Inhibition of the NAADP receptor using the selective antagonist Ned-19. (A) EA.hy926 cells were treated with vehicle alone for 20 minutes and then stimulated with TMPH followed by histamine (representative trace). (B) Cells were treated with 50μM Ned-19 for 20 minutes followed by stimulation with TMPH, histamine, and then 50μM GPN (representative trace). (C) Cells were treated with 50μM Ned-19 for 20 minutes, then stimulated or not with 2 U/mL thrombin. (D) Summary of responses to TMPH. (E) Summary of responses to histamine. Images show (F) Ned-19 staining, (G) lysotracker red staining, (H) merged image, and (I) brightfield image. Cells were viewed on a Zeiss 510 META confocal microscope with a plan apochromat objective 63× oil immersion aperture 1.4, at room temperature in Hanks buffer, and analyzed with LSM Software. Where applicable, error bars represent SEM; n = 3 independent experiments; *P < .05 by Student t test.