Extracellular ATP Induces Immediate-Early

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Extracellular ATP Induces Immediate-Early
1034

             Extracellular ATP Induces Immediate-Early
            Gene Expression but Not Cellular Hypertrophy
                    in Neonatal Cardiac Myocytes
              Jing-Sheng Zheng, Marvin         0.   Boluyt, Lydia O'Neill, Michael T. Crow, Edward G. Lakatta

  Abstract It is well-documented that norepinephrine (NE)            attenuated the ATP-stimulated increase in c-fos expression. In
induces the expression of immediate-early genes (IEGs), such         contrast, NE did not increase cytosolic Ca 2+ in quiescent
as c-fos, c-jun, and jun-B, in cultured neonatal heart cells and     myocytes, and pretreatment with BAPTA-AM did not inhibit
leads to cell growth without cell division (ie, hypertrophy).        the NE-stimulated increase in c-fos gene expression. Further-
Although purinergic receptors activated by ATP are present           more, although NE markedly increased [14C]phenylalanine
on cardiac myocytes and ATP is coreleased with NE from               incorporation into protein and myocyte hypertrophy measured
sympathetic nerve endings within the heart, the potential role       by cell size, ATP did not. These results demonstrate that
of the purinergic system in the cascade of events that leads to      stimulation of purinergic receptors by ATP activates IEGs via
cardiac hypertrophy is unknown. We report in the present             a Ca2'-dependent pathway in cardiac myocytes that differs
study that stimulation of purinergic receptors by micromolar         from the NE-stimulated activation of these genes. Since ATP
concentrations of extracellular ATP increased the levels of          activated IEG expression but did not increase the rate of
c-fos and jun-B mRNA as well as FOS and JUN-B proteins in            protein synthesis nor augment cell size, we conclude that the
neonatal cardiac myocytes. The magnitude of response to
micromolar ATP was comparable to that elicited by NE. The            activation of c-fos and jun-B expression is not sufficient to
increase in IEG expression induced by ATP was preceded by            induce cellular hypertrophy in neonatal cardiac myocytes.
a rapid transient increase in cytosolic Ca`. Pretreatment of         (Circ Res. 1994;74:1034-1041.)
myocytes with the intracellular Ca` chelator BAPTA-AM                  Key Words * purinergic receptors * c-fos * jun-B *
prevented the ATP-stimulated increase in cytosolic Ca>' and          cardiac myocytes

   Q     n nerve   stimulation, ATP is coreleased with              The responses to a,-adrenergic receptor stimulation
           norepinephrine (NE) from the endings of the              include induction of the immediate-early genes (IEGs)
           sympathetic nerves that innervate the heart.             c-fos, c-jun, jun-B, and Egr-1, followed by selective
Corelease probably accounts for the majority of extra-              upregulation of early developmental isogenes (eg, car-
cellular ATP found in the heart.1 Extracellular ATP,                diac /8-myosin heavy chain and skeletal a-actin), in-
however, can also be released by platelet degranulation             creased synthesis and release of atrial natriuretic factor
and from anoxic myocytes or damaged cells.2-5 When                  (ANF) from ventricular myocytes, and an increase in
ATP is released, it activates purinergic receptors in the           protein content and cell size of individual ventricular
cardiac cell membrane and leads to a number of bio-                 myocytes.14'17 These descriptive studies have led to the
chemical changes in the heart. Stimulation of P2-puri-              speculation that the induction of IEGs has a critical role
nergic receptors by ATP increases [Ca"] in freshly                  in the activation of the ANF gene and the increase in
isolated adult rat ventricular myocytes.6-1/ Extracellular          cell size.
ATP also increases phosphoinositide turnover in adult                  There is no current evidence that ATP invokes
ventricular myocytes12 and has been shown to stimulate              changes that could positively or negatively influence the
phosphoinositide hydrolysis and inhibit accumulation of             hypertrophic process stimulated by NE. Our purpose
cAMP in cultured ventricular myocytes isolated from                 was to determine whether extracellular ATP constitutes
fetal mice.13                                                       a stimulus sufficient to induce changes in the pattern of
   There is a growing body of evidence that NE released             IEG expression associated with cardiac myocyte hyper-
from sympathetic nerve endings may be an important                  trophy and, if so, whether ATP, like NE, stimulates
nonhemodynamic stimulus for cardiac growth.14 In neo-               cardiac hypertrophy. We used neonatal ventricular myo-
natal cardiac myocytes, NE induces cardiac hypertrophy              cytes to study the effects of extracellular ATP on
primarily via activation of a1-adrenergic receptors.'5-18           expression of the IEGs c-fos and jun-B and on the rate
                                                                    of [14C]phenylalanine incorporation into myocyte pro-
  Received April 9, 1993; accepted January 26, 1994.                teins. Our results indicate that both ATP and NE
  From the Laboratory of Cardiovascular Science, Gerontology        increase the expression of IEGs in neonatal myocytes
Research Center, National Institute on Aging, National Institutes   and suggest that the effect of ATP is mediated via a
of Health, Baltimore, Md.                                           Ca'+-dependent pathway, distinct from that stimulated
  Correspondence to Dr Michael T. Crow, Laboratory of Cardio-       by NE. Additionally, NE increased protein synthesis
vascular Science, Gerontology Research Center, National Insti-
tute on Aging, National Institutes of Health, 4940 Eastern Ave,     and cell size in cardiac myocytes, whereas ATP did not.
Baltimore, MD 21224.                                                This discordance between the induction of IEGs and

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Zheng et al ATP Dissociates IEG Expression and Myocyte Hypertrophy                 1035

cardiac hypertrophy by ATP suggests that activation of             RNA Analysis
c-fos or jun-B is not sufficient to stimulate myocyte                 Total RNA was isolated from cultured ventricular myocytes
hypertrophy.                                                        as described by Chirgwin et al.21 After denaturation with
                                                                    formamide and formaldehyde, equal amounts of RNA (10 j,g
                Materials and Methods                               per lane) were size-fractionated by electrophoresis through a
Cell Culture                                                        1% agarose gel containing 3% formaldehyde. RNA was elec-
                                                                    trophoretically transferred to a nylon (Duralon) membrane at
   Neonatal ventricular myocytes were cultured as previously        5 V/cm, cross-linked by ultraviolet irradiation (120 mJ), and
described by Iwaki et al9 with some modifications. Hearts           then hybridized at 62°C with either a 32P-radiolabeled ribo-
from 1- to 3-day-old Wistar rats were removed, and the              probe for rat c-fos or a cDNA probe for mouse jun-B22 or at
ventricles were trisected and then digested with collagenase        60°C with an end-labeled oligonucleotide to ribosomal 18S as
type 11 (0.5 mg/mL, Worthington) and pancreatin (0.6 mg/mL,         described by Church and Gilbert.23 Hybridization intensity
Sigma Chemical Co) for 20 minutes at 37°C. The cell super-          was quantified in disintegrations per minute directly from blots
natant was collected by centrifugation, and the pellet was          with a Betascope 603 (Betagen Corp). The signal from each
resuspended in 100% horse serum. The above steps were               sample was normalized to the signal obtained with a synthetic
repeated 7 to 10 times until the hearts were completely             oligonucleotide probe specific for the 18S ribosomal RNA.
digested. The cells were combined, washed, and centrifuged
through a discontinuous Percoll gradient of 1.050, 1.062, and       Probes
1.082 g/mL, respectively. The band at the 1.062/1.082 interface       The probe used to detect c-fos mRNA was an "500-bp
was used for myocyte culture. Forty-eight hours after the           ['2P]CTP-labeled riboprobe synthesized from the vector
initial plating, the purity of such cultures was >95% myocytes      pCRcfos(500) by in vitro RNA transcription.24 pCRcfos(500)
as assessed by immunohistochemistry with antibodies to sar-         encodes "500 bp of the conserved coding region of the rat
comeric myosin (MAb F5920) or sarcomeric actin (Sigma). The         c-fos cDNA and was generated by a polymerase chain reaction
myocytes were resuspended in culture medium containing 4:1          (PCR) by use of primers complementary to the published
Dulbecco's modified Eagle's medium (DMEM)/medium 199                sequence25 and cloned into the vector PCRII according to the
(M199, GIBCO Laboratories) supplemented with 10% horse              manufacture's instructions (In Vitrogen Corp). The complete
serum, 5% fetal bovine serum, and penicillin (100 U/mL)/            sequence of the cDNA insert derived by PCR was verified by
streptomycin (100 ,ug/mL, GIBCO). Myocytes were then                the dideoxy-mediated chain-termination method.26 The probe
plated on gelatin-coated 100-mm plastic tissue culture plates       used to detect jun-B was a 1.917-kb EcoRI fragment cDNA
(Costar) at a density of 1.08x 105 cells per square centimeter      from clone 465.2 (American Type Culture Collections).27 The
for RNA analyses and on gelatin-coated glass or plastic             probe for 18S RNA was a synthetic end-labeled oligonucleo-
eight-chamber culture slides for immunocytochemistry studies.       tide as previously described.28
Plating efficiency was such that at the time of study, cell-cell
contact was minimal. Addition of ATP did not increase the           Measurement of       [Ca2"]i
percentage of cultured neonatal myocytes exhibiting contrac-           Neonatal cardiac myocytes were cultured on coverslips
tion either in the short term (24 hours). NE did not increase the number of cells con-           incubation solution was changed from DMEM/M199 to a
tracting in short term, but it did increase the proportion of       HEPES buffer solution containing (mmol/L) NaCl 137, KCl
myocytes contracting from 80% after 24         5.4, CaCl2 1.5, MgSO4 1.2, and HEPES 20 at pH 7.4. Myocytes
hours of incubation. Myocyte cultures were maintained in            were then loaded with indo 1-AM (25 ,mol/L) for 20 to 30
serum-containing medium at 37°C for 24 hours in humidified          minutes at 37°C. Indo 1 fluorescence was excited at 350 nm.
air with CO2 sufficient to maintain the pH at 7.3. The medium       Paired photomultipliers collected indo 1 emission by simulta-
was then changed to serum-free DMEM/M199. All experi-               neously measuring spectral windows of 391 to 434 and 457 to
ments were initiated 24 hours after the change to serum-free        507 nm selected by bandpass interference filters.29 Since indo
culture medium.                                                     1-AM may be partially hydrolyzed and the Ca21 indicator may
                                                                    be compartmentalized in mitochondria or other organelles,
Immunocytochemistry Studies                                         calibration of [Ca2+]i in neonatal myocytes remains uncertain.
   After incubation with experimental reagents (such as ATP         The 410/490-nm ratio of emitted fluorescence was taken as an
and NE) for the designated periods of time, chamber slides          index of [Ca2"]j, as described previously.29 [Ca2+]i measure-
were rinsed twice with phosphate-buffered saline (PBS) and          ments were performed at room temperature.
fixed with 3.7% formaldehyde-PBS for 5 minutes at room
temperature. Cells were then washed three times with PBS,           [4C]Phenylalanine Incorporation Experiments and
permeabilized with 1% Triton X-100 in PBS for 15 minutes,           Cell-Size Measurement
washed three additional times in PBS, and dehydrated with             The rate of protein synthesis was assessed as described by
100% ethanol for 5 minutes. After three PBS washes, cells           Simpson,'0 with the following modifications: After cell isola-
were incubated with 2% bovine serum albumin (BSA) in PBS            tion, cells were cultured in serum-containing medium and
for 30 minutes. Incubation with the primary antibody (5             plated on six-well tissue culture plates at a density of 5x10'
,ug/mL anti-rabbit FOS IgG, Oncogene Science) in 2% BSA             per well (5 x 104 cells per square centimeter). Several experi-
was carried out overnight at 4°C. The next day, the slide           ments were done at a density range from 1 x 104 to 1 x 10' cells
chambers were washed three times with PBS for 5 minutes             per square centimeter. Similar results were obtained. To
each. Cells were then incubated with a secondary antibody (1        inhibit fibroblast growth in the culture, lX 10-4 mol/L bro-
,ug/mL anti-rabbit IgG conjugated with alkaline phosphatase,        modeoxyuridine (BrdU, Boehringer Mannheim) was added to
Promega) for 4 hours at room temperature and then washed            the cell culture the first day of cell isolation and was main-
three times with PBS. Cells were incubated with alkaline            tained in the culture media throughout the experiment. Cul-
phosphate buffer (mmol/L: NaCl 100, MgCl1 5, and Tris-HCl           tures were essentially free of fibroblasts as assessed by cell
100, pH 9.5) containing substrates, nitro blue tetrazolium (330     staining with antibodies that recognize the myocyte-specific
,ug/mL), and 5-bromo-4-chloro-3-indolyl phosphate (165 ,ug/         sarcomeric myosin (monoclonal antibody F59) and sarcomeric
mL) for 15 minutes. Photomicrographs were made at an                actin (Sigma). Serum-containing medium was removed and
original magnification of x400.                                     replaced with serum-free medium supplemented with 1

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1036      Circulation Research Vol 74, No 6 June 1994

gmol/L insulin, 5 gmol/L transferrin, and 10 nmol/L selenium
24 hours after plating. After an additional 24 hours, ['4C]phe-      A c-fos                  -
                                                                                                            *@                   C jun-B -"-
nylalanine (0.1 gCi/mL) and either ATP or NE were added to
the culture medium. Cells were analyzed for changes in cell
size or protein synthesis 3 days later. We had noticed that ATP                   18 S185--
                                                                                         0    -
                                                                                                                                         18 S -*             fl
may be degraded during long-term incubation in culture
medium. High-performance liquid chromatography measure-                                                   1-EM Z
                                                                                                                                                              D   K

ment indicated that ATP was stable for 6 hours of incubation         D                                U
and was partially degraded (
Zheng et al ATP Dissociates IEG Expression and Myocyte Hypertrophy         1037

                                                                                                 expression, we determined whether ATP increased the
                         5-                                                                      [Ca2"], in cultured neonatal myocytes. On addition of
                                                                                                 100 ,umol/L ATP, quiescent cultured neonatal cardiac
                                                                                                 myocytes exhibited a transient increase in [Ca2+]i fol-
                         4-
                                                                                  (n=9)          lowed by a sustained increase of a lesser magnitude (Fig
                                                                                                 6A). The sustained elevation of [Ca 2+] varied in dura-
                   Z     3
                                                                                                 tion, but in some cases, it lasted as long as 10 minutes.
                                                                                                 NE did not elicit an increase in [Ca 2+] in quiescent
                   E                                                                             cells, whereas ATP increased [Ca2"]I in the same myo-
                                                                                                 cytes (Fig 6B). Pretreatment of neonatal myocytes with
                   ,,    2-
                                                                                                 BAPTA-AM (10 ,umol/L) for 30 minutes prevented the
                                                                                                 ATP-induced increase of [Ca2"], (Fig 6C).
                         1-
                                                                                                 ATP-Induced Elevation in c-fos mRNA Is Mediated
                                                                                                 by a Ca2'-Dependent Pathway
                        .0-
                                                                                                    To determine whether a Ca2'-dependent pathway is
                                                 I                         l
                               0                -7      -6     -5          4       -3
                                                                                                 required for the ATP-induced increase in the expression
                                                                                                 of IEGs, we used Northern blot analysis to determine
                              log [ATP] (M)                                                      whether the intracellular Ca2' chelator BAPTA-AM
FIG 3. Graph showing the effect of different concentrations of                                   modulates the ATP-induced c-fos expression. Preincuba-
ATP on activation of c-fos mRNA. Serum-free cell cultures were                                   tion of myocyte cultures with 10 gmol/L BAPTA-AM for
treated with different concentrations of ATP for 30 minutes. Cells                               30 minutes inhibited the ATP-induced expression of c-fos
were harvested, and total mRNA was extracted. c-fos mRNA
levels were measured as described in Fig 1.                                                      but not the NE-induced c-fos expression (Fig 7). These
                                                                                                 data suggest that NE and ATP induce the expression of
                                                                                                 IEGs via different intracellular mechanisms and that the
Extracellular ATP Increases FOS Protein Levels                                                   increase in [Ca2"]i by ATP is required to maximally
  To determine whether the increase in c-fos mRNA                                                induce IEG expression in neonatal cardiac myocytes.
was followed by an increase in FOS protein, cultures
were stained with an anti-FOS antibody. The addition
                                                                                                 Effect of ATP on [4C] Phenylalanine Incorporation
of ATP (100 ,umol/L, Fig 5C) to cultured cardiac                                                 Into Myocyte Proteins and on Myocyte Cell Size
myocytes, like NE (Fig 5D), resulted in an increase of                                              Since both ATP and NE increase the expression of
FOS protein within the nuclear compartment. In-                                                  IEGs, we investigated whether ATP also increases
creased nuclear staining was observed as early as 45                                             neonatal myocyte protein synthesis as does NE. Incor-
minutes after addition of ATP to myocyte cultures and                                            poration of [14C]phenylalanine into TCA-precipitable
increased up to 2 to 3 hours. The effect of ATP was dose                                         protein was measured after incubation with experimen-
dependent, with the maximum observable increase in                                               tal reagents for 3 days. NE increased ['4C]phenylalanine
nuclear staining at 100 ,umol/L (data not shown).                                                incorporation over the 3-day period by 130% over the
                                                                                                 control value, whereas extracellular ATP did not in-
Effects of ATP on [Ca"2];                                                                        crease ['4C]phenylalanine incorporation (Fig 8). To
  Extracellular ATP is known to increase [Ca 2+]i in                                             eliminate fibroblast proliferation, these experiments
adult cardiac myocytes.6-11 As a first step to investigate                                       were performed in the presence of 100 gmol/L BrdU.
intracellular signal transduction pathways responsible                                           The addition of BrdU did not affect the ability of
for the effects of extracellular ATP                                      on   c-fos and jun-B   myocytes to activate IEG expression in response to ATP
                                                                                                 or NE (data not shown). To alleviate concern regarding
               6
                                                                                                 degradation of ATP or accumulation of ATP-degrada-
                                                                                                 tion products, additional experiments were conducted
               5
                                       P   <   0.05                                              in which ATP was replenished or replaced at 8-hour
                                                                                                 intervals. The three different ATP treatment regimens
    U:                                                                                           all decreased ['4C]phenylalanine incorporation into
    cc 4
                                                                                                 TCA-precipitable protein (Table 1). However, neither
    z                                                                                            the replenishment of ATP at 8-hour intervals nor the
         993                                                                                     replacement of ATP and media every 8 hours produced
    cq                                                                                           results different from those after treatment with a single
    CJ                                                                                           dose of ATP.
                                                                                                    We also determined whether ATP increased cell size.
                                                                                                 After treatment with ATP or NE, individual cell areas
                                                                                                 were measured by planimetry in high-power fields
                                                                                                 (x 400). Although NE increased average myocyte size by
                             control                  ATP           ADP          adsne
                                                                                                 ;50%, extracellular ATP did not significantly increase
FIG 4. Bar graph comparing the effects of ATP, ADP, and                                          cell size (Fig 9 and Table 2).
adenosine on c-fos gene expression. Serum-free cell cultures
were incubated with ATP (100 ,Lmol/L), ADP (100 ,umol/L), or                                                           Discussion
adenosine (100 ,umol/L) for 30 minutes. c-fos mRNA levels were
measured as described in Fig 1. Data are expressed as                                               The findings presented here demonstrate that extra-
mean±SEM (n=3 to 6) relative to control.                                                         cellular ATP induces the expression of the IEGs c-fos

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1038       Circulation Research Vol 74, No 6 June 1994

A                              .I
                                    'j B                  *t
                                                                                        FIG 5. Photomicrographs showing representative ex-
                                                                        *               amples of FOS protein levels in neonatal cardiac
                                                                                        myocytes. Immunocytochemistry was performed with
                                                                                        a primary antibody specific for FOS and a secondary
                                                                                        antibody conjugated with alkaline phosphatase as
                                                                        *               described in "Materials and Methods." A shows vehi-
                                                                                        cle-treated control; B, untreated myocytes incubated
c                   0
                                       D                                                without primary antibody; C, ATP-treated myocytes
    4   -^-*-                                                                           (1 00 gmol/L); and D, norepinephrine-treated myo-
                                                                                        cytes (2 gmol/L). Slides were incubated with various
                                                                   rnm.sn~              agents for 2 hours before fixation. Arrows point to the
                                             -211
                                                                                        nuclear region.
                                         A   .

                    '0100           lp
                                         l

                                                                        and jun-B. The time course of c-fos activation by ATP is
                                                                        similar to that observed for a,-adrenergic stimulation of
                                                                        cardiac myocytes'9 and is typical of IEG responses.35,36
                                                                        The present study also demonstrates that extracellular
                                                                        ATP increases [Ca2t]i in quiescent myocytes and that
                                                                        the ATP-induced increase in [Ca2t]i precedes the ATP-
                                                                        induced increase in IEG expression (Fig 2 and Fig 6).
                                                                        This suggests that the transient alteration in [Ca2tli
                                                                        induced by ATP may be a stimulus for the induction of
                                                                        IEG expression. Supporting this hypothesis is the find-
                                                                        ing that chelating intracellular Ca> with BAPTA-AM
                                                                        inhibited the ATP-induced increase in the level of c-fos
                                                                        mRNA. The dependence of ATP on [Ca2t]i is consis-
                                                                        tent with the documented effect of ATP on Ca> entry
                                                                        via L-type Ca2+ channels and nonselective cation chan-
                                                                        nels." It is not clear whether ATP activates other
                                                                        intracellular signaling mechanisms, but the failure of
                                                                        BAPTA-AM to completely inhibit c-fos induction in
                                                                        these experiments suggests that it may.

                                                                                  120   A                         s
                                                                                        A                         B
                                                                                                  < 0.05
                                                                                  100

                                                                             cn
                                                                                  80

                                                                             z
                                                                                  60-

                                                                              S40 -

                                                                                  20r
FIG 6. Tracings showing the effects of ATP or norepinephrine
and BAPTA-AM+ATP on [Ca2+1] in neonatal ventricular myo-
                                                                                   0
cytes. A, Addition of ATP (100 gmol/L) to the superfusate                                             ATP/UAPTA           NE     NE/BAPTA
entering a chamber containing cultured myocytes increased                                   ATP
[Ca2+t,. [Ca>2], was measured by loading myocytes with indo             FIG 7. Bar graphs showing the effects of BAPTA-AM on the level
1-AM (25 pmol/L) for 20 to 30 minutes at 370C. Indo 1 fluores-          of c-fos mRNA stimulated by ATP or norepinephrine (NE).
cence was excited at 350 nm, and the 410/490-nm ratio of                Serum-starved cell cultures were pretreated with the intracellular
emitted fluorescence was taken as an index of [Ca>2+], as               Ca2t chelator BAPTA-AM (10 gmol/L) for 30 minutes, and then
described previously.29 B, Addition of norepinephrine (2 umol/L)        ATP (100 gmol/L) or NE (2 gmol/L) was added for an additional
to quiescent cells did not elicit Ca2t response. C, Pretreatment        30 minutes. Total RNA was isolated as described in "Materials
with BAPTA-AM (10 gtmol/L) for 30 minutes and the subsequent            and Methods." Data are expressed as mean+SEM (n=5) as
addition of ATP (100 gmol/L) did not increase [Ca2t],.                  percent relative to ATP- or NE-treated control.

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Zheng et al ATP Dissociates IEG Expression and Myocyte Hypertrophy                    1039

                200 -
                                P c 0.01

           o.
           0
                                            -I
       M0
       0

       0X XI50'
       0 0

      0,         50

                  0

                          ATP                 NE
FIG 8. Bar graph showing different effects of norepinephrine
(NE) and ATP on [14Cjphenylalanine incorporation. Serum-
starved cell cultures were incubated with ATP (100 umol/L) or
NE (2 gmol/L) and [14C]phenylalanine (0.1 gCi/mL) for 3 days.
Cells were washed, treated with 10% trichloroacetic acid for 1
hour, and harvested with 1% sodium dodecyl sulfate. [14ClPhe-
nylalanine incorporation was determined with a beta counter.
Data are expressed relative to control (arbitrarily assigned 1) as
mean-+SEM (n =9).

  In contrast to the effects of ATP on [Ca2Jj,, NE did
not increase [Ca>]i in quiescent myocytes, although it
did activate IEG expression in the present study and in
the previous studies of others.19 The NE-induced ex-
pression of the c-fos gene was not inhibited by pretreat-
ment with BAPTA-AM. Since BAPTA-AM blocked all
detectable Ca' activity (ie, Ca' transients and beat-
ing), an increase in [Ca"]i is not required for c-fos
induction by NE in rat ventricular myocytes and sug-
gests that ATP and NE activate IEG expression by
different mechanisms. These data do not distinguish,
however, between the activation of cAMP-dependent                    FIG 9. Photomicrographs showing representative examples of
and diacylglycerol-protein kinase C signaling pathways               the different effects of norepinephrine and ATP on cell size. After
by NE, both of which are known to activate c-fos gene                24 hours in serum-free media, cells were treated without (con-
expression.                                                          trol) or with ATP (100 gmol/L) or with norepinephrine (2 gmol/L)
   A number of studies have suggested that the IEGs                  for 3 days. Photomicrographs were prepared using a Nikon
play a crucial role in cell growth and proliferation. For            phase-contrast inverted microscope in high-power fields (origi-
                                                                     nal magnification x400). Individual cell areas were measured by
example, c-fos has an apparent role in the proliferative             planimetry and are summarized in Table 2.
response induced by growth factors in BALB/c 3T3
cells37; high-level expression of c-fos in transgenic mice           with growth factors, hormones, or hemodynamic factors,
interferes with normal bone development.38 Although                  they do increase in size (ie, hypertrophy) in response to
cardiac myocytes do not proliferate when stimulated                  a number of these agents.1417,39,40 Without apparent

      TABLE 1. Effects of Different ATP Treatment Regimens on [14C]Phenylalanine Incorporation Into
      Trichloroacetic Acid-Precipitable Protein
                                                        Control           ATP1                  ATP2                    ATP3
      [14CJPhe incorporation (mean-+-SEM)                   1             0.48+0.07           0.48+0.04          0.54+0.08
      Change from control, %                              ...               -52%               -52%                -46%
      n                                                    18                 12                16                    8
      P value vs control                                  ...
1040       Circulation Research Vol 74, No 6 June 1994

TABLE 2. Effects of ATP and Norepinephrine on                              10. Zheng J-S, Christie A, De Young MB, Levy MN, Scarpa A. Ca `
Individual Cell Size                                                           mobilization by extracellular ATP in rat cardiac myocytes: regu-
                                                                               lation by protein kinase C and A. Am J Physiol. 1992;263:
                          Control        ATP         Norepinephrine            C933-C940.
                                                                           11. Zheng J-S, Christie A, De Young MB, Levy MN, Scarpa A. Mod-
Individual cell area,                                                          ulation by extracellular ATP of two distinct currents in rat
gum2                     1385+60       1330+51           2086+93               myocytes. Am J Physiol. 1993;264:C1411-C1417.
Change from                                                                12. Legssyer A, Pogglioli J, Renard D, Vassort G. ATP and other
control, %               ...          -4              +51                      adenosine compounds increase mechanical activity and inositol
                                                                               triphosphate production in rat heart. J Physiol (Lond). 1988;401:
n                        120         120              120                      185-199.
                                                                           13. Yamada M, Hamamori Y, Akita H, Yokoyama M. P2-purinoceptor
P value                  ...          NS
Zheng et al ATP Dissociates IEG Expression and Myocyte Hypertrophy                        1041

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35. Simpson PC. Proto-oncogenes and cardiac hypertrophy. Annu Rev              405-411.
    Physiol. 1988;51:189-202.                                              43. Izumo S, Nadal-Ginard B, Mahdvi V. Proto-oncogene induction
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Extracellular ATP induces immediate-early gene expression but not cellular hypertrophy
                             in neonatal cardiac myocytes.
               J S Zheng, M O Boluyt, L O'Neill, M T Crow and E G Lakatta

                                       Circ Res. 1994;74:1034-1041
                                      doi: 10.1161/01.RES.74.6.1034
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