Dehydroepiandrosterone activates endothelial cell nitric-oxide synthase by a specific plasma membrane receptor coupled to Gαi2,3

Abstract

The adrenal steroid dehydroepiandrosterone (DHEA) has no known cellular receptor or unifying mechanism of action, despite evidence suggesting beneficial vascular effects in humans. Based on previous data from our laboratory, we hypothesized that DHEA binds to specific cell-surface receptors to activate intracellular G-proteins and endothelial nitric-oxide synthase (eNOS). We now pharmacologically characterize a putative plasma membrane DHEA receptor and define its associated G-proteins. The [3H]DHEA binding to isolated plasma membranes from bovine aortic endothelial cells was of high affinity (Kd = 48.7 pM) and saturable (Bmax = 500 fmol/mg protein). Structurally related steroids failed to compete with DHEA for binding. The putative DHEA receptor was functionally coupled to G-proteins, because guanosine 5′-O-(3-thio)triphosphate (GTPγS) inhibited [3H]DHEA binding to plasma membranes by 69%, and DHEA increased [35S]GTPγS binding by 157%. DHEA stimulated [35S]GTPγS binding to Gαi2 and Gαi3, but not to Gαi1 or Gαo. Pretreatment of plasma membranes with antibody to Gαi2 or Gαi3, but not to Gαi1, inhibited the DHEA activation of eNOS. Thus, DHEA receptors are expressed on endothelial cell plasma membranes and are coupled to eNOS activity through Gαi2 and Gα1s. These novel findings should allow us to isolate the putative receptor and reevaluate the physiological role of DHEA activity.