In myocardial ischemia, pathologic release of norepinephrine (NE) from sympathetic nerves leads to arrhythmias and death. The mechanisms responsible remain obscure. The overall objective of this grant is to address the important biological issue of whether a local renin-angiotensin system (RAS) plays a significant role in exacerbating NE release in myocardial ischemia. Our preliminary results demonstrate that cardiac mast cells are a novel source of renin, the rate-limiting enzyme responsible for initiating ANG n formation. We hypothesize that degranulation of cardiac mast cells, as occurs in ischemia, is pivotal for activation of local RAS and ANG II formation in the proximity of neuronal ATi receptors (ATiR). In Specific Aim I, we will characterize mast-cell renin from congenic controls (CC) of c-Kit knock out (KO) mice, and from cultured human mast cells (HMC-1). We will also investigate in normoxic and ischemic cardiac models from guinea-pigs, and CC and KO mice, whether locally synthesized ANG n from mast-cell-derived renin activates neuronal ATiR to elicit NE release and reperfusion arrhythmias. We have already ascertained in a cultured-cell model of ischemic sympathetic nerves, that exogenous ANG n stimulates neuronal Na+/H^ exchange (NHE) leading to excessive carrier-mediated NE release. Thus, in Specific Aim n we will study, in human neuroblastoma cells transfected with the cloned rat ATjA receptor (SH-SYSY-ATu), the signal transduction of this ANG H-mediated NHE stimulation. Experiments in SH-SYSY-ATiA cells demonstrate that stimulation of NHE and carrier-mediated NE release by AT)R activation occurs via a Ca +-dependent pathway. Accordingly, our investigation will focus on elucidating the role of PLC, Ca2+, PKC, and calmodulin in this Ca2+-dependent pathway. The overall premise of these experiments is that if we understand the patho-physiological basis for the formation of ANG n in the ischemic heart, and how ANG II exacerbates NE release, then we will also have identified potential therapeutic targets to alleviate ischemic arrhythmias and associated cardiac dysfunctions.