CHARACTERIZATION OF ATRIAL NATRIURETIC FACTOR STORAGE POOLS IN HL-1 ATRIAL CARDIOMYOCYTES Asna Choudhry This Thesis is Submitted to the Faculty of Graduate and Postdoctoral Studies in Partial Fulfillment of the Master of Science program in Cellular and Molecular Medicine Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa, Ottawa, Ontario ! Asna Choudhry, Ottawa, Canada, 2011 Abstract Atrial natriuretic factor (ANF) is a cardiac hormone that helps maintain cardiovascular homeostasis. ANF secretion is linked to the constitutive, regulated and constitutive-like pathways. Presence of a monensin-sensitive pool that may follow constitutive-like secretion has previously been identified in an isolated atrial perfusion study. The intracellular ANF storage pools linked to each secretory pathway have not been identified. In this study, ANF storage and secretion was characterized in HL-1 atrial cardiomyocytes through the use of pharmacological agents, density gradient and RP- HPLC analysis. Treatment of HL-1 cells with monensin followed by cell fractionation was unsuccessful in identifying the monensin-sensitive pool. RP-HPLC analysis identified presence of low molecular weight ANF in low density gradient fractions that were defined by the presence of organelle markers of Golgi, early endosome, clathrin and corin. Since the monensin-sensitive pool was thought to be of a constitutive-like nature, targeting this pathway with pharmacological inhibitors of clathrin coat vesicle (CCV) formation and endosomal trafficking failed to prevent stimuli-independent secretion. Based on an inability to prevent ANF secretion by targeting the constitutive-like pathway and the presence of low molecular weight ANF in low density gradient fractions, stimuli- independent ANF secretion seems to be through a constitutive pathway. 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