Real time measurement of nitric oxide released from cultured endothelial cells

Direct detection of nitric oxide (NO) is essential for understanding the precise mechanism of its production from endothelial cells. Previously, we developed an NO detection system based on the chemiluminescence reaction between NO and luminol-H2O2. Here, we have applied this system to cultured endothelial cells for the direct and on-time measurement of NO. The perfusate from cultured endothelial cells was continuously mixed with luminol-H2O2. N(G)-monomethyl-L-arginine (L-NMMA) (10(-4) M) decreased the chemiluminescence signal of NO, suggesting the existence of basal NO release. Bradykinin (10(-8) M-10(-6) M) increased the NO signal (10(-6) M; 5.1+/-0.4 fmol/min, corresponding to 1.7 pM in the perfusate), and this was inhibited by 10(-4) M L-NMMA (1.8+/-0.3 fmol/min). These results corresponded to the changes in cGMP levels in RFL-6 cells, which provide an NO bioassay system. We conclude that the luminol-H2O2 system is useful for the direct and continuous measurement of NO from cultured endothelial cells.