Mitogen-activated Protein Kinase Cascade in Neonatal Rat Cardiomyocytes

J Mol Cell Cardiol 29, 2491–2501 (1997)

Protein Kinase A and Protein Kinase C Synergistically Activate the Raf-1 Kinase/Mitogen-activated Protein Kinase Cascade in Neonatal Rat Cardiomyocytes Tsutomu Yamazaki1,2, Issei Komuro1, Yunzeng Zou1, Sumiyo Kudoh1, Takehiko Mizuno1, Yukio Hiroi1, Ichiro Shiojima1, Hiroyuki Takano1, Koh-ichiro Kinugawa3, Osami Kohmoto3, Toshiyuki Takahashi3 and Yoshio Yazaki1 1

Department of Medicine III, University of Tokyo School of Medicine; 2Health Service Center, University of Tokyo; and 3Department of Medicine II, University of Tokyo School of Medicine, Tokyo, Japan (Received 29 August 1996, accepted in revised form 22 May 1997) T. Y, I. K, Y. Z, S. K, T. M, Y. H, I. S, H. T, K.-I. K, O. K, T. T  Y. Y. Protein Kinase A and Protein Kinase C Synergistically Activate the Raf1 Kinase/Mitogen-activated Protein Kinase Cascade in Neonatal Rat Cardiomyocytes. Journal of Molecular and Cellular Cardiology (1997) 29, 2491–2501. Adrenoceptor agonists play an important role in cardiac hypertrophy. In cardiomyocytes, activation of a- and b-adrenoceptors induces a variety of hypertrophic responses via activation of protein kinase C (PKC) and protein kinase A (PKA), respectively. Although PKC evokes activation of the Raf1 kinase (Raf-1)/mitogen-activated protein (MAP) kinase cascade, PKA has been shown to inhibit the activation of Raf-1 and MAP kinases induced by growth factors in various cell types. The present study was performed to elucidate the role of PKA and PKC in cardiomyocyte hypertrophy. PKA activators such as forskolin (FSK), isobutylmethylxanthine, dibutyryl cAMP and isoproterenol, significantly activated Raf-1 and MAP kinases with a peak at 2 and 8 min, respectively, followed by an increase in protein synthesis in cardiac myocytes. Similar responses were observed when cardiomyocytes were stimulated with PKC activators such as 12-O-tetradecanoylphorbol-13-acetate (TPA), angiotensin II, phenylephrine and mechanical stretch. After depleting extracellular Ca2+ with EGTA, FSK did not activate MAP kinases, while down-regulation of PKC by long exposure with TPA did not influence FSK-induced MAP kinase activation. Furthermore, FSK and TPA synergistically activated Raf-1. Similar synergistic activation of MAP kinases was observed when other PKC activators were added to cardiac myocytes with FSK at the same time. In conclusion, unlike other cell types, PKA activates Raf1 and MAP kinases followed by an increase in protein synthesis in cardiac myocytes.  1997 Academic Press Limited

K W: Cardiac hypertrophy; Protein kinase A; Protein kinase C; Raf-1 kinase; MAP kinase; cAMP.

Please address all correspondence to: I. Komuro, Department of Medicine III, University of Tokyo School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. T.Y. has been selected as a finalist for the 1995 Richard J. Bing Young Investigators Award, and presented the part of this manuscript at the competition.

0022–2828/97/092491+11 $25.00/0

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Introduction Cardiac hypertrophy is induced by neurohumoral factors such as catecholamines and angiotensin II (Ang II), as well as hemodynamic overload (Morgan and Baker, 1991; Komuro and Yazaki, 1993). Although cardiac hypertrophy is formed as a compensatory response to extracellular stimuli, resulting in maintaining the function of the heart, it is finally associated with disorders of contraction and relaxation. Recent epidemiological studies have shown that cardiac hypertrophy is a major risk factor of heart diseases, including myocardial infarction and cardiac arrhythmias (Levy et al., 1990). Therefore, it is important to prevent or treat cardiac hypertrophy, and it is necessary to fully understand the molecular mechanisms of cardiomyocyte hypertrophy. Generally, extracellular stimuli are transmitted from the membrane into the nucleus by activation of protein kinase cascades of phosphorylation. A growing body of data have suggested that protein kinase C (PKC) plays an important role in the regulation of differentiation and proliferation of various cell types as one of the major signal transduction molecules (Komuro and Yazaki, 1993). With regard to cardiomyocyte hypertrophy, we have previously reported that both mechanical stretch and Ang II serially activate multiple protein kinases in the order of PKC→Raf-1 kinase (Raf1)→mitogen-activated protein (MAP) kinase kinase→MAP kinases→90 kDa-S6 kinase, followed by an increase in specific gene expression and protein synthesis in neonatal rat cardiomyocytes (Komuro et al., 1990, 1991; Yamazaki et al., 1993, 1995a; Zou et al., 1996). Among catecholamines, norepinephrine from sympathetic nerve ends and epinephrine from the adrenal medulla have been reported to induce cardiac hypertrophy. Norepinephrine and epinephrine display their functions mainly through two types of adrenoceptors, a1 and b, in cardiac myocytes (Simpson, 1985). Stimulation of the a1 receptor of cardiomyocytes activates phosphoinositide-specific phospholipase C resulting in production of diacylglycerol, the endogenous activator of PKC (Karliner et al., 1990). On the other hand, stimulation of b receptor induces the activation of adenylyl cyclase, which increases intracellular cAMP levels and finally activates protein kinase A (PKA). Since adrenergic stimuli through both receptors produce cardiomyocyte hypertrophy (Pinson et al., 1993), it is conceivable that PKA as well as PKC acts as the activator for hypertrophic responses in cardiac myocytes. However, there have been many reports suggesting that PKA activation

inhibits cell growth induced by various growth factors (Hollenberg and Cuatrecasas, 1973; Pastan and Willingham, 1978; Nilsson and Olsson, 1984; Blomhoff et al., 1987). A number of recent reports have shown that PKA inhibits activation of Raf-1 and MAP kinases by interfering with Ras–Raf-1 association in a variety of cell types (Burgering et al., 1993; Cook and McCormick, 1993; Graves et al., 1993; Sevetson et al., 1993; Wu et al., 1993; Haefner et al., 1994; Hordijik et al., 1994; Russel et al., 1994; VanRenterghem et al., 1994). Haefner et al. (1994) have reported, by using NIH 3T3 cells, that in addition to weakening the interaction of Raf-1 with Ras, cAMP-dependent PKA directly phosphorylates Raf-1 and inhibits Raf-1 activation by PKC both in vitro and in vivo. However, we have recently indicated that norepinephrine induces hypertrophic responses such as Raf-1 and MAP kinase activation through a1- and b-adrenoceptors in cardiac myocytes (Yamazaki et al., 1997). In the present study, to understand the mechanism by which PKA induces cardiac hypertrophy, we examined the effects of PKA on activities of Raf1 and MAP kinases and on protein synthesis in cultured cardiomyocytes. We show that not only PKC activators but also PKA activators, such as dibutyryl cAMP (dbcAMP), isobutylmethylxanthine (IBMX), forskolin (FSK) and isoproterenol (ISO), induce activation of Raf-1 and MAP kinases followed by an increase in protein synthesis, and that PKA and PKC synergistically activate Raf-1 and MAP kinases in neonatal rat cardiomyocytes. These results implicate the synergistic actions between neuronal factors and mechanical stress or vasoactive peptides on cardiac hypertrophy.

Materials and Methods Culture of cardiomyocytes Primary cultures of cardiomyocytes were prepared from ventricles of 1-day-old Wistar rats, as described previously (Komuro et al., 1990), basically, according to the method of Simpson and Savion (1982). Cardiomyocytes were plated at high density (1×103 cells/mm2). Cardiomyocytes were stretched by 20%, as described previously (Komuro et al., 1990, 1991). Cardiomyocytes were also stimulated by addition of various agents, and lysed on ice with buffer A, consisting of 25 mmol/l Tris-HCl, 25 mmol/l NaCl, 1 mmol/l sodium orthovanadate, 10 mmol/l NaF, 10 mmol/l sodium pyrophosphate, 10 mmol/l okadaic acid, 0.5 mmol/l EGTA and

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1 mmol/l phenyl-methyl sulfonyl fluoride (Yamazaki et al., 1993). A series of experiments were performed simultaneously with the same pool of cells in each experiment to match for temperature, CO2 content, or pH of the medium between stimulated and control cells.

rapidly rinsed extensively with ice-cold phosphatebuffered saline (10 mmol/l sodium phosphate and 0.85% NaCl, pH 7.4) and incubated for over 20 min on ice with 1 ml of 20% trichloroacetic acid. Total radioactivity in each dish was determined by liquid scintillation counting.

Assays of MAP kinases in myelin basic protein (MBP)-containing gels

Measurement of intracellular Ca2+ concentration ([Ca2+]i)

MAP kinase activities were measured using MBPcontaining gels, as described previously (Yamazaki et al., 1993). In brief, MAP kinases were immunoprecipitated with polyclonal antibody against MAP kinases (aY91) (Tobe et al., 1991), and were electrophoresed on an SDS-polyacrylamide gel containing MBP. Phosphorylation of MBP by MAP kinases was assayed by incubating the gel with [c32 P]adenosine triphosphate (ATP) (Du Pont-New England Nuclear Co., Boston, MA, USA). After incubation, the gel was washed, dried and then subjected to autoradiography.

[Ca2+]i was measured with Ca2+ fluorescent dye Indo-1, as previously described (Kinugawa et al., 1994). In beating cardiomyocytes, data of peak systolic [Ca2+]i were collected from an average of five consecutive beats.

Assay of MAP kinase kinase kinase activity of Raf-1 Raf-1 has been reported to have MAP kinase kinase kinase activity (Kyriakis et al., 1992; Lange-Carter et al., 1993). The activities were assayed by measuring the phosphorylation of recombinant MAP kinase kinase (rMAPKK) (Crews et al., 1992), as reported previously (Yamazaki et al., 1995b). The immunoprecipitates with anti-Raf-1 antibody (a-raf) (Santa Cruz Biochemistry, Inc., Santa Cruz, CA, USA) were incubated with the substrate (rMAPKK) and 2 lCi [c-32P]ATP. After incubation, rMAPKK was collected and electrophoresed on a polyacrylamide gel. The gel was dried and subjected to autoradiography.

Amino acid uptake into cardiomyocytes After being maintained in the serum-free medium for 2 days, cardiomyocytes were stimulated by FSK (10−5 mol/l) and/or TPA (12-O-tetradecanoylphorbol-13-acetate) (10−9 mol/l) for 24 h. The relative amount of protein synthesis was determined by assessing the incorporation of the radioactivity into a trichloroacetic acid-insoluble fraction. One lCi/ml [3H]phenylalanine (Du Pont-New England Nuclear Co., Boston, MA, USA) was added to the culture medium 2h before harvest. The cells were

Statistics Differences within groups in Raf-1 and MAP kinase activities were compared by one-way ANOVA and Dunnett’s t test. Since the interaction effect in the 2×2 factorial ANOVA is the relevant test for documenting synergism, 2×2 factorial ANOVA was performed for the definition of the synergism, and P values reported in the figures were from the interaction test.

Results Both PKA and PKC activate MAP kinases in cardiac myocytes Many laboratories have reported that PKA activators as well as PKC activators induce cardiac hypertrophy (Morgan and Baker, 1991). To elucidate the mechanism of PKA-induced cardiac hypertrophy, we first measured MAP kinase activities in neonatal rat cardiomyocytes in the absence or presence of a variety of agents which raise the activity of PKA or PKC. Four chemically and mechanistically dissimilar agents such as dbcAMP, IBMX, FSK and ISO, which are capable of activating PKA by elevating intracellular cAMP levels, activated both 42-kDa and 44-kDa MAP kinases in cardiomyocytes [Fig. 1(a)]. The data on 42-kDa MAP kinase activities are shown in Figure 1(b). Similar results were obtained for the 44-kDa MAP kinase. This result suggests that cAMP-dependent PKA activates MAP kinases in cardiac myocytes. In addition, PKC activators such as TPA and phenylephrine (PHE) also induced MAP kinase activation

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Figure 1 MAP kinase activation by PKA or PKC activators in cardiac myocytes. Cardiac myocytes [(a), (b)] or cardiac non-myocytes (c) were stimulated by dbcAMP (10−5 mol/l), IBMX (10−5 mol/l), FSK (10−5 mol/l), ISO (10−5 mol/l), TPA (10−9 mol/l) or PHE (10−5 mol/l) for 8 min, and the cell lysates were subjected to the MAP kinase assay as described under Materials and Methods. (a), (c) Representative autoradiograms. (b) relative kinase activities of 42-kDa MAP kinase obtained in cardiac myocytes were determined by scanning each band on autoradiograms with a densitometer. The results were indicated as mean±.. for four independent experiments. The activities were expressed relative to that of 42-kDa MAP kinase obtained in unstimulated cardiomyocytes (∗P<0.05 v control).

[Figs 1(a), 1(b)], which is consistent with our previous results (Yamazaki et al., 1993). The levels of MAP kinase activation by PKA activators were low as compared with those by same concentration

of TPA but comparable to those by PHE. Either 10−5 mol/l dbcAMP, 10−5 mol/l IBMX, 10−5 mol/l FSK or 10−5 mol/l ISO increased activities of MAP kinases by >five-fold. Same levels of increase in MAP kinase activities were observed when cardiomyocytes were stimulated by 10−9 mol/l TPA or 10−5 mol/l PHE. On the other hand, when cardiac non-myocytes were exposed to ISO or FSK, MAP kinase activities remained unchanged [Fig. 1(c)]. These results suggest that PKA-induced these responses are specific to cardiac myocytes. Next, the time course of FSK- and TPA-induced MAP kinase activation was examined. An increase in MAP kinase activity was observed from as early as 1 min by 10−5 mol/l FSK, and the activity reached a peak at 8 min. The activity decreased sharply and returned completely to control levels within 30 min after stimulation with FSK. Fold increase in 42-kDa MAP kinase is shown in Figure 2(a), and the similar results were obtained for the 44-kDa MAP kinase (data not shown). Other cAMP-sensitive PKA activators, such as 10−5 mol/l IBMX, 10−5 mol/l dbcAMP and 10−5 mol/l ISO also activated MAP kinases with a similar time course (data not shown). When 10−9 mol/l TPA was added to cultured cardiomyocytes, almost the same levels of MAP kinase activation were observed as 10−5 mol/l FSK. The initial and maximal activations were also observed at 1 and 8 min, respectively; however, the activities were decreased gradually and sustained relatively higher levels for over 45 min. The activities returned to basal levels at 60 min after stimulation [Fig. 2(b)]. To further examine PKA-induced MAP kinase activation, cardiac myocytes were incubated with various concentrations (10−9 mol/l to 10−5 mol/l) of FSK for 8 min. No increase in MAP kinase activities was observed at 10−9 mol/l FSK. A slight increase in MAP kinase activities was observed at 10−8 mol/l. There was good correlation between activities of 42-kDa MAP kinase and concentrations of FSK, and maximal activation of MAP kinases was obtained at 10−5 mol/l FSK (Fig. 3). Similar results were obtained for the 44-kDa MAP kinase (data not shown).

PKA and PKC synergistically activate MAP kinases in cardiomyocytes As indicated above, cAMP-sensitive PKA activators as well as PKC activators induced MAP kinase activation in cardiomyocytes. Since PKA has been reported to inhibit MAP kinase activation induced by various growth factors in a number of cell

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Figure 3 Dose dependence of FSK-induced MAP kinase activation. Cardiomyocytes were stimulated by various concentrations of FSK for 8 min. Activities of 42-kDa MAP kinase were determined as described under Materials and Methods. The data were presented by mean for two independent experiments as compared with controls. The activity of 42-kDa MAP kinase in control was designated as 1.0.

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Figure 2 Time course of MAP kinase activation induced by FSK or TPA. Cardiac myocytes were stimulated by FSK (10−5 mol/l) (a) or TPA (10−9 mol/l) (b) for the indicated periods of time. MAP kinase activities were determined by MBP phosphorylation assay in the gel. Relative kinase activities of 42-kDa MAP kinase were determined by scanning each band on autoradiograms with a densitometer. The activities were expressed relative to that to 42-kDa MAP kinase obtained in unstimulated cardiomyocytes. The results are shown as the mean for two independent experiments.

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types (Burgering et al., 1993; Cook and McCormick, 1993; Graves et al., 1993; Sevetson et al., 1993; Wu et al., 1993; Haefner et al., 1994; Hordijik et al., 1994; Russel et al., 1994; VanRenterghem et al., 1994), we examined whether PKA enhances or inhibits MAP kinase activation induced by PKC in cardiomyocytes. The activity of 42-kDa MAP kinase was increased by FSK (3.9-fold) and by TPA (4.5-fold), while the simultaneous administration of both agents synergistically increased the activity by 11.7-fold (P=0.04) (Fig. 4). To elucidate

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Figure 4 Synergistic activation of MAP kinases by FSK and TPA in cardiac myocytes. Cardiomyocytes were stimulated by FSK (10−5 mol/l) and/or TPA (10−9 mol/l) for 8 min, and MAP kinase activities were determined. The intensities of 42-kDa bands were measured by densitometric scanning of the autoradiogram. Values represent the mean±.. from four independent experiments. The intensity of 42-kDa MAP kinase of unstimulated cardiomyocytes was designated as 1.0. (Χ) TPA(+); (Β) TPA(−); P=0.04, TPA(+) v TPA(−).

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Figure 5 Synergistic effects between FSK and PKC activators on the MAP kinase activation in cardiac myocytes. PKC was activated by addition of Ang II (10−8 mol/ l) (Ο), PHE (10−5 mol/l) (Μ) or ET-1 (10−9 mol/l) (Ε) or by mechanical stretch (Χ) in cultured cardiac myocytes in the absence or presence of FSK (10−5 mol/l) [control (Β)]. After stimulation for 8 min, cardiomyocytes were subjected to the MAP kinase assay. The intensities of 42kDa bands were measured by densitometric scanning of the autoradiogram. The data were presented by mean±.. from four independent experiments compared with controls. The activity of 42-kDa MAP kinase in control was designated as 1.0. P=0.02, PHE v control; P=0.01, Ang II v control; P=0.004, ET-1 v control; P= 0.01, stretch v control.

whether PKA and PKC activators generally activate MAP kinases in a synergistic manner, various kinds of PKC activators, such as Ang II (10−8 mol/l), PHE (10−5 mol/l), endothelin-1 (ET-1) (10−9 mol/l) and mechanical stretch (20%) were added to cultured cardiac myocytes with FSK (10−5 mol/l). Each PKC activator increased MAP kinase activities, and the simultaneous addition of FSK and the PKC activators provoked much greater increases in MAP kinase activities (Fig. 5).

FSK activates MAP kinases through Ca2+-dependent and PKC-independent pathways Since PKA and PKC activators increased MAP kinase activities synergistically, PKA and PKC may activate MAP kinases through different mechanisms. Therefore, we examined the involvement of Ca2+ in PKA-induced MAP kinase activation. After pretreatment with 5×10−3 mol/l EGTA for 30 min, cardiomyocytes were stimulated by 10−5 mol/l FSK for 8 min. The pretreatment with

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Figure 6 Effects of Ca2+ depletion, PKA inhibition or PKC down-regulation on cAMP-induced MAP kinase activation. After pretreated with 5×10−3 mol/l EGTA for 30 min, 3×10−5 mol/l H-89 for 30 min or 10−7 mol/ l TPA for 24 h, cardiac myocytes were stimulated by 10−5 mol/l FSK or ISO for 8 min, and MAP kinase activities were measured. The data represent the average fold increase in 42-kDa MAP kinase activity over controls (42 kDa-MAP kinase activity of unstimulated cells) for four independent experiments (mean±..). ∗P<0.05 v FSK treatment, †P<0.05 v ISO treatment.

5×10−3 mol/l EGTA completely blocked transmembrane Ca2+ influx (data not shown). As shown in Figure 6, FSK-induced MAP kinase activation was completely abolished by EGTA (P<0.05), suggesting that FSK-induced MAP kinase activation depends on transsarcolemmal Ca2+ influx. To further elucidate the involvement of Ca2+ in FSKevoked MAP kinase activation, intracellular Ca2+ levels were measured using Indo-1. FSK significantly increased peak systolic [Ca2+]i by approximately 1.7-fold (from 309.5–531.5× 10−9 mol/l, P=0.03). ISO also increased peak systolic [Ca2+]i (data not shown) and MAP kinase activities [Figs 1(a), 1(b)], and pretreatment with a specific PKA inhibitor H-89 (3×10−5 mol/l) (Chijiwa et al., 1990) completely blocked cell contraction and intracellular Ca2+ transients, and abolished ISO-induced MAP kinase activation (Fig. 6). These results suggest that PKA activates MAP kinases by increasing Ca2+ influx in cardiac myocytes. On the other hand, down-regulation of PKC by long exposure with TPA (10−7 mol/l for 24 h) did not affect FSK-evoked MAP kinase activation (Fig. 6), suggesting that PKA and PKC activate MAP kinases through different signaling pathways.

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Raf-1 has been reported to activate MAP kinases through the activation of MAP kinase kinase (Ahn et al., 1991; Gomez and Cohen, 1991; Matsuda et al., 1992), and to be activated directly by PKC (Kolch et al., 1993). Recently, PKA has been reported to inhibit MAP kinase activation by modulating Raf-1 (Burgering et al., 1993; Cook and McCormick, 1993; Graves et al., 1993; Sevetson et al., 1993; Wu et al., 1993; Haefner et al., 1994; Hordijik et al., 1994; Russel et al., 1994; VanRenterghem et al., 1994). We thus examined effects of PKA on Raf-1 in cardiomyocytes. Not only TPA (10−9 mol/l) but also FSK (10−5 mol/l) and dbcAMP (10−5 mol/l) activated Raf-1 in cardiac myocytes [Fig. 7(a)]. FSK slightly but significantly induced Raf-1 activation dose-dependently, and maximal activation (2.76-fold) was observed at the concentration of 10−5 mol/l FSK [Fig. 7(b)]. Other cAMP elevating agents such as IBMX (10−5 mol/l) and ISO (10−5 mol/l) had similar effects on Raf1 activation (data not shown). TPA (10−9 mol/l) increased Raf-1 activities by 3.48-fold. Most importantly, FSK and TPA acted as a synergistic manner on Raf-1 activities. Simultaneous administration of FSK (10−5 mol/l) and TPA (10−9 mol/l) synergistically activated Raf-1 (fold increase of 785%, P=0.0002) [Fig. 7(c)].

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Both PKA and PKC increase protein synthesis in cardiomyocytes PKA has been reported to inhibit cell growth induced by various growth factors (Hollenberg and Cuatrecasas, 1973; Pastan and Willingham, 1978; Nilsson and Olsson, 1984; Blomhoff et al., 1987). We finally examined the effects of PKA and PKC on the growth of cardiomyocyte (i.e. hypertrophy). FSK (10−5 mol/l) and TPA (10−9 mol/l) increased phenylalanine incorporation into proteins in cardiomyocytes by 1.29-fold and 1.38-fold, respectively (Fig. 8). When both FSK and TPA were added to cardiomyocytes simultaneously, phenylalanine uptake was increased by 2.06-fold (Fig. 8). These results suggest that PKA as well as PKC induces cardiomyocyte hypertrophy. However, the analysis for the amino acid uptake with the 2×2 factorial ANOVA showed that there was no statistically significant interaction of PKA and PKC in the protein synthesis levels (P=0.21).

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Figure 7 Synergistic effects of FSK and TPA on Raf-1 activity in cardiac myocytes. Cardiomyocytes were exposed to FSK (10−9–10−5 mol/l) and/or TPA (10−9 mol/l) for 2 min. Raf-1 activity was measured as described under Materials and Methods. A representative autoradiogram was presented in (a). The intensity of 74-kDa band was measured by densitometric scanning of the autoradiogram. The data are presented as mean±.. for four independent experiments compared with control [(b), (c)]. ∗P<0.05 v control (c). (Χ) TPA(+); (Β) TPA(−); P=0.0002, TPA(+) v TPA(−).

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Figure 8 Effects of FSK and TPA in phenylalanine uptake in cardiac myocytes. Cardiomyocytes were stimulated by FSK (10−5 mol/l) and/or TPA (10−9 mol/l) for 24 h, and [3H]phenylalanine (1 lCi/ml) was added 2 h before harvest. Total radioactivity of incorporated [3H]phenylalanine into proteins was determined by liquid scintillation counting. The relative phenylalanine uptake is shown as mean±.. for four independent experiments. (Χ) TPA(+); (Β) TPA(−); P=0.21, TPA(+) v TPA(−).

Discussion In the present study, we have demonstrated that unlike many other cell types, an increase in PKA activities induces activation of Raf-1 and MAP kinases in cardiac myocytes. In addition, activation of MAP kinases by PKA activators was dependent on trans-sarcolemmal Ca2+ influx and independent of PKC. Moreover, PKA activators synergistically activated Raf-1 and MAP kinases with PKC activators such as TPA followed by increased protein synthesis. Two protein kinase pathways, Raf-1/MAP kinase and cAMP/PKA pathways, have been reported to be important in cell growth (Dumont et al., 1989; Pelech and Sanghera, 1992; Daum et al., 1994; Walsh and Van, 1994). cAMP has been shown to positively regulate cell proliferation in yeast and in some differentiated mammalian cells (Dumont et al., 1989). A number of recent papers, however, have demonstrated that an increase in cAMP levels blocks activation of the Raf-1/MAP kinase cascade, which is also critical for cell proliferation in various cell types, including Raf-1 fibroblasts (Burgering et al., 1993; Cook and McCormick, 1993; Wu et al., 1993; Hordijik et al., 1994; Russel et al., 1994), human arterial smooth muscle cells (Graves et al., 1993), rat adipocytes, CHO cells (Sevetson et al., 1993), NIH 3T3 cells (Haefner et al., 1994), and

Xenopus oocytes (VanRenterghem et al., 1994). Although the mechanism of inhibition of the Raf-1/ MAP kinase cascade by cAMP-dependent PKA is not fully understood, cAMP-dependent PKA reduces the affinity of Raf-1 for Ras (Wu et al., 1993) or directly inhibits Raf-1 function (Haefner et al., 1994). A small GTP-binding protein, Rap1A (also known as Krev-1) (Graves et al., 1993; Hordijik et al., 1994), or a guanine nucleotide exchange factor for Ras, Sos (Burgering et al., 1993), may be involved in cAMP-induced inhibition of Raf-1/MAP kinase cascade. On the contrary, there have been only a few reports showing that cAMP-dependent PKA activates MAP kinases. Froedin et al. (1994) have reported that in combination with TPA, cAMP synergistically increases MAP kinase activities in PC12 cells. In addition, Faure and Bourne (1995) have recently reported that cAMP activates MAP kinases in Swiss-3T3 and COS-7 cells, but inhibits them in Rat-1 cells. In many cells, including PC12 cells, Swiss-3T3 and COS-7 cells, however, cAMP has not increased the activity of Raf-1. Thus, they have speculated that cAMP activates MAP kinases through Raf-1-independent pathways. In neonatal rat cardiomyocytes, however, cAMP-sensitive PKA activators induced the activation of Raf-1 as well as that of MAP kinases in the present study. Moreover, synergistic increases in Raf-1 and MAP kinase activities were observed when both PKA and PKC activators were added to cardiomyocytes simultaneously. Although the mechanism of how cAMP-dependent PKA activates the Raf-1/MAP kinase cascade is not clear at present, effects of PKA may be dependent on cell types. As described above, there are several cell types where PKA activates MAP kinases (Froedin et al., 1994; Faure and Bourne, 1995). It is of interest, that like other many cell types, FSK did not induce MAP kinase activation in cardiac non-myocytes of neonatal rats. How does PKA activate the Raf-1/MAP kinase cascade in cardiac myocytes? We showed, in the present study, that PKA-activating agents induce trans-sarcolemmal Ca2+ influx and that increased intracellular Ca2+ activates MAP kinases. Moreover, elevation of cAMP levels and the subsequent activation of PKA have been shown to activate voltage-dependent Ca2+ channels resulting in an increase in Ca2+ influx (Yatani and Brown, 1989). Therefore, it is suggested that PKA-induced increase in Ca2+ influx through voltage-dependent Ca2+ channels may activate the Raf-1/MAP kinase cascade. Consistent with this idea, we have previously reported that stretching of cardiomyocytes induces MAP kinase activation, which is also partially

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dependent on trans-sarcolemmal influx of Ca2+ (Yamazaki et al., 1993). On the other hand, Yatani and Brown (1989) have also shown that there is another pathway coupling b-adrenergic stimulation with Ca2+ entry, independently of PKA. In the present study, however, pretreatment with a specific PKA inhibitor, H-89, completely abolished ISOinduced MAP kinase activation as well as Ca2+ influx in cardiac myocytes. These results also support the above-mentioned hypothesis. Concerning Ca2+-induced signaling pathways, it has been reported that intracellular Ca2+ is important for Ang II-induced MAP kinase activation in vascular smooth muscle cells (Eguchi et al., 1996; Lucchesi et al., 1996). Moreover, in the brain, PYK2, a member of the focal adhesion kinase family of nonreceptor tyrosine kinases, has been isolated, and has been shown to transmit the Ca2+ signal from a G protein-coupled receptor to the formation of the Shc-Grb2-Sos complex, thereby activating MAP kinases (Lev et al., 1995). The molecular mechanism by which an increase in Ca2+ influx leads to MAP kinase activation in cardiac myocytes awaits further investigation. Recently, Sadoshima et al. (1995) have shown that b-adrenergic agonists such as ISO do not activate MAP kinases and 90-kDa S6 kinase. The reason for the discrepancy between our and their results with regard to ISO-induced MAP kinase activation is not clear at present. However, Sadoshima et al. (1995) have shown that Ca2+ influx by Ca2+ ionophore A23187 activates MAP kinases and 90-kDa S6 kinase in cardiac myocytes and that Ca2+ is critical for Ang II-induced MAP kinase activation. Phorbol esters such as TPA induce the translocation of PKC protein to the membranous fraction and increase its activity (Henrich and Simpson, 1988). However, since TPA is not an endogenous PKC activator, there are some differences in function on cardiac myocytes between TPA and physiological PKC activators such as norepinephrine. It has been reported, that TPA activates PKC more strikingly and persistently than norepinephrine, and that long exposure to TPA down-regulates PKC, whereas long exposure to norepinephrine continuously up-regulates PKC (Simpson et al., 1991). To further examine the effects of PKA activation on PKC-induced hypertrophic responses, endogenous PKC activators such as Ang II, ET-1 and mechanical stretch were also examined in the present study. All of these PKC activators increased MAP kinase activities as reported previously (Yamazaki et al., 1993, 1995a, 1995b; Sadoshima et al., 1995). When these PKC activators were added to cardiomyocytes in the presence of FSK, more remarkable increases in

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MAP kinase activities were observed. These results suggest that PKA-activating agents such as b-adrenergic agonists would synergistically induce the activation of Raf-1/MAP kinase cascade and cardiac hypertrophy with PKC-activating agents, such as Ang II, ET-1 and mechanical stretch. The analysis of the protein synthesis data with the 2×2 factorial ANOVA showed a P value of 0.21 (Fig. 8). These results indicate that there is no evidence suggesting synergistic activation of protein synthesis between PKA and PKC. Moreover, we computed and found that statistical power of this test was approximately 45%. It was calculated that if the sample size is increased to 10, the statistical power is over 80%. Therefore, the conclusion that there was no statistically significant interaction may be purely due to a lack of statistical power. There remains a possibility that contaminating non-myocytes release growth-promoting factors into the culture medium by b-adrenergic stimulation and that the paracrine factors induce hypertrophic responses in cardiac myocytes (Long et al., 1993). Further, although we have previously shown that mechanical stretch of cardiac myocytes, but not of cardiac non-myocytes, increases the release of Ang II and ET-1, and that these factors induce the hypertrophic responses of cardiac myocytes (Yamazaki et al., 1995a, 1996), ISO-induced MAP kinase activation in cardiac myocytes was not inhibited by Ang II type-1 receptor blocker CV-11974 or ET type-A receptor blocker BQ123 (unpublished observation), suggesting that b-adrenergic stimulation did not induce the release of these factors from cardiomyocytes. However, we cannot rule out the possibility that other unknown factors, including transforming growth factor b, which are released from non-myocytes, might be involved in ISO-induced MAP kinase activation in cardiac myocytes. Norepinephrine, which is secreted from the nerve end and is a key player in the heart among catecholamines, activates PKA and PKC through band a-receptors, respectively. Although the stimulation of b- and a-receptors activates different kinases, such as PKA and PKC, respectively, the stimulation of both receptors often shows similar hypertrophic phenotypes (Bishopric and Kedes, 1991; Morgan and Baker, 1991). In the present study, we have shown that PKA also induces activation of Raf-1/MAP kinase cascade, which is important for hypertrophic responses in cardiac myocytes (Thorburn et al., 1994; Glennon et al., 1996). Collectively, intracellular signals evoked by activation of PKA and PKC may converge at Raf-1

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activation and this activation may induce hypertrophy via activating MAP kinases.

Acknowledgments We are indebted to Toru Suzuki for critical reading, and Fumiko Harima, Mika Ono and Makiko Iwata for the excellent technical assistance. We also acknowledge Chikuma Hamada, Pharmacoepidemiology, Faculty of Medicine, University of Tokyo, for statistical analysis. This work was supported by a grant-in-aid for scientific research and developmental scientific research from the Ministry of Education, Science, Sports and Culture of Japan, a grant from the Kanae Foundation of Research for New Medicine (to T.Y.), Japan.

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