(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-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.
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 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.
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 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.
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 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.
RNA extracted from adult cultured rat ventricular cardiac
myocytes was subjected to a solution hybridization RNase protection
analysis using a synthetic 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.
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-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.
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.
, 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.
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-phorbol 12,13-didecanoate (Fig. 3).
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-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.
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-phorbol 12,13-didecanoate (Fig. 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-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.
Immunoblot
analyses of total extracts from untreated cardiac myocytes revealed the
expression of PKC- (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).
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-,
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.
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 () 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
.
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-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.
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- 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.
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 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.
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.