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Cellular Mechanisms of Hydrogen Peroxide (H2O2)‐induced Permeability Increases in Intact Venules: Role of H2O2‐induced Ca2+‐independent Nitric Oxide (NO) Production in Endothelial Cells

H2O2 has been known to increase NO production contributing to vasodilation in arterioles, but its role in venular microvessel permeability remains unknown. We previously showed that H2O2 induced delayed but progressive increases in EC [Ca2+]i, hydraulic conductivity (Lp), and cell apoptosis in intact venules. This study aims to explore the signaling mechanisms of H2O2‐induced Lp increases with focus on H2O2‐induced NO production in ECs of venular walls. NO and EC [Ca2+]i were measured in DAF‐2 and Fura‐2 loaded vessels and permeability was determined by Lp measurements in rat venules. H2O2 (10 μM) induced an immediate increase in NO and reached a plateau (9.1±0.4 times that of control) after 45 min, which occurred prior to the increases in EC [Ca2+]i and not affected by Ca2+influx inhibitor LaCl3 (50 μM). Immunostaining for nitrotyrosine using confocal microscopy revealed extensive tyrosine nitration in vessels exposed to H2O2 for 2h but not in untreated vessels. Blocking H2O2‐induced NO production with L‐NMMA not only abolished H2O2‐induced delayed increases in EC [Ca2+]i and tyrosine nitration, but also prevented the Lp increases. Our results indicate that H2O2 activates endothelial NOS via a Ca2+ independent pathway and the excessive NO production and subsequent peroxynitrite formation‐mediated protein nitration may be the key for EC Ca2+ overload, resulting in cell injury and Lp increases. Supported by HL56237 and HL084338.