(Received for publication, July 15, 1996, and in revised form, November 18, 1996)
From the Institute of Biological Chemistry "A.
Bonsignore," School of Medicine, University of Sassari, Viale San
Pietro 43/B, 07100 Sassari, Italy, and the § National
Laboratory of the National Institute of Biostructures and
Biosystems, Osilo, Italy
Prodynorphin mRNA and dynorphin B expression
have been previously shown to be greatly increased in cardiac myocytes
of BIO 14.6 cardiomyopathic hamsters. Here we report that exogenous
dynorphin B induced a dose-dependent increase in
prodynorphin mRNA levels and stimulated prodynorphin gene
transcription in normal hamster myocytes. Similar responses were
elicited by the synthetic selective opioid receptor agonist
U-50,488H. These effects were counteracted by the
opioid receptor
antagonist Mr-1452 and were not observed in the presence of
chelerythrine or calphostin C, two specific protein kinase C (PKC)
inhibitors. Treatment of cardiomyopathic cells with Mr-1452
significantly decreased both prodynorphin mRNA levels and
prodynorphin gene transcription. In control myocytes, dynorphin B
induced the translocation of PKC-
to the nucleus and increased
nuclear PKC activity without affecting the expression of PKC-
, -
,
or -
. Acute release of either U-50,488H or dyn B over single normal
or cardiomyopathic cells transiently increased the cytosolic
Ca2+ concentration. A sustained treatment with each opioid
agonist increased the cytosolic Ca2+ level for a more
prolonged period in cardiomyopathic than in control myocytes and led to
a depletion of Ca2+ from the sarcoplasmic reticulum in both
groups of cells. The possibility that prodynorphin gene expression may
affect the function of the cardiomyopathic cell through an autocrine
mechanism is discussed.
Dynorphin B (dyn B)1 is a biologically
active end product of the prodynorphin gene acting as a selective opioid receptor agonist (1, 2). In rat ventricular myocytes, dyn B
appears to be constitutively released shortly after synthesis, as
indicated by the observation that the levels of secreted dyn B
significantly exceeded those of the intracellular peptide (3, 4). The finding that the myocardial cell expresses
opioid receptors (5, 6)
and that the stimulation of these receptors affects the cytosolic
Ca2+ and pH homeostasis as well as the inotropic state in
isolated cardiac myocytes (4, 7, 8) suggests that prodynorphin mRNA
translation into dyn B may be part of an autocrine mechanism of
regulation of the myocyte function. Furthermore, the observation that
opioid receptor stimulation is coupled to protein kinase C (PKC)
(8) and that PKC is involved in the regulation of prodynorphin gene
expression (4) raises the possibility that the gene itself may be
regulated in an autocrine fashion by one of its peptide products. In
this regard, it may be conceivable that dyn B would affect prodynorphin
gene expression in pathological conditions characterized by an increase
in the synthesis and release of this opioid peptide from the myocardial
cell. In companion studies (50, 51), we have shown that the expression
of both prodynorphin mRNA and dyn B is markedly enhanced in cardiac
myocytes isolated from BIO 14.6 cardiomyopathic Syrian hamsters
compared with cells obtained from normal hamster hearts and that PKC
activation and/or intracellular Ca2+ loading may be
involved in the regulation of prodynorphin gene expression throughout
the cardiomyopathic process.
In this study, we aimed at investigating whether the stimulation of opioid receptors by dyn B or by U-50,488H, a synthetic selective
opioid receptor agonist (9), may affect prodynorphin mRNA
expression or the rate of transcription of the prodynorphin gene in
cardiac myocytes isolated either from normal or from cardiomyopathic hamsters. In attempting to verify whether endogenously synthesized
opioid receptor ligands may regulate prodynorphin gene expression, we
also assessed prodynorphin mRNA levels and prodynorphin gene transcription in cardiomyopathic myocytes that have been treated in the
presence of Mr-1452, a selective antagonist of
opioid receptors
(10, 11). Finally, the possible consequences of
opioid receptor
agonism were further investigated in normal and cardiomyopathic cells
by examining the effects produced by a short- or long-term exposure to
U-50,488H or dyn B on the cytosolic Ca2+ level and on the
releasable sarcoplasmic reticular Ca2+ pool.
Dyn B was purchased from Neosystem Laboratoire (Strasbourg, France). Certified peptide purity was 98% and was confirmed in our laboratory by reverse-phase high performance liquid chromatography. Dyn B was received as a lyophilized water-soluble peptide and was dissolved immediately before use in the same medium in which cardiac myocytes were resuspended, containing (mM): 116.4 NaCl, 5.4 KCl, 1.6 MgSO4, 26.2 NaHCO3, 1.0 NaH2PO4, 5.6 D-glucose, 1.0 CaCl2 (pH 7.36 ± 0.05 in the presence of 95% O2, 5% CO2).
(Trans-(dl)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide)methanesulfonate
hydrate (U-50,488H) was purchased from The Upjohn Co.
()-N-(3-Furylmethyl)-
-normetazocine methanesulfonate
(Mr-1452) was a gift from Boehringer Ingelheim Pharmaceuticals, Inc.
(Ridgefield, CO). Caffeine was purchased from Sigma. Ryanodine,
purified, was from BIOMOL Res. Labs., Inc. (Plymouth Meeting, PA).
Animals and all the other chemicals were from the sources listed in the
first article of our series of studies (50).
Cardiac myocytes were isolated from 60-day-old control (F1B) or cardiomyopathic (BIO 14.6) hamsters by using the procedure described in the first study of this series (50). The extraction of RNA, the determination of prodynorphin mRNA, the isolation of myocardial nuclei, the assessment of purity of the nuclear fraction and the nuclear run-off transcription assay were all performed as described (in the first study of this series (50)), as were the identification of dynorphin B-like material, the immunoblotting analysis and the quantitative immunoautoradiography of PKC isozymes, the measurement of PKC activity, and the estimation of cytosolic calcium in single myocardial cells.
Acute Release of Dyn B, U-50,588H, or Caffeine over Single Cardiac MyocytesEach agent was rapidly "puffed" from a micropipette positioned directly above a single resting myocyte (the concentrations of dyn B or U-50,488H in the pipette were 10 or 100 µM, respectively, while the concentration of caffeine was 10 mM). Pressure pulses of 20 p.s.i. were applied to the pipette with a picospritzer II (General Valve Corp., Fairfield, NJ). The duration of these pulses was 2 s for the experiments with dyn B or U-50,488H and 200 ms for the studies in the presence of caffeine.
Data AnalysisThe statistical analysis of the data was performed by using a one-way analysis of variance, followed by Newman Keul's test and assuming a p value less than 0.05 as the limit of significance.
Using the same methodology outlined in our companion studies (50,
51), we have examined whether dyn B or the synthetic selective opioid receptor agonist U-50,488H may affect the levels of prodynorphin
mRNA in hamster ventricular myocytes. A 4-h incubation of myocytes
isolated from 60-day-old control animals in the presence of increasing
concentrations of dyn B produced a dose-dependent stimulation of prodynorphin mRNA expression (Fig.
1). This effect was evident at a concentration as low as
0.1 µM and reached a plateau when the myocytes were
incubated in the presence of concentrations of dyn B ranging between 1 and 10 µM (Fig. 1). Similar to dyn B, U-50,488H (1 µM) markedly increased prodynorphin mRNA levels in
control cells (Fig. 1). Cell treatment with the specific
opioid
receptor antagonist Mr-1452 completely antagonized the effects produced
by dyn B or U-50,488H (Fig. 1). Dyn B or U-50,488H also failed to
affect prodynorphin mRNA levels in control myocytes that were
treated with 5 µM chelerythrine or 1 µM
calphostin C, two highly selective PKC inhibitors (12-15) (Fig.
2). In companion studies (50, 51), we have shown that
prodynorphin mRNA was significantly more expressed in myocytes
isolated from cardiomyopathic hamsters than in myocardial cells from
control animals. In the present study, the incubation for 4 h of
cardiomyopathic myocytes in the presence of 1 µM Mr-1452
markedly down-regulated the expression of prodynorphin mRNA.
Although, mRNA levels remained higher than the levels observed in
control cells or in cardiomyopathic myocytes that were treated either
with chelerythrine or with calphostin C (Fig. 3).
Similar effects were observed when cardiomyopathic myocytes were
exposed for 4 h to 5 or 10 µM Mr-1452 (not shown). The incubation of cardiomyopathic myocytes with 1 µM dyn
B elicited a further slight increase in the expression of prodynorphin
mRNA (Fig. 3). Similar results were observed when cardiomyopathic
cells were incubated for 4 h in the presence of 1 µM
U-50,488H (not shown).
We have previously shown that the increase in prodynorphin mRNA
levels observed in cardiomyopathic myocytes was attributable to an
increase in the transcription of the prodynorphin gene (50, 51). Here
we performed additional run-off experiments in isolated myocardial
nuclei to verify whether the stimulation of prodynorphin mRNA
expression elicited by dyn B or U-50,488H may also occur at the
transcriptional level. The incubation of control myocytes in the
presence of 1 µM dyn B or U-50,488H was able to induce a
marked increase in prodynorphin gene transcription that was completely
antagonized by 1 µM Mr-1452 or by myocyte treatment with
5 µM chelerythrine or 1 µM calphostin C
(Fig. 4). Exposure of myocytes isolated from
cardiomyopathic animals to the opioid receptor antagonist resulted in a
marked decrease in the transcription rate of the prodynorphin gene
which remained above that observed in nuclei that have been isolated
from control cells (Fig. 4).
The exposure of control myocytes to 1 µM dyn B for 30 min
increased the nuclear expression of PKC- (Figs. 5 and
6). A concomitant reduction in the amount of PKC-
was
observed in the cytosolic fraction from dyn B-treated control cells
(Figs. 5 and 6). The treatment with dyn B did not apparently affect the
expression of PKC-
, -
, or -
(Figs. 5 and 6). The
phosphorylation of the acrylodan-labeled myristoylated alanine-rich PKC
substrate (MARCKS) peptide, a high affinity fluorescent substrate for
PKC (16-19), occurred at a higher rate in the presence of nuclei
isolated from control myocytes exposed for 30 min to 1 µM
dyn B than in the presence of nuclei obtained from untreated control
cells (Fig. 7). Such a stimulatory effect was suppressed
by myocyte treatment with 1 µM Mr-1452 (not shown). The
rate of substrate phosphorylation was lower in the presence of nuclei
from dyn B-treated control myocytes than in the presence of nuclei
isolated from cardiomyopathic cells (Fig. 7). No significant change in
acrylodan-peptide fluorescence was observed in the presence of nuclei
which were isolated from dyn B-treated control cells and subsequently
exposed to chelerythrine (5 µM) or calphostin C (1 µM) before being added to the reaction mixture (Fig. 7).
Similar results were obtained when each PKC inhibitor was added to
nuclei isolated from untreated control or cardiomyopathic cells (not
shown).
We have previously shown that Ca2+ release from the
sarcoplasmic reticulum (SR), followed by depletion of this pool,
mediates the effect of opioid receptor stimulation in adult rat
ventricular cardiac myocytes (3, 7). In the present study, we
investigated the effects produced by acute or prolonged stimulation of
opioid receptors on the cytosolic Ca2+ level
([Ca2+]i) in control and cardiomyopathic hamster
myocytes. Fig. 8 shows the effects observed on
[Ca2+]i following an acute release of dyn B or
U-50,488H over a single control or cardiomyopathic myocyte (see
"Materials and Methods"). Confirming the results presented in a
companion study (51), resting [Ca2+]i was
significantly higher in cardiomyopathic than in control cells. Each
opioid agonist elicited an increase in [Ca2+]i of
similar magnitude in both groups of cells (Fig. 8, A and
B). The opioid effect was abolished by cell superfusion in
the presence of 1 µM Mr-1452 (Fig. 8) and was still
preserved immediately after exchanging the perfusate with a
Ca2+-free buffer containing 0.1 mM EGTA (not
shown). We next assessed the releasable SR Ca2+ pool under
basal conditions and after prolonged exposure of control myocytes
either to dyn B or to U-50,488H, by the rapid addition of a high
concentration of caffeine from a pipette above the cell. Caffeine
released Ca2+ from the SR and caused a
[Ca2+]i transient which was abolished after
superfusion with ryanodine (Fig. 9), a substance which
binds to and opens the SR Ca2+ channel (20, 21) leading to
a release and depletion of Ca2+ from this organelle (22).
Previous studies have shown that the effect of caffeine on
[Ca2+]i transient is still evident immediately
after switching the bathing medium to a Ca2+-free buffer
(21) indicating that the increase in [Ca2+]i
caused by caffeine is due to release of Ca2+ from the SR
rather than to influx from the extracellular space. Fig. 9 shows that a
prolonged superfusion of a quiescent control cell with dyn B or
U-50,488H slowly increased resting [Ca2+]i. After
approximately 15 min in the presence of each opioid agonist,
[Ca2+]i returned to the baseline and, at that
time, a rapid addition of caffeine failed to trigger a
[Ca2+]i transient (Fig. 9). When either dyn B or
U-50,488H were superfused in the presence of the
opioid receptor
antagonist Mr-1452, no increase in resting
[Ca2+]i occurred and the caffeine-induced
[Ca2+]i transient was unchanged from the control
(Fig. 9). During the continuous superfusion of a resting
cardiomyopathic myocyte with either dyn B or U-50,488H,
[Ca2+]i was persistently increased for about 40 min before returning to the basal value (Fig. 9). A subsequent rapid
addition of caffeine failed to trigger a [Ca2+]i
transient (Fig. 9). The marked difference in the time course of
[Ca2+]i increase among control and
cardiomyopathic cells exposed to the prolonged action of the two opioid
agonists was consistently observed throughout all the cells tested.
When cardiomyopathic myocytes were superfused with dyn B or U-50,488H
in the presence of Mr-1452, [Ca2+]i remained
unchanged from the basal value and the cell responded to the rapid
addition of caffeine with [Ca2+]i transient (Fig.
9).
The data presented in this report show that the exposure of
control hamster ventricular myocytes to dyn B resulted in a
dose-dependent stimulation in the expression of
prodynorphin mRNA and that prodynorphin mRNA levels were also
increased by the synthetic selective opioid receptor agonist
U-50,488H. These effects appear to be mediated by the interaction of
the opioid agonists with
opioid receptors, since they were
prevented by the specific
opioid receptor antagonist Mr-1452, and
occurred at the transcriptional level, as indicated by the results in
nuclear run-off experiments. Interestingly, Mr-1452 markedly reduced
prodynorphin gene expression in cardiomyopathic myocytes, suggesting an
autocrine function of endogenously synthesized
opioid receptor
ligands. The observation that the opioid antagonist was not able to
affect basal prodynorphin mRNA in control cells suggests, that in
cardiomyopathic myocytes due to the marked increase in the
synthesis and release of endogenous dyn B, the amount of peptide in the
medium might have been raised above a critical concentration, acting in
an autocrine fashion at the level of
opioid receptors to elicit a
tonic feed-forward stimulation of prodynorphin gene expression. Since
we observed only a slight further increase in prodynorphin mRNA
levels following the exposure of cardiomyopathic myocytes to dyn B
or to U-50,488H, we cannot exclude that the amount of dyn B being
released by cardiomyopathic cells might have approached the maximal
effect of the opioid ligand on the expression of the prodynorphin gene.
Subsequently, addition of exogenous dyn B or of the synthetic ligand to
cardiomyopathic myocytes would only produce minimal additive effects.
In companion studies (50, 51), we have shown that PKC is involved in
mediating the increase in prodynorphin gene expression observed in
cardiomyopathic cells. On the other hand, we have also previously shown
that, in the myocardial cell,
opioid receptors are coupled to PKC (7). In the present study, the finding that dyn B or U-50,488H failed
to affect prodynorphin mRNA levels and prodynorphin gene transcription in control myocytes that have been treated with chelerythrine or calphostin C indicates that
opioid receptor stimulation may have increased the expression of the prodynorphin gene
through a PKC dependent pathway. Such a possibility appears to be
confirmed by the finding that the treatment of control myocytes with
dyn B induced the translocation of PKC-
to the nucleus and increased
nuclear PKC activity. Although Mr-1452 significantly down-regulated
prodynorphin gene expression in cardiomyopathic myocytes, the levels of
prodynorphin mRNA in cardiomyopathic cells exposed to the opioid
antagonist remained higher than those detected in cardiomyopathic
myocytes treated in the presence of PKC inhibitors. This might be due
to the fact that autocrine stimulation of
opioid receptors, by
increasing the nuclear expression of PKC-
without affecting the
expression of PKC-
, -
, and -
, may only have elicited the
activation of part of the PKC isozymes available in the myocardial
cell. In this regard, the expression of both PKC-
and PKC-
,
besides that of PKC-
, were found to be increased in nuclei isolated
from cardiomyopathic myocytes and, in the presence of these nuclei, the
phosphorylation of the MARCKS peptide occurred at a higher rate than
that observed in the presence of nuclei isolated from dyn B-treated
control cells. Therefore, the total amount of activated PKC which
contributes to stimulate the expression of the prodynorphin gene in
cardiomyopathic myocytes may result from a number of different
stimuli that may share PKC activation as a common regulatory mechanism
of gene expression. This possibility is supported by the observation
that, in the myocardial cell, different receptor systems are coupled to
PKC, including muscarinic,
1-adrenergic, adenosine,
angiotensin II, and endothelin-1 receptors, as well as poorly
characterized stretching sensitive "mechanoceptors" (23-27).
Further support is the finding that PKC activation by agonists of these
receptors results in common downstream consequences, including the
stimulation of gene expression and the hypertrophic growth
(28-31).
Here we show that acute release of both dyn B or U-50,488H over single
normal or cardiomyopathic myocytes elicited a transient increase in
[Ca2+]i of similar magnitude in both groups of
cells. Such a response appeared to be mediated by opioid receptors
and to involve the release of Ca2+ from an intracellular
storage site since it was completely abolished by a specific
opioid
receptor antagonist and was preserved in a Ca2+-free
buffer. It may be of interest that this effect was observed following a
direct local exposure to the opioid agonists, as it might occur when
endogenously synthesized
opioid receptor ligands are secreted
from the myocardial cell. The results obtained following a prolonged
exposure of normal or cardiomyopathic myocytes to dyn B or U-50,488H
show that these opioid agonists ultimately led to a depletion of
Ca2+ from an intracellular pool in both groups of cells. In
previous studies we have shown that
opioid receptor stimulation
released Ca2+ from the SR in rat cardiac myocytes (3, 7, 8)
and from an intracellular pool in neuroblastoma-2 a cells
(7). The receptor activation depleted these Ca2+ storage
sites in both cell types and was coupled with a rapid and sustained
increase in phosphoinositide turnover (7, 32). These observations
suggest that the opioid-induced effects on cytosolic Ca2+
homeostasis observed in the present study may represent a general mechanism for the action of
opioid receptor agonists. The present data also show that the time course of [Ca2+]i
increase in response to a sustained exposure to the opioid agonists was
significantly more prolonged in cardiomyopathic than in normal cells.
This difference may be due to the presence of altered Ca2+
efflux rates in cardiomyopathic cells compared with normal myocytes, as
suggested by the finding that the sarcolemmal Ca2+ ATPase
activity and gene expression were both reduced in myocardial cells from
BIO 14.6 cardiomyopathic hamsters (33). Therefore, due to the abnormal
[Ca2+]i observed at rest in cardiomyopathic
myocytes, a further Ca2+ loading due the opioid-mediated
Ca2+ release from the SR may require a more prolonged time
for Ca2+ extrusion through the sarcolemma in
cardiomyopathic myocytes compared with normal cells. On the other
hand, we cannot exclude that the difference in the time course of
[Ca2+]i increase observed among cardiomyopathic
and normal cells in response to
opioid receptor stimulation may
reflect the presence of abnormalities in Ca2+ sequestration
and/or release at the level of the SR. Supporting such a hypothesis are
the observation that the SR Ca2+ ATPase activity and gene
expression are also inhibited in cardiomyopathic hamster hearts (33)
and the finding that the number of ryanodine-binding sites is increased
in cardiac membrane preparations from BIO 14.6 hamsters, suggesting a
defect in the function of the ryanodine-sensitive SR calcium release
channel (34, 35).
The possible consequences of the present results, suggesting that dyn B may be involved in an autocrine feed-back loop regulating prodynorphin gene expression, remain to be elucidated. We may only speculate that tonic release of dyn B, by depleting the SR releasable Ca2+ pool, may contribute to elicit the decrease in the amplitudes of the cytosolic Ca2+ transient and of the associated contraction previously observed in isolated cardiomyopathic cells (36). Moreover, it is now clear that opioid peptides also act as growth regulators in many normal and malignant tissues (37-43). Recently, tonic release of opioid peptides has been implicated in the regulation of neuroblastoma proliferation through the activation of specific opioid receptors (44). Interestingly, accumulating evidence show that opioid peptides may be produced in an autocrine and, probably, paracrine manner (44-46) and may influence proliferation and differentiation in a wide variety of cells and tissues, including neurones and glia in the nervous system (47) and myocardial and epicardial cells in the neonatal heart (48). These findings have led to the consideration of opioid peptides as growth factors which act by regulating cell proliferation and are also able to alter cell migration and the orchestration of cells into a specific architecture (49). We cannot exclude that these "trophic" effects of opioid peptides might also apply to primary hypertrophic cardiomyopathies, a number of diseases which, besides showing an impairment of myocyte contractility and cytosolic Ca2+ handling, also exhibit substantial alterations in processes related to the growth, differentiation and architectural assembly of cardiac myocytes. However, it must be emphasized that we have not yet demonstrated whether endogenously synthesized dynorphin-related peptides may have a trophic role in the hamster model of hypertrophic cardiomyopathy and whether, if there is such a role, they may contribute to initiate or to counteract myocyte abnormalities in growth and differentiation. Clarification of these issues must await more direct experimental approaches.
We thank Giuseppe Delogu for his excellent technical assistance.