Carlo Ventura, Elisabetta Zinellu, Emiliana Maninchedda, Margherita Maioli
Abstract—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 Bmax 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.)
Key Words: nuclear receptors nuclear signaling endorphins embryonic stem cells gene transcription
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