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(Received for publication, August 30, 1995) From the
Opioid peptide gene expression was characterized in adult rat
ventricular cardiac myocytes that had been cultured in the absence or
the presence of phorbol 12-myristate 13-acetate. The phorbol ester
induced a concentration- and time-dependent increase of prodynorphin
mRNA, the maximal effect being reached after 4 h of treatment. The
increase in mRNA expression was suppressed by incubation of
cardiomyocytes with staurosporine, a putative protein kinase C
inhibitor, and was not observed when the cells were cultured in the
presence of the inactive phorbol ester 4
Opioid peptides have been shown to have a wide range of tissue
distribution and are known to control the cardiac function through
reflex mechanisms involving the central nervous system or the
modulation of neurotransmitter release from neurons located within the
heart(1, 2) . The discovery that the mammalian
myocardial cells possess opioid receptors (3) has led to
studies aimed at investigating direct myocardial effects due to opioid
receptor stimulation and identifying possible intracellular opioidergic
pathways. In this regard, we have shown that, in adult rat ventricular
cardiac myocytes, k opioid receptor stimulation depleted the
sarcoplasmic reticulum of Ca The signal transduction pathway(s) involved in the
expression of the prodynorphin gene in cardiac myocytes is currently
unknown. However, the observed effects of k opioid receptor agonists on
myocardial cytosolic Ca In the
present study, we aimed at elucidating whether PKC activation induced
by phorbol 12-myristate 13-acetate (PMA) may influence the expression
of prodynorphin mRNA as well as the synthesis and release of dynorphin
B in adult cultured rat ventricular myocytes. The effect induced by the
phorbol ester on the transcription of the prodynorphin gene and the
presence of a PKC in isolated myocardial nuclei were also investigated.
The
hybridization reaction was allowed to run for 36 h at 65 °C. After
the hybridization, the filters were washed in 2
RNA extracted from adult cultured rat ventricular cardiac
myocytes was subjected to a solution hybridization RNase protection
analysis using a synthetic
Figure 1:
Time course of the effect of PMA on the
expression of prodynorphin mRNA in adult cultured rat ventricular
myocytes. White bars, control; black bars, 100 nM PMA; hatched bar, 2 nM staurosporine; black
bar with white hatching, 100 nM PMA and 2 nM staurosporine; shaded bar, 100 nM 4
Figure 2:
Dose-response of PMA effect on the
expression of prodynorphin mRNA. The myocardial cells were incubated in
the presence of each concentration of PMA for a period of 4 h.
Consistent amounts of ir-dyn B were found under basal conditions in
cardiac myocytes and in the culture medium (Fig. 3). Culturing
of myocytes for 4 h in the presence of 100 nM PMA resulted in
a marked increase in the level of this biologically active peptide
product of the prodynorphin gene both in the cells and in the culture
medium (Fig. 3). The same figure shows that the cellular levels
of ir-dyn B were significantly lower than those detected in the medium,
both in the absence and the presence of the phorbol ester. Myocyte
treatment with 2 nM staurosporine suppressed the PMA-mediated
increase in ir-dyn B both in the medium and in the myocardial cell (Fig. 3). No significant increase in ir-dyn B levels was
observed in cells or medium when the myocytes were cultured for 4 h in
the presence of 100 nM 4
Figure 3:
Regulation of ir-dyn B levels by PMA.
Adult cultured rat ventricular myocytes were treated for 4 h with 100
nM PMA in the absence or presence of 2 nM staurosporine (ST) or exposed for a period of 4 h to 100
nM 4
To investigate whether the increase in prodynorphin
mRNA expression elicited by PMA reflects changes in the transcriptional
status of the myocyte nucleus, we assessed the rate of transcription of
the proynorphin gene by using an in vitro run-off
transcription assay. The nuclear preparation used in the present study
lacked contamination by sarcoplasmic reticular membranes, inner or
outer mitochondrial membranes, or sarcolemmal membranes, as indicated
by the measurement of the activities of the corresponding marker
enzymes rotenone-insensitive NADPH-cytochrome c reductase,
succinate dehydrogenase, rotenone-insensitive NADH-cytochrome c reductase, and
5`-nucleotidase(33, 34, 35) , which were all
undetectable in the nuclear fraction (data not shown). We found that
incubation of the cardiac myocytes in the presence of 100 nM PMA, a concentration that proved to be the most effective in
increasing prodynorphin mRNA, resulted in a marked increase in nuclear
transcription of the prodynorphin gene (Fig. 4). Interestingly,
in nuclei isolated from untreated cells and subsequently exposed for 4
h to 100 nM PMA, prodynorphin gene transcription was found to
be enhanced at a level similar to that observed in the nuclei from
PMA-treated cardiomyocytes (Fig. 4). The pretreatment of
cardiomyocytes or isolated nuclei with staurosporine abolished the
PMA-induced increase in transcriptional activity. No increase in
transcription of the prodynorphin gene was observed following
incubation of myocytes or isolated nuclei in the presence of 100 nM 4
Figure 4:
Effect of PMA on the rate of transcription
of the prodynorphin gene in isolated myocardial nuclei. Isolation of
myocardial nuclei and run-off transcriptional analysis of the
prodynorphin gene were performed as described under ``Materials
and Methods.'' plB15, a cDNA encoding rat cyclophilin, and pBR322
plasmid DNA were included as positive and negative controls,
respectively. The values are representative of six experiments. 1, transcription of the prodynorphin gene; 2, pBR322; 3, plB15. A, nuclei were isolated from control
cardiac myocytes; B, nuclei were isolated from cardiac
myocytes that have been exposed to 100 nM PMA for 4 h; C, nuclei were isolated from untreated myocytes and
subsequently exposed to 100 nM PMA for 4 h; D, nuclei
were isolated from cardiac myocytes cultured for 4 h in the presence of
100 nM PMA and 2 nM staurosporine; E, nuclei
isolated from untreated cells were subsequently incubated for 4 h in
the presence of 100 nM PMA and 2 nM staurosporine; F, nuclei were isolated from cardiac myocytes that have been
exposed to 100 nM 4
Immunoblot
analyses of total extracts from untreated cardiac myocytes revealed the
expression of PKC-
Figure 5:
Immunoblot analysis of PKC isotypes in
isolated myocardial nuclei or total cell lysates. Nuclei and total cell
lysates were prepared from control myocytes and subjected to
immunoblotting (50 µg of protein/lane) as described under
``Materials and Methods'' with antisera to PKC-
We next investigated whether
the exposure of isolated myocardial nuclei to a PKC activator may
result in the phosphorylation of a specific PKC substrate. Fig. 6shows the effect induced on acrylodan-peptide
phosphorylation by the incubation of myocardial nuclei, isolated from
control cells, in the absence or the presence of
1,2-dioctanoyl-sn-glycerol, a diglyceride that acts as a
potent PKC activator(36) . When the nuclear fraction was
incubated with the acrylodan peptide in the absence of the PKC
activator, no significant change in the peptide fluorescence was
observed (Fig. 6). On the contrary, exposure of the myocardial
nuclei to 1,2-dioctanoyl-sn-glycerol for 10 min resulted in a
time-dependent fluorescence decrease at the 480 nm emission maximum of
the PKC substrate, corresponding to PKC-mediated acrylodan-peptide
phosphorylation (Fig. 6). The maximal decrease in the intensity
of fluorescence was achieved about 10 min from the addition of each
nuclear sample to the reaction mixture.
Figure 6:
Effect induced on acrylodan-peptide
fluorescence by exposure of isolated myocardial nuclei to a PKC
activator. Myocardial nuclei isolated from control myocytes were
incubated for 10 min in the absence (
Our data show that the prodynorphin gene was expressed in
adult rat ventricular myocytes and that prodynorphin mRNA was
translated into a biologically active end product. The observation that
the levels of ir-dyn B were significantly higher in the culture medium
than in the myocytes suggests that in the ventricular myocardial cell,
which lacks secretory granules(37) , the prodynorphin-derived
peptides may be constitutively released shortly after synthesis. PMA
elicited a marked increase in the expression of prodynorphin mRNA
within the first 4 h of treatment, which was associated to a
significant increase in levels of ir-dynB both in the cells and in the
culture medium. The finding that these effects of the phorbol ester
were inhibited by staurosporine suggests that prodynorphin mRNA
induction by PMA was a PKC-mediated event that was associated to an
increase in mRNA translation into the biologically active peptide
product. A role for PKC in prodynorphin mRNA induction is also
supported by the observation that the levels of this mRNA were
down-regulated to control values following prolonged exposures to PMA,
a treatment that is known to result in depletion or down-regulation of
PKC(38, 39) . PKC involvement in the regulation of
prodynorphin gene expression is further supported by the finding that
the inactive phorbol ester 4 The ability of isolated nuclei to phosphorylate a
specific PKC fluorescent substrate when a PKC activator was added and
the finding that among the different PKC isozymes expressed in the
myocardial cell, only PKC- The implications of
the results of the present report, showing an increase in prodynorphin
gene expression following the activation of PKC in myocardial nuclei
are still unknown. Nevertheless, the stimulation of nuclear PKC, by
eliciting increased prodynorphin gene expression and release of a
biologically active opioid peptide, may stimulate myocardial opioid
receptors, resulting in the activation of autocrine or paracrine
mechanisms that might trigger previously described effects of opioid
peptides on myocardial Ca The mechanisms by which PMA-mediated
stimulation of nuclear PKC may affect the expression of the
prodynorphin gene have not been investigated in the present study. It
has been hypothesized that a potential mechanism by which PKC may
affect gene transcription is the phosphorylation of RNA polymerase or
the phosphorylation of specific nuclear proteins acting as
transcription factors(48, 49) . However, nuclear
events initiated by PKC activation, including the activation of RNA
polymerases, have been reported to be long-lived and to persist after
the down-regulation of PKC(50) , indicating that a role for the
regulation of transcription by phosphorylation-dephosphorylation
remains to be defined. The identification of possible molecular
mechanisms involved in PMA effect on gene transcription is further
complicated by the finding that different consensus sequences that
function as PMA-responsive elements have been identified in phorbol
ester-inducible genes and by the fact that DNA-binding proteins that
are part of the PMA-activated signal transduction network have been
only in part identified(51) . Therefore, the mechanism(s) by
which the expression of the prodynorphin gene is regulated by PKC in
the myocardial cell remains to be elucidated.
Volume 270,
Number 50,
Issue of December 15, 1995 pp. 30115-30120
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ROLE OF NUCLEAR PROTEIN KINASE C (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-phorbol
12,13-didecanoate. Incubation of cardiac myocytes with phorbol
12-myristate 13-acetate also elicited a specific and
staurosporine-sensitive increase in immunoreactive dynorphin B, a
biologically active end product of the precursor, both in the
myocardial cells and in the culture medium. In vitro run-off
transcription assays indicated that transcription of the prodynorphin
gene was increased both in nuclei isolated from phorbol ester-treated
myocytes and in nuclei isolated from control cells and then exposed to
phorbol 12-myristate 13-acetate. No transcriptional effect was observed
when cardiac myocytes or isolated nuclei where exposed to
4-phorbol 12,13-didecanoate. The phorbol ester-induced increase in
prodynorphin gene transcription was prevented by pretreatment of
myocytes or isolated nuclei with staurosporine, suggesting that
myocardial opioid gene expression may be regulated by nuclear protein
kinase C. In this regard, cardiac myocytes expressed protein kinase
C-, -, -
, and -
, as shown by immunoblotting. Only
protein kinase C-
and protein kinase C-
were expressed in
nuclei that have been isolated from control myocytes, suggesting that
these two isotypes of the enzyme may be part of the signal transduction
pathway involved in the effect elicited by the phorbol ester on opioid
gene transcription in isolated nuclei. The incubation of myocardial
nuclei isolated from control cells in the presence of a protein kinase
C activator induced the phosphorylation of the myristoylated
alanine-rich protein kinase C substrate peptide, a specific fluorescent
substrate of the enzyme. The possibility that prodynorphin gene
expression may control the heart function through autocrine or
paracrine mechanisms is discussed.
and caused a marked
reduction in the amplitudes of the cytosolic Ca
transient and of the associated twitch(4) . In addition
to affecting cytosolic Ca
homeostasis, the incubation
of cardiac myocytes with k opioid receptor agonists also elicited
intracellular alkalosis and increased myofilament Ca
responsiveness(5) , an event that blunted in part the negative
inotropic effect due to the intracellular Ca
depletion. However, the myocardial cell, besides being a target for
opioid agonists, also acts as a source for opioid peptides. We have
recently shown that the prodynorphin gene is expressed in adult
cultured rat ventricular cardiac myocytes and that these cells are able
to synthesize and release dynorphin B(6) , an opioid peptide
that binds selectively to k opioid receptors(7) . Similar to
the synthetic k opioid receptor agonist U-50,488H, dynorphin B was able
to elicit Ca
depletion from an intracellular storage
site when it was acutely released over single cardiac
myocytes(6) . These findings suggest that the myocardial
function may be affected in an autocrine or paracrine fashion by an
opioid gene and by intracellular pathways that may regulate its
expression.
and pH homeostasis were
largely attributable to the capability of these opioids to increase the
formation of inositol 1,4,5-trisphosphate and inositol
1,3,4,5-tetraphosphate (8) and to elicit a protein kinase
C-dependent stimulation of the Na
/H
antiporter(5) , indicating that myocardial opioid
receptors are coupled to phosphoinositide turnover and protein kinase C
(PKC). (
)This enzyme is expressed in various forms and is
widely distributed in different tissues (9, 10, 11) . In the myocardial cell, PKC
activation, besides representing an important step in the opioidergic
pathway, has been reported to regulate the expression of different
genes, including c-fos and skeletal -actin
genes(12) . PKC activation by phorbol esters has also been
shown to induce the human proenkephalin gene transfected into CV 1
cells, and a DNA sequence on this opioid gene has been identified
conferring the sensitivity to phorbol ester induction(13) . The
role of protein kinase C in the regulation of prodynorphin gene
expression in cells naturally expressing this gene and, in particular,
in the myocardial cell, has not been investigated, however.
Isolation of Cardiac Myocytes
Hearts
from 2-3-month-old male Wistar rats were retrogradely perfused
through the aorta in the presence of a bicarbonate buffer, pH 7.4,
containing low calcium and collagenase(14) . Following the
isolation procedure, the cardiomyocytes were subjected to a short term
serum-free primary culture, according to a previously described
method(15) . Briefly, the cardiac myocytes were cultured under
5% CO
atmosphere at 37 °C in Petri dishes (60-mm Falcon
dishes, Becton Dickinson) at a density of 2 
10
cells/dish in 3 ml of M-199 medium (with Earle's salts)
containing 0.2% bovine serum albumin, 10M
insulin, 2.5
10M penicillin G, 2.5
10M streptomycin, and
10
M cytosine arabinofuranoside. One day
after plating, cells were treated under the experimental conditions
described in the legend of each figure.
RNA Extraction and Determination of Prodynorphin
mRNA
Following RNA isolation from the adult cultured myocytes (16) , the levels of prodynorphin mRNA were determined by using
a sensitive solution hybridization RNase protection
assay(17, 18) . Briefly, a 400-base pair HindIII-BamHI fragment of the main exon of rat
genomic prodynorphin clone was inserted into pGEM3. Transcription of
the plasmid linearized with BamHI generated a sense strand of
prodynorphin mRNA used to construct a standard curve of prodynorphin
mRNA, whereas transcription of the plasmid linearized with EcoRI in the presence of [P]CTP (800
Ci/mmol) gave an antisense strand used to hybridize cellular
prodynorphin mRNA. The protected fragments were recovered after phenol
chloroform extraction and electrophoretically separated in a
polyacrylamide nondenaturing gel. Autoradiographic exposure was
performed for 48 h. The individual bands were counted for radioactivity
by liquid scintillation spectrometry, and cpm values were translated to
pg values on a correlated standard curve. Data were expressed as pg of
mRNA/µg of total RNA.
Identification of Dynorphin B-like
Material
Immunoreactive dynorphin B (ir-dyn B) was measured
by a radioimmunoassay procedure that utilized the 13 S antiserum raised
against dyn B and capable of recognizing the high molecular weight
peptides cleaved from the prodynorphin precursor and containing dyn B
in their sequence(19, 20) . Acetic acid extracts from
cardiac myocytes or pooled samples from the culture medium were
processed by reverse-phase high performance liquid chromatography. The
collected fractions were radioimmunoassayed according to a previously
described method(6, 21) , and the immunoreactivity was
attributed to authentic dyn B by comparison with the elution position
of a synthetic standard.Isolation of Myocardial Nuclei
Nuclei
from control or PMA-treated cells were isolated by a modification of a
previously described method(22) . Cells were rinsed twice with
a physiological saline solution (Buffer I) containing 137 mM NaCl, 1.2 mM MgSO
, 5 mM KCl, 1.2
mM NaHPO, 20 mM HEPES, 16
mMD-glucose, and 1 mM CaCl, pH
7.4. The dishes were scraped with Buffer I, and the cells from pooled
samples were sedimented at 1000 g for 10 min at 4
°C. The cardiac myocytes were then resuspended in a hypo-osmotic
buffer (Buffer II) of the following composition: 10 mM Tris/HCl, pH 7.4, 1 mM MgCl, 10 mM NaCl, 5 mM CaCl, 0.1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1
µM leupeptin, and 10 mM 
-mercaptoethanol.
The resuspended cells were incubated for 30 min at 4 °C and then
sedimented at 1000 g for 10 min at 4 °C. The
pellet was resuspended in 20 ml of Buffer II and sonicated for 40 s at
setting 2 in a Branson sonifier W-350. The sonicated preparation was
added with Triton X-100 at a final concentration of 0.1% and then
centrifuged at 1000 g for 10 min at 4 °C. The
pellet was resuspended in a buffer (Buffer III) containing 10 mM Tris, pH 8.0, 0.3 M sucrose, 5 mM
MgCl, 0.1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, and 10
mM -mercaptoethanol. The resuspended material was layered
over an equal volume of Buffer III containing 0.6 M sucrose
and centrifuged at 1500 g for 10 min at 4 °C. The
resulting nuclear pellet was resuspended in a buffer (Buffer IV)
containing 50 mM Tris/HCl, pH 8.0, 5 mM MgCl, 0.1 mM EDTA, 40% glycerol, 0.1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1
µM leupeptin, and 10 mM -mercaptoethanol.Nuclear Run-off Transcription
Assay
Nuclear run-off experiments were carried out by using
a modification of the method described by Greenberg(23) .
Nuclei resuspended in 90 µl of Buffer IV were added with 100 µl
of 2 reaction buffer (10 mM Tris/HCl, pH 7.5, 5
mM MgCl, 0.3 M KCl, 5 mM dithiothreitol, 1 mM each of ATP, GTP, and CTP), and 10
µl of [-P]UTP (3000 Ci/mmol), followed
by incubation at room temperature for 15 min. DNA was digested by
incubating the transcription mixture for 5 min at room temperature in
the presence of 1 µl of 20,000 units/ml RNase-free DNase. Nuclear
RNA was isolated by using guanidine thiocyanate and acid phenol
extraction (16) , followed by purification on
RNAMATRIX
(BIO 101, Inc. Vista, CA). The
P-labeled RNA pellet was resuspended in 1 ml of TES
solution containing 10 mM TES, pH 7.4, 10 mM EDTA,
and 0.2% SDS. Samples were eventually diluted by adding TES solution to
equalize the cpm of
P-labeled RNA/ml (about 5
10
). 1 ml of RNA solution was then mixed with 1 ml of
TES/NaCl solution (TES solution containing 0.6 M NaCl). A
total volume of 2 ml of RNA solution was then hybridized to 1 µg of
heat-denatured DNA insert corresponding to a 400-base pair fragment of
the main exon of rat genomic prodynorphin clone that has been
immobilized on nitrocellulose filters following isolation from a pGEM3
plasmid. In this regard, it has been shown that the use of a DNA insert versus the whole plasmid-containing insert can considerably
increase the efficiency of hybridization(17) . SSC for 1 h at
65 °C with several changes and treated with RNase A for 30 min at
37 °C. The filters were washed again in 2 SSC for 1 h at 37
°C and exposed to Kodak X-Omat film at -70 °C. The amount
of prodynorphin gene transcript was quantitated by liquid scintillation
counting. Denatured plB15, a cDNA clone encoding for rat
cyclophilin(24) , was slot blotted onto nitrocellulose and used
as a positive control. pBR322 plasmid DNA was also hybridized with the
labeled RNA as a negative control.Immunoblotting Analysis of Nuclear
PKC
Samples were electrophoresed on 10% SDS-polyacrylamide
gels and were then transferred to nitrocellulose membrane at 250 mA for
1 h according to Towbin et al.(25) . After transfer,
the blot was saturated for 1 h at room temperature with 2% fat-free
milk powder in PBST (80 mM NaHPO, 20
mM NaHPO, 100 mM NaCl
containing 0.05% Tween 20, pH 7.5). The immunoreaction was carried out
for 1 h at room temperature in PBST and 2% milk powder containing the
primary antibody diluted 1:1000 (antisera to PKC-, PKC-,
PKC-
, and PKC-
were from Calbiochem). After being washed
three times with PBST (5 min each wash), the blot was incubated for 1 h
at room temperature in the presence of the secondary antibody (1:5000
horseradish peroxidase-linked donkey anti-rabbit immunoglobulin) in
PBST containing 1% fat-free milk powder. Finally, the nitrocellulose
membranes were washed three times in PBST, developed by the ECL method,
and exposed to films for 5 min (the secondary antibody as well as the
ECL Western blotting detection reagents and the autoradiography film
Hyperfilm MP were all from Amersham International).
Measurement of Nuclear PKC Activity
PKC
activity from isolated myocardial nuclei was measured by using a
continuous fluorescence assay (26, 27) that utilized
the acrylodan-labeled myristoylated alanine-rich protein kinase C
substrate (MARCKS) peptide (Boehringer Mannheim), a high affinity
substrate in vitro for
PKC(28, 29, 30, 31) . The MARCKS
peptide consists of a conserved sequence of 24 amino acids from the
MARCKS protein, which includes four sites for PKC
phosphorylation(27) . The fluorescing acrylodan label is
introduced N-terminally via an additional cysteine group. In the
presence of PKC activators, maximum fluorescence is measured at 480 nm
with excitation at 370 nm. In the course of phosphorylation by PKC, the
intensity of the fluorescence decreases about 20%(27) . In the
present study, the fluorescence changes during phosphorylation of the
MARCKS peptide were monitored at 25 °C. The reaction mixture
contained, in a final volume of 1 ml, 10 mM Tris/HCl, pH 7.0,
90 mM KCl, 3 mM MgCl, 0.3 mM
CaCl, 0.1 mM EGTA, 100 µM ATP, 10%
ethylene glycol, 0.5 µg phosphatidylserine, 0.1 µg
1,2-dioctanoyl-sn-glycerol, and 75 nM acrylodan-labeled MARCKS peptide. Phosphorylation of the
acrylodan-labeled peptide was started by adding 20 µg of nuclear
protein.Data Analysis
The statistical analysis of
the data was performed by using the unpaired Student's t test, assuming a p value less than 0.05 as the limit of
significance.
P-antisense probe for
detection. Incubation of the cultured myocytes in the presence of 100
nM PMA induced a marked increase in the amount of detectable
mRNA. This stimulatory effect was evident after 1 h of cell exposure to
the phorbol ester and peaked at 4 h of treatment. (Fig. 1). On
the contrary, incubation of the cardiac myocytes for 4 h in the
presence of 100 nM 4
-phorbol 12,13-didecanoate, a
biochemically inactive phorbol, failed to affect the expression of
prodynorphin mRNA (Fig. 1). When the cardiac myocytes were
pretreated for 30 min with 2 nM staurosporine, a putative PKC
inhibitor(32) , and subsequently treated for 4 h with PMA in
the presence of this PKC inhibitor, the levels of prodynorphin mRNA did
not differ significantly from those observed in control cardiac
myocytes (Fig. 1). After 6 h of treatment with PMA, the levels
of prodynorphin mRNA progressively declined and resulted in being
down-regulated to the control value between 12 and 24 h (Fig. 1). Fig. 2reports the dose-response curve of the
effect of PMA on prodynorphin mRNA expression, following a 4-h exposure
of the cultured myocytes to the phorbol ester. A significant increase
in mRNA levels was observed at a concentration as low as 1 nM.
The amount of prodynorphin mRNA was progressively increased in a
dose-dependent manner by cell treatment in the presence of the phorbol
ester, reaching a plateau when the myocytes where cultured in the
presence of PMA concentrations ranging between 50 and 100 nM.
-phorbol
12,13-didecanoate. The data are expressed as the mean values ±
S.E. (n = 6). *, significantly different from the
control value. Representative autoradiograms of the ribonuclease
protection analysis of myocardial prodynorphin mRNA are shown in the inset. A, control; B, 100 nM PMA
for 1 h; C, 100 nM PMA for 4 h; D, 4 h of
exposure to 100 nM PMA in the presence of 2 nM staurosporine; E, 4 h of incubation in the presence of
100 nM 4-phorbol 12,13-didecanoate. Autoradiographic
exposure was for 2 days on Kodak X-Omat film with an intensifying
screen. The mark indicates the position of a 400 base pair
radiolabeled DNA marker, showing that the single protected fragment
migrates with a molecular size of 400 bases, corresponding to
prodynorphin mRNA.
, control; 
, PMA. The data are expressed as the mean
values ± S.E. (n = 6). *, significantly
different from the control value. Representative autoradiograms of the
ribonuclease protection analysis of myocardial prodynorphin mRNA are
shown in the inset. Autoradiographic exposure was carried out
as described in Fig. 1.
-phorbol 12,13-didecanoate (Fig. 3).
-phorbol 12,13-didecanoate (4-phorbol). Shaded bars, ir-dyn B in cultured myocytes; white
bars, ir-dyn B in the medium. Each single value in the medium was
calculated in a final volume of 15 ml, corresponding to the volume of
pooled samples of the culture medium from 10 cells. Each
experiment was performed in the presence of a peptidase inhibitor
mixture containing 20 µM bestatin, 1 mM leucyl-L-leucine, 3 µM poly-L-lysine, 0.3 µM thiorphan, 30
µM 1-10-phenanthroline, and 6 µM
1,4-dithiothreitol. The data are expressed as the mean values ±
S.E. (n = 6). *, significantly different from its own
control value; , significantly different from its own control
value; , the value of the white bar is significantly different
from that of the shaded bar.
-phorbol 12,13-didecanoate (Fig. 4).
-phorbol 12,13-didecanoate for 4 h; G, nuclei were isolated from untreated myocytes and then
incubated in the presence of 100 nM 4-phorbol
12,13-didecanoate for 4 h.
(80 kDa), PKC- (78 kDa), PKC-
(97
kDa), and PKC-
(75 kDa) (Fig. 5). PKC-
and PKC-
were not detected (not shown). Western blot analysis of nuclear samples
from untreated cells revealed that only PKC-
and PKC-
were
expressed in the myocardial nucleus (Fig. 5). The densitometric
analysis of the bands corresponding to PKC-
and PKC-
indicated that each of these two PKC isotypes was almost completely
expressed at nuclear level (not shown).
,
PKC-, PKC-
, and PKC-
. The arrows to the left of each panel indicate PKC immunoreactivity as confirmed
in peptide antigen competition experiments (results not shown). The
numbers to the right of each panel refer to the molecular mass
(kDa) of marker proteins. Lane 1, myocardial nuclei; lane
2, total cell lysates. The values are representative of four
separate experiments.
) or the presence ()
of the PKC activator 1,2-dioctanoyl-sn-glycerol (0.1
µg/ml). Each nuclear sample was added at the time indicated by the arrow. As the acrylodan-peptide becomes phosphorylated, it
undergoes a time-dependent decrease in its fluorescence at 480 nm.
Changes in the rate of this decrease in fluorescence correspond to
different rates of PKC-dependent phosphorylation. The figure also shows
the time course of the fluorescence of the acrylodan-peptide alone
(
). The data are expressed as the mean values ± S.E. (n = 6). From 500-1200 s, was significantly
different from
or .
-phorbol 12,13-didecanoate did not
alter both prodynorphin mRNA and dyn B levels. The nuclear run-off
experiments performed in myocardial nuclei isolated from PMA-treated
cells indicate that the level of PMA-mediated stimulation of
prodynorphin mRNA expression was transcriptional. Specificity and PKC
dependence of this stimulatory action of PMA are suggested by the lack
of an effect by 4-phorbol 12,13-didecanoate and by the capability
of staurosporine to suppress the increase in transcription due to
myocyte exposure to the phorbol ester. However, PMA increased nuclear
transcription of the prodynorphin gene even when it was directly
applied to isolated nuclei. This direct effect of PMA also appears to
be specific in nature, because it was not observed following exposure
of isolated myocardial nuclei to the inactive phorbol ester
4-phorbol 12,13-didecanoate. The observation that staurosporine
was capable of abolishing the effect produced by PMA in nuclei that
have been subjected to direct treatment with the phorbol ester suggests
that myocyte nucleus may harbor PKC and that activation of this nuclear
PKC may be responsible for the stimulation of prodynorphin gene
transcription. The presence of PKC at nuclear level was confirmed by
the discovery of PKC- and PKC-
in the nucleus of untreated
myocytes. These results are in agreement with other studies that used
immunofluorescent techniques to determine the subcellular localization
of different PKC isozymes in cardiac myocytes, showing that PKC-
and -
immunostaining patterns were mainly detectable in the
nucleus of unstimulated cells(40) . Failure to detect PKC-
and PKC- in our myocyte preparation is consistent with a previous
study that utilized a reverse transcriptase-polymerase chain reaction
approach and reported the absence of both PKC-
and PKC-
transcripts in both neonatal and adult cardiomyocytes(41) .
Further, Kosaka et al.(42) failed to detect any
PKC-
following chromatographic resolution of PKC activity from
heart extracts. and PKC-
were detected in the
nucleus suggest that these two PKC isotypes may play an important role
in regulating gene expression in the myocardial cell and may be part of
the signal transduction pathway involved in the stimulation of
prodynorphin gene transcription observed when PMA was directly added to
intact isolated nuclei. The phosphorylation of the MARCKS peptide by
``nuclear embedded'' PKC also seems to indicate that the
biochemical machinery necessary for activation of PKC is present in the
myocardial nucleus. This hypothesis is in agreement with other studies
showing that several enzymes and substrates associated with
diacylglycerol production are present in the nuclei of rat liver (43) and that inositolphospholipids are synthesized in nuclei
of Friend cells(44) . Whether the PKC here described in
isolated myocardial nuclei might be activated by endogenous factors
remains to be defined. However, receptors for prolactin and various
growth hormones have been identified in isolated nuclei from rat liver
and splenic mononuclear cells, and these agonists have been shown to
increase the activity of a nuclear PKC to a similar extent of that
elicited by PMA(45) , suggesting that the phorbol ester effect
might be mimicked by endogenous agonists and that PKC activation
through specific nuclear receptors might by involved in the regulation
of nuclear events, including gene transcription.
and pH homeostasis as well
as on the inotropic state of the cardiac
myocyte(4, 5) . Moreover, in several tissues
endogenous opioids have been shown to act on plasma membrane opioid
receptors in such a way to inhibit cell proliferation and promote cell
differentiation(46, 47) . We cannot exclude that
myocardial opioid gene expression and its stimulation via nuclear PKC
may also play an autocrine role in the regulation of myocardial cell
growth and differentiation.
)
We thank Giuseppe Delogu for excellent technical
assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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