Abstract 116: Regulation Of Nampt Secretion From Human Neutrophils By The Trpv2 Channel

Abstract

Introduction: Nampt functions both as an intracellular enzyme critical for NAD + salvage and a novel extracellular damage-associated molecular pattern. In a mouse model of cardiac arrest, increased plasma Nampt correlates with inflammation, cardiovascular dysfunction and death. The mechanism that regulates Nampt secretion during cardiac arrest is not known. Hypothesis: We hypothesize that Nampt secretion is regulated by a non-selective cation channel TRPV2. Methods: Extracellular Nampt (eNampt) concentration was measured by ELISA. Neutrophils were isolated from human peripheral blood. Results: In non-stimulated human neutrophils incubated at 37 o C, eNampt was observed in cell supernatants at 10 min and plateaued after 30 min. eNampt is not released by the ER-Golgi classical pathway. Brefeldin A, an inhibitor of protein transport from ER to Golgi had no effect on eNampt release. Similarly, nocodazole, which disrupts microtubule assembly, had no effect on eNampt release either. eNampt release was markedly reduced at 4 o C, suggesting that the release is active. eNampt release was regulated by extracellular pH. The decline in pH from 7.4 to 6.5 suppressed the secretion of eNampt, while the increase of pH from 7.4 to 7.8 enhanced the amounts of eNampt release. We therefore reasoned that ion channels sensitive to pH and/or involved in intracellular pH homeostasis might play a role in Nampt secretion. 10 μM 5-(N,N-Hexamethylene) amiloride had no effect on eNampt release, which indicates that Na + /H + exchanger is not involved in eNampt release. In contrast, eNampt release was upregulated by the TRPV2 agonists 200 μM H 2 O 2 , 1 mM 2-aminoethoxydiphenyl borate and10 μM anandamide, and downregulated byTRPV2 inhibitors 0.5 mM EGTA, 5 mM NAM, 100 μM tranilast and 100 μM piperlongumine. Conclusion: Nampt is secreted from neutrophils via a non-classical pathway and its secretion is regulated by the pH sensitive TRPV2 channel.