Abstract
The neurosteroid dehydroepiandrosterone (DHEA) at 1 nM protects NMDA-/GABAA-receptor negative neural crest-derived PC12 cells from apoptosis. We now report that membrane-impermeable DHEA-BSA conjugate replaces unconjugated DHEA in protecting serum-deprived PC12 cells from apoptosis (IC50=1.5 nM). Protection involves phosphorylation of the prosurvival factor Src and induction of the anti-apoptotic protein Bcl-2 and is sensitive to pertussis toxin. Binding assays of [3H]DHEA on isolated PC12 cell membranes revealed saturation within 30 min and binding of DHEA with a Kd of 0.9 nM. A similar binding activity was detectable in isolated membranes from rat hippocampus and from normal human adrenal chromaffin cells. The presence of DHEA-specific membrane binding sites was confirmed by flow cytometry and confocal laser microscopy of DHEA-BSA-FITC stained cells. In contrast to estrogens and progestins, glucocorticoids and androgens displaced DHEA from its membrane binding sites but with a 10-fold lower affinity than DHEA (IC50=9.3 and 13.6 nM, respectively). These agents acted as pure antagonists, blocking the antiapoptotic effect of DHEA as well as the induction of Bcl-2 proteins and Src kinase activation. In conclusion, our findings suggest that neural crest-derived cells possess specific DHEA membrane binding sites coupled to G proteins. Binding to these sites confers neuroprotection.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adrenal Medulla / cytology
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Androgens / pharmacology
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Animals
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Apoptosis / drug effects
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Cell Membrane / drug effects
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Chromaffin Cells / drug effects
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Chromaffin Cells / metabolism
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Culture Media, Serum-Free / pharmacology
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Dehydroepiandrosterone / analogs & derivatives
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Dehydroepiandrosterone / metabolism
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Dehydroepiandrosterone / pharmacology*
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Estrogens / pharmacology
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Glucocorticoids / pharmacology
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Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
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Hippocampus / cytology
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Hippocampus / drug effects
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Humans
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Male
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Microscopy, Confocal
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Neuroprotective Agents / metabolism
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Neuroprotective Agents / pharmacology*
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PC12 Cells / drug effects
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Pertussis Toxin / pharmacology
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Proto-Oncogene Proteins c-bcl-2 / physiology
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Rats
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Receptors, G-Protein-Coupled / drug effects*
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Receptors, G-Protein-Coupled / metabolism
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Receptors, GABA-A / physiology
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Receptors, N-Methyl-D-Aspartate / physiology
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Receptors, Steroid / drug effects*
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Receptors, Steroid / metabolism
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Serum Albumin, Bovine / pharmacology
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bcl-X Protein / physiology
Substances
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Androgens
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Culture Media, Serum-Free
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Estrogens
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Glucocorticoids
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Neuroprotective Agents
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Proto-Oncogene Proteins c-bcl-2
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Receptors, G-Protein-Coupled
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Receptors, GABA-A
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Receptors, N-Methyl-D-Aspartate
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Receptors, Steroid
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bcl-X Protein
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dehydroepiandrosterone receptor
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dehydroepiandrosterone-bovine serum albumin
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Serum Albumin, Bovine
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Guanosine 5'-O-(3-Thiotriphosphate)
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Dehydroepiandrosterone
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Pertussis Toxin