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(Circulation Research. 2003;92:623.)
© 2003 American Heart Association, Inc.

Molecular Medicine

Dynorphin B Is an Agonist of Nuclear Opioid Receptors Coupling Nuclear Protein Kinase C Activation to the Transcription of Cardiogenic Genes in GTR1 Embryonic Stem Cells

Carlo Ventura, Elisabetta Zinellu, Emiliana Maninchedda, Margherita Maioli

From the Department of Biomedical Sciences, Center for Biotechnology Development and Biodiversity Research, Division of Biochemistry, University of Sassari, Sassari, Italy, and the National Laboratory of the National Institute of Biostructures and Biosystems, Osilo, Italy.

Correspondence to Carlo Ventura, MD, PhD, Department of Biomedical Sciences, Division of Biochemistry, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy. E-mail cvent{at}libero.it

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.

Key Words: nuclear receptors • nuclear signaling • endorphins • embryonic stem cells • gene transcription

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