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Circulation Research. 1995;76:1-15

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(Circulation Research. 1995;76:1-15.)
© 1995 American Heart Association, Inc.

Articles

Angiotensin II and Other Hypertrophic Stimuli Mediated by G Protein–Coupled Receptors Activate Tyrosine Kinase, Mitogen-Activated Protein Kinase, and 90-kD S6 Kinase in Cardiac Myocytes

The Critical Role of Ca2+-Dependent Signaling

Junichi Sadoshima, Zhihua Qiu, James P. Morgan, Seigo Izumo

From the Cardiovascular Research Center, Division of Cardiology, University of Michigan Medical Center, Ann Arbor (J.S., S.I.), and the Cardiovascular Division, Beth Israel Hospital, and Department of Medicine, Harvard Medical School, Boston, Mass (J.S., Z.Q., J.P.M., S.I.).

Correspondence to Dr Seigo Izumo, Division of Cardiology, University of Michigan Medical Center, Ann Arbor, MI 48109-0644.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Abstract Many hypertrophic stimuli such as angiotensin II (Ang II) activate phospholipases through G protein–coupled receptors in cardiac myocytes. However, it is not known whether these stimuli also activate the tyrosine phosphorylation–dependent signaling pathway, which plays an essential role in growth factor–induced mitogenic responses in other cell types. Serine/threonine kinases such as mitogen-activated protein (MAP) kinases and 90-kD S6 kinase (RSK) are activated in response to many growth stimuli and are important downstream signaling pathways of tyrosine kinases. Therefore, we examined whether Ang II activates these protein kinases in primary cultures of cardiac myocytes and fibroblasts from neonatal rats. Ang II rapidly induced tyrosine phosphorylation of multiple proteins, including 42-, 44-, 75- to 80-, and 120- to 130-kD proteins, in both cardiac myocytes and fibroblasts. This was accompanied by an increase in tyrosine kinase activity. The 42- and 44-kD proteins were immunologically related to an extracellular signal-regulated kinase family (MAP kinases). Ang II rapidly increased kinase activity of MAP kinases and their downstream kinase, RSK. The Ang II–induced tyrosine phosphorylation and activation of MAP kinases and RSK were AT1 receptor–mediated. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate or an increase in intracellular Ca2+ by the Ca2+ ionophore A23187 was sufficient to cause tyrosine phosphorylation of multiple proteins and activation of MAP kinase and RSK. Although downregulation of PKC did not suppress Ang II–induced activation of MAP kinase and RSK, chelating intracellular Ca2+ by BAPTA-AM completely abolished Ang II–induced activation of these kinases. Activation of MAP kinases and RSK was also observed in myocytes stimulated with other agonists for Gq protein–coupled receptors, such as phenylephrine, norepinephrine, and endothelin 1, but not with agonists to Gs protein–coupled receptors, such as isoproterenol. These results suggest that Ang II and other hypertrophic stimuli, known to act through Gq protein–coupled receptors, rapidly cause tyrosine phosphorylation of several intracellular substrates through activation of tyrosine kinase and activate MAP kinases and RSK in cardiac myocytes as well as in cardiac fibroblasts. Furthermore, intracellular Ca2+, rather than PKC, seems to be critical for Ang II–induced activation of these protein kinases in cardiac myocytes.


Key Words: mitogen-activated protein kinase • 90-kD ribosomal S6 kinase • angiotensin II


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Many growth stimuli cause tyrosine phosphorylation of various intracellular substrates through receptors that are either directly or indirectly coupled to tyrosine kinases. Tyrosine phosphorylation of the intracellular substrates plays an essential role not only in cellular mitogenic responses but also in many other functions, including differentiation of neuronal, endocrine, and lymphoid cells.1 In terminally differentiated cardiac muscle, myocytes are known to respond to mechanical load and growth factors by increasing their mass (hypertrophy). This process consists of an increase in protein synthesis and alteration of muscle phenotype but is not accompanied by DNA synthesis or cell division.2 3 Although tyrosine phosphorylation has been shown to play an essential role in the mitogenic response, it is not known whether it is also important for the hypertrophic growth response in terminally differentiated cells.

Recently, several protein serine/threonine kinases that may participate in regulating cell growth have been identified. Among these are the mitogen-activated protein (MAP) kinase family. MAP kinases are rapidly activated in response to stimulation of various receptors, including growth factor/tyrosine kinase receptors (such as insulin receptor) and G protein–coupled receptors (such as the thrombin receptor). Importantly, activity of MAP kinase is critically regulated by both tyrosine and threonine phosphorylation.4 5 It has been recently shown that MAP kinase kinases (MEKs) phosphorylate MAP kinase on both tyrosine and threonine residues; this phosphorylation results in activation of MAP kinase.6 It has also been shown that raf-1 and MEK kinase phosphorylate and activate MEK.7 8 MAP kinase phosphorylates and activates 90-kD ribosomal S6 protein kinase (RSK), a member of the S6 kinase family.5 9 Interestingly, recent studies suggest that MAP kinase and RSK can phosphorylate nuclear transcriptional factors, such as JUN, MYC, p62TCF, FOS, and the serum response factor, and regulate expression of downstream genes.5 10 11 Thus, the protein kinase cascade can potentially regulate a wide variety of cellular phenomena not restricted to mitogenesis. Therefore, it is of interest whether hypertrophic stimuli use this protein kinase cascade, which critically depends on tyrosine phosphorylation.

Many vasoactive substances, including angiotensin II (Ang II), norepinephrine (NE), phenylephrine (Phe), and endothelin 1 (ET-1), cause hypertrophic responses in cardiac myocytes.12 13 14 These humoral factors are known to activate several second-messenger systems through heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors. It has been recently shown that Ang II causes tyrosine phosphorylation and activates MAP kinases in vascular smooth muscle cells.15 16 17 More recently, Bogoyevitch and coworkers18 19 reported that Phe, ET-1, and phorbol ester activate MAP kinases in cultured ventricular cardiac myocytes. These data suggest that tyrosine kinases or MAP kinases can be activated in cellular growth responses initiated by agonists for G protein–coupled receptors.

Several studies have suggested that Ang II may be a critical factor in mediating cardiac hypertrophy in vivo.20 We and others have reported that Ang II causes a hypertrophic response in neonatal rat cardiac myocytes and a mitogenic response in cardiac fibroblasts in vitro.12 21 We have recently demonstrated that mechanical stretch causes a secretion of Ang II from cardiac myocytes and that Ang II acts as an initial mediator of stretch-induced hypertrophy through activation of AT1 receptors in neonatal rat cardiac myocytes.22 cDNA cloning of the AT1 receptor has indicated that the receptor putatively has seven-membrane-spanning regions, a typical feature of a G protein–coupled receptor, but has no apparent tyrosine kinase domain.23 Interestingly, we24 and others25 have shown that mechanical stretch of neonatal rat ventricular cardiac myocytes causes tyrosine phosphorylation of several proteins24 and activates MAP kinases and RSK.24 25 However, it remains to be determined whether Ang II activates tyrosine kinases or other tyrosine phosphorylation–dependent signal transduction pathways in cardiac myocytes and nonmyocytes. Moreover, it has not been clear, in any other systems, how stimulation of G protein–coupled receptors leads to activation of tyrosine phosphorylation and tyrosine phosphorylation–dependent signaling. Therefore, the experiments described in the present study were conducted (1) to examine whether Ang II and other hypertrophic stimuli activate tyrosine kinases in cardiac cells, (2) to examine whether Ang II and other hypertrophic stimuli activate MAP kinases and RSK, and (3) to examine the mechanism of how Ang II activates MAP kinase and RSK. We demonstrate that Ang II and other hypertrophic stimuli cause tyrosine phosphorylation of several intracellular proteins and activate MAP kinases and RSK in both cardiac myocytes and nonmyocytes. Interestingly, a Ca2+-dependent signaling mechanism seems to play an essential role in this Ang II–induced activation of the protein kinase cascade.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
All culture reagents were purchased from GIBCO. Glass coverslips were from Fisher Scientific. [{gamma}-32P]ATP (10 and 6000 Ci/mmol) was obtained from Du-Pont-New England Nuclear. 125I–protein A (30 mCi/mg) was from ICN; gel electrophoresis reagents were from Bio-Rad; nitrocellulose membrane was from Schleicher & Schuell; protein A–Sepharose CL-4B was from Pharmacia Biotech; pertussis toxin, pansorbin, and BAPTA-AM were from Calbiochem; H-7, calphostin C, and chelerythrine were from LC Services: okadaic acid was from Moana BioProducts; S6 peptide (RRLSSLRA) was from UBI; phosphocellulose paper (2.5 cm) was from Whatman; and Ang II was from Peninsula. Losartan and PD123319 were gifts from Du Pont–Merck and Parke-Davis, respectively. All other chemicals were purchased from Sigma Chemical Co. Stock solutions of the chemicals were prepared freshly just before experiments as 100- to 1000-fold concentrated solutions. Stock solutions of calphostin C, phorbol 12-myristate 13-acetate (PMA), A23187, and BAPTA-AM were made by dissolving these compounds in dimethyl sulfoxide (DMSO). The final concentration of DMSO was <0.1%, which did not affect basal tyrosine phosphorylation and activities of MAP kinases and RSK. Monoclonal anti-phosphotyrosine antibody (4G10)26 was a gift from Dr T. Roberts (Harvard Medical School, Boston, Mass). Recombinant anti-phosphotyrosine antibody (RC20H)27 was purchased from Transduction Laboratories. Anti-MAP kinase antibody ({alpha}-cMAPK)10 and anti-RSK antibody ({alpha}RSK)28 were gifts from Dr J. Blenis (Harvard Medical School). Other anti-MAP kinase polyclonal antibodies (anti–ERK-1 [K-23] and anti–ERK-2 [C-14], where ERK indicates extracellular signal–related kinase-1) were purchased from Santa Cruz Biotechnology, and an anti-rat MAP kinase polyclonal antibody (erk1-CT) was from UBI. Normal rabbit serum (nonimmune serum) was from Jackson Immuno Research.

Cell Culture and Immunofluorescent Staining
Primary cultures of the neonatal rat cardiac myocytes were prepared as previously described.29 After an enzymatic dissociation, the cells were preplated for 1 hour to selectively enrich for cardiac myocytes. The resultant suspension of myocytes was plated onto gelatin-coated 60-mm tissue culture dishes at a density of 1.35x105 cells per cm2 and cultured in Dulbecco's modified Eagle medium/F-12 (GIBCO) (1:1 [vol/vol]) supplemented with 5% horse serum, 2 g/L bovine serum albumin (fraction V), 4 mmol/L glucose, 3 mmol/L pyruvic acid, 15 mmol/L HEPES (pH 7.6), 100 µmol/L ascorbic acid, 100 µg/mL ampicillin, and 100 µmol/L bromodeoxyuridine. The culture medium was changed 24 to 36 hours after seeding to a defined serum-free Dulbecco's modified Eagle medium/F-12, which had the same composition as described above, except that 5% horse serum and bromodeoxyuridine were not added. Using this method, we routinely obtained myocyte cultures with 90% to 95% myocytes, as assessed by immunofluorescence staining with a monoclonal antibody against sarcomeric myosin heavy chain (MF20).30 For immunofluorescence studies, cardiac myocytes were grown on uncoated glass coverslips, and the indirect immunofluorescent staining was performed as described previously.29 Immunofluorescence studies of MAP kinases were performed as previously described10 by using 10 µg/mL of an anti–MAP kinase antibody (erk1-CT). Cardiac nonmyocyte (fibroblast) culture was prepared by two passages of the cells adherent to the culture dish during the preplating procedure.29 All experiments were done in serum-free medium 48 hours after changing to the serum-free medium.

Immunoblotting
Cell stimulation was terminated by a rapid aspiration of the medium and addition of 120 µL of ice-cold buffer A containing 25 mmol/L Tris-HCl (pH 7.4), 25 mmol/L NaCl, 1 mmol/L sodium orthovanadate, 10 mmol/L sodium fluoride, 10 mmol/L sodium pyrophosphate, 10 nmol/L okadaic acid, 0.5 mmol/L EGTA, 1 mmol/L phenylmethysulfonyl fluoride (PMSF), 0.8 µg/mL leupeptin, 10 µg/mL aprotinin, and 10 mg/mL p-nitrophenylphosphate. Cell lysates were incubated on ice for 20 minutes and centrifuged for 20 minutes at 4°C. In some experiments, 1% Triton X-100, 1% deoxycholic acid, and 0.1% sodium dodecyl sulfate (SDS) were added to buffer A (modified buffer A). The lysate from one 60-mm dish (3x106 cells) contained 485±28 µg (mean of four randomly picked samples) of total protein. After addition of 5x Laemmli's SDS–polyacrylamide gel electrophoresis (PAGE) sample buffer, the lysates were boiled for 5 minutes. Lysates containing equal amounts of protein (200 µg) or immunoprecipitates (see below) were electrophoresed on an 8% or 10% polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were blocked by using 5% bovine serum albumin in TBST (20 mmol/L Tris [pH 8.0], 150 mmol/L NaCl, and 0.05% Tween 20) for 2 hours and were then incubated with 4G10 at a concentration of 2 µg/mL,26 RC20H (Transduction Laboratory) at a dilution of 1:2500,27 or polyclonal antisera raised against C-terminal MAP kinase peptide ({alpha}-cMAPK) at a dilution of 1:20010 in TBST. Immunoreactive bands were probed with 125I–protein A (30 mCi/mg). In experiments using RC20H, an enhanced chemiluminescence system (Amersham) was used as a detection method, which gave almost identical results as the 4G10 and 125I–protein A system. Blots were washed and subjected to autoradiography. Molecular weights of proteins were estimated by using prestained markers (Bio-Rad, 161-0324). Each lane presented in a single panel of the gel picture was from the same gel and the same exposure of the autoradiogram, although in some cases lanes were cut for the final figure production. In some blots, Ponceau S staining was performed according to the manufacturer's instruction (Sigma, P-7767) to confirm that the equal amount of protein was loaded in each lane.

Immune Complex Tyrosine Kinase Assay
Tyrosine kinase assays were performed as previously described.31 Cell-free lysates were prepared as above by using buffer A. Lysates containing equal amounts of protein (100 µg) were incubated with 10 vol RIPA buffer (10 mmol/L Tris [pH 7.2], 150 mmol/L NaCl, 1% Triton X-100, 1% deoxycholic acid, and 0.1 % SDS) and 10 µg of 4G10 for 3 hours at 4°C. Protein A–Sepharose CL-4B was then added, and immunoprecipitates were washed once with phosphate-buffered saline, twice with 20 mmol/L Tris (pH 7.8) and 0.5 mol/L LiCl, and once with 50 mmol/L Tris (pH 7.4) and 10 mmol/L MnCl2. Ten microliters of immunoprecipitate was incubated with 160 µg of an acid-insoluble synthetic tyrosine kinase substrate [EY(4:1), a polymer of glutamate (E) and tyrosine (Y) with a ratio of 4:1; Sigma P0275] and 40 µL of 2x kinase buffer containing 100 mmol/L HEPES (pH 7.6), 60 mmol/L MgCl2, 2 mmol/L MnCl2, 0.2 mmol/L sodium orthovanadate, and 0.2% Nonidet P-40. After addition of 5 µCi of [{gamma}-32P]ATP (6000 Ci/mmol) and 5 µmol/L of cold ATP, samples (80 µL per sample) were incubated at 25°C for 5 minutes. Reactions were terminated by spotting the samples onto Whatman 3MM papers. The papers were immediately washed with 5% trichloroacetic acid (wt/vol) containing 1% sodium pyrophosphate (wt/vol) four times (15 minutes each). The papers were placed into scintillation vials, and the radioactivity was counted. The rates of tyrosine phosphorylation of EY(4:1) were linear for at least 20 minutes in this assay condition. Control immunoprecipitates were prepared by using the anti-phosphotyrosine antibody preabsorbed by phosphotyrosine (50 mmol/L). The 32P count obtained without EY(4:1) was subtracted from that with EY(4:1) to eliminate background.

MAP Kinase Assays in Myelin Basic Protein–Containing Polyacrylamide Gels
Cell-free lysates were prepared in 60 µL of buffer A, and immune complex was prepared as previously described.10 Briefly, lysates containing equal amounts of protein (300 µg) were adjusted to 1% SDS, denatured by heating to 85°C to 90°C for 5 minutes, and diluted with 10 vol of RIPA buffer. The lysates were incubated with 2 µL of {alpha}-cMAPK,10 1 µg of K-23, or 1 µg of C-14 (Santa Cruz Biotechnology) overnight at 4°C. Pansorbin or protein A–Sepharose CL-4B was then added, and the immunoprecipitates were washed twice with buffer A. The immunoprecipitates (immune complex assays) or whole-cell lysates (direct assays) were electrophoresed on a 10% SDS-polyacrylamide gel containing 0.5 mg/mL myelin basic protein (MBP, Sigma M2016). Kinase assays in MBP-containing polyacrylamide gel were performed at room temperature as previously described.32 Briefly, after electrophoresis, SDS was removed by washing the gel with two changes of 20% 2-propanol in 50 mmol/L Tris (pH 8.0) for 1 hour and then with two changes of 50 mmol/L Tris (pH 8.0) containing 5 mmol/L 2-mercaptoethanol for 1 hour. The enzyme was denatured by incubating the gel with two changes of 6 mol/L guanidine-HCl for 1 hour and then renatured with five changes of 50 mmol/L Tris (pH 8.0) containing 0.04% Tween 40 and 5 mmol/L 2-mercaptoethanol for 1 hour (5 times for 12 minutes each). The gel was then incubated with 40 mmol/L HEPES (pH 8.0) containing 2 mmol/L dithiothreitol and 10 mmol/L MgCl2. The kinase reaction was performed in conditions inhibitory to cyclic nucleotide–dependent protein kinases and Ca2+-dependent protein kinases by incubating the gel at 25°C for 1 hour with 40 mmol/L HEPES (pH 8.0) containing 0.5 mmol/L EGTA, 10 mmol/L MgCl2, 2 µmol/L protein kinase inhibitor peptide (rabbit sequence, Sigma P0300), 40 µmol/L ATP, and 2.5 µCi/mL of [{gamma}-32P]ATP (6000 Ci/mmol). After incubation, the gel was washed with a 5% (wt/vol) trichloroacetic acid solution containing 1% (wt/vol) sodium pyrophosphate until the radioactivity of the solution became negligible. The gel was dried and then subjected to autoradiography.

RSK Assay
RSK activity was measured by an immune complex kinase assay using S6 peptide (RRLSSLRA) as a substrate as previously described.28 33 The cell-free lysates were prepared as those for MAP kinase assay except that buffer B was used. Buffer B contained 10 mmol/L KPO4 (pH 7.4), 1 mmol/L EDTA, 5 mmol/L EGTA, 10 mmol/L MgCl2, 50 mmol/L ß-glycerophosphate, 1 mmol/L sodium orthovanadate, 2 mmol/L dithiothreitol, 40 µg/mL PMSF, 10 nmol/L okadaic acid, 0.8 µg/mL leupeptin, 10 mg/mL p-nitrophenylphosphate, and 10 µg/mL aprotinin. The lysate from one 60-mm dish (3x106 cells) contained 490±23 µg (mean of four randomly picked samples) of total protein. The lysates containing equal amounts of protein (300 µg) were diluted with 10 vol of RIPA buffer and were incubated with 2 µL of {alpha}RSK28 for 2 hours at 4°C. Pansorbin was then added, and the immunoprecipitates were washed with buffer B without p-nitrophenylphosphate. Ten microliters of immunoprecipitate was incubated with 12.5 µL of 2x kinase buffer containing 50 mmol/L MOPS (pH 7.2), 120 mmol/L ß-glycerophosphate, 60 mmol/L p-nitrophenylphosphate, 10 mmol/L EGTA, 30 mmol/L MgCl2, 2 mmol/L dithiothreitol, 2 mmol/L sodium orthovanadate, 2 µmol/L protein kinase inhibitor (rabbit sequence), 12.5 µCi (6.25 µL) of [{gamma}-32P]ATP (10 Ci/mmol), and 1.25 µL of 5 mmol/L S6 peptide at 30°C for 15 minutes. To terminate the reaction, samples were spotted onto Whatman P81 phosphocellulose paper (2.5 cm) and washed five times (5 minutes each) with 180 mmol/L phosphoric acid and once with 95% ethanol. The papers were then placed into scintillation vials, and the radioactivity was counted.

Measurement of [Ca2+]i
Cardiac myocytes were grown on coverslips in serum-free medium. The cells were loaded with indo 1 by incubation with 10 µmol/L of acetoxymethyl ester indo 1 for 30 minutes at 37°C as described previously.34 After three washes with serum-free culture medium, the coverslips were incubated at 37°C for 1 hour and then mounted on the stage of a fluorescence microscope. Ca2+ monitoring was performed as described previously.34 The excitation wavelength was 360 nm. Fluorescence signals at 405- and 480-nm emissions from single cells were monitored continuously. Free [Ca2+]i was calculated by the ratio method (405/480 nm), with correction for background fluorescence as described previously.34

Statistics
Data are given as mean±SEM. Statistical analysis was performed by using ANOVA or unpaired t test as appropriate. Significance was accepted at P<.05.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Ang II Causes Tyrosine Phosphorylation in Cardiac Myocytes and Nonmyocytes
We examined whether Ang II induces protein tyrosine phosphorylation in neonatal rat cardiac myocytes by immunoblot analysis using 4G10.26 Ang II (100 nmol/L) caused an increase in phosphotyrosine content of several proteins with apparent molecular sizes of {approx}42-, 75- to 80-, and 120- to 130-kD in cardiac myocytes (Fig 1ADown, left). Ponceau S staining of the filter confirmed that almost equal amounts of protein were loaded in each lane (Fig 1ADown, right). These signals are specific to phosphotyrosyl residues, because preabsorption of the antibody with 50 mmol/L phosphotyrosine abolished the signals (Fig 1BDown; see figure legend). The bands at 75- to 80- and 120- to 130-kD are broad and may consist of multiple proteins. A similar pattern of tyrosine phosphorylation was observed when RC20H27 or a polyclonal anti-phosphotyrosine antibody35 was used (data not shown). The tyrosine phosphorylation of p120 to p130 was observed even in the control state, and the level increased after Ang II stimulation, having a peak at {approx}1 minute. On the other hand, p42 and p75 to p80 have low or no baseline tyrosine phosphorylation, and their levels reached a peak after 5 minutes. Although less evident in Fig 1ADown, an increase in tyrosine phosphorylation of p44 by Ang II was clearly observed in multiple experiments (for example see Figs 1BDown and 1CDown, 3B, and 4A). When detergents were included in a lysis buffer (modified buffer A), tyrosine phosphorylation of p90 was observed in the control state, and Ang II caused a decrease in the phosphotyrosine content of p90 (eg, see Fig 3BDown).



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  		Figure 1. Angiotensin II and serum increase protein tyrosine phosphorylation in cardiac myocytes and nonmyocytes. A, Cardiac myocytes were treated with angiotensin II (Ang II, 100 nmol/L) for the times (minutes) indicated, and cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on an 8% gel. On the left, tyrosine phosphorylation was assessed by immunoblotting with an anti-phosphotyrosine antibody (4G10). Proteins with increased tyrosine phosphorylation are indicated by arrows (n=17 experiments). On the right, the same filter was stained with Ponceau S solution after the autoradiograph was taken. B, Cardiac myocytes were treated with or without Ang II (100 nmol/L) for 5 minutes. Duplicate samples were electrophoresed on the same gel. After transfer, a filter was cut in half so that each filter had an identical set of samples. One filter was immunoblotted with an untreated anti-phosphotyrosine antibody (4G10, unblocked), and the other was immunoblotted with an anti-phosphotyrosine antibody that had been incubated with 50 mmol/L phosphotyrosine at 4°C for 2 hours (blocked). All other procedures were performed in an identical manner, except that two different exposures were taken for autoradiograms for the unblocked antibody. On the left and center, proteins with increased tyrosine phosphorylation are indicated by arrows. In these blots, the increase in phosphotyrosine content of p120 to p130 was less evident. On the right, faint bands were observed around 25, 30, 40, 48, 55, and 70 kD, but none of them comigrated with those detected with the unblocked anti-phosphotyrosine antibody. (Note that left and right blots are from the same gel.) These nonspecific bands were broad and much weaker than those observed with the unblocked antibody (n=2 experiments). C, Cardiac myocytes (left) or cardiac nonmyocytes (fibroblasts, right) were treated with Ang II (100 nmol/L) or fetal calf serum (FCS, 20%) for 5 minutes. Tyrosine phosphorylation was assessed by immunoblotting with anti-phosphotyrosine antibody (4G10) after SDS-PAGE on a 10% gel. Short exposures (6 hours) and long exposures (24 hours for myocytes and 30 hours for nonmyocytes) are shown for each autoradiograph (n=4 experiments [myocytes] and n=4 experiments [nonmyocytes]). Proteins with increased tyrosine phosphorylation are indicated by arrows. In panels A and C, cell lysates were prepared by using buffer A; in panel B, modified buffer A was used. Cell lysates containing equal amounts of protein (200 µg) were loaded in each lane.



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  		Figure 3. The roles of AT1 receptors, G proteins, Ca2+, and protein kinase C in angiotensin II (Ang II)–induced tyrosine phosphorylation in cardiac myocytes. A, Myocytes were pretreated with or without losartan (10 µmol/L) or PD123319 (10 µmol/L) for 30 minutes and then treated with Ang II (100 nmol/L) for the times indicated (n=5 experiments). B, Cardiac myocytes were pretreated with or without pertussis toxin (PTX, 100 ng/mL) for 24 hours and were then treated with or without Ang II (100 nmol/LM) for 5 minutes (n=3 experiments). C, On the left, cardiac myocytes were treated with or without phorbol 12-myristate 13-acetate (PMA, 1 µmol/L) for 5 minutes (n=4 experiments). On the right, cardiac myocytes were treated with or without A23187 (30 µmol/L) for 5 minutes (n=3 experiments). In panels A through C, cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on an 8% or 10% gel. Cell lysates were prepared using buffer A, except in panel B, where modified buffer A containing detergents was used. Cell lysates containing equal amounts of protein (200 µg) were loaded in each lane. Tyrosine phosphorylation was assessed by immunoblotting with anti-phosphotyrosine antibody (4G10). Proteins with an increase in tyrosine phosphorylation after the drug treatment are indicated by arrows. In panel B, p90, which seems to be tyrosine dephosphorylated by Ang II, is indicated by white arrowhead. Although the gel pictures have been cut in panel C, each lane in the same panel was from the same gel and the same exposure of the autoradiogram.

We and others have previously shown that Ang II increases both DNA synthesis and cell number (hyperplasia) in cardiac nonmyocytes, the majority of which are cardiac fibroblasts.12 21 In nonmyocyte culture, a treatment with Ang II (100 nmol/L) caused an increase in the phosphotyrosine content of several proteins similar to those observed in myocytes culture, including p75 to p80 and p120 to p130. In a longer exposure of the autoradiogram, tyrosine phosphorylation of p42 and p44 was also observed (Fig 1CUp, right).

A similar pattern of tyrosine phosphorylation (p42, p44, p75 to p80, and p120 to p130) was observed when cardiac myocytes or fibroblasts were stimulated with 20% fetal calf serum (FCS, Fig 1CUp), except that in FCS-stimulated cardiac fibroblasts tyrosine phosphorylation of {approx}p170 was also observed (Fig 1CUp, right).

Ang II–Induced Increase in Tyrosine Phosphorylation Is Accompanied by an Increase in Tyrosine Kinase Activity
Increased tyrosine phosphorylation can be caused by activation of tyrosine kinases or by suppression of tyrosine phosphatases. To confirm that tyrosine kinases are activated by Ang II, tyrosine kinase activity in the cell lysates from cardiac myocytes was measured by using an acid-insoluble synthetic tyrosine kinase substrate, EY(4:1).31 Because most, if not all, tyrosine kinases are autophosphorylated when they are activated,1 the kinase assay was performed after immunoprecipitation of cell lysates with 4G10. This removes high background activities of serine/threonine kinases toward endogenous substrates.31 As shown in Fig 2Down, Ang II (100 nmol/L) treatment significantly increased the incorporation of 32P into EY(4:1) within 1 minute (open circles). This increase in 32P incorporation was specific to tyrosine-phosphorylated protein, because no significant increase in 32P incorporation was observed when 4G10 was preabsorbed with 50 mmol/L phosphotyrosine (filled circle). This suggests that the Ang II–induced increase in phosphotyrosine content in cardiac myocytes is due, at least in part, to an Ang II–induced increase in tyrosine kinase activity.



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  		Figure 2. Time course of angiotensin II–induced activation of tyrosine kinase activity. Cardiac myocytes were stimulated with angiotensin II (100 nmol/L) for the times indicated. Tyrosine kinase activity was determined by the anti-phosphotyrosine immune complex kinase assay by using a polymer of glutamate and tyrosine (4:1 ratio) as a substrate. The results are expressed as stimulated tyrosine kinase activity divided by unstimulated activity and are mean±SEM from three experiments. Filled circle represents the tyrosine kinase activity of immunoprecipitates with preabsorbed (blocked) anti-phosphotyrosine antibody (see the legend of Fig 1BUp). *P<.01 and **P<.001 vs unstimulated control; {dagger}P<.001 vs the immunoprecipitate with preabsorbed antibody (filled circle).

Ang II–Induced Tyrosine Phosphorylation Is AT1 Receptor–Mediated and Mimicked by Ca2+ and Protein Kinase C Activation
We next examined which Ang II receptor subtype (AT1 or AT2) mediates protein tyrosine phosphorylation. The Ang II–induced increase in tyrosine phosphorylation of p42 and p120 to p130 was inhibited by the AT1 receptor antagonist losartan (10 µmol/L) (Fig 3AUp). Losartan also suppressed Ang II–induced increase in tyrosine phosphorylation of p75 to p80, although losartan slightly increased the baseline tyrosine phosphorylation (Fig 3AUp). In contrast, the AT2 receptor antagonist PD123319 (10 µmol/L) did not suppress Ang II–induced tyrosine phosphorylation of p42 and p75 to p80 (Fig 3AUp). The effect of PD123319 on p120 to p130 was less clear, because PD123319 itself also increased the basal level of tyrosine phosphorylation of p120 to p130 (Fig 3AUp). This result suggests that Ang II–induced tyrosine phosphorylation of p42 and p75 to p80 is mediated predominantly by the AT1 receptor subtype. In experiments using a detergent-containing lysis buffer (modified buffer A), Ang II caused a decrease in phosphotyrosine content of p90 (Fig 3BUp, open triangle). This Ang II–induced dephosphorylation of p90 was also inhibited by losartan but not by PD123319, suggesting that it is also AT1 receptor–mediated (data not shown).

We next examined how stimulation of the AT1 receptor by Ang II leads to an increase in phosphotyrosine content in cardiac myocytes. Ang II–induced tyrosine phosphorylation was not inhibited but augmented (Fig 3BUp) by pretreating cardiac myocytes with pertussis toxin (100 ng/mL) for 24 hours, which has been shown to fully ADP-ribosylate the Gi{alpha} subunit in neonatal rat ventricular cardiac myocytes.36 This suggests that pertussis toxin–sensitive G protein may not be involved in the Ang II–induced tyrosine phosphorylation. We have previously shown that Ang II activates multiple phospholipases, including phospholipase C (PLC), through an AT1 receptor–mediated mechanism in cardiac myocytes.37 Activation of PLC leads to production of inositol trisphosphate (IP3) and diacylglycerol through hydrolysis of phosphatidylinositol 4,5-disphosphate. These second messengers are known to cause release of Ca2+ from intracellular stores and activation of protein kinase C (PKC).38 39 A 5-minute treatment of cardiac myocytes with PMA (1 µmol/L), a direct activator of PKC, or A23187 (30 µmol/L), a Ca2+ ionophore, caused an increase in phosphotyrosine content of proteins that have molecular sizes similar to those observed with Ang II stimulation, such as p42, p44, p75 to p80, and p120 to p130 (Fig 3CUp). These data suggest that activation of each component of the PLC-derived second-messenger system seems to be sufficient to cause an increase in phosphotyrosine content of proteins that are tyrosine-phosphorylated in response to Ang II stimulation.

p42 and p44 Are Immunologically Related to ERK-1
Among several tyrosine-phosphorylated proteins, p42 and p44 may correspond to MAP kinases.4 5 40 An immunoblot of cell lysates from cardiac myocytes with {alpha}-cMAPK, directed against the rat 44-kD form of MAP kinase (also called ERK-1),10 showed the existence of two proteins immunologically related to ERK-1 (Fig 4ADown, right). Comparison of phosphotyrosine and MAP kinase blots of the same cell lysates electrophoresed on the same SDS-PAGE gel shows that the proteins recognized by {alpha}-cMAPK comigrate with tyrosine-phosphorylated p42 and p44 (Fig 4ADown). The ERK-1–related proteins were observed in the control state. However, after stimulation with Ang II, parts of p42 and p44 bands shifted slightly toward slower mobility (Fig 4ADown, right), in accordance with the appearance of tyrosine-phosphorylated proteins (Fig 4ADown, left). Because phosphorylated forms of MAP kinase are known to migrate more slowly in SDS–polyacrylamide gel than the unphosphorylated form,10 these results are consistent with the notion that Ang II induces tyrosine phosphorylation of ERK-1–related 42- and 44-kD MAP kinases in cardiac myocytes.



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  		Figure 4. Angiotensin II–induced tyrosine phosphorylation of mitogen-activated protein (MAP) kinases. A, Cardiac myocytes were treated with angiotensin II (100 nmol/L) for the times (minutes) indicated. Duplicate cell lysates electrophoresed on the same gel were immunoblotted with anti-phosphotyrosine antibody (anti-Ptyr [4G10], left) and anti–MAP kinase antibody (anti-MAPK, right). Tyrosine-phosphorylated proteins are indicated by arrows. Note tyrosine-phosphorylated 42- and 44-kD proteins comigrated with 42- and 44-kD proteins detected by anti-MAPK. Note also that angiotensin II induced a partial shift of the p42 and p44 bands to slower mobility, suggesting that these proteins were phosphorylated after treatment with angiotensin II (n=3 experiments). B, Cardiac myocytes were treated with angiotensin II (100 nmol/L) for the times indicated. MAP kinases were immunoprecipitated (IP) with anti-MAPK ({alpha}-cMAPK) and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on an 8% gel. After the transfer, the filter was probed with anti-Ptyr antibody (4G10). + and - below the autoradiogram indicate presence and absence, respectively, of {alpha}-cMAPK in the immunoprecipitation procedure. In lane 4, {alpha}-cMAPK was omitted, and only protein A–Sepharose was added in the immunoprecipitation procedure (n=2 experiments).

To further confirm that both 42- and 44-kD MAP kinases are tyrosine-phosphorylated after Ang II stimulation, MAP kinases were immunoprecipitated by {alpha}-MAPK, and anti-phosphotyrosine immunoblotting was performed. As shown in Fig 4BUp, in cardiac myocytes both 42- and 44-kD MAP kinases were tyrosine-phosphorylated after stimulation with Ang II.

Ang II Activates MAP Kinase
To examine whether MAP kinases are activated by Ang II, we next measured MAP kinase activity. To estimate the molecular sizes of the MAP kinases at the same time, the "in-gel MAP kinase assay" was performed by using an SDS–polyacrylamide gel containing MBP.32 As shown at the left in Fig 5Down, a 5-minute treatment with Ang II (100 nmol/L) increased MAP kinase activity of proteins at 42- and 44-kD in cardiac myocytes, although the kinase activity of p42 seems to have a higher background level than that of p44 in this assay condition. An additional band was observed at {approx}62 kD, and intensity of this band increased by treatment with Ang II. Boulton et al40 have reported that neonatal rat hearts express mRNA of ERK-3, a member of the ERK family that has a predicted molecular size of 62.6 kD. It is possible that the band at {approx}62 kD may correspond to ERK-3. FCS (20%) also increased MAP kinase activity of p42, p44, and p62 (Fig 5Down, left). In cardiac nonmyocytes (fibroblasts), Ang II (100 nmol/L) and FCS (20%) increased p42 MAP kinase activity (Fig 5Down, right). Interestingly, activation of p44 MAP kinase was much less pronounced, and p62 MAP kinase activity was not detectable in nonmyocytes.



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  		Figure 5. Angiotensin II (Ang II)–induced activation of mitogen-activated protein (MAP) kinases in cardiac myocytes and nonmyocytes. Cardiac myocytes (left) or nonmyocytes (fibroblasts, right) were treated with Ang II (100 nmol/L) or fetal calf serum (FCS, 20%) for 5 minutes. MAP kinase activity was assessed by the in-gel myelin basic protein kinase assay without prior immunoprecipitation with MAP kinase antibody (direct in-gel kinase assay). The autoradiograms of myocyte and nonmyocyte samples were exposed for the same duration (n=5 experiments for cardiac myocytes; n=3 experiments for nonmyocytes).

To confirm the identity of the 42- and 44-kD kinase activity, the in-gel MAP kinase assay was performed after immunoprecipitation with MAP kinase antibodies. In immunoprecipitates with the anti–ERK-1 antibody ({alpha}-MAPK), a significant increase in kinase activity at 42 and 44 kD was observed after a 5-minute treatment of cardiac myocytes with Ang II (Fig 6ADown). Immunoprecipitates with nonimmune serum had no kinase activity to MBP, indicating that kinase activity in the MAP kinase immunoprecipitates was in fact due to MAP kinase (Fig 6ADown, lane 6). The 42- and 44-kD forms of MAP kinase activities were also specifically immunoprecipitated by other polyclonal antibodies, anti–ERK-2 (C-14) and anti–ERK-1 (K-23), respectively (Fig 6BDown). These results suggest that Ang II activates MAP kinase activity of p42erk2 and p44erk1, members of the ERK family.40 The Ang II–induced increase in MAP kinase activity was observed at 100 pmol/L in p44erk1 and at 1 nmol/L in p42erk2 and reached a peak around 100 nmol/L in both cases (Fig 6BDown). Ang II–induced activation of 42- and 44-kD MAP kinases are inhibited by losartan but not by PD 123319, indicating that Ang II–induced MAP kinase activation is mediated predominantly by the AT1 receptor (Fig 6CDown). A 5-minute treatment with PMA (1 µmol/L) or A23187 (30 µmol/L) also caused a significant increase in MAP kinase activity at 42 and 44 kD, suggesting that both PKC- and Ca2+-dependent stimuli can activate 42- and 44-kD MAP kinases in cardiac myocytes (Fig 6DDown).



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  		Figure 6. Activation of 42- and 44-kD mitogen-activated protein (MAP) kinases in cardiac myocytes as assessed by the immune complex in-gel MAP kinase assay. A, Cardiac myocytes were treated with angiotensin II (100 nmol/L) for the times indicated. MAP kinases were immunoprecipitated with anti-MAP kinase antibody ({alpha}-cMAPK) and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 10% gel containing myelin basic protein. The gel was then subjected to the kinase reaction (in-gel MAP kinase assay). In lane 6, nonimmune rabbit serum was used instead of {alpha}-cMAPK in the immunoprecipitation (IP). The molecular masses of the bands correspond to 42 and 44 kD (n=3). B, Myocytes were treated with various concentrations of angiotensin II for 5 minutes, and the immune complex in-gel MAP kinase assay was performed. The concentrations of angiotensin II used were expressed as logarithmic scales. c indicates control; ERK, extracellular signal–related kinase-1. In these experiments, anti-ERK 1 (K-23, left) and anti-ERK 2 (C-14, right) were used as anti-MAP kinase antibodies in the IP. In lanes 7 and 14, {alpha}-cMAPKs preabsorbed with excess antigen peptides (Santa Cruz Biotechnology) were used (n=2). C, Myocytes were pretreated with or without losartan (10 µmol/L) or PD123319 (10 µmol/L) for 30 minutes and then treated with or without angiotensin II (100 nmol/L) for 5 minutes. MAP kinases were immunoprecipitated with {alpha}-cMAPK, and the in-gel MAP kinase assay was performed. In lane 7, nonimmune rabbit serum was used instead of {alpha}-cMAPK in the immunoprecipitation (n=3 experiments). D, Cardiac myocytes were treated with angiotensin II (Ang II, 100 nmol/L), phorbol 12-myristate 13-acetate (PMA, 1 µmol/L), or A23187 (30 µmol/L) for 5 minutes. MAP kinase activity was assessed by the in-gel myelin basic protein kinase assay after immunoprecipitation with {alpha}-cMAPK. Immunoprecipitates with nonimmune serum did not show an increase in the 42- and 44-kD bands (not shown). Although the gel picture has been cut, each lane was from the same gel and the same exposure of the autoradiogram (n=3 experiments).

Fig 7ADown shows time courses of Ang II (100 nmol/L)–induced activation of 42- and 44-kD MAP kinases in cardiac myocytes. Both MAP kinases were activated within 1 minute with treatment with Ang II. Their activity reached a peak at {approx}5 minutes and then decreased to below the control level within 30 minutes. It is interesting to note that the activity at 30 minutes after Ang II stimulation is consistently lower than that before Ang II stimulation (p42, 44±9%, P<.05, n=3; p44, 57±6%, P<.05, n=3; percent MAP kinase activity at 30 minutes compared with that at 0 minute). Panels B and C of Fig 7Down show the time course of PMA- and A23187- induced MAP kinase activation in cardiac myocytes, respectively. PMA (1 µmol/L) rapidly activated MAP kinases, and their activation persisted for >45 minutes. A23187 (30 µmol/L) also rapidly activated p42 and p44 MAP kinases, but their activity returned to the control level in {approx}30 minutes.



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  		Figure 7. Time courses of mitogen-activated protein (MAP) kinase (p42 and p44) activation. Cardiac myocytes were treated with angiotensin II (A), phorbol 12-myristate 13-acetate (PMA, B) or A23187 (C) for the times indicated. MAP kinase activity was assessed by the immune complex in-gel MAP kinase assay using an anti-MAP kinase antibody. Radioactivity of the phosphorylated bands at 42 and 44 kD was counted, and the maximum activity during the time course (at 5 or 10 minutes) is designated as 1.0. Note that the maximum induction in A23187 was lower than that in angiotensin II or PMA, resulting in an apparently higher baseline value in the A23187 experiment. Similar results were obtained from two additional experiments. pp42MAPK indicates 42-kD MAP kinase (circles); pp44MAPK, 44-kD MAP kinase (triangles).

Ang II Activates RSK
The 40S ribosomal protein (S6) is phosphorylated in response to mitogens. S6 phosphorylation has been correlated with growth and increased protein synthesis.9 The S6 kinase consists of at least two distinct families, the 70- to 85-kD S6 kinase (pp70S6K) and 90-kD S6 kinase (RSK). Although the upstream kinase capable of activating the pp70S6K has not been identified, it has been demonstrated that the RSK is activated by p42 and p44 MAP kinases in vitro.5 9 To examine whether Ang II activates RSK, we performed an immune complex RSK assay using an S6 peptide (RRLSSLRA) as a substrate.10 33 As shown in Fig 8Down (circles), Ang II (100 nmol/L) activated RSK in cardiac myocytes. The time course of activation of the RSK followed that of p42 and p44 MAP kinases, being activated within 1 minute, and it reached a peak at {approx}10 minutes, but it decreased much more slowly than that of the MAP kinases. Phosphorylation of the S6 peptide in this assay condition was specific to RSK, because no significant increase in S6 peptide phosphorylation was observed when anti-RSK antibody was preabsorbed with an excess amount of antigen peptide (Fig 8Down, triangles). This Ang II–induced S6 peptide phosphorylation was inhibited by losartan (10 µmol/L) but not by PD123319 (10 µmol/L), suggesting that RSK activation is AT1 receptor-mediated (data not shown). A 10-minute treatment of cardiac myocytes with FCS (20%), PMA (1 µmol/L), or A23187 (30 µmol/L) also caused a significant increase in RSK activity (TableDown). The increase in RSK activity was also observed when cardiac nonmyocytes (fibroblasts) were stimulated with Ang II (100 nmol/L) or FCS (20%) (TableDown).



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  		Figure 8. Angiotensin II–induced activation of 90-kD S6 kinase (RSK). The time course of the angiotensin II–induced activation of RSK is shown. Cardiac myocytes were treated with angiotensin II (100 nmol/L) for the times indicated. Immune complex RSK assay was performed using S6 peptide (RRLSSLRA) as a substrate. Phosphorylated S6 peptide was adsorbed onto Whatman P81 phosphocellulose paper and counted for radioactivity after washing. The triangles represent the S6 peptide kinase activity of immunoprecipitates with anti-RSK antibody, which was preabsorbed with excess antigen peptide. Data are the mean of three independent experiments. The activity at time zero is designated as 1.0. In immune complex kinase reactions without S6 peptide, mean background counts on the phosphocellulose paper were 0.08±0.02 (n=3 experiments) after 5-minute stimulation with angiotensin II.


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  		Table 1. Activation of 90-kD S6 Kinase by Various Stimuli in Cardiac Myocytes and Nonmyocytes (Fibroblasts)

Roles of PKC in Ang II–Induced Tyrosine Phosphorylation, MAP Kinase, and RSK Activation in Cardiac Myocytes
The precise mechanism of how agonists for G protein–coupled receptors activate the protein tyrosine kinase cascade has not been fully elucidated. The data presented so far indicate that activation of PKC or an increase in intracellular Ca2+ is sufficient to cause an increase in tyrosine phosphorylation and activation of MAP kinases and RSK. Therefore, we examined whether PKC is essential for Ang II–induced activation of these protein kinases in cardiac myocytes. We have previously shown that a 48-hour treatment of cardiac myocytes with PMA (2 µmol/L) causes complete functional downregulation of PKC activity, as assessed by PKC assay using a synthetic peptide as a substrate.37 We first examined the effect of a 48-hour treatment with PMA on Ang II–induced tyrosine phosphorylation. As shown in Fig 9ADown, prolonged pretreatment with PMA (2 µmol/L) alone increased the basal level of tyrosine phosphorylation of p75 to p80 and p120 to p130, but it did not block the Ang II–induced increase in protein tyrosine phosphorylation, including p42 and p44 (arrows). The immune complex in-gel MAP kinase assay showed that the 48-hour pretreatment with PMA prevented both 42- and 44-kD MAP kinase activation by a subsequent addition of the direct PKC activators, phorbol 12,13-dibutyrate (2 µmol/L) or PMA (2 µmol/L) (see Fig 9BDown, upper panel, and 9C), confirming that at least the phorbol ester–sensitive component of PKC has been completely downregulated in this experiment. However, under this condition, Ang II–induced activation of 42- and 44-kD MAP kinases was not inhibited (Fig 9BDown and 9CDown). General protein kinase inhibitors that are reported to be relatively specific to PKC, such as H-7 (50 µmol/L), calphostin C (1 µmol/L), and chelerythrine (10 µmol/L),41 42 43 did not block Ang II–induced MAP kinase activation (data not shown), which is consistent with the results of the PMA pretreatment experiments. Similarly, pretreatment with PMA did not affect FCS (20%)–induced MAP kinase activation (Fig 9BDown). A 48-hour pretreatment of myocytes with PMA slightly increased the baseline RSK activity but did not block subsequent Ang II–induced RSK activation (Fig 9DDown). These results suggest that Ang II seems to use PKC-independent pathways to induce tyrosine phosphorylation and activate MAP kinases and RSK. However, some isoforms of PKC, the activities of which cannot be detected in our PKC assay, may not have been downregulated. For example, it has been shown that the {xi} form of PKC is resistant to downregulation by the prolonged treatment with PMA in neonatal rat cardiac myocytes.19 Therefore, we cannot exclude the possibility that a PMA-insensitive component of PKC may play a role in Ang II–induced activation of MAP kinases.



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  		Figure 9. Roles of protein kinase C (PKC) and Ca2+ in angiotensin II (Ang II)–induced tyrosine phosphorylation and activation of mitogen-activated protein (MAP) kinases and 90-kD S6 kinase (RSK) in cardiac myocytes. A, Effects of PKC downregulation and intracellular Ca2+ chelation on Ang II–induced tyrosine phosphorylation. Downregulation of PKC was performed by treating cardiac myocytes with 2 µmol/L of phorbol 12-myristate 13-acetate (PMA) for 48 hours (Pre PMA). Intracellular Ca2+ was chelated by treating cardiac myocytes with 10 µmol/L BAPTA-AM (BAPTA) for 30 minutes. Cardiac myocytes were then treated with or without Ang II (100 nmol/L) for 5 minutes. Tyrosine phosphorylation was assessed by immunoblotting with anti-phosphotyrosine antibody (4G10) after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Each lane was from the same gel and exposed for the same duration. Tyrosine-phosphorylated 42- and 44-kD proteins are indicated by arrows. Note that the Ang II–induced increase in tyrosine phosphorylation of p42, p44, p75 to p80, and p120 to p130 was completely inhibited by BAPTA but not by a prolonged pretreatment with PMA (n=5 experiments). B, Effect of prolonged treatment with PMA and BAPTA on MAP kinase activation induced by Ang II, fetal calf serum (FCS), and PMA. Kinase activity of 42- and 44-kD MAP kinases was measured by the immune complex in-gel kinase assay. Pre PMA and/or BAPTA was administered as in panel A. Ang II (100 nmol/L), FCS (20%), phorbol 12,13-dibutyrate (2 µmol/L), or PMA (2 µmol/L) was then applied for 5 minutes. The intensity of both 42- and 44-kD bands corresponds to activity of 42- and 44-kD MAP kinases. Although pretreatment with PMA seemed to upregulate the subsequent Ang II–induced or FCS-induced MAP kinase activation in this example, this upregulation was not clear in other experiments (see bar graph in panel C). C, Bar graph showing the radioactivity of the phosphorylated bands at 44 kD obtained in the immune complex in-gel MAP kinase assay counted and expressed as a ratio of the experimental value to that obtained from control (without pretreatment), which was set as 1. Pre PMA or BAPTA was administered as in panel A. Subsequently, Ang II (100 nmol/L), PMA (2 µmol/L), or phorbol 12,13-dibutyrate (2 µmol/L) (not shown) was applied for 5 minutes. Results were from three to five independent experiments. *P<.01 vs control. Phosphorylations at 42 kD showed a tendency similar to those at p44 kD (not shown). D, Bar graph showing the effects of prolonged treatment with PMA or BAPTA on Ang II–induced RSK activation. Pre PMA or BAPTA was administered as in panel A. Ang II (100 nmol/L) was applied for 10 minutes. RSK activity was obtained by the immune complex RSK assay using S6 peptide as a substrate as in Fig 8Up. The activity observed without any drug (control) is designated as 1.0. Results are the mean of three independent experiments. *P<.01 vs respective control value.

Roles of Ca2+ in Ang II–Induced Tyrosine Phosphorylation, MAP Kinase, and RSK Activation in Cardiac Myocytes
It is known that Ang II not only increases Ca2+ influx through the L-type Ca2+ channel44 but also induces Ca2+ release from intracellular Ca2+ stores.45 46 As mentioned earlier, treatment of cardiac myocytes with A23187, a Ca2+ ionophore, induced an increase in protein tyrosine phosphorylation and activated 42- and 44-kD MAP kinases and RSK. To block an increase in [Ca2+]i, irrespective of its origin, cardiac myocytes were incubated with a membrane-permeable Ca2+-chelating compound, BAPTA-AM (10 µmol/L), for 30 minutes.47 To confirm that the concentration of BAPTA-AM used was sufficient to inhibit the Ang II–induced rise in [Ca2+]i, continuous measurements of [Ca2+]i by fluorescence microscopy were performed at the single-cell level. Fig 10ADown shows a typical time course of the Ang II (100 nmol/L)–induced change in [Ca2+]i. A 30-minute pretreatment with BAPTA-AM significantly suppressed the Ang II–induced increase in [Ca2+]i. Fig 10BDown shows absolute values of peak [Ca2+]i obtained by each treatment. BAPTA-AM (10 µmol/L) significantly lowered the resting level of [Ca2+]i and suppressed the Ang II–induced increase in [Ca2+]i to the control resting level. A 30-minute treatment of cardiac myocytes with BAPTA-AM (10 µmol/L) completely inhibited the Ang II–induced increase in tyrosine phosphorylation of multiple proteins, including p42 and p44 (Fig 9AUp, arrows). BAPTA-AM also markedly suppressed the Ang II–induced activation of 42- and 44-kD MAP kinases (Fig 9BUp and 9CUp). However, BAPTA-AM did not affect PMA-induced MAP kinase activation (Fig 9BUp, lower panel, and 9C), suggesting that BAPTA-AM, at the concentration used (10 µmol/L), did not affect the PKC-dependent pathway of MAP kinase activation. BAPTA-AM also significantly suppressed Ang II–induced RSK activation, as assessed by the immune complex RSK assay (Fig 9DUp). These results suggest that Ang II–induced protein tyrosine phosphorylation and activation of MAP kinases and RSK are critically dependent on [Ca2+]i.



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  		Figure 10. Inhibition of angiotensin II (Ang II)–induced Ca2+ mobilization by BAPTA-AM. A, Tracing showing [Ca2+]i in a single nonbeating cardiac myocyte as measured by microfluorometry. Ang II (100 nmol/L) was applied at the time point indicated by the arrow. In the lower tracing, cardiac myocytes were pretreated with BAPTA-AM (10 µmol/L) for 30 minutes before Ang II application. [Ca2+]i is reported as the ratio of indo 1 fluorescence at 405- and 480-nm emission. Autofluorescence produced by BAPTA-AM was subtracted. B, Bar graph showing calculated intracellular concentration attained by each treatment. Peak Ca2+ in each treatment in panel A was calculated by the ratio method, with correction for background fluorescence. The results shown represent mean±SEM from four experiments. (-) indicates the absence of Ang II. **P<.01 vs control without BAPTA-AM.

Other Gq Protein–Coupled Hypertrophic Stimuli Also Activate Tyrosine Kinase, MAP Kinase, and RSK in Cardiac Myocytes
Finally, we examined whether other hypertrophic stimuli acting through G protein–coupled receptors, such as Phe, NE, ET-1, and isoproterenol (ISO) activate tyrosine kinase, MAP kinases, and RSK in cardiac myocytes. As shown in Fig 11ADown, a 5-minute treatment of cardiac myocytes with NE (1 µmol/L), Phe (100 µmol/L), or ET-1 (50 nmol/L), agonists for Gq protein–coupled receptors, produced an increase in phosphotyrosine content of p42, p75 to p80, and p120 to p130. Interestingly, ISO (10 µmol/L), an agonist for Gs protein–coupled receptor, induced an increase in phosphotyrosine content of p55, p75 to p80, and p120 to p125, but it did not increase that of p42 and p130 (Fig 11ADown, arrows with asterisk). NE, Phe, and ET-1 also increased 42- and 44-kD forms of MAP kinase activity in cardiac myocytes, as assessed by the immune complex in-gel kinase assay (Fig 11BDown), in agreement with recent reports that Phe and ET-1 stimulate MAP kinase activity in cardiac myocytes.18 However, ISO (10 µmol/L) did not increase MAP kinase activity (Fig 11BDown), corresponding to the lack of tyrosine phosphorylation of p42. Similarly, a 10-minute treatment of cardiac myocytes with NE, Phe, or ET-1 caused a significant increase in RSK activity, as assessed by the immune complex RSK assay using S6 peptide as a substrate, but stimulation with ISO did not (TableUp).



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  		Figure 11. Effects of various hypertrophic stimuli on tyrosine phosphorylation (A) and mitogen-activated protein (MAP) kinase activity (B) in cardiac myocytes. Cardiac myocytes were treated with norepinephrine (1 µmol/L), phenylephrine (100 µmol/L), isoproterenol (10 µmol/L), endothelin-1 (50 nmol/L), or angiotensin II (100 nmol/L) for 5 minutes. A, Tyrosine phosphorylation was assessed by anti-phosphotyrosine immunoblotting after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins with increased phosphorylation are indicated by arrows. Although the gel picture has been cut in the left panel, each lane was from the same gel and the same exposure of the autoradiogram. In the shorter exposure, the thick band at p120 to p130 seems to consist of at least two bands. Asterisks indicate the proteins that were not tyrosine-phosphorylated by isoproterenol. The small circle indicates p55 that was tyrosine-phosphorylated more strongly by isoproterenol than by the others. Note tyrosine phosphorylation of p42 by hypertrophic stimuli, except by isoproterenol (n=3 experiments). B, MAP kinases were immunoprecipitated with anti-MAP kinase antibody. MAP kinase activity was assessed by the immune complex in-gel MAP kinase assay using myelin basic protein as a substrate. Molecular weights of the bands correspond to 42 and 44 kD (n=2 experiments).


*    Discussion
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We have demonstrated that Ang II and other hypertrophic stimuli known to act through Gq protein–coupled receptors cause tyrosine phosphorylation of several intracellular proteins and activate MAP kinases and RSK. Activation of these protein kinases was observed in both cardiac myocytes and nonmyocytes (fibroblasts), suggesting that these second-messenger systems may be used in both hypertrophic and mitogenic responses to Ang II. Interestingly, a Ca2+-dependent, rather than PKC-dependent, mechanism seems to be critical in Ang II–induced tyrosine phosphorylation and activation of MAP kinases and RSK in cardiac myocytes.

Because our cardiac myocyte culture contains 5% to 10% of contaminating nonmyocytes, which are primarily fibroblasts,12 29 one may argue that the Ang II–induced increase in tyrosine phosphorylation and activation of MAP kinases and RSK might have originated solely from the "contaminating" fibroblasts without contribution from myocytes. To address this question, we examined Ang II–induced activation of these protein kinases in both myocyte-rich culture and cardiac fibroblast culture. We performed experiments on myocytes and fibroblasts in identical conditions and observed comparable levels of activation of these second-messenger systems in both cell types. If the fibroblasts were the sole source of the activation of protein kinases, the contaminating fibroblasts (which are <10% of the total cells) in the myocyte culture could not have accounted for the levels of activation seen in the myocyte culture.

Our results also indicate that a downstream signaling molecule of PLC (either elevation of intracellular Ca2+ or activation of PKC) is sufficient to cause activation of MAP kinases and RSK in cardiac myocytes. However, among these two signaling pathways, the Ca2+-dependent pathway seems to play a more critical role in Ang II–induced activation of MAP kinases and RSK in cardiac myocytes. The following lines of evidence support this conclusion. First, downregulation of PKC by prolonged treatment with PMA had no effect on Ang II–induced activation of MAP kinases or RSK. In contrast, phorbol ester–induced MAP kinase activation was completely inhibited by PKC downregulation. Second, three different classes of protein kinase inhibitors reported to be (relatively) specific to PKC also failed to inhibit Ang II–induced activation of MAP kinases. Third, BAPTA-AM, at a concentration that completely suppressed the Ang II–induced increase in [Ca2+]i, fully suppressed activation of these protein kinases. Because treatment with BAPTA-AM did not completely inhibit Ang II–induced production of IP337 or phorbol ester–induced activation of MAP kinase (Fig 9BUp and 9CUp), the effect of BAPTA-AM was unlikely to be due to a direct inhibition of PLC, PKC, or MAP kinases. Fourth, the time course of Ang II–induced MAP kinase activation was more similar to an A23187-induced MAP kinase activation than a PMA-induced one. MAP kinase activation by Ang II was rapid and transient, and its activity returned to the levels lower than the control level within 30 minutes. The mechanism of this more rapid inactivation of MAP kinases observed in Ang II–treated or A23187-treated cells remains to be determined. Ca2+-dependent activation of phosphatase may be one of the mechanisms, as has been suggested by deactivation of epidermal growth factor–activated MAP kinase in human foreskin fibroblasts.48 It is interesting to note that Duff et al49 recently reported that Ang II rapidly upregulates mRNA of 3CH134, a protein tyrosine phosphatase that specifically dephosphorylates MAP kinase in vascular smooth muscle cells.

It has been shown that activation of MAP kinase by thapsigargin also requires Ca2+, and the Ca2+ released from internal stores appears to be sufficient for mediating the MAP kinase activation in human foreskin fibroblasts.48 At present, we do not know what step in Ang II–induced MAP kinase activation is critically dependent on [Ca2+]i. It is interesting to note that Huckle and coworkers31 50 have reported that Ang II–induced tyrosine kinase activation is Ca2+ dependent in WB and GN4 liver epithelial cells. This raises the possibility that there exists an intermediate tyrosine kinase(s) that plays an important role in Ang II–induced MAP kinase activation. Although our results suggest that Ca2+-dependent signaling is essential, we found that the magnitudes of tyrosine phosphorylation and activation of MAP kinases and RSK were higher in Ang II stimulation than in A23187 stimulation alone. In physiological conditions, both Ca2+ and PKC are likely to act synergistically for the Ang II–induced activation of MAP kinases and RSK in cardiac myocytes.

We have shown that MAP kinases are tyrosine-phosphorylated by Ang II treatment. However, we do not know the molecular identities of other proteins that are tyrosine-phosphorylated by hypertrophic stimuli. In the case of the platelet-derived growth factor and epidermal growth factor receptors, several cellular proteins have been identified as substrates of the receptor tyrosine kinases. These include phospholipase C{gamma} (molecular mass, {approx}148 kD), PI3 kinase (85 and 110 kD), and GTPase activating protein (120 kD).1 Recently, Molloy et al15 reported that p75 and p120, which are tyrosine-phosphorylated by Ang II in vascular smooth muscle, are different from these known tyrosine kinase substrates. We also observed tyrosine phosphorylation at {approx}75 to 80 kD and 120 to 130 kD in cardiac myocytes and fibroblasts. Further studies are necessary to identify whether either or both of these proteins are known substrates.

In the present experiments, we have shown that in cardiac myocytes both hypertrophic stimuli such as Ang II, NE, Phe, and ET-112 13 14 and mitogenic stimuli such as FCS51 seem to use a similar tyrosine kinase–MAP kinase cascade. This is reminiscent of PC12 cells, in which agonists promoting differentiation (nerve growth factor) and proliferation (epidermal growth factor) both activate the tyrosine kinase–MAP kinase cascade.52 This indicates that differences in the cellular response to stimuli that cause hypertrophy or hyperplasia may depend on differences in cellular processes downstream from these protein kinases. Alternatively, each specific form of receptor activation may stimulate other parallel signaling pathways that determine the specificity of the stimulation.

We have previously shown that Ang II–induced activation of various phospholipid-derived second-messenger systems is mediated by the AT1 receptor subtype.37 Our results indicate that tyrosine phosphorylation and activation of MAP kinases and RSK are also mediated by the AT1 receptor subtype. The AT2 receptor in rat adrenal glomerulosa and PC12W cells has recently been suggested to modulate tyrosine phosphatase activity.53 54 In our experiments, tyrosine phosphorylation of p90 was observed by using a detergent-containing lysis buffer (modified buffer A), and the tyrosine phosphorylation of p90 was decreased by the treatment with Ang II (Fig 3BUp). However, this decrease was not affected in the presence of PD123319, indicating that the AT2 receptor does not seem to play a major role in the regulation of phosphotyrosine content in cardiac myocytes.

We have previously shown that after prolonged treatment with PMA, Ang II–induced c-fos expression is significantly, if not completely, suppressed, indicating that the classical PKC pathway plays an essential role in Ang II–induced c-fos expression in cardiac myocytes.37 This indicates that Ang II activates c-fos and MAP kinases mainly through different signaling mechanisms. However, it should be noted that this Ang II–induced c-fos expression was not completely inhibited by the prolonged treatment with PMA.37 This suggests that there may be a PKC-independent mechanism in Ang II–induced c-fos expression, although we cannot exclude a contribution from a PMA-insensitive component of PKC.19 39 We have previously shown that a binding site for p62TCF in the c-fos promoter is necessary for the Ang II–induced c-fos expression in cardiac myocytes.37 Interestingly, a recent report showed that MAP kinase can phosphorylate the nuclear transcription factor p62TCF and regulate transcription of c-fos.11 Ang II rapidly activates MAP kinase, and our preliminary results using immunofluorescence staining suggest that Ang II causes translocation of MAP kinase into the nucleus of cardiac myocytes (J. Sadoshima and S. Izumo, unpublished data). Thus, MAP kinase may play a role in Ang II–induced immediate-early gene expression in cardiac myocytes, although further investigation is necessary to prove this hypothesis.

It is known that some agonists for G protein–coupled receptors, such as thrombin and lysophosphatidic acid, are capable of stimulating DNA synthesis in some cell types. However, activation of the PLC pathway is neither required nor sufficient for lysophosphatidic acid–induced and thrombin-induced DNA synthesis, and the importance of other downstream signaling mechanisms has been suggested.55 Some other agonists for G protein–coupled receptors (such as bombesin and serotonin) are capable of stimulating DNA synthesis only in the presence of other growth factors that activate tyrosine kinase (such as insulin and epidermal growth factor).55 Because Ang II alone induces a mitogenic response in cardiac fibroblasts, it would be interesting to examine whether tyrosine kinase activation is critical in Ang II–induced mitogenic response in these cells. Moreover, by analogy to mitogenic response, it would be interesting to examine whether tyrosine kinase activation is necessary for hypertrophic response of cardiac myocytes. Further studies are necessary to identify the role of tyrosine kinases and downstream signaling in the cardiac hypertrophic response caused by G protein–coupled receptors.


*    Acknowledgments
 
This study was supported in part by the Jacob D. Indursky Memorial Fund to Beth Israel Hospital, Boston. Dr Sadoshima is a Fellow of the Charles A. King Trust, Boston, Mass. Dr Izumo is an Established Investigator of the American Heart Association. We thank Dr J. Blenis for MAP kinase and RSK antibodies, Dr T. Roberts for anti-phosphotyrosine antibodies, and Dr T.J. Kulik for critical reading of the manuscript.

Received September 22, 1993; accepted September 19, 1994.


*    References
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1. Cantley LC, Auger KR, Carpenter C, Duckworth B, Graziani A, Kapeller R, Soltoff S. Oncogenes and signal transduction. Cell. 1991;64:281-302. [Medline] [Order article via Infotrieve]

2. Morgan HE, Baker KM. Cardiac hypertrophy. Circulation. 1991;83:13-25. [Free Full Text]

3. Parker TG, Schneider MD. Growth factors, proto-oncogenes, and plasticity of cardiac phenotype. Annu Rev Physiol. 1991;53:179-200. [Medline] [Order article via Infotrieve]

4. Cobb MH, Boulton TG, Robbins DJ. Extracellular signal-regulated kinases: ERKs in progress. Cell Regul. 1991;2:965-978. [Medline] [Order article via Infotrieve]

5. Blenis J. Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl Acad Sci U S A. 1993;90:5889-5892. [Abstract/Free Full Text]

6. Crews CM, Erikson RL. Extracellular signals and reversible protein phosphorylation: what to Mek of it all. Cell. 1993;74:215-217. [Medline] [Order article via Infotrieve]

7. Kyriakis JM, App H, Zhang X, Banerjee P, Brautigan DL, Rapp UR, Avruch J. Raf-1 activates MAP kinase-kinase. Nature. 1992;358:417-421. [Medline] [Order article via Infotrieve]

8. Lange-Carter CA, Pleiman CM, Gardner AM, Blumer KJ, Johnson GL. A divergence in the MAP kinase regulatory network defined by MEK kinase and raf. Science. 1993;260:315-319. [Abstract/Free Full Text]

9. Erikson RL. Structure, expression, and regulation of protein kinases involved in the phosphorylation of ribosomal protein S6. J Biol Chem. 1991;266:6007-6010. [Free Full Text]

10. Chen R-H, Sarnecki C, Blenis J. Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol Cell Biol. 1992;12:915-927. [Abstract/Free Full Text]

11. Gille H, Sharrocks AD, Shaw PE. Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c-fos promoter. Nature. 1992;358:414-417. [Medline] [Order article via Infotrieve]

12. Sadoshima J, Izumo S. Molecular characterization of angiotensin II-induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts: a critical role of the AT1 receptor subtype. Circ Res. 1993;73:413-423. [Abstract/Free Full Text]

13. Simpson PC. Norepinephrine-stimulated hypertrophy of cultured rat myocardial cells is an {alpha}1-adrenergic response. J Clin Invest. 1983;72:732-738.

14. Shubeita HE, McDonough PM, Harris AN, Knowlton KU, Glembotski CC, Brown JH, Chien KR. Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular hypertrophy. J Biol Chem.. 1990;265:20555-20562. [Abstract/Free Full Text]

15. Molloy CJ, Taylor DS, Weber H. Angiotensin II stimulation of rapid protein tyrosine phosphorylation and protein kinase activation in rat aortic smooth muscle cells. J Biol Chem. 1993; 268:7388-7345.

16. Tsuda T, Kawahara Y, Ishida Y, Koide M, Shii K, Yokoyama M. Angiotensin II stimulates two myelin basic protein/microtubule–associated protein 2 kinases in cultured vascular smooth muscle cells. Circ Res. 1992;71:620-630. [Abstract/Free Full Text]

17. Duff JL, Berk BC, Corson MA. Angiotensin II stimulates the pp44 and pp42 mitogen-activated protein kinases in cultured rat aortic smooth muscle cells. Biochem Biophys Res Commun. 1992;188:257-264. [Medline] [Order article via Infotrieve]

18. Bogoyevitch MA, Glennon PE, Sugden PH. Endothelin-1, phorbol esters and phenylephrine stimulate MAP kinase activities in ventricular cardiomyocytes. FEBS Lett. 1993;317:271-275. [Medline] [Order article via Infotrieve]

19. Bogoyevitch MA, Glennon PE, Andersson MB, Clerk A, Lazou A, Marshall CJ, Parker PJ, Sugden PH. Endothelin-1 and fibroblast growth factor stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes: the potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy. J Biol Chem. 1994;269:1110-1119. [Abstract/Free Full Text]

20. Baker KM, Booz GW, Dostal DE. Cardiac actions of angiotensin II: role of an intracardiac renin-angiotensin system. Annu Rev Physiol. 1992;54:227-241. [Medline] [Order article via Infotrieve]

21. Schorb W, Booz GW, Dostal DE, Conrad KM, Chang KC, Baker KM. Angiotensin II is mitogenic in neonatal rat cardiac fibroblasts. Circ Res. 1993;72:1245-1254. [Abstract/Free Full Text]

22. Sadoshima J, Xu Y, Slayter HS, Izumo S. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell. 1993;75:977-984. [Medline] [Order article via Infotrieve]

23. Inagami T, Iwai N, Sasaki K, Yamano Y, Bardhan S, Chaki S, Guo D-F, Furuta H. Cloning, expression, and regulation of angiotensin II receptors. In: Raizada MK, Phillips MI, Sumners C, eds. Cellular and Molecular Biology of the Renin-Angiotensin System. Ann Arbor, Mich: CRC Press; 1993:273-291.

24. Sadoshima J, Izumo S. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autorine/paracrine mechanism. EMBO J. 1993;12:1681-1692. [Medline] [Order article via Infotrieve]

25. Yamazaki T, Tobe K, Hoh E, Maemura K, Kaida T, Komuro I, Tanemoto H, Kadowaki T, Nagai R, Yazaki Y. Mechanical loading activates mitogen-activated protein kinase and S6 peptide kinase in cultured rat cardiac myocytes. J Biol Chem. 1993;268:12069-12076. [Abstract/Free Full Text]

26. Druker BJ, Mamon HJ, Roberts TM. Oncogenes, growth factors, and signal transduction. N Engl J Med. 1989;321:1383-1391. [Medline] [Order article via Infotrieve]

27. Ruff-Jamison S, Chen K, Cohen S. Induction by EGF and interferon-gamma of tyrosine phosphorylated DNA binding proteins in mouse liver nuclei. Science. 1993;261:1733-1736. [Abstract/Free Full Text]

28. Chen R-H, Blenis J. Identification of Xenopus S6 protein kinase homo- logs (pp90rsk) in somatic cells: phosphorylation and activation during initiation of cell proliferation. Mol Cell Biol. 1990;10:3204-3215. [Abstract/Free Full Text]

29. Sadoshima J, Jahn L, Takahashi T, Kulik TJ, Izumo S. Molecular characterization of the stretch-induced adaptation of cultured cardiac cells: an in vitro model of load-induced cardiac hypertrophy. J Biol Chem. 1992;267:10551-10560. [Abstract/Free Full Text]

30. Bader D, Masaki T, Fischman D. Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro. J Cell Biol. 1982;95:763-770. [Abstract/Free Full Text]

31. Huckle WR, Dy RC, Earp HS. Calcium-dependent increase in tyrosine kinase activity stimulated by angiotensin II. Proc Natl Acad Sci U S A. 1992;89:8837-8841. [Abstract/Free Full Text]

32. Gotoh Y, Nishida E, Yamashita T, Hoshi M, Kawakami M, Sakai H. Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells: identity with the mitogen-activated MAP kinase of fibroblastic cells. Eur J Biochem. 1990;193:661-669. [Medline] [Order article via Infotrieve]

33. Pelech SL, Krebs EG. Mitogen-activated S6 kinase is stimulated via protein kinase C-dependent and independent pathways in Swiss 3T3 cells. J Biol Chem. 1987;262:11598-11606. [Abstract/Free Full Text]

34. Qiu Z, Morgan JP. Differential effects of cocaine and cocaethylene on intracellular Ca2+ and myocardial contraction in cardiac myocytes. Br J Pharmacol. 1993;109:293-298. [Medline] [Order article via Infotrieve]

35. Kamps MP, Sefton BM. Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene. 1988;2:305-315. [Medline] [Order article via Infotrieve]

36. Allen IS, Gaa ST, Rogers TB. Changes in expression of a functional Gi protein in cultured rat heart cells. Am J Physiol. 1988;255(Cell Physiol 24):C51-C59.

37. Sadoshima J, Izumo S. Signal transduction pathways of angiotensin II–induced c-fos gene expression in cardiac myocytes in vitro: roles of phospholipid-derived second messengers. Circ Res. 1993;73:424-438. [Abstract/Free Full Text]

38. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature. 1993;361:315-325. [Medline] [Order article via Infotrieve]

39. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992;258:607-614. [Abstract/Free Full Text]

40. Boulton TG, Nye SH, Robbins DJ, Ip NY, Radziejewska E, Morgenbesser SD, DePinho RA, Panayotatos N, Cobb MH, Yancopoulos GD. ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell. 1991;65:663-675. [Medline] [Order article via Infotrieve]

41. Hidaka H, Inagaki M, Kawamoto S, Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 1984;23:5036-5041. [Medline] [Order article via Infotrieve]

42. Kobayashi E, Nakano H, Morimoto M, Tamaoki T. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun. 1989;159:548-553. [Medline] [Order article via Infotrieve]

43. Herbert JM, Augereau JM, Gleye J, Maffrand JP. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun. 1990;172:993-999. [Medline] [Order article via Infotrieve]

44. Allen IS, Cohen NM, Dhallan RS, Gaa ST, Lederer WJ, Rogers TB. Angiotensin II increases spontaneous contractile frequency and stimulates calcium current in cultured neonatal rat heart myocytes: insights into the underlying biochemical mechanism. Circ Res. 1988;62:524-534. [Abstract/Free Full Text]

45. Baker KM, Singer HA, Aceto JF. Angiotensin II receptor-mediated stimulation of cytosolic-free calcium and inositol phosphates in chick myocytes. J Pharmacol Exp Ther. 1989;251:578-585. [Abstract/Free Full Text]

46. Kem DC, Johnson EIM, Capponi AM, Chardonnens D, Lang U, Blondel B, Koshida H, Vallotton MB. Effect of angiotensin II on cytosolic free calcium in neonatal rat cardiomyocytes. Am J Physiol. 1991;261:C77-C85. [Abstract/Free Full Text]

47. Tsien RY. New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry. 1980;19:2396-2404. [Medline] [Order article via Infotrieve]

48. Chao T-SO, Byron KL, Lee K-M, Villereal M, Rosner MR. Activation of MAP kinases by calcium-dependent and calcium-independent pathways: stimulation by thapsigargin and epidermal growth factor. J Biol Chem. 1992;267:19876-19883. [Abstract/Free Full Text]

49. Duff JL, Marrero MB, Paxton WG, Charles CH, Lau LF, Bernstein KE, Berk BC. Angiotensin II induces 3CH134, a protein-tyrosine phosphatase, in vascular smooth muscle cells. J Biol Chem. 1993;268:26037-26040. [Abstract/Free Full Text]

50. Huckle WR, Prokop CA, Dy RC, Herman B, Earp S. Angiotensin II stimulates protein-tyrosine phosphorylation in a calcium-dependent manner. Mol Cell Biol. 1990;10:6290-6298. [Abstract/Free Full Text]

51. Ueno H, Perryman MB, Roberts R, Schneider MD. Differentiation of cardiac myocytes after mitogen withdrawal exhibits three sequential states of the ventricular growth response. J Cell Biol. 1988;107:1911-1918. [Abstract/Free Full Text]

52. Chao MV. Growth factor signaling: where is the specificity? Cell. 1992;68:995-997. [Medline] [Order article via Infotrieve]

53. Bottari SP, King IN, Reichlin S, Dahlstroem I, Lydon N, de Gasparo M. The angiotensin AT2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase. Biochem Biophys Res Commun. 1992;183:206-211. [Medline] [Order article via Infotrieve]

54. Kambayashi Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H, Hamakubo T, Inagami T. Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. J Biol Chem. 1993;268:24543-24546. [Abstract/Free Full Text]

55. Moolenaar WH. G-protein-coupled receptors, phosphoinositide hydrolysis, and cell proliferation. Cell Growth Differ. 1991;2:359-364.[Medline] [Order article via Infotrieve]




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Home page
 EndocrinologyHome page
F. Rossi, A. Ferraresi, P. Romagni, L. Silvestroni, and V. Santiemma
Angiotensin II Stimulates Contraction and Growth of Testicular Peritubular Myoid Cells in Vitro
Endocrinology, August 1, 2002; 143(8): 3096 - 3104.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Q. Wang, M. Liu, B. Mullah, D. P. Siderovski, and R. R. Neubig
Receptor-selective Effects of Endogenous RGS3 and RGS5 to Regulate Mitogen-activated Protein Kinase Activation in Rat Vascular Smooth Muscle Cells
J. Biol. Chem., July 5, 2002; 277(28): 24949 - 24958.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Iijima, M. Laser, H. Shiraishi, C. D. Willey, B. Sundaravadivel, L. Xu, P. J. McDermott, and D. Kuppuswamy
c-Raf/MEK/ERK Pathway Controls Protein Kinase C-mediated p70S6K Activation in Adult Cardiac Muscle Cells
J. Biol. Chem., June 14, 2002; 277(25): 23065 - 23075.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Seta, M. Nanamori, J. G. Modrall, R. R. Neubig, and J. Sadoshima
AT1 Receptor Mutant Lacking Heterotrimeric G Protein Coupling Activates the Src-Ras-ERK Pathway without Nuclear Translocation of ERKs
J. Biol. Chem., March 8, 2002; 277(11): 9268 - 9277.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
K. R Sipido, P. G.A Volders, M. A Vos, and F. Verdonck
Altered Na/Ca exchange activity in cardiac hypertrophy and heart failure: a new target for therapy?
Cardiovasc Res, March 1, 2002; 53(4): 782 - 805.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
W. G. Thomas, Y. Brandenburger, D. J. Autelitano, T. Pham, H. Qian, and R. D. Hannan
Adenoviral-Directed Expression of the Type 1A Angiotensin Receptor Promotes Cardiomyocyte Hypertrophy via Transactivation of the Epidermal Growth Factor Receptor
Circ. Res., February 8, 2002; 90(2): 135 - 142.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
S. Wei, E. C. Rothstein, L. Fliegel, L. J. Dell'Italia, and P. A. Lucchesi
Differential MAP kinase activation and Na+/H+ exchanger phosphorylation by H2O2 in rat cardiac myocytes
Am J Physiol Cell Physiol, November 1, 2001; 281(5): C1542 - C1550.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. Tian, X. Gong, and Z. Xie
Signal-transducing function of Na+-K+-ATPase is essential for ouabain's effect on [Ca2+]i in rat cardiac myocytes
Am J Physiol Heart Circ Physiol, November 1, 2001; 281(5): H1899 - H1907.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
S. Sanada, M. Kitakaze, K. Node, S. Takashima, A. Ogai, H. Asanuma, Y. Sakata, M. Asakura, H. Ogita, Y. Liao, et al.
Differential Subcellular Actions of ACE Inhibitors and AT1 Receptor Antagonists on Cardiac Remodeling Induced by Chronic Inhibition of NO Synthesis in Rats
Hypertension, September 1, 2001; 38(3): 404 - 411.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. D. Everett, T. D. Stoops, A. C. Nairn, and D. Brautigan
Angiotensin II regulates phosphorylation of translation elongation factor-2 in cardiac myocytes
Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H161 - H167.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
M. Henaff, S. N. Hatem, and J.-J. Mercadier
Low Catecholamine Concentrations Protect Adult Rat Ventricular Myocytes against Apoptosis through cAMP-Dependent Extracellular Signal-Regulated Kinase Activation
Mol. Pharmacol., April 13, 2001; 58(6): 1546 - 1553.
[Abstract] [Full Text]

Home page
 Physiol. Rev.Home page
G. Vassort
Adenosine 5'-Triphosphate: a P2-Purinergic Agonist in the Myocardium
Physiol Rev, April 1, 2001; 81(2): 767 - 806.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
C. Kishimoto, H. Kawamata, S. Sakai, H. Shinohara, and H. Ochiai
Enhanced Production of Macrophage Inflammatory Protein 2 (MIP-2) by In Vitro and In Vivo Infections with Encephalomyocarditis Virus and Modulation of Myocarditis with an Antibody against MIP-2
J. Virol., February 1, 2001; 75(3): 1294 - 1300.
[Abstract] [Full Text]

Home page
 CirculationHome page
J. N. Muth, I. Bodi, W. Lewis, G. Varadi, and A. Schwartz
A Ca2+-Dependent Transgenic Model of Cardiac Hypertrophy : A Role for Protein Kinase C{{alpha}}
Circulation, January 2, 2001; 103(1): 140 - 147.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
T. Aoyagi and S. Izumo
Hemodynamic Overload-Induced Activation of Myocardial Mitogen-Activated Protein Kinases In Vivo : Augmented Responses in Young Spontaneously Hypertensive Rats and Diminished Responses in Aged Fischer 344 Rats
Hypertension, January 1, 2001; 37(1): 52 - 57.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
J. W. Adams, A. L. Pagel, C. K. Means, D. Oksenberg, R. C. Armstrong, and J. H. Brown
Cardiomyocyte Apoptosis Induced by G{alpha}q Signaling Is Mediated by Permeability Transition Pore Formation and Activation of the Mitochondrial Death Pathway
Circ. Res., December 8, 2000; 87(12): 1180 - 1187.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. G. Meszaros, R. Raphael, F. M. Lio, and L. L. Brunton
Protein kinase C contributes to desensitization of ANG II signaling in adult rat cardiac fibroblasts
Am J Physiol Cell Physiol, December 1, 2000; 279(6): C1978 - C1985.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
H. T. Nguyen, R. M. Adam, S. H. Bride, J. M. Park, C. A. Peters, and M. R. Freeman
Cyclic stretch activates p38 SAPK2-, ErbB2-, and AT1-dependent signaling in bladder smooth muscle cells
Am J Physiol Cell Physiol, October 1, 2000; 279(4): C1155 - C1167.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
H. Kodama, K. Fukuda, J. Pan, M. Sano, T. Takahashi, T. Kato, S. Makino, T. Manabe, M. Murata, and S. Ogawa
Significance of ERK cascade compared with JAK/STAT and PI3-K pathway in gp130-mediated cardiac hypertrophy
Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1635 - H1644.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
M. de Gasparo, K. J. Catt, T. Inagami, J. W. Wright, and Th. Unger
International Union of Pharmacology. XXIII. The Angiotensin II Receptors
Pharmacol. Rev., September 1, 2000; 52(3): 415 - 472.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
J. M. Colomer and A. R. Means
Chronic Elevation of Calmodulin in the Ventricles of Transgenic Mice Increases the Autonomous Activity of Calmodulin-Dependent Protein Kinase II, Which Regulates Atrial Natriuretic Factor Gene Expression
Mol. Endocrinol., August 1, 2000; 14(8): 1125 - 1136.
[Abstract] [Full Text]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
I. Hamanaka, Y. Saito, T. Nishikimi, T. Magaribuchi, S. Kamitani, K. Kuwahara, M. Ishikawa, Y. Miyamoto, M. Harada, E. Ogawa, et al.
Effects of cardiotrophin-1 on hemodynamics and endocrine function of the heart
Am J Physiol Heart Circ Physiol, July 1, 2000; 279(1): H388 - H396.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
C. Ruwhof and A. van der Laarse
Mechanical stress-induced cardiac hypertrophy: mechanisms and signal transduction pathways
Cardiovasc Res, July 1, 2000; 47(1): 23 - 37.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
A. Dana, M. Skarli, J. Papakrivopoulou, and D. M. Yellon
Adenosine A1 Receptor Induced Delayed Preconditioning in Rabbits : Induction of p38 Mitogen-Activated Protein Kinase Activation and Hsp27 Phosphorylation via a Tyrosine Kinase- and Protein Kinase C-Dependent Mechanism
Circ. Res., May 12, 2000; 86(9): 989 - 997.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Morisco, D. Zebrowski, G. Condorelli, P. Tsichlis, S. F. Vatner, and J. Sadoshima
The Akt-Glycogen Synthase Kinase 3beta Pathway Regulates Transcription of Atrial Natriuretic Factor Induced by beta -Adrenergic Receptor Stimulation in Cardiac Myocytes
J. Biol. Chem., May 5, 2000; 275(19): 14466 - 14475.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
S. Kim and H. Iwao
Molecular and Cellular Mechanisms of Angiotensin II-Mediated Cardiovascular and Renal Diseases
Pharmacol. Rev., March 1, 2000; 52(1): 11 - 34.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
M. A. Bogoyevitch
Signalling via stress-activated mitogen-activated protein kinases in the cardiovascular system
Cardiovasc Res, March 1, 2000; 45(4): 826 - 842.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
A. Nakano, C. P. Baines, S. O. Kim, S. L. Pelech, J. M. Downey, M. V. Cohen, and S. D. Critz
Ischemic Preconditioning Activates MAPKAPK2 in the Isolated Rabbit Heart : Evidence for Involvement of p38 MAPK
Circ. Res., February 4, 2000; 86(2): 144 - 151.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
K.-i. Nakahara, S. Matsushita, H. Matsuoka, T. Inamatsu, M. Nishinaga, M. Yonawa, T. Aono, T. Arai, Y. Ezaki, and H. Orimo
Insertion/Deletion Polymorphism in the Angiotensin-Converting Enzyme Gene Affects Heart Weight
Circulation, January 18, 2000; 101(2): 148 - 151.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. G. Meszaros, A. M. Gonzalez, Y. Endo-Mochizuki, S. Villegas, F. Villarreal, and L. L. Brunton
Identification of G protein-coupled signaling pathways in cardiac fibroblasts: cross talk between Gq and Gs
Am J Physiol Cell Physiol, January 1, 2000; 278(1): C154 - C162.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
J.-Y. Min, S. Sandmann, A. Meissner, T. Unger, and R. Simon
Differential Effects of Mibefradil, Verapamil, and Amlodipine on Myocardial Function and Intracellular Ca2+ Handling in Rats with Chronic Myocardial Infarction
J. Pharmacol. Exp. Ther., December 1, 1999; 291(3): 1038 - 1044.
[Abstract] [Full Text]

Home page
 Circ. Res.Home page
S. Gunasegaram, R. S. Haworth, D. J. Hearse, and M. Avkiran
Regulation of Sarcolemmal Na+/H+ Exchanger Activity by Angiotensin II in Adult Rat Ventricular Myocytes : Opposing Actions via AT1 Versus AT2 Receptors
Circ. Res., November 12, 1999; 85(10): 919 - 930.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
L. Kim, T. Lee, J. Fu, and M. E. Ritchie
Characterization of MAP kinase and PKC isoform and effect of ACE inhibition in hypertrophy in vivo
Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H1808 - H1816.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
R. R. Baliga, D. R. Pimental, Y.-Y. Zhao, W. W. Simmons, M. A. Marchionni, D. B. Sawyer, and R. A. Kelly
NRG-1-induced cardiomyocyte hypertrophy. Role of PI-3-kinase, p70S6K, and MEK-MAPK-RSK
Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H2026 - H2037.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
R. A. Hunt, G. J. Bhat, and K. M. Baker
Angiotensin II-Stimulated Induction of sis-Inducing Factor Is Mediated by Pertussis Toxin-Insensitive Gq Proteins in Cardiac Myocytes
Hypertension, October 1, 1999; 34(4): 603 - 608.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
K. C Wollert and H. Drexler
The renin-angiotensin system and experimental heart failure
Cardiovasc Res, September 1, 1999; 43(4): 838 - 849.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
C. M. Ford, S. Li, and J. G. Pickering
Angiotensin II Stimulates Collagen Synthesis in Human Vascular Smooth Muscle Cells : Involvement of the AT1 Receptor, Transforming Growth Factor-{beta}, and Tyrosine Phosphorylation
Arterioscler Thromb Vasc Biol, August 1, 1999; 19(8): 1843 - 1851.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
R. Malhotra, J. Sadoshima, F. C. Brosius III, and S. Izumo
Mechanical Stretch and Angiotensin II Differentially Upregulate the Renin-Angiotensin System in Cardiac Myocytes In Vitro
Circ. Res., July 23, 1999; 85(2): 137 - 146.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Chiloeches, H. F. Paterson, R. Marais, A. Clerk, C. J. Marshall, and P. H. Sugden
Regulation of Ras{middle dot}GTP Loading and Ras-Raf Association in Neonatal Rat Ventricular Myocytes by G Protein-coupled Receptor Agonists and Phorbol Ester. ACTIVATION OF THE EXTRACELLULAR SIGNAL-REGULATED KINASE CASCADE BY PHORBOL ESTER IS MEDIATED BY Ras
J. Biol. Chem., July 9, 1999; 274(28): 19762 - 19770.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
Y. Takeuchi, N. Pausawasdi, and A. Todisco
Carbachol activates ERK2 in isolated gastric parietal cells via multiple signaling pathways
Am J Physiol Gastrointest Liver Physiol, June 1, 1999; 276(6): G1484 - G1492.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
Y. Moriguchi, H. Matsubara, Y. Mori, S. Murasawa, H. Masaki, K. Maruyama, Y. Tsutsumi, Y. Shibasaki, Y. Tanaka, T. Nakajima, et al.
Angiotensin II–Induced Transactivation of Epidermal Growth Factor Receptor Regulates Fibronectin and Transforming Growth Factor-ß Synthesis via Transcriptional and Posttranscriptional Mechanisms
Circ. Res., May 14, 1999; 84(9): 1073 - 1084.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
K.-D. Schluter, A. Simm, M. Schafer, G. Taimor, and H. M. Piper
Early response kinase and PI 3-kinase activation in adult cardiomyocytes and their role in hypertrophy
Am J Physiol Heart Circ Physiol, May 1, 1999; 276(5): H1655 - H1663.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
P. H. Sugden
Signaling in Myocardial Hypertrophy : Life After Calcineurin?
Circ. Res., April 2, 1999; 84(6): 633 - 646.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
R. H. Ritchie, J. D. Marsh, and R. J. Schiebinger
Bradykinin-stimulated protein synthesis by myocytes is dependent on the MAP kinase pathway and p70S6K
Am J Physiol Heart Circ Physiol, April 1, 1999; 276(4): H1393 - H1398.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
S. G. Finn, S. G. Plonk, and S. J. Fuller
G{alpha}13 stimulates gene expression and increases cell size in cultured neonatal rat ventricular myocytes
Cardiovasc Res, April 1, 1999; 42(1): 140 - 148.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Montessuit and A. Thorburn
Transcriptional Activation of the Glucose Transporter GLUT1 in Ventricular Cardiac Myocytes by Hypertrophic Agonists
J. Biol. Chem., March 26, 1999; 274(13): 9006 - 9012.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
T. Shimizu, K.-i. Kinugawa, A. Yao, Y. Sugishita, K. Sugishita, K. Harada, H. Matsui, O. Kohmoto, T. Serizawa, and T. Takahashi
Platelet-derived growth factor induces cellular growth in cultured chick ventricular myocytes
Cardiovasc Res, March 1, 1999; 41(3): 641 - 653.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Z. Gao, T. Chen, M. J. Weber, and J. Linden
A2B Adenosine and P2Y2 Receptors Stimulate Mitogen-activated Protein Kinase in Human Embryonic Kidney-293 Cells. CROSS-TALK BETWEEN CYCLIC AMP AND PROTEIN KINASE C PATHWAYS
J. Biol. Chem., February 26, 1999; 274(9): 5972 - 5980.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. A. Maloney, O. M. Tsygankova, L. Yang, Q. Li, A. Szot, K. Baysal, and J. R. Williamson
Activation of ERK by Ca2+ store depletion in rat liver epithelial cells
Am J Physiol Cell Physiol, January 1, 1999; 276(1): C221 - C230.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
M. R. Rosen, I. S. Cohen, P. Danilo Jr., and S. F. Steinberg
The heart remembers
Cardiovasc Res, December 1, 1998; 40(3): 469 - 482.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. M. Park, J. G. Borer, M. R. Freeman, and C. A. Peters
Stretch activates heparin-binding EGF-like growth factor expression in bladder smooth muscle cells
Am J Physiol Cell Physiol, November 1, 1998; 275(5): C1247 - C1254.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
M. O. Gray, C. S. Long, J. E. Kalinyak, H.-T. Li, and J. S. Karliner
Angiotensin II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-{beta}1 and endothelin-1 from fibroblasts
Cardiovasc Res, November 1, 1998; 40(2): 352 - 363.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
M. Yano, S. Kim, Y. Izumi, S. Yamanaka, and H. Iwao
Differential Activation of Cardiac c-Jun Amino-Terminal Kinase and Extracellular Signal-Regulated Kinase in Angiotensin II–Mediated Hypertension
Circ. Res., October 5, 1998; 83(7): 752 - 760.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
A. Nagahara, L. Wang, J. Del Valle, and A. Todisco
Regulation of c-Jun NH2-terminal kinases in isolated canine gastric parietal cells
Am J Physiol Gastrointest Liver Physiol, October 1, 1998; 275(4): G740 - G748.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
S. Murasawa, Y. Mori, Y. Nozawa, H. Masaki, K. Maruyama, Y. Tsutsumi, Y. Moriguchi, Y. Shibasaki, Y. Tanaka, T. Iwasaka, et al.
Role of Calcium-Sensitive Tyrosine Kinase Pyk2/CAKß/RAFTK in Angiotensin II–Induced Ras/ERK Signaling
Hypertension, October 1, 1998; 32(4): 668 - 675.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
K.-D. Schluter, Y. Goldberg, G. Taimor, M. Schafer, and H. Michael Piper
Role of phosphatidylinositol 3-kinase activation in the hypertrophic growth of adult ventricular cardiomyocytes
Cardiovasc Res, October 1, 1998; 40(1): 174 - 181.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
H. Kai, A. Muraishi, Y. Sugiu, H. Nishi, Y. Seki, F. Kuwahara, A. Kimura, H. Kato, and T. Imaizumi
Expression of Proto-oncogenes and Gene Mutation of Sarcomeric Proteins in Patients With Hypertrophic Cardiomyopathy
Circ. Res., September 21, 1998; 83(6): 594 - 601.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Kudoh, I. Komuro, Y. Hiroi, Y. Zou, K. Harada, T. Sugaya, N. Takekoshi, K. Murakami, T. Kadowaki, and Y. Yazaki
Mechanical Stretch Induces Hypertrophic Responses in Cardiac Myocytes of Angiotensin II Type 1a Receptor Knockout Mice
J. Biol. Chem., September 11, 1998; 273(37): 24037 - 24043.
[Abstract] [Full Text] [PDF]

Home page
 J Am Coll CardiolHome page
S. M. Dodge, M. A. Beardslee, B. J. Darrow, K. G. Green, E. C. Beyer, and J. E. Saffitz
Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes
J. Am. Coll. Cardiol., September 1, 1998; 32(3): 800 - 807.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
K. McBride and M. Nemer
The C-Terminal Domain of c-fos Is Required for Activation of an AP-1 Site Specific for jun-fos Heterodimers
Mol. Cell. Biol., September 1, 1998; 18(9): 5073 - 5081.
[Abstract] [Full Text]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. Goussev, V. G. Sharov, H. Shimoyama, M. Tanimura, M. Lesch, S. Goldstein, and H. N. Sabbah
Effects of ACE inhibition on cardiomyocyte apoptosis in dogs with heart failure
Am J Physiol Heart Circ Physiol, August 1, 1998; 275(2): H626 - H631.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
J. W. Adams, V. P. Sah, S. A. Henderson, and J. H. Brown
Tyrosine Kinase and c-Jun NH2-Terminal Kinase Mediate Hypertrophic Responses to Prostaglandin F2{alpha} in Cultured Neonatal Rat Ventricular Myocytes
Circ. Res., July 27, 1998; 83(2): 167 - 178.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. J. Fuller, J. Gillespie-Brown, and P. H. Sugden
Oncogenic src, raf, and ras Stimulate a Hypertrophic Pattern of Gene Expression and Increase Cell Size in Neonatal Rat Ventricular Myocytes
J. Biol. Chem., July 17, 1998; 273(29): 18146 - 18152.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
N. O. Dulin, L. D. Alexander, S. Harwalkar, J. R. Falck, and J. G. Douglas
Phospholipase A2-mediated activation of mitogen-activated protein kinase by angiotensin II
PNAS, July 7, 1998; 95(14): 8098 - 8102.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
R. S. Ross, C. Pham, S.-Y. Shai, J. I. Goldhaber, C. Fenczik, C. C. Glembotski, M. H. Ginsberg, and J. C. Loftus
ß1 Integrins Participate in the Hypertrophic Response of Rat Ventricular Myocytes
Circ. Res., June 15, 1998; 82(11): 1160 - 1172.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. Kometiani, J. Li, L. Gnudi, B. B. Kahn, A. Askari, and Z. Xie
Multiple Signal Transduction Pathways Link Na+/K+-ATPase to Growth-related Genes in Cardiac Myocytes. THE ROLES OF Ras AND MITOGEN-ACTIVATED PROTEIN KINASES
J. Biol. Chem., June 12, 1998; 273(24): 15249 - 15256.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
S. McKay, J. C. de Jongste, P. R. Saxena, and H. S. Sharma
Angiotensin II Induces Hypertrophy of Human Airway Smooth Muscle Cells: Expression of Transcription Factors and Transforming Growth Factor-beta 1
Am. J. Respir. Cell Mol. Biol., June 1, 1998; 18(6): 823 - 833.
[Abstract] [Full Text]

Home page
 Cardiovasc ResHome page
S. T Rapundalo
Cardiac protein phosphorylation: functional and pathophysiological correlates
Cardiovasc Res, June 1, 1998; 38(3): 559 - 588.
[Abstract] [Full Text] [PDF]

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