COMMUNICATION
Nuclear Opioid Receptors Activate Opioid Peptide Gene Transcription in Isolated Myocardial Nuclei*

Carlo VenturaDagger §, Margherita MaioliDagger , Gianfranco PintusDagger §, Anna Maria PosadinoDagger , and Bruna TadoliniDagger §

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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 kappa  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 kappa  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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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-delta and -epsilon 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 kappa  opioid receptors (6) elicited a tonic feed-forward stimulation of the expression of its coding gene (7). Moreover, kappa  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 kappa  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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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). [alpha -32P]UTP and 5alpha ,7alpha ,8beta -(-)-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)-alpha -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 beta -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 beta -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 [alpha -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 kappa  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 beta -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.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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 kappa  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 kappa  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 kappa  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 kappa  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 kappa  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 kappa  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 kappa  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 kappa -binding sites may reflect changes in opioid receptor turnover and/or gene expression occurring during the cardiomyopathic process.


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Fig. 1.   Prodynorphin gene transcription in isolated nuclei exposed to dyn B or U-50. Nuclear run-off assays were performed in myocardial nuclei isolated from myocytes of 60-day-old normal (A) or cardiomyopathic (B) hamsters. Autoradiograms are representative of six separate experiments. 1, transcription of the prodynorphin gene; 2, cyclophilin mRNA. Autoradiographic exposure was for 2 days on Kodak X-Omat film with an intensifying screen. The marks on the right indicate the position of 400- or 220-base pair (bp) radiolabeled DNA markers, showing that the single protected fragments migrated with a molecular size of 400 or 270 bases, corresponding to prodynorphin or cyclophilin mRNA, respectively. Isolated nuclei were exposed for 4 h to the indicated concentrations of dyn B or U-50. In separate experiments, nuclei were treated with dyn B (1 µM) or U-50 (1 µM) in the presence of 1 µM Mr. A 4-h treatment with 1 µM dyn B or U-50 was also performed in the presence of 5 µM chelerythrine (Chele) or 1 µM calphostin C (Cal).


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Fig. 2.   PKC activity in myocardial nuclei exposed to dyn B. Nuclei were obtained from myocytes of 60-day-old normal or cardiomyopathic hamsters. Nuclear PKC activity was measured in the presence of the acrylodan-labeled MARCKS peptide. Peptide phosphorylation was started by the addition of 10 µg of nuclear protein (arrow) and was followed at 37 °C. As the acrylodan peptide becomes phosphorylated, it undergoes a time-dependent decrease in its fluorescence at 480 nm. black-diamond  and bullet , nuclei were isolated from normal myocytes and exposed for 30 min in the absence or presence of 1 µM dyn B, respectively; diamond  and open circle , nuclei were isolated from cardiomyopathic cells and exposed for 30 min in the absence or presence of 1 µM dyn B, respectively. black-triangle and triangle , nuclei were isolated from untreated cardiomyopathic cells and incubated for 30 min with 1 µM dyn B in the presence of 5 µM chelerythrine or 1 µM Mr, respectively. The time course of the fluorescence of the acrylodan peptide alone (black-square) is also reported. The data are expressed as mean values ± S.E. (n = 6). From 600 to 1200 s, black-diamond , bullet , diamond , and open circle  were significantly different from black-square, black-triangle, and triangle ; from 600 to 900 s, bullet  was significantly different from black-diamond ; from 500 to 600 s, open circle  was significantly different from diamond ; no significant difference was observed between black-square, black-triangle, and triangle  (one-way analysis of variance, Newman-Keul's test).


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Fig. 3.   Scatchard analysis of the specific [3H]U-69 binding to myocardial nuclei and F40 membranes. Samples were isolated from 60-day-old normal (open circle ) or cardiomyopathic (bullet ) myocytes. The data are expressed as mean values ± S.E. (n = 6). *, significantly different from open circle . Bmax values in F40 membranes from normal or cardiomyopathic cells were significantly different from the values observed in myocardial nuclei isolated from the corresponding group of cells (one-way analysis of variance, Newman-Keul's test).

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.

    FOOTNOTES

* 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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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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