AJP - Heart AJP: Cell Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 276: H2109-H2116, 1999;
0363-6135/99 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, D.
Right arrow Articles by Klip, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, D.
Right arrow Articles by Klip, A.
Vol. 276, Issue 6, H2109-H2116, June 1999

Participation of PI3K and atypical PKC in Na+-K+-pump stimulation by IGF-I in VSMC

Dailin Li1,2, Gary Sweeney1, Qinghua Wang1, and Amira Klip1

1 Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; and 2 Department of Woman and Child Health, Pediatric Unit, Astrid Lindgren Children's Hospital, Karolinska Hospital, S-171 76 Stockholm, Sweden


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The activity of the Na+-K+-pump is intricately linked to the maintenance of vascular tone. Here we demonstrate that insulin-like growth factor I (IGF-I) increases Na+-K+-pump activity in the vascular smooth muscle cell (VSMC) clone A7r5 in a time- and dose-dependent manner. This stimulatory effect of IGF-I was prevented by the tyrosine kinase inhibitor genistein (5 µM) and by the specific phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin (100 nM) and LY-294002 (25 µM). IGF-I activated a wortmannin-sensitive PI3K and its purported effector, the atypical protein kinase C (PKC)-zeta . Stimulation of PKC-zeta was prevented by the generic PKC inhibitor GF109203x (bisindolylmaleimide, 10 µM). Downregulation of diacylglycerol-sensitive (conventional and novel) PKCs by 24-h pretreatment with 1 µM phorbol 12-myristate 13-acetate had no effect on IGF-I-stimulated Na+-K+-pump activity. Similarly, inhibition of only conventional and novel PKCs with GF109203x (1 µM) had no effect on IGF-I-stimulated Na+-K+-pump activity. In contrast, a concentration of GF109203x (10 µM) that also inhibits the atypical PKCs abolished Na+-K+-pump stimulation by IGF-I. Neither the Na+-K+-2Cl- cotransporter inhibitor bumetanide (100 µM) nor the Na+/H+ exchanger inhibitor HOE-694 (5 µM) affected the Na+-K+-pump stimulation by IGF-I, suggesting that a rise in intracellular Na+ concentration is not necessary for increased Na+-K+-pump activity. These results suggest that IGF-I directly stimulates the Na+-K+ pump via a signaling pathway involving PI3K and atypical PKC (zeta ).

sodium-potassium adenosine 5'-triphosphatase; tyrosine kinase; vascular resistance


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CONTROL of vascular tone depends largely on the gradients of Na and Ca ions across the cell membrane (3, 23, 34). The Na+-K+ pump is the sole membrane protein responsible for Na+ efflux, extruding 3 Na ions and taking up 2 K ions against their concentration gradient, utilizing ATP hydrolysis to drive this process (39). The Na+-K+ pump maintains low intracellular Na+ concentrations ([Na+]i) required for adequate removal of Ca2+ from the cell via Na+/Ca2+ exchange. If agonist-induced increases in intracellular Ca2+ concentrations ([Ca2+]i) via voltage-operated channels were not adequately counteracted by Na+/Ca2+ exchange, sustained vasoconstriction would ensue. Maintenance of a low [Na+]i is therefore essential in supporting normal vascular tone.

Insulin-like growth factor I (IGF-I) is a circulating hormone produced in the liver. It is also locally generated in vascular smooth muscle cells (VSMC), where it plays an important role in the regulation of cell proliferation (14, 22). In addition, increasing evidence shows that IGF-I and insulin attenuate vasoconstrictive responses and increase blood flow, a process likely due to several components including stimulation of the Na+-K+ pump in VSMC (6, 42). Indeed, the vasodilator effects of IGF-I are more potent than those of insulin, and this correlates with the ability of these two hormones to stimulate the Na+-K+ pump (38). The elevated Na+-K+-pump activity could explain observations from single-cell analysis showing that insulin and IGF-I attenuate rises in [Ca2+]i induced by vasoconstrictor agents (33). Although various mechanisms to explain the attenuation of agonist-induced vasoconstriction by IGF-I and insulin have been proposed, the precise mode of action remains unclear.

The signaling pathway involved in the short-term regulation of Na+-K+-pump activity by IGF-I remains unknown. IGF-I and insulin are two structurally related hormones that produce similar physiological effects. Their receptors also share similarities in both structure and function, such as possessing intrinsic tyrosine kinase activity and having common downstream substrates such as insulin receptor substrate (IRS)-1 and IRS-2 (19, 36). Indeed, IRS-2 phosphorylation by insulin or interleukin-4 can desensitize bovine fibroblasts to the mitogenic effects of IGF-I (11). Phosphorylated IRS proteins directly bind to and activate another common downstream signaling molecule, phosphatidylinositol 3-kinase (PI3K). Atypical PKC isoforms have been suggested to lie downstream of PI3K because they are stimulated by the products of PI3K in vitro (30, 45) and because their activation and translocation are prevented by inhibitors of PI3K (13, 27). The PKC-zeta is required for stimulation of protein synthesis by insulin (26), and this hormone also leads to a rapid translocation of both PKC-zeta and PI3K in fibroblasts (28). The aim of this work, therefore, was elucidation of the signaling molecules employed by IGF-I in mediating Na+-K+-pump stimulation in VSMC, with particular emphasis on PI3K and atypical PKCs.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemical and biochemical reagents. Ouabain, bumetanide, phorbol 12-myristate 13-acetate (PMA), wortmannin, and myelin basic protein were purchased from Sigma (St. Louis, MO). GF109203x (bisindolylmaleimide) was from Calbiochem (La Jolla, CA). Genistein, LY-294002, and okadaic acid were from Biomol Research Laboratories (Plymouth Meeting, PA). HOE-694 was a kind gift from Dr. H. Urbach (Hoechst, Frankfurt, Germany). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and other tissue culture materials were from GIBCO (Burlington, ON, Canada). 86Rb+ and autoradiography film were from NEN Research Products (Boston, MA). Protein A-Sepharose CL-4B and Protein G-Sepharose were from Pharmacia (Uppsala, Sweden). Purified phosphatidylinositol was from Avanti Polar Lipids (Alabaster, AL). Potassium oxalate (1%)-treated Silica gel H thin-layer chromatography (TLC) plates (20 × 20 cm, 250 µm) were from Analtech (Newark, DE). Monoclonal antibodies McK1 and McB2 against alpha 1- and alpha 2-subunits of Na+-K+ pump, respectively, were kind gifts from Dr. Kathleen J. Sweadner (Laboratory of Membrane Biology, Massachusetts General Hospital, Boston, MA). Monoclonal antibody 6H against the alpha 1-subunit of Na+-K+ pump was a kind gift from Dr. Michael J. Caplan (Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT). The polyclonal antibody HERED against the alpha 2-subunit of Na+-K+-pump was a kind gift from Dr. Thomas Pressley (Department of Physiology, Texas Tech University Health Science Center, Lubbock, TX). The polyclonal antibodies SpETbeta 1, SpETbeta 2, and SpETbeta 3 were used to detect, respectively, the beta 1-, beta 2-, and beta 3-isoforms of Na+-K+ pump (18). Anti-phosphotyrosine monoclonal antibody was from Upstate Biotechnology (Lake Placid, NY). Monoclonal antibodies against the PKC alpha -, beta -, gamma -, delta -, epsilon -, theta -, iota -, lambda -, and µ-isoforms were from Transduction Laboratories (Lexington, KY). Polyclonal antibody against PKC-zeta was from Santa Cruz Biotechnology (Santa Cruz, CA). [gamma -32P]ATP (6,000 Ci/mmol) and enhanced chemiluminescence (ECL) immunoblotting detection reagents were from Amersham (Oakville, ON, Canada). Prestained SDS-PAGE molecular weight standards were from Bio-Rad Laboratories (Hercules, CA). Bicinchoninic acid protein assay reagents were from Pierce (Rockford, IL). Human recombinant IGF-I was a gift from Dr. Mladen Vranic (Dept. of Physiology, University of Toronto, Toronto, ON, Canada). A7r5 smooth muscle cells derived from embryonic rat aorta were obtained from American Type Culture Collection (Rockville, MD). Bumetanide, GF109203x, genistein, HOE-694, LY-294002, okadaic acid, PMA, and wortmannin were dissolved in DMSO at final DMSO concentrations of 0.05%, with the same concentration of solvent being used in control cells.

Cell culture. A7r5 cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2 in air and used between passages 2 and 15. Cells were fed every other day. At 1 day postconfluence, cells were made quiescent by a 19- to 20-h incubation in DMEM containing 0.1% FBS.

Immunoblotting of Na+-K+ pump. Quiescent A7r5 cells cultured in six-well plates were rinsed three times with ice-cold PBS and then harvested at 4°C in lysis buffer A containing 115 mM Tris · HCl (pH 6.8), 100 µM PMSF, 1 µM leupeptin, 1 µM pepstatin A, 4% SDS, 10 mM dithiothreitol (DTT), and 10% (vol/vol) glycerol. Protein samples (20 µg/lane) were loaded onto 7.5% SDS-PAGE gels. Electrophoresis, protein transferring, and immunoblotting were performed as described previously (37). Na+-K+-pump isoform-specific antibodies were diluted at 1:1,000 (McK1, 6H, SpETbeta 1, SpETbeta 2, and SpETbeta 3), 1:2,000 (HERED), or 1:500 (McB2).

Ouabain-sensitive 86Rb+ uptake. Na+-K+-pump activity was measured as ouabain-sensitive 86Rb+ uptake (37). Quiescent A7r5 cells cultured in 24-well plates were rinsed once with 1 ml of HEPES-buffered saline solution (HBSS) containing (in mM) 140 NaCl, 2.4 MgSO4, 5 KCl, 1 CaCl2, and 20 Na-HEPES, pH 7.4. The cells were then incubated at 37°C for 30 min with 0.25 ml of HBSS in the presence or absence of 100 nM IGF-I. Where indicated, cells were preincubated for 20 min with the tyrosine kinase inhibitor genistein (5 µM), the PI3K inhibitors wortmannin (100 nM) or LY-294002 (25 µM), or the PKC inhibitor GF109203x (1 or 10 µM). The Na+-K+-2Cl- cotransporter inhibitor bumetanide (100 µM) or the Na+/H+ exchanger inhibitor HOE-694 (5 µM) was added before the addition of IGF-I for 30 min. 86Rb+ was then added in 50 µl of HBSS to a final concentration of 5 µCi/ml in the presence or absence of a final concentration of 1.6 mM ouabain, and uptake was allowed to proceed for 20 min. Na+-K+-pump activity was calculated as the difference between total 86Rb+ uptake and ouabain-insensitive 86Rb+ uptake and was linear over this 20-min period. After this period, the radioactive solution was aspirated and the cells were rinsed twice with 1 ml of ice-cold 0.9% NaCl. The cells were lysed with 1 ml of 0.05 N NaOH, and a 0.8-ml aliquot was counted using a liquid scintillation counter. The protein concentration of the lysate was determined using the Bradford method (4).

Assay of PI3K activity associated with phosphotyrosine. To determine PI3K activity, cell extracts were prepared exactly the same way as for IRS-1 immunoprecipitation, and PI3K activity was measured on phosphotyrosine immunoprecipitates as described previously (46). Briefly, the ability of PI3K associated with phosphotyrosine to convert phosphatidylinositol to phosphatidylinositol monophosphate (PIP) was detected by separation of these lipids by TLC. Detection and quantitation of [32P]PIP on the TLC plates were done using a Molecular Dynamics PhosphorImager System (Sunnyvale, CA).

PKC-zeta activity assay. Quiescent A7r5 cells cultured in 10-cm dishes were rinsed once with 10 ml of serum-free DMEM. The cells were then incubated at 37°C for 10 min with 10 ml of serum-free DMEM in the presence or absence of 100 nM IGF-I. Where indicated, cells were preincubated with the PKC inhibitor GF109203x (10 µM) for 20 min before IGF-I treatment. After treatment, cells were rinsed twice with ice-cold PBS and then harvested at 4°C in 1 ml of lysis buffer B containing 50 mM HEPES, pH 7.6, 150 mM NaCl, 10% (vol/vol) glycerol, 1% (vol/vol) Triton X-100, 30 mM Na4P2O7, 10 mM NaF, 1 mM Na3VO4, 100 nM okadaic acid, 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine, and 1 mM DTT. Cells were homogenized by being passed five times through a 25-gauge syringe. Cell lysates were subjected to immnoprecipitation with anti-PKC-zeta polyclonal antibody that was precoupled to a mixture of Protein A- and Protein G-Sepharose beads by incubating 2 µg of the antibody with 20 µl of the Protein A-/Protein G-Sepharose beads (100 mg/ml) at 4°C overnight. Antibody-coupled beads were washed twice with PBS and once with lysis buffer B. PKC-zeta was immunoprecipitated by incubating the cell lysates (250 µg of total protein) with the anti-PKC-zeta antibody-bead complex at 4°C for 3 h under constant rotation. The immunocomplex beads were sedimented and then washed four times with 1 ml of wash buffer [25 mM HEPES, pH 7.8, 1 M NaCl, 10% (vol/vol) glycerol, 1% (vol/vol) Triton X-100, 0.1% bovine serum albumin (BSA), 1 mM DTT, 1 mM PMSF, and 100 nM okadaic acid] and twice with 1 ml of kinase buffer (50 mM Tris · HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT, and 100 nM okadaic acid). To measure PKC-zeta activity, the beads were incubated under constant agitation at 30°C for 10 min with 30 µl of the kinase buffer supplemented with 5 µg of myelin basic protein and [gamma -32P]ATP (25 µM, 5 µCi). The reaction was stopped by brief centrifugation, and 30 µl of supernatant was spotted onto Whatman P81 phosphocellulose paper that was washed four times for 10 min with 3 ml of 175 mM phosphoric acid and one time for 5 min with distilled water. Papers were air-dried and then subjected to liquid scintillation counting for measurement of 32P incorporation into myelin basic protein.

Downregulation of PKC isoforms and immunoblotting. A7r5 cells cultured in six-well plates or 10-cm dishes were incubated with 1 µM PMA at 37°C for 24 h (5 h in DMEM-10% FBS, 19 h in DMEM-0.1% FBS). After treatment, cells were rinsed three times with ice-cold PBS and then harvested at 4°C in lysis buffer A as described in Immunoblotting of Na+-K+ pump. Samples were homogenized using a 25-gauge syringe and then boiled for 5 min. Protein samples (20-35 µg/lane) were separated by electrophoresis on 7.5% SDS-PAGE gels and then transferred to polyvinylidene difluoride transfer membranes. Membranes were blocked for 1 h with 3% BSA in Tris-buffered saline containing 0.05% (vol/vol) Tween 20 and 0.05% (vol/vol) NP-40 and then incubated for 1 h with anti-PKC isoform-specific monoclonal antibodies at a 1:1,000 (PKC-alpha , -beta , -gamma , and -zeta ) or 1:750 (PKC-delta , -epsilon , -theta , -iota , -lambda , and -µ) dilution. After incubation with appropriate horseradish peroxidase-linked secondary antibody, specific binding was detected by means of the ECL immunoblotting detection system.

Statistical analysis. Values are given as means ± SE. Data were analyzed by the Student's t-test or analysis of variance (ANOVA, Fisher Scientific) as indicated. P values <0.05 were considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of IGF-I on Na+-K+-pump activity. It has been reported that A7r5 VSMC express Na+-K+ pump alpha 1- and alpha 2-subunits mRNA (44). The present study confirmed the presence of alpha 1-subunit protein, which migrated at ~95 kDa, with the use of two different monoclonal antibodies, 6H and McK1. Skeletal muscle membranes were used as reference control (Fig. 1). In carotid artery VSMC, a truncated alpha 1-isoform (65 kDa) is expressed (25). In our study, a 65-kDa band was detectable by antibody McK1 in A7r5 cells but not by antibody 6H (data not shown). In contrast, we could not detect the alpha 2-subunit protein in these cells using two different antibodies specific for alpha 2, HERED, and McB2 (Fig. 1). Of the beta -subunit isoforms, beta 1 was readily visualized in A7r5 cells, and a protein of somewhat higher molecular mass was detected by anti-beta 3 antibody. The beta 2-isoform was not detected despite being abundant in (white) skeletal muscle (Fig. 1).


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1.   Expression of Na+-K+ pump alpha 1- alpha 2-, beta 1-, beta 2-, and beta 3-isoforms in A7r5 vascular smooth muscle cells (VSMC). Protein (20 µg) of whole cell lysates prepared as described in MATERIALS AND METHODS was separated by SDS-PAGE and immunoblotted with Na+-K+-pump isoform-specific antibodies (6H, McK1, HERED, and McB2). Total membranes from skeletal muscle (SKM; 25 µg) were used as positive control. Results are from 1 experiment and are representative of 3 individual experiments.

The activity of the Na+-K+ pump in A7r5 cells, measured as ouabain-sensitive 86Rb+ (K+) uptake, constituted ~70% of total 86Rb+ uptake (Fig. 2A). Incubation with 100 nM IGF-I for 30 min increased Na+-K+-pump activity by ~80% (Fig. 2A). Ouabain-insensitive 86Rb+ uptake was not altered by IGF-I (data not shown). Acute incubation (10 min) with 100 nM IGF-I rapidly induced Na+-K+-pump stimulation with slightly higher levels apparent after 20 or 30 min of IGF-I incubation (Fig. 2B). When several doses of IGF-I were tested, maximal stimulation was achieved with 10 and 100 nM IGF-I (Fig. 2C).


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 2.   Effect of insulin-like growth factor I (IGF-I) on K+ (86Rb+) uptake in A7r5 VSMC. A: total 86Rb+ uptake over a 20-min period under unstimulated conditions was 6.6 ± 0.5 nmol/mg protein, whereas ouabain-sensitive 86Rb+ uptake for the same period was 4.5 ± 0.4 nmol/mg protein. Cells were treated with 100 nM IGF-I for 30 min, and then increased total and ouabain-sensitive 86Rb+ uptake was detected. Results are means ± SE of n > 4 separate experiments performed in duplicate or triplicate. * P < 0.05 compared with basal value (Student's t-test). B: time course of Na+-K+-pump stimulation by IGF-I. Cells were treated with 100 nM IGF-I for 10-30 min. In basal group, ouabain-sensitive 86Rb+ uptake over 20 min was 2.4 ± 0.6 nmol/mg protein. In B and C, basal values were assigned a value of 100%, and other results were expressed relative to this value. C: dose-response of IGF-I-stimulated Na+-K+-pump activity. Cells were treated with 1-100 nM IGF-I for 30 min. In the basal group, ouabain-sensitive 86Rb+ uptake over 20 min was 2.3 ± 0.5 nmol/mg protein. All data in B and C are means ± SE of 4 separate experiments performed in triplicate. * P < 0.05 compared with basal value (ANOVA).

Effect of tyrosine kinase inhibition on IGF-I-induced Na+-K+-pump stimulation. Tyrosine kinase activity has been established as an important component of IGF-I-triggered signaling cascades. Therefore, we first investigated the effect of the specific tyrosine kinase inhibitor genistein (1) on stimulation of the Na+-K+-pump by IGF-I. Figure 3 shows that genistein (5 µM) did not alter basal ouabain-sensitive 86Rb+ uptake but completely prevented the stimulatory effect of IGF-I, suggesting that activation of tyrosine kinase(s) is required for IGF-I-mediated stimulation of Na+-K+-pump activity.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3.   Effect of tyrosine kinase inhibitor genistein (Gen) on IGF-I-induced stimulation of Na+-K+ pump in A7r5 VSMC. Cells were preincubated with genistein (5 µM) for 20 min before incubation with 100 nM IGF-I for 30 min in continued presence of inhibitor. In basal group, ouabain-sensitive 86Rb+ uptake over 20 min was 4.8 ± 0.3 nmol/mg protein. This value was assigned a value of 100%, and other results were expressed relative to this value. All data are means ± SE of 3 separate experiments performed in duplicate. * P < 0.05 compared with basal value (ANOVA).

Involvement of PI3K in IGF-I-induced Na+-K+-pump stimulation. We then examined the role of PI3K in the stimulation of the Na+-K+ pump by IGF-I. Inhibition of PI3K with wortmannin (100 nM) completely blocked IGF-I-induced Na+-K+-pump stimulation in these cells (Fig. 4A). A second PI3K inhibitor, LY-294002 (25 µM), was also able to completely prevent Na+-K+-pump stimulation by IGF-I (Fig. 4A). Neither of these inhibitors had any significant effect on basal Na+-K+-pump activity. To confirm that PI3K is indeed stimulated by IGF-I in these cells, PI3K activity associated with phosphotyrosine immunoprecipitates was measured using purified phosphatidylinositol as substrate. As shown in Fig. 4, B and C, incubation with IGF-I for 5 min caused maximal activation of PI3K (~12-fold). The activation was sustained to a significant extent in the continued presence of IGF-I for 50 min. In vitro incubation with the specific PI3K inhibitor wortmannin (100 nM) abolished the measured PI3K activity (Fig. 4, B and C).


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4.   Analysis of role of phosphatidylinositol 3-kinase (PI3K) in IGF-I-induced Na+-K+-pump stimulation in A7r5 VSMC. A: effect of PI3K inhibitors LY-294002 and wortmannin on IGF-I-induced stimulation of Na+-K+ pump in A7r5 cells. LY-294002 (LY; 25 µM) or wortmannin (WM; 100 nM) was added 20 min before incubation with 100 nM IGF-I for 30 min in continued presence of inhibitor. In basal group, ouabain-sensitive 86Rb+ uptake over 20 min was 6.7 ± 1.0 nmol/mg protein. Basal values were assigned a value of 100%, and other results were expressed relative to this value. All data are means ± SE of 3-6 separate experiments performed in triplicate. * P < 0.05 compared with corresponding basal value (ANOVA). B: effect of IGF-I on PI3K activity. Cells were treated with 100 nM IGF-I for 5-50 min, and PI3K activity was then measured as described in MATERIALS AND METHODS. Where indicated, phosphotyrosine immunoprecipitates (PI3P) were incubated with wortmannin at room temperature for 15 min in vitro before assay of lipid kinase activity. Immunoprecipitation with nonspecific IgG is shown as control. C: quantitative analysis of PI3K activity shown in B. Basal values were assigned a value of 1, and other results were expressed relative to this value. All data are means ± SE of 4-5 separate experiments. * P < 0.05 compared with corresponding basal value (ANOVA).

Involvement of atypical PKCs in Na+-K+-pump stimulation. It has been reported that certain PKCs are effectors of PI3K. As a preamble to determining the possible involvement of members of the PKC family in IGF-I action, we examined the complement of PKC isoforms expressed in A7r5 cells. Twelve PKC isoforms have been identified in mammalian cells, and these are classed in three groups: Ca2+- and diacylglycerol (DAG)-dependent "conventional" PKCs (alpha -, beta I-, beta II-, and gamma -isoforms); Ca2+-independent and DAG-dependent "novel" PKCs (delta -, epsilon -, eta -, and theta -isoforms); and Ca2+- and DAG-insensitive "atypical" PKCs (zeta -, iota -, lambda -, and µ-isoforms) (15, 31). We have demonstrated, in accordance with a previous report, that A7r5 cells express PKC-alpha , -gamma , -delta , -epsilon , -zeta , -iota , -lambda , and -µ but not PKC-beta or -theta (10).

Twenty-four hour treatment with the phorbol ester PMA (1 µM) fully downregulated PKC-alpha , -gamma , and -delta and partially downregulated PKC-epsilon , whereas atypical PKC-zeta , -iota , -lambda , and -µ were unaffected (Fig. 5A). The upper band in the PKC-zeta immunoblot is most likely due to PKC-alpha , which cross-reacts with the PKC-zeta antibody (20). This is supported by the fact that this band, like that of PKC-alpha , was completely downregulated on PMA treatment.




View larger version (63K):
[in this window]
[in a new window]
  		Fig. 5.   Analysis of role of protein kinase C (PKC) in IGF-I-induced Na+-K+-pump stimulation in A7r5 VSMC. A: expression of PKC isoforms in A7r5 cells and their susceptibility to downregulation. Downregulation of PKC was achieved by 24-h treatment (5 h in DMEM-10% FBS, 19 h during serum deprivation) with 1 µM phorbol 12-myristate 13-acetate (PMA). Each lane contained 20 µg of total protein, except for PKC-zeta (35 µg/lane). Results are from 1 experiment representative of 3. Molecular mass (kDa) of PKC isoforms are as follows: alpha , 82; gamma , 80; delta , 78; epsilon , 90; µ, 115; iota , 74; lambda , 74; and zeta , 74. B: effect of IGF-I on PKC-zeta activity in A7r5 cells. Cells were treated with 100 nM IGF-I for 10 min. PKC-zeta activity was then measured as described in MATERIALS AND METHODS. Where indicated, cells were preincubated with GF109203x (10 µM) for 20 min before IGF-I treatment. Basal cells were assigned a value of 1, and other results were expressed relative to this value. All data are means ± SE of 3 separate experiments performed in duplicate. * P < 0.05 compared with corresponding basal value (ANOVA). C: effect of PKC downregulation on IGF-I-induced stimulation of Na+-K+ pump in A7r5 cells. Cells were treated with 1 µM PMA for 24 h as described in MATERIALS AND METHODS and then with 100 nM IGF-I for 30 min. In basal group, ouabain-sensitive 86Rb+ uptake over a 20-min period was 4.3 ± 0.8 nmol/mg protein. Basal values were assigned a value of 100%, and other results were expressed relative to this value. All data are means ± SE of 3 separate experiments performed in triplicate. * P < 0.05 compared with corresponding basal value (ANOVA). D: effect of PKC inhibitor GF109203x on IGF-I-induced stimulation of Na+-K+ pump in A7r5 VSMC. Cells were preincubated with GF109203x (GF; 1 or 10 µM) for 20 min before incubation with 100 nM IGF-I for 30 min in continued presence of inhibitor. In basal group, ouabain-sensitive 86Rb+ uptake over a 20-min period was 6.0 ± 1.1 nmol/mg protein. Basal values were assigned a value of 100%, and other results were expressed relative to this value. All data are means ± SE of 4 separate experiments performed in duplicate. * P < 0.05 relative to corresponding basal value (ANOVA).

Having shown the activation of PI3K by IGF-I and realizing that atypical PKC-zeta has been shown to lie downstream of PI3K (30, 45), we investigated whether IGF-I activates atypical PKC-zeta in A7r5 cells. It should be noted here that the polyclonal antibody used to immunoprecipitate the kinase is marketed as being specific for PKC-zeta but may also detect PKC-lambda . Figure 5B shows that IGF-I activated PKC-zeta activity ~twofold and that the activation was blocked by a high concentration (10 µM) of the PKC inhibitor GF109203x. We then used two different approaches to investigate the role of atypical PKCs in the stimulation of Na+-K+-pump activity by IGF-I. Downregulation of PKCs by PMA did not inhibit IGF-I-induced Na+-K+-pump stimulation and had no effect on basal Na+-K+-pump activity (Fig. 5C). This suggests that phorbol ester-sensitive PKC isoforms are not involved in mediating IGF-I action. However, the PKC inhibitor GF109203x (24) abolished Na+-K+-pump stimulation by IGF-I when used at 10 µM (inhibiting all PKCs) but not at 1 µM (inhibiting only conventional and novel PKCs) (Fig. 5D). No significant effect of GF109203x on basal Na+-K+-pump activity was observed (Fig. 5D). These results suggested that atypical PKC (zeta ) participates in the signaling pathway mediating stimulation of the Na+-K+ pump by IGF-I.

Role of [Na+]i in IGF-I-induced Na+-K+-pump stimulation. The contribution of entry of extracellular Na+ to IGF-I-induced Na+-K+-pump stimulation was evaluated by using specific inhibitors of the Na+-K+-2Cl- cotransporter and Na+/H+ exchanger. These inhibitors [bumetanide (100 µM) and HOE-694 (5 µM), respectively] did not alter Na+-K+-pump stimulation by IGF-I and had no effect on basal Na+-K+-pump activity (Fig. 6). This suggests that the observed stimulation of the Na+-K+ pump by IGF-I is not secondary to changes in [Na+]i.


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 6.   Effect of inhibitors of Na+-K+-2Cl- cotransporter and Na+/H+ exchanger on IGF-I-induced stimulation of Na+-K+ pump in A7r5 vascular smooth muscle cells. Cells were preincubated with bumetanide (Bumet; 100 µM) for 15 min or HOE-694 (H694; 5 µM) for 10 min before incubation with 100 nM IGF-I for 30 min in continued presence of inhibitor. In basal group, ouabain-sensitive 86Rb+ uptake over a 20-min period was 3.0 ± 0.4 nmol/mg protein. Basal values were assigned a value of 100%, and other results were expressed relative to this value. All data are means ± SE of 4-8 separate experiments performed in triplicate. * P < 0.05 compared with corresponding basal value (ANOVA).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Physiological role of the regulation of the Na+-K+ pump by IGF-I in smooth muscle. IGF-I was first shown to stimulate Na+-K+-pump activity in VSMC in vitro in 1997 (42), and it was hypothesized that locally produced IGF-I might have vasodilating activity in vivo. Very recently, IGF-I, but not insulin, was shown to increase blood flow via a vasodilating effect in the human forearm (32). Consistent with these findings, we show here that IGF-I increased Na+-K+-pump activity in A7r5 VSMC derived from rat aorta. Unlike IGF-I, insulin did not stimulate the Na+-K+ pump within <= 50 min (results not shown), although exposure to supraphysiological insulin concentrations for several hours can elevate pump content and, hence, net pump activity in these cells (44). In addition, IGF-I is more potent than insulin in stimulating glucose transport in VSMC cells (41), highlighting the importance of IGF-I in these cells. Locally produced IGF-I may act as an autocrine and/or paracrine factor by inducing vascular relaxation through activation of the Na+-K+-pump activity and consequently increasing the transmembrane Na+ gradient that drives Ca2+ efflux via Na+/Ca2+ exchange. In this way, stimulation of the Na+-K+ pump could lead to lowering vascular cytosolic Ca2+ levels, thereby counteracting the effect of vasoconstriction stimuli. In addition, IGF-I promotes the production and release of nitric oxide from endothelium and VSMC (29, 48). Both IGF-I and the Na+-K+ pump have been implicated in diverse forms of hypertension. Indeed, several antihypertensive agents are thought to act, at least in part, by altering IGF-I expression (7). The alpha 1-Na+-K+-pump gene was recently shown to be a susceptibility hypertension gene in the Dahl salt-sensitive Harlan Sprague Dawley rat (12), in part via its participation in renal Na+ handling. Moreover, pressure overload induces transcription of the alpha 2-Na+-K+-pump gene in rat and human aorta (35), presumably in an attempt to counteract the insult under conditions in which alpha 1-pump activity is insufficient. Given these facts, it is attractive to pursue the possibility that physiologically IGF-I promotes Na+-K+-pump activity in vascular smooth muscle as an integral part of the maintenance of blood flow and counteraction of vasoconstriction. Ultimately, it is conceivable that the insulin resistance observed in type 2 diabetes and several forms of hypertension may also confer IGF-I resistance, compromising Na+-K+-pump stimulation by IGF-I.

Signals involved in the stimulation of the Na+-K+ pump by IGF-I. Despite the importance of IGF-I in regulating ion fluxes in vascular smooth muscle, the mechanism of communication between the occupied IGF-I receptor and Na+-K+ pump is not known. Therefore, in the present study we investigated the signaling pathways involved in the stimulation of Na+-K+-pump activity by IGF-I in VSMC. Signaling from the IGF-I receptor mirrors that for insulin in many ways, including activation of the receptor tyrosine kinase activity. We found that activation of tyrosine kinase(s) is necessary for the stimulation of the Na+-K+ pump by IGF-I, based on its prevention by genistein. This compound also prevented the stimulation by epidermal growth factor and insulin of Na+-K+-pump activity in kidney tubular cells (9). In contrast, the tyrosine kinase inhibitors lavendustin A (1 µM) and herbimycin (0.5 µM) were unable to attenuate IGF-I regulation of Na+-K+-pump activity in primary cultures of VSMC (42). A plausible reason for this unexpected discrepancy is the differential selectivity of these inhibitors for individual tyrosine kinases (40).

In a similar fashion to insulin action, it has been established that activation of PI3Ks is essential in mediating several effects of IGF-I (16). Indeed, activation of N- and L-type Ca2+ channels in cerebellar granule neurons by IGF-I requires PI3K activity (2). We recently reported that insulin stimulates Na+-K+-pump activity in 3T3-L1 fibroblasts by a pathway involving PI3K (43). In the present study we found that IGF-I stimulated and maintained an elevated PI3K activity for <= 50 min in A7r5 VSMC. PI3K stimulation was inhibited by wortmannin, and both wortmannin and LY-294002 also inhibited Na+-K+-pump stimulation. These results suggest an essential role for PI3K in IGF-I-mediated stimulation of the Na+-K+ pump in VSMC.

The PKC family members have been proposed as both mediators and inhibitors of insulin action (5). Recent studies have strongly implicated the atypical isoforms of the PKC family as substrates of PI3K and mediators of insulin action (8). Most recently, promotion of macrophage differentiation by IGF-I was found to involve activation of PI3K and PKC-zeta (21). Several studies in cultured cells have shown that reduced Na+-K+-pump activity is a common consequence of hyperglycemia as observed in individuals with type 2 diabetes (47). Hyperglycemia increases vascular tone by increasing DAG levels and, therefore, DAG-sensitive PKC activity (17). Thus we designed our next set of experiments to examine the role of both DAG-sensitive and atypical PKC isoforms in IGF-I-mediated stimulation of the Na+-K+ pump in VSMC.

The results presented in this study show that atypical, but not conventional or novel, isoforms of the PKC family are indeed required for IGF-I-mediated stimulation of the Na+-K+ pump in VSMC. To establish this fact, we first exploited the fact that DAG-sensitive PKC isoforms can be downregulated by long-term phorbol ester treatment. We confirmed that this method was effective by immunoblotting with isoform-specific antibodies. When Na+-K+-pump activity was assessed in cells no longer expressing DAG-sensitive PKC isoforms, there was no change in basal or IGF-I-stimulated activity. However, the ability to discriminate between atypical and other PKC isoforms with the inhibitor GF109203x allowed us to study the role of these PKC isoforms in IGF-I action (24). Interestingly, we found that inhibition of atypical isoforms with 10 µM GF109203x prevented IGF-I action. Moreover, we showed that IGF-I activated atypical PKC-zeta and that this activation was prevented by 10 µM GF109203x. Previous studies (42) using staurosporine to inhibit PKC failed to show a role for PKC in IGF-I-stimulated VSMC Na+-K+-pump activity. However, only 10 nM staurosporine was used in that study, and it is appreciated that much higher concentrations of this rather nonspecific inhibitor are needed to inhibit the atypical PKC-zeta (IC50 1.3 µM).

Stimulation of Na+-K+ pump in VSMC does not require prior Na+ entry. We have shown recently (43) that insulin-stimulated increases in Na+-K+-pump activity in 3T3-L1 fibroblasts required antecedent stimulation of the Na+-K+-2Cl- cotransporter. The ensuing increased [Na+]i was sufficient to increase Na+-K+-pump activity. In contrast, here we show that the stimulatory effect of IGF-I on the Na+-K+ pump in A7r5 cells was not secondary to the changes in Na+ entry via the Na+-K+-2Cl- cotransporter or Na+/H+ exchanger. The mechanism of Na+-K+-pump activation in this case may be via either direct activation or translocation of alpha -subunits to the plasma membrane. In preliminary experiments using HEK cells overexpressing hemagglutinin (HA)-tagged alpha 1-Na+-K+-pump isoforms (the same isoform found in A7r5 cells) we have observed increased HA-tagged alpha 1-subunit exposure at the cell surface in response to IGF-I. Whereas these findings are indicative of a potential mechanism, the relevance of this system to smooth muscle cells remains to be established.

In summary, IGF-I provokes increases in VSMC Na+-K+-pump activity by a pathway involving tyrosine kinase(s), PI3K, and atypical PKC (possibly the zeta -isoform). Defects in Na+-K+-pump activity have been associated with primary essential hypertension as well as hypertension related to diabetes. We hypothesize that in insulin resistant states there may be defects in the pathway of stimulation of the Na+-K+ pump by IGF-I, contributing to the development of hypertension.


    ACKNOWLEDGEMENTS

We thank Dr. Anita Aperia and the Karolinska Institute for supporting Dailin Li via an exchange program with The Hospital for Sick Children.


    FOOTNOTES

This work was supported by Medical Research Council of Canada Grant MT-12601 (to A. Klip).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. Klip, Programme in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8 (E-mail: amira{at}sickkids.on.ca).

Received 8 June 1998; accepted in final form 3 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akiyama, T., J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shibuya, and Y. Fukami. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262: 5592-5595, 1987[Abstract/Free Full Text].

2.   Blair, L. A., and J. Marshall. IGF-I modulates N and L calcium channels in a PI3K-dependent manner. Neuron 19: 421-429, 1997[Medline].

3.   Bova, S., W. F. Goldman, X. J. Yauan, and M. P. Blaustein. Influence of Na+ gradient on Ca2+ transients and contraction in vascular smooth muscle. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H409-H423, 1990[Abstract/Free Full Text].

4.   Bradford, M. M. A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principal of protein-dye binding. Anal. Biochem. 72: 247-254, 1976.

5.   Considine, R. V., and J. F. Caro. Protein kinase C: mediator or inhibitor of insulin action? J. Cell. Biochem. 52: 8-13, 1993[Medline].

6.   Copeland, K. C., and K. S. Nair. Recombinant human insulin-like growth factor-I increases forearm blood flow. J. Clin. Endocrinol. Metab. 79: 230-232, 1994[Abstract].

7.   Donohue, T. J., L. D. Dworkin, J. Ma, M. N. Lango, and V. M. Catanese. Antihypertensive agents that limit ventricular hypertrophy inhibit cardiac expression of insulin-like growth factor-I. J. Investig. Med. 45: 584-591, 1997[Medline].

8.   Farese, R. V. Insulin-sensitive phospholipid signaling systems and glucose transport: an update. Proc. Soc. Exp. Biol. Med. 213: 1-12, 1996[Medline].

9.   Feraille, E., M. L. Carranza, M. Rousselot, and H. Favre. Modulation of Na+-K+-ATPase activity by a tyrosine phosphorylation process in rat proximal convoluted tubule. J. Physiol. (Lond.) 498: 99-108, 1997[Medline].

10.   Fiorani, M., O. Cantoni, A. Tasinato, D. Boscoboinik, and A. Azzi. Hydrogen peroxide- and fetal bovine serum-induced DNA synthesis in vascular smooth muscle cells: positive and negative regulation by protein kinase C isoforms. Biochim. Biophys. Acta 1269: 98-104, 1995[Medline].

11.   Haddad, T. C., and C. A. Conover. Insulin and interleukin-4 induce desensitization to the mitogenic effects of insulin-like growth factor-I. Pivotal role for insulin receptor substrate-2. J. Biol. Chem. 272: 19525-19531, 1997[Abstract/Free Full Text].

12.   Herrera, V. L. M., H. X. Xie, L. V. Lopez, N. J. Schork, and N. Ruiz-Opazo. The alpha 1 Na,K-ATPase gene is a susceptibility hypertension gene in the Dahl salt-sensitive HSD rat. J. Clin. Invest. 102: 1102-1111, 1998[Medline].

13.   Herrera-Velit, P., K. L. Knutson, and N. E. Reiner. Phosphatidylinositol 3-kinase-dependent activation of protein kinase C-z in bacterial lipopolysaccharide-treated human monocytes. J. Biol. Chem. 272: 16445-16452, 1997[Abstract/Free Full Text].

14.   Humbel, R. E. Insulin-like growth factors I and II. Eur. J. Biochem. 190: 445-462, 1990[Medline].

15.   Jaken, S. Protein kinase C isozymes and substrates. Curr. Opin. Cell Biol. 8: 168-173, 1996[Medline].

16.   Kaliman, P., J. Canicio, P. R. Shephers, C. A. Beeton, X. Testar, M. Palacin, and A. Zorzano. Insulin-like growth factors require phosphatidylinositol 3-kinase to signal myogenesis: dominant negative p85 expression blocks differentiation of L6E9 muscle cells. Mol. Endocrinol. 12: 66-77, 1998[Abstract/Free Full Text].

17.   King, G. L., M. Kunisaki, Y. Nishio, T. Inoguchi, T. Shiba, and P. Xia. Biochemical and molecular mechanisms in the development of diabetic vascular complications. Diabetes 45: S105-S108, 1996.

18.   Lecuona, E., S. Luquin, J. Avila, L. M. Garcia-Segura, and P. Martin-Vasallo. Expression of the beta 1 and beta 2(AMOG) subunits of the Na,K-ATPase in neural tissues: cellular and developmental distribution patterns. Brain Res. Bull. 40: 167-174, 1996[Medline].

19.   LeRoith, D., H. Werner, D. Beitner-Johnson, and C. T. Roberts, Jr. Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr. Rev. 16: 143-163, 1995[Medline].

20.   Liao, D. F., B. Monia, N. Dean, and B. C. Berk. Protein kinase C-zeta mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells. J. Biol. Chem. 272: 6146-6150, 1997[Abstract/Free Full Text].

21.   Liu, Q., W. Ning, R. Dantzer, G. G. Freund, and K. W. Kelley. Activation of protein kinase C-zeta and phosphatidylinositol 3'-kinase and promotion of macrophage differentiation by insulin-like growth factor-I. J. Immunol. 160: 1393-1401, 1998[Abstract/Free Full Text].

22.   Lou, H., Y. Zhao, P. Delafontaine, T. Kodama, N. Katz, P. W. Ramwell, and M. L. Foegh. Estrogen effects on insulin-like growth factor-I (IGF-I)-induced cell proliferation and IGF-I expression in native and allograft vessels. Circulation 96: 927-933, 1997[Abstract/Free Full Text].

23.   Magyar, C. E., J. Wang, K. K. Azuma, and A. A. McDonough. Reciprocal regulation of cardiac Na-K-ATPase and Na/Ca exchanger: hypertension, thyroid hormone, development. Am. J. Physiol. 269 (Cell Physiol. 38): C675-C682, 1995[Abstract/Free Full Text].

24.   Martiny-Baron, G., M. G. Kazanietz, H. Mischak, P. M. Blumberg, G. Kochs, H. Hug, D. Marme, and C. Schachtele. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976. J. Biol. Chem. 268: 9194-9197, 1993[Abstract/Free Full Text].

25.   Medford, R. M., R. Hyman, M. Ahmad, J. C. Allen, T. A. Pressley, P. D. Allen, and B. Nadal-Ginard. Vascular smooth muscle expresses a truncated Na+-K+-ATPase alpha-1 subunit isoform. J. Biol. Chem. 266: 18308-18312, 1991[Abstract/Free Full Text].

26.   Mendez, R., G. Kollmorgen, M. F. White, and R. E. Rhoads. Requirement of protein kinase C for stimulation of protein synthesis by insulin. Mol. Cell. Biol. 17: 5184-5192, 1997[Abstract].

27.   Mizukami, Y., T. Hirata, and K. Yoshida. Nuclear translocation of PKC zeta during ischemia and its inhibition by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. FEBS Lett. 401: 247-251, 1997[Medline].

28.   Mosthaf, L., M. Kellerer, A. Muhlhofer, J. Mushack, E. Seffer, and H. U. Haring. Insulin leads to a parallel translocation of PI3K and protein kinase C. Exp. Clin. Endocrinol. 104: 19-24, 1996.

29.   Muniyappa, R., M. F. Walsh, J. S. Rangi, R. M. Zayas, P. R. Standley, J. L. Ram, and J. R. Sowers. Insulin like growth factor 1 increases vascular smooth muscle nitric oxide production. Life Sci. 61: 925-931, 1997[Medline].

30.   Nakanishi, H., K. A. Brewer, and J. H. Exton. Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 268: 13-16, 1993[Abstract/Free Full Text].

31.   Newton, A. C. Regulation of protein kinase C. Curr. Opin. Cell Biol. 9: 161-167, 1997[Medline].

32.   Pendergrass, M., E. Fazioni, D. Collins, and R. A. DeFronzo. IGF-I increases forearm blood flow without increasing forearm glucose uptake. Am. J. Physiol. 275 (Endocrinol. Metab. 38): E345-E350, 1998[Abstract/Free Full Text].

33.   Ram, J. L., M. A. Fares, P. R. Standley, and J. R. Sowers. Insulin inhibits vasopressin elicited contraction of vascular smooth muscle cells. J. Vasc. Biol. 4: 250-254, 1993.

34.   Reuter, H. Sodium-calcium exchange. Ins and outs of Ca2+ transport. Nature 349: 567-568, 1991[Medline].

35.   Ruiz-Opazo, N., J.-F. Cloix, M.-G. Melis, X. H. Xiang, and V. L. M. Herrera. Characterization of a sodium-response transcriptional mechanism. Hypertension 30: 191-198, 1997[Abstract/Free Full Text].

36.   Saltiel, A. R. Diverse signaling pathways in the cellular actions of insulin. Am. J. Physiol. 270 (Endocrinol. Metab. 33): E375-E385, 1996[Abstract/Free Full Text].

37.   Sargeant, R. J., Z. Liu, and A. Klip. Action of insulin on Na+-K+-ATPase and the Na+-K+-2Cl- cotransporter in 3T3-L1 adipocytes. Am. J. Physiol. 269 (Cell Physiol. 38): C217-C225, 1995[Abstract/Free Full Text].

38.   Simmons, D. A., and A. I. Winegrad. Insulin does not regulate vascular smooth muscle Na+-K+-ATPase activity in rabbit aorta. Diabetologia 36: 212-217, 1993[Medline].

39.   Skou, J. C., and M. Esmann. The Na,K-ATPase. J. Bioenerg. Biomembr. 24: 249-261, 1992[Medline].

40.   Srinivas, P. R., and G. Grunberger. Inhibitors of the insulin receptor tyrosine kinase. Pharmacol. Ther. 64: 23-35, 1994[Medline].

41.   Standley, P. R., and K. A. Rose. Insulin and insulin-like growth factor-I modulation of glucose transport in arterial smooth muscle cells: implication of GLUT-4 in the vasculature. Am. J. Hypertens. 7: 357-362, 1994[Medline].

42.   Standley, P. R., F. Zhang, R. M. Zayas, R. Muniyappa, M. F. Walsh, E. Cragoe, and J. R. Sowers. IGF-I regulation of Na+-K+-ATPase in rat arterial smooth muscle. Am. J. Physiol. 273 (Endocrinol. Metab. 36): E113-E121, 1997[Abstract/Free Full Text].

43.   Sweeney, G., R. Somwar, T. Ramlal, P. Martin-Vasallo, and A. Klip. Insulin stimulation of K+ uptake in 3T3-L1 fibroblasts involves phosphatidylinositol 3-kinase and protein kinase C-zeta. Diabetologia 41: 1199-1204, 1998[Medline].

44.   Tirupattur, P. R., J. L. Ram, P. R. Standley, and J. R. Sowers. Regulation of Na+-K+-ATPase gene expression by insulin in vascular smooth muscle cells. Am. J. Hypertens. 6: 626-629, 1993[Medline].

45.   Toker, A., M. Meyer, K. K. Reddy, J. R. Falck, R. Aneja, S. Aneja, A. Parra, D. J. Burns, L. M. Ballas, and L. C. Cantley. Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3. J. Biol. Chem. 269: 32358-32367, 1994[Abstract/Free Full Text].

46.   Tsakiridis, T., H. E. McDowell, T. Walker, C. P. Downes, H. S. Hundal, M. Vranic, and A. Klip. Multiple roles of phosphatidylinositol 3-kinase in regulation of glucose transport, amino acid transport, and glucose transporters in L6 skeletal muscle cells. Endocrinology 136: 4315-4322, 1995[Abstract].

47.   Xia, P., R. M. Kramer, and G. L. King. Identification of the mechanism for the inhibition of Na+-K+-adenosine triphosphatase by hyperglycemia involving activation of protein kinase C and cytosolic phospholipase A2. J. Clin. Invest. 96: 733-740, 1995.

48.   Zeng, G., and M. J. Quon. Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells. J. Clin. Invest. 98: 894-898, 1996[Medline].

Am J Physiol Heart Circ Physiol 276(6):H2109-H2116
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
A. Carranza, P. L. Musolino, M. Villar, and S. Nowicki
Signaling cascade of insulin-induced stimulation of L-dopa uptake in renal proximal tubule cells
Am J Physiol Cell Physiol, December 1, 2008; 295(6): C1602 - C1609.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. R. Sowers
Insulin resistance and hypertension
Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1597 - H1602.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
K. A. Buhagiar, P. S. Hansen, B. Y. Kong, R. J. Clarke, C. Fernandes, and H. H. Rasmussen
Dietary cholesterol alters Na+/K+ selectivity at intracellular Na+/K+ pump sites in cardiac myocytes
Am J Physiol Cell Physiol, February 1, 2004; 286(2): C398 - C405.
[Abstract] [Full Text]

Home page
 Am. J. Physiol. Cell Physiol.Home page
G. Sweeney, W. Niu, V. A. Canfield, R. Levenson, and A. Klip
Insulin increases plasma membrane content and reduces phosphorylation of Na+-K+ pump alpha 1-subunit in HEK-293 cells
Am J Physiol Cell Physiol, December 1, 2001; 281(6): C1797 - C1803.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
A. G. Therien and R. Blostein
Mechanisms of sodium pump regulation
Am J Physiol Cell Physiol, September 1, 2000; 279(3): C541 - C566.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
P. S. Hansen, K. A. Buhagiar, D. F. Gray, and H. H. Rasmussen
Voltage-dependent stimulation of the Na+-K+ pump by insulin in rabbit cardiac myocytes
Am J Physiol Cell Physiol, March 1, 2000; 278(3): C546 - C553.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. S. Feschenko, E. Stevenson, and K. J. Sweadner
Interaction of Protein Kinase C and cAMP-dependent Pathways in the Phosphorylation of the Na,K-ATPase
J. Biol. Chem., October 27, 2000; 275(44): 34693 - 34700.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
<