Long-term complications of diabetes have a devastating impact on the lives of millions of Americans. While microvascular dysfunction plays a key role in the development of complications, the pathophysiology of microvascular changes in diabetes is poorly understood. The aging-regulated, adrenal steroid hormone and nutritional supplement, dehydroepiandrosterone (DHEA), improves microvascular dysfunction in diabetes. The molecular mechanism of this action is unknown. Our long-term objective is to decrease the morbidity of diabetic complications, by developing novel drugs that target diabetic vascular dysfunction. Our short-term goal is to define the cellular mechanism for the vascular action of DHEA. We hypothesize that DHEA affects vascular function by activating an endothelial receptor. This hypothesis is based on our discovery of a vascular endothelial, G-protein coupled, DHEA-specific receptor, linked to nitric oxide production. Isolating this receptor is a crucial first step in testing our hypothesis. We plan to achieve the following specific aims: 1 Isolate, sequence and clone the vascular endothelial DHEA receptor. Evaluate functional aspects of the cloned receptor in transiently transfected COS-7 cells. ? ? Our research design involves two strategies:(1) affinity chromatographic purification and isolation of a DHEA binding protein from solubilized bovine aortic plasma membranes and (2) cDNA microarray to identify candidate orphan G-protein coupled receptors, expressed on vascular endothelial cells. The candidate cDNAs will be tested in cellular expression and DHEA binding assays. We expect to show definitively how DHEA interacts with vascular endothelial cells and, for the first time, begin to provide a molecular basis for some of the widespread health effects claimed for DHEA. The result will allow us to determine the role of the receptor-DHEA interaction in vascular function, development, aging and disease. We will be able to screen for high potency receptor activating molecules to evaluate therapeutically in human vascular disease, including diabetic microvascular dysfunction. The results will facilitate the study of signaling pathways to important vascular functions such as nitric oxide production, cell proliferation and apoptosis. Thus, the successful completion of our project will advance our long-term goal of developing novel therapeutic vasoactive agents, to decrease the morbidity related to diabetic vascular dysfunction. ? ?
