Intracrine Endorphinergic Systems in Modulation of Myocardial Differentiation (original) (raw)
Related papers
Circulation Research, 2003
The cardiac differentiation of embryonic stem (ES) cells was found to involve prodynorphin gene and dynorphin B expression and was associated with the interaction of secreted dynorphin B with cell surface opioid receptors coupled with protein kinase C (PKC) signaling and complex subcellular redistribution patterning of selected PKC isozymes. Here, confocal microscopy revealed the presence of immunoreactive dynorphin B-like material in GTR1 ES cells, suggesting that dynorphin peptides may also act intracellularly. Opioid binding sites were identified in ES cell nuclei, with a single dissociation constant in the low nanomolar range. A significant increase in B max for a opioid receptor ligand was observed in nuclei isolated from ES-derived cardiomyocytes compared with nuclei from undifferentiated cells. Direct exposure of nuclei isolated from undifferentiated ES cells to dynorphin B or U-50,488H, a synthetic opioid receptor agonist, time-and dose-dependently activated the transcription of GATA-4 and Nkx-2.5, 2 cardiac lineage-promoting genes. Nuclear exposure to dynorphin B also enhanced the rate of prodynorphin gene transcription. These responses were abolished in a stereospecific fashion by the incubation of isolated nuclei with selective opioid receptor antagonists. Nuclei isolated from undifferentiated cells were able to phosphorylate the acrylodan-labeled MARCKS peptide, a high-affinity fluorescent PKC substrate. Exposure of isolated nuclei to dynorphin B induced a remarkable increase in nuclear PKC activity, which was suppressed by opioid receptor antagonists. Nuclear treatment with PKC inhibitors abolished the capability of dynorphin B to prime the transcription of cardiogenic genes. (Circ Res. 2003;92:623-629.)
Dynorphin gene expression and release in the myocardial cell
The Journal of biological chemistry, 1994
The expression of the prodynorphin gene was investigated in adult cultured rat ventricular cardiac myocytes by using a sensitive solution hybridization RNase protection assay for the quantitative analysis of prodynorphin mRNA. Myocyte culture in high KCl resulted, after 4 h, in a marked increase in cellular prodynorphin mRNA, while a KCl treatment for 6, 12, or 24 h progressively down-regulated the levels of prodynorphin mRNA below the control value. Immunoreactive dynorphin B, a biologically active end product of the precursor, was found to be present in the culture medium in significantly higher amounts than in the cardiac myocytes. The levels of this biologically active K opioid receptor agonist significantly increased after 4 h of KCl treatment and were markedly reduced following a 24-h exposure of the cardiac myocytes to KCl. These KCl-induced effects were all abolished by cell incubation in the presence of the calcium channel blocker verapamil. In single cardiac myocytes, acut...
Elf-pulsed magnetic fields modulate opioid peptide gene expression in myocardial cells
Cardiovascular Research, 2000
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 runoff 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.
Protein Kinase C Signaling Transduces Endorphin-Primed Cardiogenesis in GTR1 Embryonic Stem Cells
Circulation Research, 2003
The prodynorphin gene and its product, dynorphin B, have been found to promote cardiogenesis in embryonic cells by inducing the expression of GATA-4 and Nkx-2.5, two transcription factor-encoding genes essential for cardiogenesis. The molecular mechanism(s) underlying endorphin-induced cardiogenesis remain unknown. In the present study, we found that GTR1 embryonic stem (ES) cells expressed cell surface opioid receptors, as well as protein kinase C (PKC)-␣, - 1 , - 2 , -␦, -⑀, and -. Cardiac differentiation was associated with a marked increase in the B max value for a selective opioid receptor ligand and complex subcellular redistribution of selected PKC isozymes. PKC-␣, - 1 , - 2 , -␦, and -⑀ all increased in the nucleus of ES-derived cardiac myocytes, compared with nuclei from undifferentiated cells. In both groups of cells, PKC-␦ and -⑀ were mainly expressed at the nuclear level. The nuclear increase of PKC-␣, - 1 , and - 2 was due to a translocation from the cytosolic compartment. In contrast, the increase of both PKC-␦ and PKC-⑀ in the nucleus of ES-derived cardiomyocytes occurred independently of enzyme translocation, suggesting changes in isozyme turnover and/or gene expression during cardiogenesis. No change in PKC-expression was observed during cardiac differentiation. Opioid receptor antagonists prevented the nuclear increase of PKC-␣, PKC- 1 , and PKC- 2 and reduced cardiomyocyte yield but failed to affect the nuclear increase in PKC-␦ and -⑀. PKC inhibitors prevented the expression of cardiogenic genes and dynorphin B in ES cells and abolished their development into beating cardiomyocytes. (Circ Res. 2003;92:617-622.)
Opioid Peptide Gene Expression Primes Cardiogenesis in Embryonal Pluripotent Stem Cells
Circulation Research, 2000
Zinc finger-containing transcription factor GATA-4 and homeodomain Nkx-2.5 govern crucial developmental fates and have been found to promote cardiogenesis in embryonic cells exposed to the differentiating agent DMSO. Nevertheless, intracellular activators of these transcription factors are largely unknown. In this study, pluripotent P19 cells expressed the prodynorphin gene, an opioid gene encoding for the dynorphin family of opioid peptides. P19 cells were also able to synthesize and secrete dynorphin B, a biologically active end product of the prodynorphin gene. DMSO-primed GATA-4 and Nkx-2.5 gene expression was preceded by a marked increase in prodynorphin gene expression and dynorphin B synthesis and secretion. The DMSO effect occurred at the transcriptional level. In the absence of DMSO, dynorphin B triggered GATA-4 and Nkx-2.5 gene expression and led to the appearance of both ␣-myosin heavy chain and myosin light chain-2V transcripts, two markers of cardiac differentiation. Moreover, dynorphin B-exposed cells were positively stained in the presence of MF 20, a mouse monoclonal antibody raised against the ␣-myosin heavy chain. Opioid receptor antagonism and inhibition of opioid gene expression by a prodynorphin antisense phosphorothioate oligonucleotide blocked DMSO-induced cardiogenesis, suggesting an autocrine role of an opioid gene in developmental decisions.
Nuclear Opioid Receptors Activate Opioid Peptide Gene Transcription in Isolated Myocardial Nuclei
Journal of Biological Chemistry, 1998
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.
Phorbol Ester Regulation of Opioid Peptide Gene Expression in Myocardial Cells
Journal of Biological Chemistry, 1995
Opioid peptide gene expression was characterized in adult rat ventricular cardiac myocytes that had been cultured in the absence or the presence of phorbol 12myristate 13-acetate. The phorbol ester induced a concentration-and time-dependent increase of prodynorphin mRNA, the maximal effect being reached after 4 h of treatment. The increase in mRNA expression was suppressed by incubation of cardiomyocytes with staurosporine, a putative protein kinase C inhibitor, and was not observed when the cells were cultured in the presence of the inactive phorbol ester 4␣-phorbol 12,13-didecanoate. Incubation of cardiac myocytes with phorbol 12-myristate 13-acetate also elicited a specific and staurosporine-sensitive increase in immunoreactive dynorphin B, a biologically active end product of the precursor, both in the myocardial cells and in the culture medium. In vitro run-off transcription assays indicated that transcription of the prodynorphin gene was increased both in nuclei isolated from phorbol estertreated myocytes and in nuclei isolated from control cells and then exposed to phorbol 12-myristate 13-acetate. No transcriptional effect was observed when cardiac myocytes or isolated nuclei where exposed to 4␣phorbol 12,13-didecanoate. The phorbol ester-induced increase in prodynorphin gene transcription was prevented by pretreatment of myocytes or isolated nuclei with staurosporine, suggesting that myocardial opioid gene expression may be regulated by nuclear protein kinase C. In this regard, cardiac myocytes expressed protein kinase C-␣, -␦, -⑀, and -, as shown by immunoblotting. Only protein kinase C-␦ and protein kinase C-⑀ were expressed in nuclei that have been isolated from control myocytes, suggesting that these two isotypes of the enzyme may be part of the signal transduction pathway involved in the effect elicited by the phorbol ester on opioid gene transcription in isolated nuclei. The incubation of myocardial nuclei isolated from control cells in the presence of a protein kinase C activator induced the phosphorylation of the myristoylated alanine-rich protein kinase C substrate peptide, a specific fluorescent substrate of the enzyme. The possibility that prodynorphin gene expression may control the heart function through autocrine or paracrine mechanisms is discussed.
Opioid receptors and opioid peptides in the cardiomyogenesis of mouse embryonic stem cells
Journal of Cellular Physiology, 2018
The stimulation of myocardium repair is restricted due to the limited understanding of heart regeneration. Interestingly, endogenous opioid peptides such as dynorphins and enkephalins are suggested to support this process. However, the mechanism—whether through the stimulation of the regenerative capacity of cardiac stem cells or through effects on other cell types in the heart—is still not completely understood. Thus, a model of the spontaneous cardiomyogenic differentiation of mouse embryonic stem (mES) cells via the formation of embryoid bodies was used to describe changes in the expression and localization of opioid receptors within cells during the differentiation process and the potential of the selected opioid peptides, dynorphin A and B, and methionin‐enkephalins and leucin‐enkephalins, to modulate cardiomyogenic differentiation in vitro. The expressions of both κ‐ and δ‐opioid receptors significantly increased during mES cell differentiation. Moreover, their primary colocal...
Cytotoxic Effects of Dynorphins through Nonopioid Intracellular Mechanisms
Experimental Cell Research, 2001
Dynorphin A, a prodynorphin-derived peptide, is able to induce neurological dysfunction and neuronal death. To study dynorphin cytotoxicity in vitro, prodynorphin-derived peptides were added into the culture medium of nonneuronal and neuronal cells or delivered into these cells by lipofection or electroporation. Cells were unaffected by extracellular exposure when peptides were added to the medium. In contrast, the number of viable cells was significantly reduced when dynorphin A or "big dynorphin," consisting of dynorphins A and B, was transfected into cells. Big dynorphin was more potent than dynorphin A, whereas dynorphin B; dynorphin B-29; [Arg 11,13 ]dynorphin A(1-13)-Gly-NH-(CH 2 ) 5 -NH 2 , a selective -opioid receptor agonist; and poly-L-lysine, a basic peptide more positively charged than big dynorphin, failed to affect cell viability. The opioid antagonist naloxone did not prevent big dynorphin cytotoxicity. Thus, the toxic effects were structure selective but not mediated through opioid receptors. When big dynorphin was delivered into cells by lipofection, it became localized predominantly in the cytoplasm and not in the nuclei. Big dynorphin appeared to induce toxicity through an apoptotic mechanism that may involve synergistic interactions with the p53 tumor-suppressor protein. It is proposed that big dynorphin induces cell death by virtue of its net positive charge and clusters of basic amino acids that mimic (and thereby perhaps interfere with) basic domains involved in protein-protein interactions. These effects may be relevant for a pathophysiological role of dynorphins in the brain and spinal cord and for control of death of tumor cells, which express prodynorphin at high levels.
Creating prodynorphin-expressing stem cells alerted for a high-throughput of cardiogenic commitment
Regenerative Medicine, 2007
Background: The development of cell therapy for the rescue of damaged heart muscle is a major area of inquiry. Within this context, the establishment of a cardiogenic cell line may remarkably facilitate the molecular dissection of cardiac fate specification, a low-efficiency and still poorly understood process, paving the way for novel approaches in the use of stem cells for cardiac repair. Methods & results: We used GTR1 cells, a derivative of mouse R1 embryonic stem cells bearing the puromycin-resistance gene driven by the cardiomyocyte-specific α-myosin heavy chain promoter, affording a gene trapping selection of a virtually pure population of embryonic stem cell-derived cardiomyocytes. Third-generation lentiviral vectors were used to overexpress the prodynorphin gene, previously shown to orchestrate a dynorphinergic system acting as a major conductor of embryonic stem cell cardiogenesis. Lentiviral prodynorphin transduction remarkably enhanced the transcription of GATA-4 and Nkx...