M, Holzmeister J 1996 Molecular biology of angiotensin receptors and their role in human cardiovascular disease. J Mol Med 74:233–251[CrossRef][Medline]
5. Rabkin SW, Goutsouliak V, Kong JY 1997 Angiotensin II induces activation of phosphatidylinositol 3-kinase in cardiomyocytes. J Hypertens 15:891–899[CrossRef][Medline]
6. Saad MJA, Velloso LA, Carvalho CRO 1995 Angiotensin II induces tyrosine phosphorylation of insulin receptor substrate 1 and its association with phosphatidylinositol 3-kinase in rat heart. Biochem J 310:741–744
7. Funaki M, Katagiri H, Inukai K, Kikuchi M, Asano T 2000 Structure and function of phosphatidylinositol-3, 4 kinase. Cell Signal 12:135–142[CrossRef][Medline]
8. Hemmings BA 1997 PtdIns (3, 4, 5) P3 gets its message across. Science 277:534–567[Abstract/Free Full Text]
9. Viedt C, Soto U, Krieger-Brauer HI, Fei J, Elsing C, Kubler W, Kreuzer J 2000 Differential activation of mitogen-activated protein kinases in smooth muscle cells by angiotensin II. Arterioscler Thromb Vasc Biol 20:940–948[Abstract/Free Full Text]
10. Shah BH, Reyes J, Yesilkaya A, Catt KJ 2002 Independence of angiotensin II-induced MAP kinase activation from angiotensin type 1 receptor internalization in clone 9 hepatocytes. Mol Endocrinol 16:610–620[Abstract/Free Full Text]
11. Minden A, Lin A, McMahon M, Lange-Carter C, Derijard B, Davis RJ, Johnson GL, Karin M 1994 Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK. Science 266:1719–1723[Abstract/Free Full Text]
12. Hill CS, Treisman R 1995 Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 80:199–211[CrossRef][Medline]
13. Blanco G, Mercer WR 1998 Isozymes of the Na⁺, K⁺-ATPase: heterogeneity in structure, diversity in function. Am J Physiol 275:F633–F650
14. Crambert C, Hasler U, Beggah AT, Yu C, Modyanov NN, Horisberger J, Lelièvre L, Geering K 2000 Transport and pharmacological properties of nine different human Na⁺, K⁺-ATPase isozymes. J Biol Chem 275:1976–1986[Abstract/Free Full Text]
15. Krug LM, Berk BC 1992 Na⁺, K⁺-adenosine triphosphatase regulation in hypertrophied vascular smooth muscle cells. Hypertension 20:144–150[Abstract/Free Full Text]
16. Brock TA, Lewis LJ, Smith JB 1982 Angiotensin increases Na⁺ entry and Na⁺K⁺ pump activity in cultures of smooth muscle from rat aorta. Proc Natl Acad Sci USA 79:1438–1442[Abstract/Free Full Text]
17. Isenovic RE, Meng Y, Divald A, Milivojevic N, Sowers JR 2003 Role of PI3K/Akt pathway in angiotensin II and insulin-like growth factor-1 modulation of nitric oxide synthase in vascular smooth muscle cells. Endocrine 19:287–291
18. Standley PR, Zhang F, Ram JL, Zemel MB, Sowers JR 1991 Insulin attenuates vasopressin-induced calcium transients and a voltage-dependent calcium response in rat vascular smooth muscle cells. J Clin Invest 88:1230–1236
19. Standley PR, Zhang F, Zayas RM, Muniyappa R, Walsh MF, Cragoe E, Sowers JR 1997 IGF-I regulation of Na⁺-K⁺-ATPase in rat arterial smooth muscle. Am J Physiol 273:E113–E121
20. Suzuki-Yagawa Y, Kawakami K, Nagano K 1992 Housekeeping Na⁺, K⁺-ATPase ₁ subunit gene promoter is composed of multiple cis elements to which common and cell type-specific factors bind. Mol Cell Biol 12:4046–4055[Abstract/Free Full Text]
21. Isenovic E, Muniyappa R, Milivojevic N, Rao Y, Sowers, J 2001 Role of PI3-Kinase in isoproterenol and IGF-1 induced ecNOS activity. Biochem Biophys Res Commun 285:954–958[CrossRef][Medline]
22. Qin X, Liu B, Gick G 1994 Low external K⁺ regulates Na, K-ATPase 1 and ß1 gene transcription in rat cardiac myocytes. Am J Hypertens 7:96–99[Medline]
23. Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, Trzaskos JM 1998 Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273:18623–18632[Abstract/Free Full Text]
24. Touyz RM, Schiffrin EL 1999 Activation of the Na⁺-H⁺ exchange modulates angiotensin II-stimulated Na⁺-dependent Mg2⁺ transport in vascular smooth muscle cells in genetic hypertension. Hypertension 34:442–449[Abstract/Free Full Text]
25. Grahnquist L, Chen M, Gerasev A, Aizman R, Celsi G 2001 Regulation of K⁺ transport in the rat distal colon via angiotensin II subtype receptors and K⁺-pathways. Acta Physiol Scand 171:145–151[CrossRef][Medline]
26. Muscella A, Aloisi F, Marsigliante S, Levi G 2000 Angiotensin II modulates the activity of Na⁺, K⁺-ATPase in cultured rat astrocytes via the AT₁ receptor and protein kinase C-activation. J Neurochem 74:1325–1331[Medline]
27. Saward I, Zahradka P 1997 Angiotensin II activates phospholatidylinositol 3-kinase in vascular smooth muscle cells. Circ Res 81:249–257[Abstract/Free Full Text]
28. Villoso LA, Folli F, Sun XJ, White MF, Saad MJA, Kahn CR 1996 Cross talk between the insulin and angiotensin signaling systems. Proc Natl Acad Sci USA 93:12490–12495[Abstract/Free Full Text]
29. Folli F, Kahn CR, Hansen H, Bouchie JL, Feener EP 1997 Angiotensin II inhibits insulin signaling in aortic smooth muscle cells at multiple levels. A potential role for serine phosphorylation in insulin/ angiotensin II cross-talk. J Clin Invest 100:2158–2169[Medline]
30. Muthalif MM, Karzoum AN, Gaber L, Khandekar Z, Benter FI, Saeed EA, Parmentier J, Estes A, Malik UK 2000 Angiotensin II-induced hypertension contribution of Ras GTPase mitogen-activated protein kinase and cytochrome 450 metabolites. Hypertension 36:604–609[Abstract/Free Full Text]
31. Feraille E, Carranza ML, Rousselot M, Favre H 1997 Modulation of Na⁺, K (⁺)-ATPase activity by a tyrosine phosphorylation process in rat proximal convoluted tubule. J Physiol 498:99–108[CrossRef][Medline]
32. Muscella A, Marsigliante S, Carluccio MA, Vinson GP, Storelli C 1997 Angiotensin II AT₁ receptors and Na⁺/K⁺ ATPase in human umbilical vein endothelial cells. J Endocrinol 155:587–593[Abstract]
33. Muscella A, Greco S, Elia MG, Storelli C, Marsigliante S 2002 Angiotensin II stimulation of Na⁺/K⁺ATPase activity and cell growth. J Endocrinol 173:315–323[Abstract]
34. Unger T 1999 The angiotensin type 2 receptor: variations on an enigmatic theme. J Hypertension 17:1775–1786[CrossRef][Medline]
35. Gallinat S, Busche S, Raizada MK, Sumners C 2000 The angiotensin II type 2 receptor: an enigma with multiple variations. Am J Physiol Endocrinol Metab 278:E357–E374
36. Wu L, Iwai M, Nakagami H, Li Z, Chen R, Suzuki J, Akishita M, de Gasparo M, Horiuchi M 2001 Roles of angiotensin II type 2 receptor stimulation associated with selective angiotensin II type 1 receptor blockade with valsartan in the improvement of inflammation-induced vascular injury. Circulation 104:2716–2721[Abstract/Free Full Text]
37. Weissberg PL, Little PJ, Cragoe Jr EJ, Bobik A 1987 Na-H antiport in cultured rat aortic smooth muscle: its role in cytoplasmic pH regulation. Am J Physiol 253:C193–C198
38. Matsuda T, Murata Y, Kawamura N, Hayashi M, Tamada K, Takuma K, Maeda S, Baba A 1993 Selective induction of 1 isoform of Na⁺, K⁺-ATPase by insulin/insulin-like growth factor-I in cultured rat astrocytes. Arch Biochem Biophys 307:175–182[CrossRef][Medline]
39. Yamamoto K, Ikeda U, Okada K, Saio T, Kawakami K, Schimada S 1994 Sodium ion-mediated regulation of Na/K-ATPase gene transcription in vascular smooth muscle cells. Cardiovasc Res 128:957–962
40. Azuma KK, Hensley CB, Tang MJ, McDonough A 1993 Thyroid hormone specifically regulates skeletal muscle Na⁺-K⁺-ATPase 2- and ß2-isoforms. Am J Physiol 265:C680–C687
41. Simmons DA, Winegrad AI 1993 Insulin does not regulate vascular smooth muscle Na⁺, K⁺-ATPase activity in rabbit aorta. Diabetologia 36:212–217[CrossRef][Medline]
42. Therien AG, Blostein R 2000 Mechanisms of sodium pump regulation. Am J Physiol 279:C541–C566

This article has been cited by other articles:

				S. A. Cooper, A. Whaley-Connell, J. Habibi, Y. Wei, G. Lastra, C. Manrique, S. Stas, and J. R. Sowers Renin-angiotensin-aldosterone system and oxidative stress in cardiovascular insulin resistance Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2009 - H2023. [Abstract] [Full Text] [PDF]

				D. D Vadysirisack, A. Venkateswaran, Z. Zhang, and S. M Jhiang MEK signaling modulates sodium iodide symporter at multiple levels and in a paradoxical manner Endocr. Relat. Cancer, June 1, 2007; 14(2): 421 - 432. [Abstract] [Full Text] [PDF]

				O. Kotova, L. Al-Khalili, S. Talia, C. Hooke, O. V. Fedorova, A. Y. Bagrov, and A. V. Chibalin Cardiotonic Steroids Stimulate Glycogen Synthesis in Human Skeletal Muscle Cells via a Src- and ERK1/2-dependent Mechanism J. Biol. Chem., July 21, 2006; 281(29): 20085 - 20094. [Abstract] [Full Text] [PDF]

				I. Vadasz, R. E. Morty, A. Olschewski, M. Konigshoff, M. G. Kohstall, H. A. Ghofrani, F. Grimminger, and W. Seeger Thrombin Impairs Alveolar Fluid Clearance by Promoting Endocytosis of Na+,K+-ATPase Am. J. Respir. Cell Mol. Biol., October 1, 2005; 33(4): 343 - 354. [Abstract] [Full Text] [PDF]

				S. J. Khundmiri, W. L. Dean, K. R. McLeish, and E. D. Lederer Parathyroid Hormone-mediated Regulation of Na+-K+-ATPase Requires ERK-dependent Translocation of Protein Kinase C{alpha} J. Biol. Chem., March 11, 2005; 280(10): 8705 - 8713. [Abstract] [Full Text] [PDF]

				A. Maestroni, D. Ruggieri, G. Dell'Antonio, L. Luzi, and G. Zerbini C-peptide increases the expression of vasopressin-activated calcium-mobilizing receptor gene through a G protein-dependent pathway Eur. J. Endocrinol., January 1, 2005; 152(1): 135 - 141. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 145/3/1151    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Isenovic, E. R. Articles by Sowers, J. R. Search for Related Content PubMed PubMed Citation