PROJECT SUMMARY Erectile dysfunction (ED) is one of the most prevalent and impactful complications of diabetes mellitus (DM). The combination of diabetes and sexual dysfunction affects family planning and overall quality of life, and ED is now considered an early marker for cardiovascular diseases and consequently could predate the development of a life-threatening condition. Hyperglycemia, hyperlipidemia and inflammation are all associated with DM and induce the generation of vascular reactive oxygen species (ROS). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) is the main source of ROS in the vasculature and ROS activate intracellular signaling pathways that induce constriction of the penile vasculature. In vascular smooth muscle cells (VSMCs), this signaling cascade begins with extracellular superoxide (O2-•) production by Nox1 at the plasma membrane. Extracellular O2-• from Nox1 can alter vascular tone by inactivating endothelium-derived nitric oxide (NO). But O2-• also triggers activation of RhoA/Rho-kinase signaling to cause constriction of the penile vasculature. In diabetes, activation of RhoA/Rho-kinase signaling is exaggerated leading to ED. We have demonstrated functional and structural inter-dependence between Nox1 and volume-regulated anion channels (VRACs) composed of leucine-rich repeat-containing 8 (LRRC8) family proteins. LRRC8A is required for all VRACs and combines with one of four closely related LRRC8 isoforms (LRRC8B through E) to produce channels with unique biophysical properties. We speculate and provide preliminary data that VRACs provide O2-• access to the cytoplasm. Our preliminary data demonstrate that LRRC8 knockout (KO) or channel inhibition enhances dilator function (or reduces constriction) in control blood vessels and null mice are protected against impairment of vasodilation by TNFα in vitro and angiotensin II (AngII) in vivo. This proposal will provide a structural framework to understand how extracellular O2-• produces intracellular signals that lead to contraction of the penile vasculature. We hypothesize that in diabetes, erectile dysfunction is partly due to the entry of extracellular O2-• from Nox1 via LRRC8 anion channels to activate RhoA/Rho-kinase resulting in vascular contraction. Three aims address this central hypothesis: 1) to test the hypothesis that in diabetes, ED is caused by excess Nox1 and LRRC8A-dependent production of extracellular O2-• which then enters VSMCs via LRRC8A anion channels, activates RhoA/Rho kinase and increases vascular tone; 2) to test the hypothesis that diabetic mice that lack the LRRC8 channel in the vasculature will not develop ED; and 3) to test the hypothesis that drugs that selectively inhibit LRRC8 channels will be novel therapeutic agents for the treatment of ED in diabetes. This work will provide a comprehensive approach to understanding the role of LRRC8/VRACs, O2-• and RhoA in the etiology and development of ED in diabetic males and provide a...