From the Department of Biomedical Sciences, Division
of Biochemistry, Laboratory of Cardiovascular Research, University of
Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy and the
§ National Laboratory of the National Institute of
Biostructures and Biosystems, 07100 Osilo, Italy
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ABSTRACT |
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Opioid-binding sites were identified in highly
purified nuclei isolated from hamster ventricular myocardial cells. A
significant increase in the maximal binding capacity for a opioid
receptor ligand was observed in myocardial nuclei from BIO 14.6 cardiomyopathic hamsters, as compared with nuclei obtained from normal
myocytes of the F1B strain. The exposure of isolated nuclei to
dynorphin B, a natural agonist of
opioid receptors, markedly
increased opioid peptide gene transcription. The transcriptional effect was mediated by nuclear protein kinase C activation and occurred at a
higher rate in nuclei from cardiomyopathic myocytes than in nuclei
isolated from normal cells. Thus, a nuclear endorphinergic system may
play an intracrine role in the regulation of gene transcription under
both normal and pathological conditions.
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INTRODUCTION |
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It is increasingly becoming evident that cell nuclei harbor the
potential for intrinsic signal transduction pathway(s), as indicated by
the presence of nuclear enzymes and substrates associated with the
synthesis of diacylglycerol and inositol phospholipids (1, 2). We have
previously shown that the direct exposure of isolated myocardial nuclei
to a protein kinase C (PKC)1
activator stimulated opioid gene transcription through a mechanism mediated by nuclear PKC activation (3). Recently, we observed that
PKC- and -
and PKC activity were increased in nuclei from myocytes of cardiomyopathic hamsters (4), leading to an overexpression of the prodynorphin gene and of its peptide product dynorphin B (dyn B)
(4, 5). Interestingly, myocyte exposure to this agonist of
opioid
receptors (6) elicited a tonic feed-forward stimulation of the
expression of its coding gene (7). Moreover,
opioid receptor
stimulation modified both cytosolic Ca2+ and pH homeostasis
and remarkably affected myocardial contractility (8, 9). Thus, nuclear
PKC-mediated activation of opioid gene transcription may be part of an
autocrine circuit of regulation of cellular homeostasis under normal or
pathological conditions. Although most of myocardial dyn B was targeted
for secretion (10), consistent amounts of dyn B were also found at
myocyte level (4, 5), suggesting that the opioid peptide may also act
intracellularly.
In the present study, we investigated whether the exposure of isolated
myocardial nuclei to opioid receptor agonists may trigger nuclear
signaling and changes in gene transcription. Nuclei were isolated from
myocytes of 60-day-old normal (F1B) or BIO 14.6 Syrian hamsters, an
experimental model of hereditary cardiomyopathy (11-13) and were
either exposed to dyn B or incubated in the presence of the synthetic
selective opioid receptor ligand U-50,488H (U-50) (14).
Opioid-treated nuclei were processed for the analysis of both
prodynorphin gene transcription and nuclear PKC activity.
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MATERIALS AND METHODS |
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Control (F1B) and cardiomyopathic (BIO 14.6) male Syrian
hamsters were purchased from Bio Breeders (Fitchburg, MA).
EcoRI, NcoI, ATP, CTP, GTP, UTP, collagenase B,
and the acrylodan-labeled myristoylated alanine-rich protein kinase C
substrate (MARCKS) peptide were from Boehringer Mannheim. RNAMATRIXTM
was from BIO 101, Inc. (Vista, CA). Chelerythrine and calphostin C were
from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA).
[-32P]UTP and
5
,7
,8
-(
)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl]-[phenyl-3H(n)]benzene
acetamide ([3H]U-69,593) were from Amersham Pharmacia
Biotech.
(Trans-(DL)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzene-acetamide)methanesulfonate hydrate (U-50,488H) was from The Upjohn Co. (Kalamazoo, MI).
(
)-N-(3-furylmethyl)-
-normetazocine methanesulfonate
(Mr-1452) was a gift from Boehringer Ingelheim Pharmaceuticals, Inc.
(Ridgefield, CO). Dynorphin 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. 1,2-Dioctanoyl-sn-glycerol and all the other
chemicals were from Sigma.
Ventricular cardiac myocytes and myocardial nuclei were isolated as described (4) from 60-day-old normal (F1B) or cardiomyopathic (BIO 14.6) male Syrian hamsters. The nuclear preparation lacked contamination by sarcoplasmic reticular membranes, inner or outer mitochondrial membranes, or sarcolemmal membranes as indicated by the measurement of the activity of the corresponding marker enzymes rotenone-insensitive NADPH-cytochrome c reductase, succinate dehydrogenase, rotenone-insensitive NADH-cytochrome c reductase, 5'-nucleotidase, and ouabain-sensitive Na+,K+-ATPase, which were undetectable in the nuclear fraction (4). Animal care and experimentation was approved by the local Animal Care and Use Committee and complied with the standards for care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, MD).
Preparation of Sarcolemmal Membrane-enriched Fraction--
In
order to prepare a fraction (referred to as F40) enriched
in sarcolemmal membranes, myocytes obtained from 60-day-old normal or
cardiomyopathic hamsters were first homogenized with a Dounce homogenizer (three strokes of an A pestle) in a medium containing 50 mM Tris/HCl, pH 7.4, 250 mM sucrose, 1 mM EDTA, 0.1 mM DTT, 0.5 mM PMSF, 1 µM leupeptin, and 10 mM -mercaptoethanol.
The homogenate was centrifuged at 1000 × g at 4 °C
for 15 min. The supernatant was further centrifuged at 40,000 × g at 4 °C for 30 min. The resulting pellet
(F40) was then resuspended in the binding buffer (see under
"Opioid Binding Assay"). In the F40 fraction and in
cell homogenates from normal cells, the ouabain-sensitive Na+,K+-ATPase activity, assessed as reported by
Lamers and Stinis (15), was 18.3 ± 0.8 and 1.7 ± 0.06 µmol/mg protein/h, respectively, as estimated by the measure of the
inorganic phosphate released (16).
Nuclear Run-off Transcription Assay--
Prodynorphin gene
transcription was assessed by nuclear run-off assays performed as
reported in detail elsewhere (4) in nuclei isolated from both normal or
cardiomyopathic cells. Myocardial nuclei were resuspended in a buffer
containing 50 mM Tris/HCl, pH 8.0, 5 mM
MgCl2, 0.1 mM EDTA, 40% glycerol, 0.1 mM DTT, 0.5 mM PMSF, 1 µM
leupeptin, and 10 mM -mercaptoethanol. 90 µl of the
nuclear preparation were added with 100 µl of 2× reaction buffer
containing 10 mM Tris/HCl, pH 7.5, 5 mM
MgCl2, 0.3 M KCl, 5 mM DTT, 1 mM each of ATP, GTP, and CTP, and 5 µl of
[
-32P]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 (17), followed
by purification on RNAMATRIXTM. Purified RNA was then subjected to a
solution hybridization RNase protection assay (4). Briefly, equal
counts of 32P-labeled RNA (about 5 × 106
cpm) were hybridized for 12 h at 55 °C in the presence of
unlabeled antisense prodynorphin mRNA that was synthesized
following transcription of an EcoRI-linearized pGEM3 plasmid
bearing a 400-base pair HindIII-BamHI fragment of
the main exon of rat genomic prodynorphin clone. Finally, samples were
incubated with a combination of RNase A and T1 and exposed to
proteinase K. The protected fragments were recovered after phenol
chloroform extraction and electrophoretically separated in a
polyacrylamide nondenaturing gel. Autoradiographic exposure was for
48 h. 32P-Labeled nuclear RNA was also hybridized with
unlabeled antisense cyclophilin mRNA synthesized from a
NcoI-linearized pBS vector containing a 270-base pair
fragment of plB15, a cDNA clone encoding for rat cyclophilin (18).
Cyclophilin mRNA was utilized as a constant mRNA for
control.
Measurement of Nuclear PKC Activity-- PKC activity from isolated myocardial nuclei was measured by the aid of a continuous fluorescence assay (4) in the presence of the acrylodan-labeled MARCKS peptide, a high affinity fluorescent substrate in vitro for PKC (19-22). 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% (23). The reaction mixture contained, in a final volume of 1 ml, 10 mM Tris/HCl, pH 7.0, 90 mM KCl, 3 mM MgCl2, 0.3 mM CaCl2, 0.1 mM EGTA, 100 µM ATP, 10% ethylene glycol, 0.5 µg of phosphatidylserine, 0.1 µg of 1,2-dioctanoyl-sn-glycerol, and 75 nM acrylodan-labeled MARCKS peptide. The phosphorylation of the acrylodan-labeled peptide was followed at 37 °C and was started by adding 10 µg of nuclear protein.
Opioid Binding Assay--
[3H]U-69,593 (55.0 Ci/mmol) was used as a selective opioid receptor ligand in binding
assays. Each sample (300 µg of protein) was incubated with the
radiolabeled ligand (1, 2, 3, 4, 5, 10, 15, 20, or 30 nM)
in 0.25 ml of a binding buffer containing 50 mM Tris/HCl,
pH 7.4, 5 mM MgCl2, 250 mM sucrose,
0.1 mM DTT, 0.5 mM PMSF, 1 µM
leupeptin, and 10 mM
-mercaptoethanol. The specific binding was measured as the difference between binding in the absence
and binding in the presence of 10 µM of the unlabeled ligand (The Upjohn Co.). The binding reactions were allowed to run for
45 min at 25 °C and stopped by diluting five times with ice-cold
binding buffer. The incubation media were then filtered over vacuum on
Whatman GF/B glass fiber filters, followed by three washes with 10 ml
of ice-cold binding buffer. Filters were finally counted for
radioactivity by liquid scintillation spectrometry. Kd and Bmax values were calculated with
the LIGAND program (24).
Proteins-- Protein concentration was determined by the method of Lowry et al. (25) using bovine serum albumin as a standard.
Data Analysis-- The 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.
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RESULTS AND DISCUSSION |
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A 4-h exposure of isolated myocardial nuclei obtained from normal
hamster myocytes to increasing concentrations of dyn B produced a
dose-dependent stimulation of prodynorphin gene
transcription (Fig. 1A). Such
an effect was evident at a concentration as low as 0.1 µM
and reached a maximum when the isolated nuclei were incubated in the
presence of concentrations of dyn B ranging between 1 and 10 µM. Similar to dyn B, 1 µM U-50 markedly
stimulated prodynorphin gene transcription in these control nuclei
(Fig. 1A). Basal prodynorphin gene transcription was
considerably increased in nuclei isolated from cardiomyopathic myocytes
as compared with nuclei obtained from control cells (Fig.
1B). As it has been described in detail elsewhere (4, 5),
such an increase was related both to a rise in nuclear PKC activity and
to intracellular Ca2+ overload occurring during the
cardiomyopathic process. Here we show that the direct exposure to dyn B
or U-50 of nuclei isolated from cardiomyopathic myocytes led to a
further increase in the transcription rate of the prodynorphin gene
(Fig. 1B). Fig. 1 shows that both dyn B and the synthetic
opioid receptor ligand failed to affect prodynorphin gene
transcription when nuclei isolated from either normal or
cardiomyopathic cells were exposed to 1 µM Mr-1452 (Mr),
a selective
opioid receptor antagonist (26). Opioid-mediated
stimulation of prodynorphin gene transcription was also suppressed by
chelerythrine or calphostin C, two highly selective PKC inhibitors
(27-30) (Fig. 1). Moreover, in cardiomyopathic nuclei these inhibitors
down-regulated prodynorphin gene transcription at a rate below that
observed under basal conditions (Fig. 1B). Such a result is
consistent with previous observations (4) showing that inhibition of
nuclear PKC activity in nuclei from cardiomyopathic myocytes resulted
in a decrease in the basal rate of prodynorphin gene transcription. In
the current investigation, we show that a 30-min exposure to 1 µM dyn B of nuclei isolated from normal hamster myocytes
induced a significant increase in nuclear PKC activity, as revealed by
the analysis of the phosphorylation rate of the acrylodan-labeled
MARCKS peptide (Fig. 2). Confirming our
previous investigations (4), nuclear PKC activity was increased in
nuclei from cardiomyopathic myocytes compared with nuclei isolated from
normal cells. The current experimental data show that the exposure to
dyn B of nuclei isolated from cardiomyopathic cells elicited a further
increase in the phosphorylation rate of the fluorescent PKC substrate
(Fig. 2). The opioid-induced stimulation of nuclear PKC activity could
be abolished by the
opioid receptor antagonist Mr as well as by the
exposure of the isolated nuclei to chelerythrine (Fig. 2) or calphostin
C (not shown). To further investigate whether the opioid agonists may
have exerted their effects through specific nuclear
opioid
receptors, nuclei isolated from both normal and cardiomyopathic
myocytes were incubated in the presence of [3H]U-69,593
(U-69), a highly selective radiolabeled
opioid receptor ligand
(31). Receptor binding parameters for this opioid agonist were then
compared with those determined in a sarcolemmal membrane-enriched fraction (F40) obtained from both groups of myocytes. The
presence of
opioid receptors in myocardial cells has been
demonstrated in our previous studies performed in isolated cardiac
sarcolemma (32) and has been extensively confirmed in subsequent
investigations (33-35). The binding experiments performed in the
current study revealed the presence of highly specific
opioid-binding sites in myocardial nuclei (Fig.
3). The specific binding in nuclei and
F40 membranes ranged between 75 and 85% of the total
bound. The Scatchard plots of [3H]U-69 binding in each
subcellular fraction were linear and were characterized by a single
dissociation constant (Kd) in the low nanomolar
range (Fig. 3). A marked increase in the maximal binding capacity
(Bmax) for [3H]U-69 was evident in nuclei
isolated from cardiomyopathic myocytes, as compared with nuclei from
normal cells (Fig. 3). Similarly, in F40 membranes isolated
from cardiomyopathic myocytes the Bmax value was
significantly higher than that detected in the F40 fraction from normal cells. On the whole, the Bmax values in
F40 membranes isolated from either normal or
cardiomyopathic myocytes were about 4-fold higher than the values
observed in nuclei obtained from the corresponding group of cells (Fig.
3). No significant difference in the Kd values was
found among nuclear and F40 fractions obtained from normal
or cardiomyopathic cells (Fig. 3). The cellular mechanisms regulating
the expression of various neurotransmitter receptors in the myocardium
are mostly enigmatic. It is presently unknown whether the observed
increase in the amount of
-binding sites may reflect changes in
opioid receptor turnover and/or gene expression occurring during the
cardiomyopathic process.
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Our results demonstrate the involvement of nuclear opioid receptors in the transcriptional regulation of the prodynorphin gene in myocardial cells. Interestingly, the myocardial nuclei were isolated from the myocytes of cardiomyopathic animals of 60 days, an age that corresponds to an early phase in the development of the cardiomyopathy and precedes the onset of heart failure (11-13). It is also noteworthy that opioid peptides regulate crucial determinants of the myocardial cell function (8, 9). Hence, the nuclear system described here may be part of a signaling mechanism triggering the activation of nuclear PKC and PKC-mediated gene transcription under both normal and pathological conditions. The present data appear to disclose a previously undefined intracrine role for an endorphinergic system and may abet future investigations on the biological role of nuclear opioid-binding sites in both myocardial and nonmyocardial cells.
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FOOTNOTES |
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* This work was supported by Telethon-Italy Grant 958 and by grants from the Ministero Università-Ricerca Scientifica e Tecnologica.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 39-79-228121 or 39-79-228279; Fax: 39-79-228120; E-mail: Chim_med{at}SSmain.UniSS.It.
1 The abbreviations used are: PKC, protein kinase C; dyn B, dynorphin B; MARCKS, myristoylated alanine-rich protein kinase C substrate; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; U-50, U-50,488H; U-69, U-69,593; Mr, Mr-1452.
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REFERENCES |
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