PROJECT SUMMARY/ABSTRACT The importance of reactive oxygen species (ROS) signaling in cell biology is well recognized. ROS act as second messengers that transduce growth and inflammatory signals thereby regulating the balance between proliferation, differentiation and apoptosis. ROS signals are coupled to activation of transcription factors such as nuclear factor-kappa B (NF-[unreadable]B) which controls patterns of gene expression related to vascular inflammation. So while cells must produce ROS to live and grow, overproduction is clearly linked to cardiovascular diseases such as atherosclerosis and hypertension. The distinction between normal "redox signaling" and "oxidative stress" is not clear. We have shown that in vascular smooth muscle (VSM) cells, superoxide (O2-") production in response to cytokines (TNF[unreadable] and IL-1[unreadable]) occurs in early endosomes following receptor endocytosis. Endosomal O2-" production by the Nox1 NADPH oxidase (originally termed the "mitogenic oxidase" because it is linked to growth and proliferation) requires both Nox1 and ClC-3, a member of the ClC family of anion "channels". ClC-3 has recently been demonstrated to function as a Cl-/H+ antiporter. Loss of ClC-3 impairs cell proliferation in vitro and reduces neointima formation after vascular injury in vivo. We hypothesize that ClC-3 provides charge neutralization for early endosomal NADPH oxidase activity. We have developed methods to assess interrelated aspects of the ROS signaling pathway in HEK293 and VSM cells including;1) ClC-3 Cl-/H+ antiporter ion currents, 2) ROS production from purified Nox1-containing early endosomes, and 3) ROS-dependent NF-[unreadable]B activation by luciferase reporter assay. The proposal has 3 Specific Aims. Aim #1 will determine the effect of specific structural modifications of ClC-3 on these 3 end-points. Aim #2 will use fluorescent probes and confocal microscopy to define the contribution of Nox1 and ClC-3 to controlling endosomal pH, Cl- concentration and voltage across the vesicular membrane. Aim #3 will determine if O2-" or H2O2 exit endosomes and reach the cytoplasm via regulated channels. As we establish the functional relationship between ClC-3 and Nox1, we will be defining the electrophysiology of a completely novel intracellular compartment, the ROS signaling endosome. We predict that these vesicles will be more alkaline than traditional endosomes, thus prolonging the half life of O2-" and facilitating specific signaling reactions by this molecule. Understanding the "nuts and bolts" of endosomal ROS production will generate new hypotheses and new therapeutic targets for the treatment of the many inflammatory diseases that are linked to abnormalities in cytokine signaling and NADPH oxidase-dependent ROS production.