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(Circulation. 1997;95:1260-1268.)
© 1997 American Heart Association, Inc.

Articles

Norepinephrine Induces the raf-1 Kinase/Mitogen-Activated Protein Kinase Cascade Through Both ₁- and ß-Adrenoceptors

Tsutomu Yamazaki, MD, PhD; Issei Komuro, MD, PhD; Yunzeng Zou, MD, PhD; Sumiyo Kudoh, MD, PhD; Ichiro Shiojima, MD, PhD; Yukio Hiroi, MD; Takehiko Mizuno, MD; Ryuichi Aikawa, MD; Hiroyuki Takano, MD; Yoshio Yazaki, MD, PhD

the Department of Medicine III (T.Y., I.K., Y.Z., S.K., I.S., Y.H., T.M., R.A., H.T., Y.Y.), University of Tokyo School of Medicine, and Health Service Center (T.Y.), University of Tokyo, Japan.

Correspondence to Issei Komuro, MD, PhD, Department of Medicine III, University of Tokyo School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. E-mail komuro-tky@umin.u-tokyo.ac.jp.

	Abstract

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Background Although norepinephrine induces cardiac hypertrophy by activating protein kinase A and C through ß- and ₁-adrenoceptors, respectively, protein kinase A has been reported to inhibit cell growth in many other cell types.

Methods and Results To elucidate the molecular mechanism of norepinephrine-induced hypertrophic responses, we examined the effects of protein kinase A and protein kinase C on the activities of raf-1 kinase and mitogen-activated protein (MAP) kinases and on protein synthesis rates using cultured cardiomyocytes of neonatal rats. Norepinephrine-induced activation of MAP kinases was partially inhibited by either an ₁-adrenoceptor blocker (prazosin) or a ß-adrenoceptor blocker (propranolol) and was completely abolished by both blockers. Both a ß-adrenoceptor agonist, isoproterenol, and an ₁-adrenoceptor agonist, phenylephrine, increased the activities of raf-1 kinase and MAP kinases and phenylalanine incorporation into proteins. Furthermore, isoproterenol and phenylephrine synergistically activated these kinases and protein synthesis. Similar synergistic activation of MAP kinases was observed when other protein kinase A–activating agents such as forskolin, dibutyryl cAMP, and isobutylmethylxanthine were used with a protein kinase C–activating agent at the same time. Chelation of extracellular Ca²⁺ completely abolished isoproterenol- and phenylephrine-evoked MAP kinase activation.

Conclusions Norepinephrine activates the raf-1 kinase/MAP kinase cascade through both ₁- and ß-adrenergic stimulation, and signaling pathways from the two receptors synergistically induce cardiomyocyte hypertrophy.

Key Words: hypertrophy • receptors, adrenergic, alpha • receptors, adrenergic, beta • kinase

	Introduction

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Because patients with left ventricular hypertrophy, even if asymptomatic, have increased mortality,¹ elucidating the molecular mechanisms of the development of cardiac hypertrophy is vital to reduce the incidence of heart disease. Many lines of evidence have suggested that two factors, mechanical stress² and neurohumoral factors,³ play an important role in inducing cardiac hypertrophy. Recently, it has been reported that these two factors promote cardiac hypertrophy in concert.^{4 5 6 7 8} NE has been reported to be a potent growth factor for cardiac myocytes.⁹ Because prolonged infusion of subhypertensive doses of NE in dogs induces an increase in the mass of the myocardium and the thickness of the left ventricular wall,^{10 11 12} it has been postulated that NE has a direct hypertrophic effect on cardiac myocytes without affecting afterload.¹⁰ There are two major subtypes, and ß, in NE receptors. Among -adrenoceptors, the ₁-adrenoceptor but not the ₂-adrenoceptor exists in cardiomyocytes (for a review, see Reference 13). Stimulation of the ₁-adrenoceptor activates phosphoinositide-specific phospholipase C via a G protein, Gq.¹³ Activated phospholipase C hydrolyzes phosphoinositide 4,5-bisphosphate into inositol 1,4,5-triphosphate and diacylglycerol, and the latter evokes activation of PKC.¹⁴ On the other hand, both subtypes of ß-adrenoceptors, ß₁- and ß₂-adrenoceptors, are expressed in the human heart and activate effector enzyme adenylyl cyclase through a different G protein, Gs. The activation of adenylyl cyclase results in the production of a second messenger, cAMP, which activates cAMP-dependent PKA.³ Simpson¹⁵ showed that NE induces hypertrophy in cultured cardiomyocytes of neonatal rats through ₁-adrenoceptor stimulation. On the other hand, Dubus et al¹⁶ reported that protein synthesis could be stimulated through ß- but not -adrenoceptors in cultured cardiac myocytes of adult rats, and Pinson et al¹⁷ showed that an increase in protein synthesis is induced by both ₁- and ß-adrenoceptor agonists in cardiac myocytes. Thus, the pathways through which NE induces cardiomyocyte hypertrophy are as yet controversial. Quite recently, many laboratories have reported that the activation of cAMP-dependent PKA inhibits cell growth by blocking the activation of the raf-1 kinase/MAP kinase cascade, which plays a critical role in cell proliferation.^{18 19 20 21 22 23 24 25 26} Although cardiac myocytes do not divide after birth, intracellular signals evoked by a variety of extracellular stimuli in cardiac myocytes are similar to those in other cell types that have proliferative ability.^{2 6} Thus, it is of interest to examine whether PKA inhibits raf-1 kinase/MAP kinase activation and cell growth (hypertrophy) in cardiac myocytes.

MAP kinases are serine/threonine protein kinases^{27 28} and can be activated by a variety of growth factors.²⁹ We have demonstrated³⁰ by using neonatal rat cardiomyocytes cultured on deformable silicone dishes that MAP kinases are activated by mechanical stress at the early stage of cardiomyocyte hypertrophy. It has been shown in many cell types that the proto-oncogene raf-1 kinase activates MAPKKs, which sequentially activate MAP kinases.^{31 32 33 34 35} We have also reported that the activities of raf-1 kinase and MAPKKs are sequentially increased by mechanical stretch in neonatal rat cardiomyocytes.³⁶ Furthermore, activated MAP kinases have been reported to be required for PHE-induced gene expressions and cardiomyocyte hypertrophy.^{37 38}

The goal of the present study was to determine which receptor, ₁ or ß, is important for generating hypertrophic signals including raf-1 kinase and MAP kinase activation. In this study, we show that NE induces activation of raf-1 kinase and MAP kinases followed by an increase in protein synthesis in cardiac myocytes of neonatal rats. The activation of these kinases is induced by cAMP-dependent PKA through ß-adrenoceptors as well as by PKC through ₁-adrenoceptors. Furthermore, ₁- and ß-adrenergic stimulation synergistically activate the raf-1 kinase/MAP kinase cascade, leading to cardiomyocyte hypertrophy.

	Methods

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[-³²P]ATP and [³H]phenylalanine were purchased from Du Pont-NEN Co, DMEM and FBS from Gibco BRL Co, a polyclonal antibody against raf-1 kinase from Santa Cruz Biochemistry, Inc, and a cAMP antagonist, RpcAMP, from Biolog. Other reagents were purchased from Sigma Chemical Co.

Culture of Cardiomyocytes
Primary cultures of cardiomyocytes were prepared from ventricles of 1-day-old Wistar rats as described previously,³⁹ basically according to the method of Simpson and Savion.⁴⁰ Cardiomyocytes were plated at a field density of 1x10³ cells/mm² on 35-mm culture dishes and cultured in DMEM with 10% FBS for the first 24 hours. The culture medium was replaced with DMEM containing 0.1% FBS. Cardiac myocytes were cultured in this low-serum condition for 48 hours, when cardiomyocytes continued beating. Cardiomyocytes were stimulated by various agents and lysed on ice with buffer A (25 mmol/L Tris-HCl, 25 mmol/L NaCl, 1 mmol/L sodium orthovanadate, 10 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 10 nmol/L okadaic acid, 0.5 mmol/L EGTA, and 1 mmol/L phenylmethylsulfonyl fluoride). A series of experiments was performed simultaneously with the same pool of cells to match for temperature, CO₂ content, or pH of the medium between stimulated and control cells.

MAP Kinase Assay in MBP-Containing Gels
MAP kinase activities were assayed with the use of MBP-containing gels as described previously.³⁰ In brief, immunoprecipitates obtained with a polyclonal antibody against MAP kinase Y91⁴¹ were electrophoresed on an SDS-polyacrylamide gel containing MBP. MAP kinases were denatured by guanidine/HCl and renatured in Tris-HCl solution. The gel was incubated with [-³²P]ATP, washed extensively, dried, and subjected to autoradiography.

Assay of raf-1 Kinase Activities
raf-1 kinase has been reported to have MAPKK phosphorylation activity.^{35 42} Activities were analyzed by measuring the phosphorylation of rMAPKK⁴³ as described previously.³⁶ The immunoprecipitates obtained with an anti-raf-1 kinase antibody⁴⁴ were incubated with a specific substrate rMAPKK and [-³²P]ATP in buffer B (25 mmol/L Tris-HCl [pH 7.4], 10 mmol/L MgCl₂, 1 mmol/L DTT, 40 µmol/L ATP, 2 µmol/L protein kinase inhibitor peptide [rabbit sequence, Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp], and 0.5 mmol/L EGTA) for 30 minutes at 25°C. After incubation, rMAPKK was collected and electrophoresed on an SDS-polyacrylamide gel. The gel was dried and subjected to autoradiography.

Amino Acid Uptake Into Cardiomyocytes
Cardiomyocytes were stimulated with ISO (10^-5 mol/L) and/or PHE (10^-5 mol/L) for 2, 12, or 24 hours. The relative amount of protein synthesis was determined by assessing the incorporation of the radioactivity into a trichloroacetic acid–insoluble fraction. The pulse-labeling method was used. One microcurie per milliliter [³H]phenylalanine was added to the culture medium 2 hours before harvest. After rinsing with PBS (10 mmol/L sodium phosphate and 0.85% NaCl, pH 7.4), cells were incubated for >20 minutes on ice in trichloroacetic acid. Total radioactivity in each dish was determined by liquid scintillation counting.

Quantification of Total Protein and DNA From Cardiac Myocytes
An aliquot of the cell suspension was solubilized in 1N NaOH at 60°C for 30 minutes. Total protein content was determined by use of the BCA protein assay reagent (Pierce Chemicals) with a bovine serum albumin standard according to the manufacturer's directions. To quantify DNA, cultured cardiomyocytes were scraped into Tris-buffered saline and centrifuged. The cells were lysed by adding SDS and proteinase K, and extraction of DNA was performed with phenol. The absorbance of the DNA was measured at 260 nm.

Statistics
Differences within groups in raf-1 activities, phenylalanine uptake, and protein/DNA ratio were compared by one-way ANOVA and Dunnett's t test. The accepted level of significance was P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
NE Activates MAP Kinases
It has been reported that both 42- and 44-kD MAP kinases are activated by mechanical stretch and humoral factors such as Ang II, ET, and fibroblast growth factors in cardiac myocytes.^{7 8 30 45} The activation of MAP kinases plays an important role in gene regulation during cardiac hypertrophy^{37 38} and is a sensitive and quantitative marker for hypertrophic responses of cardiac myocytes.³⁶ To elucidate the signaling pathways through which NE induces cardiomyocyte hypertrophy, we first examined whether NE induces MAP kinase activation (Fig 1). The intensity of the phosphorylated bands corresponding to 42- and 44-kD MAP kinases in MBP-containing gels was increased by the incubation with various concentrations (10^-10 to 10^-4 mol/L) of NE for 8 minutes (Fig 1A and 1B). There was a clear dose dependency in NE-induced MAP kinase activation. No significant increase in MAP kinase activity was observed at 10^-11 mol/L NE, and a slight but significant increase in MAP kinase activity was observed at 10^-10 mol/L compared with that of unstimulated cardiomyocytes. The EC₅₀ was 5x10^-8 mol/L, which was in good agreement with the EC₅₀ of NE-induced protein accumulation (2 to 20x10^-8 mol/L in neonatal rat heart cells).¹⁵ Maximal activation of MAP kinases was obtained at 10^-5 mol/L of NE (Fig 1B). Next, the time course of NE-induced MAP kinase activation was examined (Fig 2). The increase in MAP kinase activities induced by 10^-6 mol/L NE was detectable as early as 2 minutes, and the activity reached a peak at 8 to 12 minutes (Fig 2A and 2C). The activity decreased thereafter and returned to basal levels at 60 minutes after stimulation with NE (Fig 2B and 2C).

View larger version (32K): [in this window] [in a new window]   Figure 1. MAP kinase activation by NE in cardiac myocytes. Cardiac myocytes were stimulated by various concentrations of NE (10-11 to 10-4 mol/L) for 8 minutes, and the total cell lysates were subjected to the MAP kinase assay described in "Methods." A, Representative autoradiogram. B, Relative kinase activities were determined by scanning each band with a densitometer. Results are indicated as mean±SE of four independent experiments. Activities are expressed relative to those of 44-kD MAP kinase obtained in unstimulated cardiomyocytes.

View larger version (75K): [in this window] [in a new window]   Figure 2. Time course of MAP kinase activation induced by NE. Cardiomyocytes were stimulated by NE (10-6 mol/L) for the indicated periods of time. MAP kinase activities were determined by using MBP phosphorylation assay in the gel. A and B, Representative autoradiograms. C, Relative kinase activities were determined by scanning each band with a densitometer. Activities are expressed relative to those of 44-kD MAP kinase obtained in unstimulated cardiomyocytes. Results are indicated as mean±SE of three independent experiments.

NE Activates MAP Kinases Through Both ₁- and ß-Adrenoceptors
In cardiac myocytes, NE activates at least two types of receptors, ₁- and ß-adrenoceptors. To examine which receptor mediates MAP kinase activation by NE, we preincubated cardiac myocytes with a specific blocker of the ₁-adrenoceptor, prazosin (10^-5 mol/L), and/or a specific blocker of the ß-adrenoceptor, propranolol (10^-5 mol/L), for 30 minutes and then exposed cardiomyocytes to 10^-6 mol/L NE for 8 minutes. Both prazosin and propranolol partially suppressed NE-induced MAP kinase activation by 70% and 45%, respectively, and preincubation with both blockers completely abolished MAP kinase activation evoked by NE (Fig 3A and 3C).

View larger version (118K): [in this window] [in a new window]   Figure 3. MAP kinase activation by NE through both 1- and ß-adrenoceptors. After pretreatment with propranolol (10-5 mol/L) and/or prazosin (10-5 mol/L), cardiomyocytes were stimulated by NE (10-6 mol/L) for 8 minutes (A). ISO (10-5 mol/L) and/or PHE (10-5 mol/L) were added to the cultured cardiac myocytes as ß- and 1-adrenoceptor stimulators, respectively (B). MAP kinase activities were determined by using MBP phosphorylation assay in the gel. A and B, Representative autoradiograms. C, Relative kinase activities were determined by scanning each band with a densitometer. Activities are expressed relative to those of 44-kD MAP kinase obtained in unstimulated cardiomyocytes. Results are indicated as mean of two independent experiments.

The results with specific blockers in Fig 3A suggest that stimuli through both ₁- and ß-adrenoceptors are involved in activating MAP kinases in cardiomyocytes. We further examined whether stimulation of either adrenoceptor activates MAP kinases by using receptor-specific agonists such as ISO (a ß-adrenoceptor–specific agonist) and PHE (an ₁-adrenoceptor–specific agonist). The addition of ISO (10^-5 mol/L) or PHE (10^-5 mol/L) evoked MAP kinase activation by 3- to 4-fold (ISO, 3.4- and 3.5-fold; PHE, 4.4- and 4.0-fold, compared with control 42- and 44-kD MAP kinases, respectively) (Fig 3B and 3C). Simultaneous addition of both agents synergistically increased activities of 42- and 44-kD MAP kinases by 8.4- and 8.0-fold, respectively (Fig 3B and 3C). These results suggest that stimulation of both ₁- and ß-adrenoceptors is involved in NE-induced MAP kinase activation in a synergistic manner.

Both PKA and PKC Activate MAP Kinases in Cardiac Myocytes
Stimulation of ₁-adrenoceptor in cardiomyocytes activates phosphoinositide-phospholipase C, causing production of diacylglycerol, the endogenous activator of PKC.¹⁴ On the other hand, stimulation of ß-adrenoceptor induces activation of adenyl cyclase, which increases intracellular cAMP levels resulting in PKA activation.³ Therefore, NE is supposed to activate both PKA and PKC through different receptors. To examine the effects of PKA and PKC on MAP kinase activation, various kinds of PKA activators, such as FSK (10^-5 mol/L), dbcAMP (10^-5 mol/L), and IBMX (10^-5 mol/L), and a PKC activator, TPA, were added to cultured cardiac myocytes (Fig 4). All of these PKA activators and the PKC activator increased the activities of MAP kinases by 5-fold at the used concentrations, and the simultaneous addition of these PKA activators and the PKC activator showed much stronger activation of MAP kinases (11- to 12-fold) (Fig 4).

View larger version (53K): [in this window] [in a new window]   Figure 4. Synergistic activation of MAP kinases in cardiac myocytes stimulated by PKA and PKC. Cardiac myocytes were stimulated by FSK (10-5 mol/L), dbcAMP (10-5 mol/L), IBMX (10-5 mol/L), and/or TPA (10-9 mol/L) for 8 minutes, and MAP kinase activities were determined. The intensities of the 42-kD and 44-kD bands were measured by densitometric scanning of the autoradiogram. Values represent the mean of two independent experiments. The intensity of 44-kD MAP kinase of unstimulated cardiomyocytes was designated as 1.0.

To examine whether ISO and PHE increase the activities of MAP kinases through activation of PKA and PKC, respectively, cardiac myocytes were stimulated by either agonist after preincubation with a cAMP inhibitor, RpcAMP (10^-4 mol/L), for 10 minutes or TPA (10^-7 mol/L) for 24 hours. As shown in Fig 5, inhibition of PKA by RpcAMP and downregulation of PKC by long exposure with TPA completely abolished ISO- and PHE-induced MAP kinase activation, respectively. On the other hand, pretreatment with RpcAMP did not affect the PHE-evoked MAP kinase activation, and downregulation of PKC by TPA had no effects on the ISO-induced MAP kinase activation (data not shown). These results suggest the existence of two signaling pathways, ISOPKAMAP kinases and PHEPKCMAP kinases, in NE-induced hypertrophic responses of cardiac myocytes.

View larger version (37K): [in this window] [in a new window]   Figure 5. Involvement of PKA, PKC, and Ca2+ in ISO- or PHE-induced MAP kinase activation. After pretreatment with 10-4 mol/L RpcAMP for 10 minutes, 10-7 mol/L TPA for 24 hours, or 5x10-3 mol/L EGTA for 30 minutes, cardiomyocytes were stimulated by 10-6 mol/L ISO or 10-6 mol/L PHE for 8 minutes, and MAP kinase activities were measured as described in "Methods." The data represent the average amount by which the results were increased versus control (44-kD MAP kinase) values of three independent experiments (mean±SE).

Transsarcolemmal Ca²⁺ Influx Is Important for ISO- and PHE-Induced MAP Kinase Activation
It has been shown that ß-adrenergic agonists phosphorylate and activate voltage-dependent Ca²⁺ channels through PKA activation⁴⁶ and that ₁-adrenoceptor stimulation also induces Ca²⁺ influx through Ca²⁺ channels in cardiac myocytes.⁴⁷ Therefore, we examined the role of transsarcolemmal Ca²⁺ influx in MAP kinase activation through ₁- and ß-adrenoceptors (Fig 5). Cardiac myocytes were pretreated with 5x10^-3 mol/L EGTA for 30 minutes and then stimulated with 10^-6 mol/L PHE or ISO for 8 minutes. Although a Ca²⁺ channel agonist, BAYK8644, significantly increases the activity of MAP kinases in cardiac myocytes,³⁰ it did not activate MAP kinases under this condition (data not shown). Both PHE- and ISO-induced MAP kinase activations were completely blocked by pretreatment with EGTA (Fig 5). Moreover, pretreatment with 10^-6 mol/L nifedipine for 60 minutes completely abolished 10^-6 mol/L PHE-induced or 10^-6 mol/L ISO-induced MAP kinase activation (data not shown), suggesting that transsarcolemmal Ca²⁺ influx through voltage-dependent Ca²⁺ entry channels is important for PHE- and ISO-induced MAP kinase activation.

Ang II Is Not Involved in NE-Induced MAP Kinase Activation
Recently, we and others^{4 5 7 8} reported that autocrine Ang II and ET-1 are involved in mechanical stretch–induced hypertrophic responses in cardiac myocytes. Thus, we also examined whether such vasoactive peptides mediate NE-induced hypertrophic responses (Fig 6). Cardiomyocytes were stimulated with 10^-6 mol/L NE, 10^-5 mol/L ISO, or 10^-5 mol/L PHE for 8 minutes with or without pretreatment with an Ang II type-1 receptor–specific antagonist, CV11974 (10^-5 mol/L),⁵ for 30 minutes. This pretreatment completely abolishes 10^-5 mol/L Ang II–induced MAP kinase activation.⁷ NE, ISO, and PHE markedly increased the activities of both 42- and 44-kD MAP kinases even after pretreatment with CV11974 (Fig 6). In addition, an ET type-A receptor–specific antagonist, BQ123 (10^-5 mol/L),⁸ also showed no inhibitory effects on NE-, ISO-, or PHE-induced MAP kinase activation (data not shown). Collectively, it was suggested that MAP kinase activation induced by ₁- and ß-adrenergic agonists is independent of Ang II and ET-1.

View larger version (31K): [in this window] [in a new window]   Figure 6. Lack of involvement of Ang II in NE-, ISO-, and PHE-induced MAP kinase activation. After pretreatment with 10-6 mol/L CV11974 for 30 minutes, cardiac myocytes were stimulated with 10-6 mol/L NE, 10-6 mol/L ISO, or 10-6 mol/L PHE for 8 minutes. A representative autoradiogram from two independent experiments is shown.

ISO and PHE Synergistically Activate raf-1 Kinase and Increase Protein Synthesis in Cardiomyocytes
The direct upstream enzymes of MAP kinases are MAPKKs,^{31 32 33} and these can be phosphorylated and activated by raf-1 kinase.³⁵ raf-1 kinase has been reported to be a target of PKA during PKA-induced inhibition of cell proliferation.^{19 23 25} Therefore, we examined the effects of PKA on raf-1 kinase in cardiac myocytes (Fig 7). Like Ang II and ET-1,^{7 8} a PKC-activating agent, PHE (10^-5 mol/L), increased activation of raf-1 kinase in cardiomyocytes by 251% (Fig 7A and 7B). Moreover, a PKA-activating agent, ISO (10^-5 mol/L), also increased the activity of raf-1 kinase by 263%. Interestingly, ISO and PHE acted in a synergistic manner on raf-1 kinase activation. Simultaneous addition of ISO (10^-5 mol/L) and PHE (10^-5 mol/L) increased activation of raf-1 kinase by 716% (Fig 7A and 7B).

View larger version (98K): [in this window] [in a new window]   Figure 7. Synergistic activation of raf-1 kinase by ISO and PHE in cardiac myocytes. A, Cardiac myocytes were exposed to ISO (10-5 mol/L) and/or PHE (10-5 mol/L) for 2 minutes. raf-1 Kinase activities were measured as described in "Methods." B, The intensities of 74-kD bands were measured by densitometric scanning of the autoradiogram. Data are presented as mean±SE of four independent experiments compared with control (*P<.05 versus control).

A similar synergistic effect was also observed on the level of protein synthesis (Fig 8). Although neither ISO (10^-5 mol/L) or PHE (10^-5 mol/L) increased phenylalanine incorporation at 12 and 24 hours after treatment, the simultaneous addition of ISO and PHE significantly increased amino acid uptake at 12 (135%) and 24 hours (195%) (Fig 8A). We also assayed phenylalanine-uptake/DNA-content ratios. Treatment for 24 hours by ISO, PHE, and ISO plus PHE increased the phenylalanine-incorporation/DNA ratio by 137%, 151%, and 196%, respectively. The degree of increase was almost the same as that of phenylalanine uptake, suggesting that there was no change in DNA content in cardiac myocytes with these treatments. Furthermore, total protein content/DNA ratios were obtained in cardiomyocytes after stimulation with ISO (10^-5 mol/L) and/or PHE (10^-5 mol/L). After 24 hours of stimulation, both ISO and PHE significantly increased the protein/DNA ratios to 111% and 107%, respectively, compared with no treatment. Simultaneous stimulation by ISO and PHE showed an increase in the ratio to 118% (Fig 8B). Hence, PKA and PKC synergistically activated the raf-1 kinase/MAP kinase cascade and induced cardiomyocyte hypertrophy.

View larger version (108K): [in this window] [in a new window]   Figure 8. Synergistic effect of ISO and PHE on phenylalanine uptake and protein synthesis in cardiac myocytes. A, Cardiomyocytes were stimulated by ISO (10-5 mol/L) and/or PHE (10-5 mol/L) for 2, 12, or 24 hours. Cardiomyocytes were pulse labeled by [3H]phenylalanine (1 µCi/mL) for 2 hours before harvest. Total radioactivity of incorporated [3H]phenylalanine into proteins was determined by liquid scintillation counting. The relative phenylalanine uptake of stimulated myocytes compared with that of unstimulated myocytes is shown as mean±SE of four independent experiments. B, Cardiac myocytes were exposed to ISO (10-5 mol/L) and/or PHE (10-5 mol/L) for 24 hours. Total protein contents were measured by use of the BCA protein assay reagent and corrected by DNA contents. Data are shown as mean±SE of four independent experiments compared with control. *P<.05 versus unstimulated myocytes.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Many reports^{10 11 12 15 16 17} have demonstrated that NE strongly induces cardiac hypertrophy. Results in the present study showed that NE activates protein kinases such as raf-1 kinase and MAP kinases and increases protein synthesis in cultured cardiomyocytes of neonatal rats. ß-Adrenoceptor stimulation as well as ₁-adrenoceptor stimulation was involved in NE-induced MAP kinase activation. It is noteworthy that unlike in other cell types, both PKC activation and PKA activation increased the activities of raf-1 kinase and MAP kinases in cardiac myocytes and induced cell growth. Moreover, PKA-activating agents synergistically activated MAP kinases and raf-1 kinase and induced cardiomyocyte hypertrophy with PKC-activating agents. It has been reported that activation of MAP kinases is required for hypertrophic responses.^{37 38} Therefore, hypertrophic signals evoked by PKA and PKC activation may converge at the raf-1 kinase/MAP kinase cascade and may induce a variety of hypertrophic responses. The presence of multiple signal-transduction pathways causing synergistic activation of hypertrophic responses may be favorable for cardiomyocytes to quickly and efficiently respond to external stimuli such as neurohumoral factors and mechanical overload.

Simpson⁹ reported that NE stimulates hypertrophy of neonatal rat cardiomyocytes in culture through ₁-adrenoceptors but not through ß-adrenoceptors. Simpson¹⁵ also showed that an increase in beating rate requires both ₁- and ß₁-adrenoceptor activation. From these results, he and his colleagues⁴⁸ have argued that the hypertrophic effect of ß-adrenoceptor agonists could be secondary to the release of catecholamines that have ₁-stimulatory properties. It has also been reported by Clark et al⁴⁹ that ß-adrenergic activation produces cardiomyocyte hypertrophy by activating the beating of cardiac myocytes. On the other hand, Bishopric and Kedes⁵⁰ showed that NE-induced hypertrophy, as well as contractility and skeletal -actin gene expression, is mediated by ß-adrenoceptor in high-density cultures (1x10³ cells/mm²). They also reported that when cardiomyocytes are plated at a low density (3x10² cells/mm²), minimizing cell contact, NE-induced skeletal -actin gene expression and hypertrophy are mediated by ₁-adrenoceptor, and they postulated that factors related to cell communication influence the pathways mediating NE-regulated gene transcription during cardiomyocyte hypertrophy.⁵⁰ In the present study, NE evoked hypertrophic responses in cardiomyocytes cultured at a high density (1x10³ cells/mm²) through both ₁- and ß-adrenoceptors. ₁-Adrenergic activation and ß-adrenergic activation synergistically increased protein synthesis, suggesting that the pathways through which ₁- and ß-adrenergic agonists induce hypertrophy are different. Moreover, because an ₁-adrenoceptor antagonist, prazosin, did not inhibit ISO-induced hypertrophy (data not shown), the cardiomyocyte hypertrophy induced by ß-adrenoceptor agonists may not be secondary to the release of ₁-stimulatory molecules. Although the reason for these differences among laboratories is not clear at present, Decker et al⁵¹ reported that by using cultured ventricular myocytes of rabbits, NE or the combination of ISO and PHE induces greater hypertrophic responses than ISO or PHE alone, which is consistent with our results.

Unlike in many other cell types, PKA did not inhibit but activated the raf-1 kinase/MAP kinase cascade in cardiac myocytes in the present study. PKA has been shown to inhibit activation of the raf-1 kinase/MAP kinase cascade in various cell types,^{18 19 20 21 22 23 24 25 26} and several possible mechanisms have been proposed. Phosphorylation of raf-1 kinase on serine 43 by PKA reduces the affinity of raf-1 kinase for ras,¹⁸ or phosphorylation of the small G protein, rap1A (also known as Krev-1), or Sos by PKA inhibits raf-1 kinase activation by ras.^{20 22 24} It has also been reported that PKA phosphorylates the guanine exchange factor CDC25 and that phosphorylated CDC25 is predominantly localized in the cytosol and thus cannot activate ras.²¹ Inhibitory effects of PKA on the ras-related pathway conceivably cause the reduction of raf-1 kinase/MAP kinase activities. Recently, it has been demonstrated that in neonatal rat ventricular cardiomyocytes, Gq is essential to ₁-adrenergic stimulation of hypertrophic responses, such as production of phosphoinositide hydrolysis, atrial natriuretic factor gene expression, and an increase in cell size, but that ras is not required for such Gq-mediated events.⁵² We observed that Ang II activates the raf-1 kinase/MAP kinase cascade through PKC activation but not through ras.⁵³ These observations suggest that PKA does not inhibit PKC-induced raf-1 kinase/MAP kinase activation in cardiac myocytes because ras, a target of PKA, may not be involved in this signaling pathway.

It has been reported previously⁵⁴ that ISO does not increase MAP kinase activities in cultured cardiac myocytes. Conversely, we observed in the present study that ISO significantly activated MAP kinases depending on Ca²⁺ influx. Although we do not know why there are discrepancies, we think that our results are reasonable on the basis of the following evidence. ß-Adrenoceptor agonists have been shown to phosphorylate and activate voltage-dependent Ca²⁺ entry channels via activation of PKA and to increase intracellular Ca²⁺ levels.⁴⁶ Ca²⁺ influx induced by a Ca²⁺ ionophore, A23187, or a Ca²⁺ channel opener, BAYK8644, activated MAP kinases and 90-kD S6 kinase in cardiac myocytes (Reference 54 and T.Y., MD, et al, unpublished observations, 1996). These results suggest that ISO would activate MAP kinases in cardiac myocytes by increasing Ca²⁺ influx through activated Ca²⁺ channels. Actually, MAP kinase activation by ISO was completely blocked by pretreatment with EGTA (Fig 5). In accordance with our results, Bishopric et al⁵⁵ reported that the regulation of skeletal -actin gene expression during ß-adrenoceptor–mediated cardiomyocyte hypertrophy requires both G protein–coupled Ca²⁺ entry channel activation and resultant Ca²⁺ release from sarcoplasmic reticulum.

How does a rapid activation and inactivation of the raf-1 kinase/MAP kinase pathway contribute to cardiac cellular hypertrophy? Many lines of evidence^{56 57} suggest that MAP kinases are key molecules in intracellular signal transduction and play an essential role in cellular proliferation and differentiation in many cell types. Although there is a report⁵⁸ showing that there are discrepancies between MAP kinase activation and hypertrophic responses and that interfering mutants of MAP kinases do not block PHE-induced activation of the atrial natriuretic peptide promoter, activation of MAP kinases has been reported to be necessary for PHE-induced transactivation of immediate-early genes such as c-fos and fetal-type genes such as atrial natriuretic peptide in cardiac myocytes.³⁷ Glennon et al³⁸ demonstrated that depletion of MAP kinases using an antisense oligonucleotide approach completely blocks the increase in myocyte size and sarcomerogenesis in response to PHE. It has recently been reported^{59 60} that MAP kinases activate phosphorylated heat- and acid-stable protein regulated by insulin (PHAS-I), resulting in its dissociation from a cap-binding protein, elF-4E, which initiates and increases cap-dependent protein synthesis. Quite recently, elF-4E has been demonstrated to be phosphorylated by electrically stimulated contraction of adult feline cardiomyocytes in vitro and by short-term pressure overload on canine hearts in vivo.⁶¹ These observations suggest that MAP kinases may play an important role in cardiac hypertrophy. We^{30 36 62} previously showed that mechanical stretch of cardiomyocytes activates the protein kinase cascade of phosphorylation and induces specific gene expression and increased protein synthesis. The serial activation of protein kinases and immediate-early gene expressions may trigger long-term events such as the increase in protein synthesis and the expression of fetal-type genes.

Many signaling molecules other than MAP kinases, such as PKA, PKC, and receptor tyrosine kinases, are also candidates for inducing hypertrophic responses. Bishopric and Kedes⁵⁰ indicated that ISO potently induces expression of skeletal -actin gene, possibly through activation of PKA, and Kariya et al⁶³ showed that transcription of the ß-myosin heavy chain isogene is stimulated by PKC. With respect to receptor tyrosine kinases, many growth factors, such as fibroblast growth factors and insulin-like growth factor, have been shown to induce specific gene expressions, such as expressions of ß-myosin heavy chain, skeletal -actin, and atrial natriuretic peptide genes, which resemble the expressions induced by mechanical overload in vitro and in vivo.⁶⁴ As shown in many reports and in the present study, MAP kinases may also contribute to these kinase-induced hypertrophic responses as downstream kinases. We are now investigating the involvement of MAP kinases in these pathways by using a dominant negative MAP kinase construct and MAP kinase phosphatase. Recently, we⁶⁵ observed in cardiac myocytes that mechanical stretch and Ang II induced the activation of JNK, a divergent molecule of MAP kinase families. Because JNK has been reported to phosphorylate and activate c-jun, this kinase may also be involved in gene expression during cardiac hypertrophy. In addition, a newly isolated cytokine, cardiotrophin-1, has been shown to induce cardiomyocyte hypertrophy through the gp130 signaling pathway.^{66 67} Stimulation of gp130 activates MAP kinases,⁶⁷ and it would be interesting to examine the role of Janus kinase signal transducers and activators of the transcription pathway in cardiac gene regulation. Understanding the regulatory mechanisms of protein kinases, including MAP kinases, may pave the way for treating cardiac hypertrophy.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II dbcAMP = dibutyryl cAMP ET = endothelin FBS = fetal bovine serum FSK = forskolin G protein = guanine nucleotide-binding regulatory protein IBMX = isobutylmethylxanthine ISO = isoproterenol JNK = c-jun aminoterminal kinase MAP = mitogen-activated protein MAPKK = mitogen-activated protein kinase kinase MBP = myelin basic protein NE = norepinephrine PHE = phenylephrine PKA = protein kinase A PKC = protein kinase C rMAPKK = recombinant mitogen-activated protein kinase kinase fused to glutathione S transferase TPA = 12-O-tetradecanoylphorbol-13-acetate

	Acknowledgments

 
We acknowledge Banyu Pharmaceutical Co Ltd (Tokyo, Japan) for providing BQ123 and Takeda Chemical Industries, Ltd (Osaka, Japan) for providing CV11974. This work was supported by a grant-in-aid for scientific research, developmental scientific research and scientific research on priority areas from the Ministry of Education, Science, Sports and Culture; a grant from the Japan Cardiovascular Foundation; the Sankyo Life Science and the Study Group of Molecular Cardiology (to Dr Komuro), Japan; and a research grant from the Japan Heart Foundation (to Dr Yamazaki). We are indebted to Toru Suzuki for critical reading of the manuscript and Fumiko Harima, Makiko Iwata, and Mika Ono for excellent technical assistance.

Received August 4, 1996; revision received September 25, 1996; accepted October 5, 1996.

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