Abstract Su107: The Role of TRPV2 Ion Channel in NAMPT Secretion During Cardiac Arrest

Introduction: Extracellular NAMPT functions as a novel damage-associated molecular pattern. It has been shown to be released from many cell types including neutrophils and cardiomyocytes. In a mouse model of cardiac arrest, increased plasma NAMPT correlates with inflammation, cardiovascular dysfunction and poor survival, and neutrophils are the major contributor to plasma NAMPT during cardiac arrest. However, the mechanisms of NAMPT release are currently not well understood. Hypothesis: We hypothesize that NAMPT release is regulated by a non-selective cation channel TRPV2. Methods: Human blood neutrophils were isolated by Percoll density gradient. TRPV2 channel activity was measured by calcium influx using Furo-4. The effect of TRPV2 agonist and inhibitor on the intracellular free Ca 2+ flux was performed by monitoring fluorometric kinetic readings. Extracellular NAMPT concentration in cell supernatants was measured by ELISA. Results: NAMPT release is affected by extracellular pH. The decrease in pHo from 7.4 to 7.0 significantly reduced the amount of eNAMPT from 8.31 ± 0.3 ng/ml to 1.72 ± 0.4 ng/ml. By contrast, the increase of the pHo from 7.4 to 7.8 enhanced NAMPT secretion into supernatant from 8.31 ± 0.3 ng/ml to 12.04 ± 0.3 ng/ml. We therefore reasoned that ion channels sensitive to pH change might play a role in NAMPT secretion. Several transient receptor potential (TRP) channels are exquisitely sensitive to extracellular pH. Calcium chelator EGTA at 1 mM completely blocked NAMPT release, which indicates a calcium channel is involved. 2-aminoethoxydiphenyl borate (2-APB) has been shown to stimulate many TRPV channels. 2-APB increased NAMPT release in a dose-dependent manner, and maximal release was induced by 1 mM 2-APB. Tranilast and piperlongumine, two structurally unrelated TRPV2 antagonists, significantly inhibited NAMPT release from neutrophils. By contrast, neither TRPV1 inhibitor SB366791 nor TRPV4 inhibitor GSK219384 has any effect on NAMPT release. Furthermore, TRPV2 inhibitors tranilast and piperlongumine blocked calcium influx induced by 2-APB. Conclusion: NAMPT release from neutrophils is regulated by TRPV2 channel-mediated calcium influx.