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Cardiovascular Research 2000 45(4):1054-1064; doi:10.1016/S0008-6363(99)00408-3
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Copyright © 2000, European Society of Cardiology

Elf-pulsed magnetic fields modulate opioid peptide gene expression in myocardial cells

Carlo Venturaa,b,*, Margherita Maiolia, Gianfranco Pintusa,b, Giovanni Gottardic and Ferdinando Bersanic,d

aDepartment of Biomedical Sciences, Division of Biochemistry, Laboratory of Cardiovascular Research, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy
bNational Laboratory of the National Institute of Biostructures and Biosystems, 07033 Osilo, Italy
cDepartment of Physics, University of Bologna, 40127 Bologna, Italy
dCentro Interuniversitario Interazione Campi Elettromagnetici e Biosistemi (ICEmB), 16145 Genova, Italy

* Corresponding author. Tel.: +39-79-228-121, +39-79-228-279; fax: +39-79-228-120 chim_med{at}ssmain.uniss.it

Received 28 May 1999; accepted 15 November 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Magnetic fields have been shown to affect cell proliferation and growth factor expression in cultured cells. Although the activation of endorphin systems is a recurring motif among the biological events elicited by magnetic fields, compelling evidence indicating that magnetic fields may modulate opioid gene expression is still lacking. We therefore investigated whether extremely low frequency (ELF) pulsed magnetic fields (PMF) may affect opioid peptide gene expression and the signaling pathways controlling opioid peptide gene transcription in the adult ventricular myocyte, a cell type behaving both as a target and as a source for opioid peptides. Methods: Prodynorphin gene expression was investigated in adult rat myocytes exposed to PMF by the aid of RNase protection and nuclear run-off transcription assays. In PMF-exposed nuclei, nuclear protein kinase C (PKC) activity was followed by measuring the phosphorylation rate of the acrylodan-labeled MARCKS peptide. The effect of PMF on the subcellular distribution of different PKC isozymes was assessed by immunoblotting. A radioimmunoassay procedure coupled to reversed-phase high performance liquid chromatography was used to monitor the expression of dynorphin B. Results: Here, we show that PMF enhanced myocardial opioid gene expression and that a direct exposure of isolated myocyte nuclei to PMF markedly enhanced prodynorphin gene transcription, as in the intact cell. The PMF action was mediated by nuclear PKC activation but occurred independently from changes in PKC isozyme expression and enzyme translocation. PMF also led to a marked increase in the synthesis and secretion of dynorphin B. Conclusions: The present findings demonstrate that an opioid gene is activated by myocyte exposure to PMF and that the cell nucleus and nuclear embedded PKC are a crucial target for the PMF action. Due to the wide ranging importance of opioid peptides in myocardial cell homeostasis, the current data may suggest consideration for potential biological effects of PMF in the cardiovascular system.

KEYWORDS Myocytes; Gene expression; Cell communication; Signal transduction; Protein kinases


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Magnetic fields (MF) may elicit multiple effects in biological systems, including behavioural changes in intact organisms or the modulation of proliferation and growth factor release in cultured cells [1–3]. Among the regulatory systems that appear to be targeted for the action of MF are endogenous opioid peptides. In vivo studies performed in different experimental animals revealed that MF can produce analgesic effects through an opioid receptor-mediated mechanism [4] and are able to affect the spontaneous electrical brain activity by interfering with the action of both exogenous and endogenous opioids [5]. In mice, MF have been found to enhance the duration of pharmacologically-induced anaesthesia by releasing endogenous opioids and/or enhancing the activity of opioid signaling pathways [6]. The capability of MF of controlling the central cholinergic system also appears to depend on the activation of an opioidergic pathway, since the opioid receptor antagonist naloxone was capable of suppressing the decrease in high-affinity choline uptake elicited by MF in the rat brain [7]. Opioid receptor antagonism also attenuated MF-induced antiparkinsonian effects in man [8].

The possibility that the observed effects of MF on opioid-related events may have important implications in cellular homeostasis is further supported by the finding that opioid peptides may act as growth modulators and may control both cell differentiation and architecture in a wide variety of tissues [9–11]. Nevertheless, the molecular mechanism(s) underlying the MF action remain to be fully elucidated. In particular, evidence that MF may affect opioid gene expression is still lacking. Unraveling this matter may be of particular relevance, since, due to the conflicting results achieved so far on the effect of MF on early gene transcription [12,13], the possibility that MF may act at the level of gene expression remains an open issue.

We have previously shown that the myocardial cell responds to opioid receptor stimulation with deep changes in cytosolic Ca2+/pH homeostasis and contractility [14,15]. Myocytes are also a source for opioid peptides since they express the prodynorphin gene and are able to synthesise and secrete the natural {kappa} opioid receptor agonist dynorphin B [16,17]. Interestingly, the prodynorphin gene and dynorphin B are overexpressed in myocytes isolated from BIO 14.6 Syrian cardiomyopathic hamsters [18]. Moreover, in cardiomyopathic myocytes dynorphin B released Ca2+ from an intracellular store [19] and acted in an autocrine fashion to stimulate the transcription of its coding gene [20]. These findings suggest that the myocardial cell is a valuable tool for the analysis of opioid gene expression under normal conditions and throughout processes involving an impairment of cell growth and differentiation.

In the present study, we aimed at investigating the effect of MF on opioid peptide gene expression and on the molecular patterning controlling opioid gene transcription. For this purpose, we exposed adult cultured rat ventricular cardiac myocytes to extremely low frequency (ELF) pulsed magnetic fields (PMF) and investigated their effects on prodynorphin mRNA levels, the transcription rate of the prodynorphin gene and the expression of a biologically active end-product of the opioid gene.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell isolation and PMF exposure
Ventricular cardiac myocytes were isolated as previously described [15] from 2 to 3 month-old male Wistar rats and were subjected to a short-term, serum-free primary culture [16,17]. 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, USA).

PMF were generated as described [2] by a pair of rectangular horizontal coils (4x23 cm; 1400 turns per coil) in a pseudo-Helmholtz configuration (distance between coils, 10 cm) powered by a pulse generator (Igea, Italy). The wave shape of the magnetic signal was approximately triangular, the rise time was 1.34 ms, the repetition frequency 50 Hz and the peak amplitude of the magnetic field 1.74 mT; the pulses were unidirectional and the minimum magnetic field was 0.196 mT, corresponding to the baseline of the signal. The induced electric field depended on the position inside the culture dishes. Taking into account the size (30 mm in diameter) of the culture dishes, the maximum electric field close to the internal walls was estimated to be approximately 0.02 mV/cm.

2.2 RNase protection assay
Prodynorphin mRNA was assessed by the aid of a solution hybridization RNase protection, as described in detail elsewhere [16–18]. 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, while transcription of the plasmid linearized with EcoRI in the presence of [32P]CTP (800 Ci/mmol) gave an antisense strand used to hybridize cellular prodynorphin mRNA. Unlabeled antisense prodynorphin mRNA was also synthesized from the plasmid and was used in nuclear run-off experiments (see below) to hybridize 32P-labeled RNA synthesized by isolated myocardial nuclei. 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 picogram values on a correlated standard curve. Data were expressed as picograms of mRNA per microgram of total RNA.

2.3 Nuclear run-off transcription assay
Nuclear run-off was performed as described [18] in isolated myocardial nuclei lacking contamination by sarcoplasmic reticular membranes, inner or outer mitochondrial membranes or sarcolemmal membranes [17,18]. 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 dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 µM leupeptin, and 10 mM β-mercaptoethanol. Ninety µl of the nuclear preparation were added with 100 µl of 2x 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) (Amersham International), 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 per ml RNase-free DNase. Nuclear RNA was isolated by using guanidine thiocyanate and acid phenol extraction, followed by purification on RNAMATRIXTM (BIO 101, Vista, CA) [18]. Equal counts of 32P-labeled RNA (about 5x106 cpm) were then subjected to a solution hybridization RNase protection assay and were hybridized for 12 h at 55°C with unlabeled antisense prodynorphin mRNA. Samples were incubated with a combination of RNase A and T1 and exposed to proteinase K. The protected fragments were 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 [21]. Cyclophilin mRNA was utilized as a constant mRNA for control.

2.4 Nuclear PKC activity
PKC activity from isolated myocardial nuclei was measured with the aid of a continuous fluorescence assay [17,18] in the presence of the acrylodan-labeled MARCKS peptide, a high affinity fluorescent substrate in vitro for PKC [22,23]. 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.

2.5 Immunoblotting analysis of PKC
Nuclear samples, total cell lysates, or cytosolic fractions were prepared as described [18] and electrophoresed on 8% SDS–polyacrylamide gels. After protein transfer to nitrocellulose, the blot was saturated for 1 h at room temperature with 3% BSA in Tris-buffered saline containing Tween (TBS-T) (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20). The immunoreaction was carried out overnight at 4°C in TBS containing 1% BSA, 0.02% Tween 20 and the primary antibody (antisera to PKC-{alpha}, PKC-{delta}, PKC-{varepsilon}, or PKC-{zeta}) diluted 1:100. After being washed three times with TBS-T the membranes were incubated with 125I-labeled donkey anti-rabbit IgG antibodies (106 cpm/ml) in TBS-T with 1% BSA for 1 h at room temperature. Then, the nitrocellulose membranes were dried and exposed to Kodak X-OMAT AR films with an intensifying screen for 48 h at –70°C. The intensities of the autoradiographic bands were measured with a laser densitometer (Image Quant-Computing Densitometer 300/325, Molecular Dynamics, Sunnyvale, CA) and, for each PKC isozyme, the data were expressed as percentage changes in the autoradiographic intensity in each sample (total lysates, cytosolic fraction, or nuclear fraction) from PMF-exposed myocytes relative to the intensity in the corresponding sample obtained from untreated cells (considered as 100%).

2.6 Dynorphin B-like material
Immunoreactive dynorphin B (ir-dyn B) was measured by a previously described radioimmunoassay procedure [16–18] that utilized the 13 S antiserum raised against dynorphin B and capable of recognizing the high molecular weight peptides cleaved from the prodynorphin precursor and containing dynorphin B in their sequence [24]. Acetic acid extracts from control or PMF-exposed cardiomyocytes, or pooled samples from the incubation medium were processed by reverse-phase high performance liquid chromatography. The collected fractions were radioimmunoassayed and the immunoreactivity was attributed to authentic dynorphin B by comparison with the elution position of a synthetic standard [16–18].

2.7 Proteins
Protein concentration was determined by the method of Lowry et al. [25], using bovine serum albumin as a standard.

2.8 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.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Ventricular myocytes were exposed to PMF of the type previously proved to be effective in promoting bone repair and modulating immune cell function [1–3].

The RNase protection analysis of prodynorphin mRNA revealed a marked increase in mRNA levels in PMF-treated cells. In particular, prodynorphin mRNA was already enhanced above the control value after 1 h of exposure to PMF (Fig. 1). The stimulatory effect elicited by PMF peaked at 4 h and was still evident following 8 h of treatment (Fig. 1). A 4-h exposure to PMF was able to elicit a marked increase in prodynorphin mRNA expression even when the coil orientation was shifted from a horizontal to a vertical alignment (not shown).


Figure 1
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  		Fig. 1 Effect of PMF on prodynorphin mRNA. The upper panel shows representative autoradiograms of the RNase protection analysis of myocardial prodynorphin mRNA. Autoradiographic exposure was for 2 days on Kodak X-Omat film with an intensifying screen. The bar indicates the position of a 400-base pair radiolabeled DNA marker, showing that the single protected fragment migrated with a molecular size of 400 bases, corresponding to prodynorphin mRNA. (A) Untreated myocytes; (B), (C) and (D) cells were exposed to PMF for 1, 4, and 8 h, respectively. Averaged values of prodynorphin mRNA levels are reported in the lower panel of the figure. The data are expressed as mean values±S.E. (n=6); * significantly different from the control value; — * —, significant difference between two groups (one-way analysis of variance, Newman Keul's test).

 
To investigate whether the increase in prodynorphin mRNA expression evoked by PMF may reflect changes in the transcriptional status of the myocyte nucleus, we assessed the rate of transcription of the prodynorphin gene by using an in vitro nuclear run-off transcription assay. We found that prodynorphin gene transcription was increased in nuclei isolated from myocytes that had been exposed for 4 h to PMF (Fig. 2). Interestingly, a direct exposure of isolated nuclei to PMF for 4 h elicited an increase in prodynorphin gene transcription that was similar to that observed in nuclei that had been isolated from PMF-exposed myocytes (Fig. 2). We have recently shown that opioid gene transcription could be enhanced following the direct exposure of myocardial nuclei to a PKC activator and that nuclear-embedded PKC isozymes and nuclear PKC activation were part of the signal transduction pathway involved in the transcriptional response [17,18]. We therefore examined whether nuclear PKC activation may be a signaling mechanism responsible for the transcriptional effect of PMF. Noteworthy, nuclear exposure to PMF failed to affect prodynorphin gene transcription in the presence of 5 µM chelerythrine (Fig. 2), a selective PKC inhibitor [26], which has been previously shown to suppress PKC activity in isolated myocardial nuclei [18]. Similar results (not shown) were observed when isolated nuclei were exposed to PMF in the presence of 1 µM calphostin C, another PKC inhibitor [26]. Consistent with our previous studies [17,18], the exposure of isolated nuclei to chelerythrine did not affect basal prodynorphin gene transcription (Fig. 2). We next investigated whether nuclear exposure to PMF may result in the phosphorylation of a specific PKC substrate. Fig. 3 shows that the phosphorylation rate of the acrylodan-labeled MARCKS peptide was significantly higher in the presence of nuclei that had been exposed for 30 min to PMF than in the presence of unexposed nuclei. A stimulatory effect of comparable magnitude was observed following a more prolonged exposure of isolated nuclei to PMF for periods of 1, 2 or 4 h (not shown). Both basal and PMF-stimulated nuclear PKC activity were suppressed by the incubation of isolated nuclei in the presence of 5 µM chelerythrine (Fig. 3) or 1 µM calphostin C (not shown). Immunobolt analyses of total extracts from untreated myocytes revealed the expression of PKC-{alpha} (80 kDa), PKC-{delta} (78 kDa), PKC-{varepsilon} (97 kDa), and PKC-{zeta} (75 kDa) (Fig. 4). PKC-β and PKC-{gamma} were not detected (not shown). Confirming previous studies performed in adult ventricular rat myocytes [17,18], western blot analysis of the subcellular distribution of PKC isozymes in untreated cells revealed that only PKC-{delta} and PKC-{varepsilon} were constitutively expressed in the nucleus (Fig. 4). On the contrary, both PKC-{alpha} and -{zeta} were mainly expressed at the cytosolic level (only a faint immunoreactivity against the anti PKC-{alpha}-specific antibody was detected in the nuclear fraction) (Fig. 4). Western blot experiments and the quantitative analysis of isozyme distribution in total cellular extracts, cytosolic and nuclear fractions indicated that the subcellular patterning of PKC isozyme expression was not affected by the exposure to PMF either for 30 min or 1 h (Figs. 4 and 5Go). PMF exposure for 2 h or 4 h was also ineffective (not shown).


Figure 2
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  		Fig. 2 Analysis of the rate of transcription of the prodynorphin gene in isolated myocardial nuclei. The upper panel shows representative autoradiograms of a nuclear run-off transcription assay. Row 1, transcription of the prodynorphin gene; row 2, cyclophilin mRNA. The bars on the right indicate the position of 400- or 220-base pair 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. (A, B) Nuclei were isolated from myocytes cultured for 4 h in the absence or presence of PMF, respectively; (C, D) nuclei, isolated from unexposed cells, were subsequently exposed for 4 h in the absence or presence of PMF, respectively; (E, F) nuclei, isolated from unexposed myocytes, were subsequently exposed for 4 h to 5 µM chelerythrine in the absence or presence of PMF, respectively. Autoradiographic exposure was for 2 days on Kodak X-Omat film with an intensifying screen. The intensities of the autoradiographic bands were measured with a laser densitometer (Image Quant-Computing Densitometer 300/325, Molecular Dynamics, Sunnyvale, CA). Individual results relative to prodynorphin (Prodyn) gene transcription were then normalized to cyclophilin (Cyclo) mRNA expression. Mean values±S.E. (n=6) of normalized data are reported in the lower panel. * significantly different from A, C, E and F.

 

Figure 3
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  		Fig. 3 Nuclear PKC activity in isolated myocardial nuclei. Myocardial nuclei were exposed for 30 min in the absence ({diamond}) or presence ({circ}) of PMF, or treated for 30 min with 5 µM chelerythrine in the absence ({blacksquare}) or presence of PMF ({bigtriangleup}). The phosphorylation of the MARCKS peptide was started by the addition of 10 µg of nuclear protein at the time indicated by the arrow. The time course of the fluorescence of the acrylodan-peptide alone ({square}) is also reported. The data are expressed as mean values±S.E. (n=6). From 10 to 15 min, ({circ}) was significantly different from ({diamond}). No significant difference was observed between ({blacksquare}), ({square}) and ({bigtriangleup}) (one-way analysis of variance, Newman Keul's test).

 

Figure 4
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  		Fig. 4 Subcellular distribution of PKC isozymes in myocytes exposed to PMF. Total cell lysates, cytosolic and nuclear fractions were prepared from myocytes cultured in the absence or presence of PMF for periods of 30 min (panel A) or 1 h (panel B). Equal amounts of protein (20 µg) from each sample were subjected to 8% SDS–polyacrylamide gel electrophoresis and analyzed by immunoblotting as described under Methods. Autoradiograms are representative of six separate experiments. The arrow to the left of each panel indicates 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 (kilodaltons) of marker proteins. Lanes 1, 3, and 5 correspond, respectively to total cell lysates, cytosolic or nuclear fractions isolated from unexposed cells; lanes 2, 4 and 6 correspond, respectively, to total cell lysates, cytosolic or nuclear fractions isolated from PMF-exposed myocytes.

 

Figure 5
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  		Fig. 5 Quantitative analysis of the subcellular distribution of PKC isozymes in PMF-exposed myocytes. Data are expressed as percentage changes in the intensity of autoradiographic bands of total extracts (T), cytosolic (C) or nuclear (N) fractions from myocytes exposed to PMF for 30 min (panel A) or 1 h (panel B) relative to the intensity in the autoradiographs of the corresponding samples from unexposed cells, considered as 100%. The data are expressed as mean values±S.E. (n=6).

 
We finally investigated whether the increase in prodynorphin mRNA levels elicited by PMF may result in an increase in the expression of a biologically active end-product of the prodynorphin gene. Consistent amounts of immunoreactive dynorphin B (ir-dyn B) were found in control cells and in their incubation media (Fig. 6). In both untreated cells and in myocytes exposed for 4 h to PMF ir-dyn B was significantly higher in the medium than at cellular level. A significant increase in the level of both intracellular and secreted ir-dyn B was observed in PMF-treated myocytes, as compared with unexposed control cells (Fig. 6). Both chelerythrine (Fig. 6) and 1 µM calphostin C (not shown) completely abolished the stimulatory effect of PMF on dynorphin B expression.


Figure 6
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  		Fig. 6 Effect of PMF on dynorphin B expression in myocardial cells and in their incubation media. Cardiac myocytes were untreated or exposed for 4 h to PMF, in the absence or presence of 5 µM chelerythrine. Black bars, immunoreactive dynorphin B (ir-dyn B) in cells; white bars, ir-dyn B in the medium. (A) Myocytes were cultured for 4 h in the absence of PMF; (B) and (C), myocytes were exposed for 4 h to PMF, in the absence or presence of 5 µM chelerythrine, respectively. Each single value in the medium was calculated in a final volume of 15 ml, corresponding to the volume of pooled samples of the incubation medium from 106 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, 6 µM 1,4-dithiothreitol. The data are expressed as mean values±S.E. (n=6). § The value of the white bar is significantly different from that of the black bar. * Significantly different from the control value.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In recent years, a large number of multidisciplinary investigations led to the increasing awareness of the existence of multiple effects of MF in biological systems. The possibility that PMF may affect signal transduction processes is supported by the observation that PKC activity was enhanced in PMF-exposed HL-60 cells [27] and by the finding that the exposure to PMF of B-lineage lymphoid cells stimulated the protein tyrosine kinase Lyn and ultimately led to PKC activation [28]. However, though endorphin systems may be affected by PKC activation [14,17,18], compelling evidence indicating that changes in opioid peptide gene expression may be a downstream consequence of the effects elicited by PMF on PKC signaling is still lacking.

Our data show that the expression of an opioid gene was markedly increased following the exposure of myocardial cells to PMF and that the PMF effect occurred at the transcriptional level. We have previously demonstrated that isolated myocardial nuclei harbored both PKC-{delta} and -{varepsilon} and were able to phosphorylate the PKC substrate MARCKS peptide [17,18]. In these studies, nuclear PKC activation was identified as a major molecular trigger stimulating prodynorphin gene transcription. Here, we sought evidence that nuclear PKC may be a signaling sensor for PMF, transducing PMF interaction with the nucleus into a transcriptional response. First, the direct exposure of myocardial nuclei to PMF elicited an increase in prodynorphin gene transcription that was superimposable to that observed in nuclei that had been isolated from PMF-treated myocytes. This finding indicates that the nucleus may represent a crucial site for the interaction among PMF and subcellular environments. Second, such a transcriptional response could be abolished by nuclear treatment with two different PKC inhibitors. Third, the exposure of isolated myocardial nuclei to PMF-enhanced nuclear PKC activity. Fourth, PMF-associated MARCKS peptide phosphorylation could be abolished by the same PKC inhibitors that suppressed the transcriptional effect of PMF on the prodynorphin gene.

According to previous investigations [17,18], adult myocytes expressed PKC-{alpha}, -{delta}, -{varepsilon} and -{zeta}. Failure to detect PKC-β and -{gamma} is consistent with other investigations that reported the absence of both isozymes in myocardial cells utilizing either the RT–PCR analysis of selected PKC transcripts [29] or the chromatographic resolution of PKC activity from heart extracts [30]. The observation that PKC-{delta} and -{varepsilon} were constitutively expressed in the nucleus of untreated myocytes is consistent with our previous findings [17,18] and agrees with other studies that detected PKC-{delta} and -{varepsilon} immunostaining patterns in the nucleus of unstimulated cells by the aid of immunofluorescent techniques [31]. Failure of PMF to affect the subcellular distribution or expression of PKC isozymes suggests that the PMF effect might have occurred independently of enzyme translocation to the nucleus and may not involve changes in isozyme turnover and/or gene expression. Nevertheless, the finding that PKC activation mediated the transcriptional response of isolated nuclei to PMF and the observation that only PKC-{delta} and -{varepsilon} were detected in the nucleus suggest that these two PKC isotypes may be part of the signal transduction pathway involved in the PMF effect on gene transcription. The ability of PMF to stimulate nuclear PKC activity raises the possibility that PMF may affect a nuclear-embedded biochemical machinery necessary for PKC activation. In this regard, several enzymes and substrates associated with diacylglycerol production have been found in rat liver nuclei [32]. Moreover, evidence for the synthesis of inositol phospholipids has been provided in the nuclei of Friend cells [33]. However, though PKC-{delta} and/or PKC-{varepsilon} appear to be the molecules directly involved in the transcriptional response elicited by PMF, the molecular mechanism(s) underlying the activation of nuclear PKC by PMF remain to be elucidated. It has been recently shown that PMF can affect the distribution and aggregation of intramembrane proteins [34]. It is known that such proteins include a variety of different specialized molecules, such as receptors, G proteins, ion channels and integrins that are essential for signal transduction processes. Moreover, PMF have been found to affect ligand binding with membrane receptors and receptor-mediated signaling [35,36]. It is now recognized that receptors for various growth hormones [37], as well as orphan receptors [38] are expressed at nuclear level and are implicated in the modulation of nuclear signaling and gene transcription. In this regard, we have recently discovered the presence of nuclear opioid receptors in hamster ventricular myocytes [39]. Noteworthy, the exposure of isolated nuclei to dynorphin B was able to trigger prodynorphin gene transcription through the activation of nuclear PKC, suggesting the involvement of a nuclear endorphinergic system in the regulation of gene transcription. Whether the effects of PMF on intramembrane protein clustering may affect nuclear signaling remains to be established. Similarly, the possibility that PMF may affect agonist binding at nuclear receptors or trigger nuclear receptor dynamics even in the absence of a ligand remains to be explored. So far no clear and unequivocal biophysical mechanisms are able to explain the variety of biological effects elicited by PMF. It is also presently unknown whether the effect of PMF on nuclear PKC may lead to the transcriptional modulation of non-opioid genes. Nevertheless, PKC activation represents a proximal common signaling event in the activation of a large number of target genes during myocardial hypertrophy [40]. Moreover, an overexpression of PKC-{delta} and -{varepsilon} isozymes has been detected in nuclei of myocytes isolated from Syrian cardiomyopathic hamsters [18] and an increase in PKC gene expression and activity has been associated with proliferative responses in a wide variety of normal and malignant cells [41–43]. Hence, the data presented here may prompt the hypothesis that a delicate growth regulatory balance may be altered following nuclear PKC activation by PMF and may contribute to delineate new molecular plight(s) through which PMF may impact biological systems.

The analysis of dynorphin B expression provides further information on the possible consequences of the PMF effect on prodynorphin gene expression. According to previous results [16–18], ir-dyn B was significantly higher in the medium than in the myocardial cell, indicating that in ventricular myocytes, which lack secretory granules, consistent amounts of prodynorphin-derived peptides may be constitutively released shortly after synthesis. The present finding that dynorphin B was overexpressed by PMF and that this effect was abolished by PKC inhibitors indicates that in myocytes exposed to PMF, PKC-mediated events enhancing prodynorphin gene expression were associated with an increase in mRNA translation into a biologically active end-product of the gene. The finding that PMF-induced prodynorphin gene transcription resulted in the increase of both intracellular and secreted dynorphin B may be of particular biological relevance. Dynorphin B is known to bind selectively {kappa} opioid receptors and the stimulation of these receptors in cardiac myocytes has been shown to promote phosphoinositide turnover, depletion of Ca2+ in the sarcoplasmic reticulum and leading to a marked decrease in the amplitude of the cytosolic Ca2+ transient and in that of the associated contraction [15]. {kappa} opioid receptor stimulation also elicited intracellular alkalosis and changes in myofilament responsiveness to Ca2+ through a PKC-dependent activation of the Na+/H+ antiporter [14]. Moreover, endogenous opioids may act in an autocrine or paracrine fashion to influence the proliferation and differentiation in normal cells including neurons [9], myocardial and epicardial cells [10], as well as in malignant tissues [44–46]. Similar to the effects elicited by opioid agonists in myocardial cells, these growth regulatory responses were mediated by specific opioid receptors and involved the modulation of both PKC activity and phosphoinositide turnover. These results may have further implications in view of the observation that magnetic fields stimulate phoshpoinositide turnover [47] and elicit cytosolic Ca2+ oscillations by triggering the release of Ca2+ from an intracellular storage site [48–50]. Therefore, opioid peptides and magnetic fields may induce relevant biological effects by acting through common signal transduction pathways.

The present finding that PMF, besides affecting the amount of secreted dynorphin B also enhanced the intracellular level of the opioid peptide is worth considering. The presence of nuclear opioid receptors in myocardial cells [39] and their capability to trigger PKC-mediated gene transcription in response to the binding of dynorphin B suggests the possibility that the PMF-mediated increase in prodynorphin gene and dynorphin B expression may also activate an intracrine circuit of regulation of gene transcription.

The biological implication(s) of a myocardial cell having a system capable of reacting to PMF remain to be elucidated. Nevertheless, evidence is now accumulating that low frequency magnetic fields may alter human cardiac rhythm [51] and that chronic exposure to magnetic fields may enhance the occurrence of arrhythmia-related heart diseases [52]. On the other hand, the exposure of chick embryos to PMF has been recently found to induce stress responses that protect the embryonic myocardium from anoxia damage [53,54]. This finding has led to the proposal that exposure to magnetic fields may be a useful, noninvasive means of minimizing damage during surgery, transplantation or heart attack in humans [54]. Clarification of the exact consequences produced on myocardial cell homeostasis by the observed effects of PMF must await more direct functional approaches and is the subject of further investigations. Although considerably more work is needed, the results presented here show that in isolated nuclei an opioid gene can be independently and fully activated by PMF, as in the intact cell. The property of conveying nuclear signaling to the modulation of gene transcription may disclose new perspectives in the molecular dissection of the biological effects elicited by PMF and in the analysis of the medical implications in the use of electromagnetic energy.

Time for primary review 35 days.


   Acknowledgements
 
This work was supported by grants from ‘Ministero dell’ Università-Ricerca Scientifica e Tecnologica (ex 40%), Ministero della Sanità (Ricerca Finalizzata 1998), Consiglio Nazionale delle Ricerche (CNR) and Regione Autonoma della Sardegna.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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