Fig. 3

Activation of NMDA receptor induces increase in the early endosomal trafficking pathway and suppression in the lysosomal trafficking pathway mediated through CaMKII activation. A NMDA receptor activation (NMDA 25 μM) causes increases in the Rab5 (Red), a marker for the early endosome that was blocked by MK801 (1 μM). Cells were counter stained with phalloidin for actin staining (Green). Scale bar indicates 25 μm. B Activation of NMDA receptor (NMDA 25 μM) induces suppression of LAMP1 (Green), that was recovered with the treatment of MK801 (1 μM). Scale bar indicates 25 μm. C Western blot analysis of Rab 5 and LAMP1 show rapid upregulation of Rab 5 and down modulation in LAMP1. GAPDH was used for loading control. D–E Stimulation of NMDA receptor induces activation of CaMK II. D Suppression of CAMKII activity decreases level of early endosome and reverses surface re-localization of clathrin heavy chain triggered by NMDA receptor stimulation. CaMKII activity was suppressed with pretreatment of KN93 (2.5 μM, 15 min) and further treated with NMDA (25 μM) for 30 s. Cells were stained for Rab 5 (Green) and Clathrin (Red). Scale bar indicates 25 μm. E Suppression of CaMKII activity reverses the decreased LAMP1 positive signal caused by activation of NMDA receptor in the brain endothelial cells. CaMKII activity was suppressed with pretreatment of KN93 (2.5 μM, 15 min) further treated with NMDA (25 μM) for 30 s. Cells were fixed and further stained with LAMP 1 (Red). Scale bar indicates 25 μm