Received for publication January 3, 2008.
Revised April 23, 2008.
Accepted for publication April 23, 2008.

RNAi-Mediated Knockdown of Dynamin 2 Reduces Endocannabinoid Uptake Into Neuronal dCAD Cells

Abstract

The precise mechanism by which the cellular uptake of the endocannabinoid anandamide occurs has been the source of much debate. In the current study, we show that neuronal dCAD cells accumulate anandamide by a process that is inhibited in a dose-dependent manner by AM404. We also show that dCAD cells express functional fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide metabolism. Previous data from our laboratory indicated that anandamide uptake occurs by a caveolae-related endocytic mechanism in RBL-2H3 cells. In the current study, we show that anandamide uptake by dCAD cells may also occur by an endocytic process that is associated with detergent-resistant membrane microdomains or lipid rafts. Nystatin and progesterone pretreatment of dCAD cells significantly inhibited anandamide accumulation. Furthermore, RNAi-mediated knockdown of dynamin 2, a protein involved in endocytosis, blocked the internalization of the fluorescently labeled anandamide analog SKM 4-45-1. RNAi-mediated knockdown of the 2 subunit of the clathrin-associated AP2 complex had no effect on SKM 4-45-1 internalization. Surprisingly, dynamin 2 knockdown in dCAD cells did not affect [³H]AEA uptake. However, dynamin 2 knockdown caused a significant increase in the overall levels of intact [³H]AEA associated with the cells suggesting that trafficking of [³H]AEA to FAAH had been disrupted. This finding may be the result of an accumulation of the anandamide carrier protein in detergent-resistant membranes following dynamin 2 knockdown. Our studies provide evidence that the cellular uptake of anandamide may occur by a dynamin 2-dependent, caveolae-related endocytic process in dCAD cells.

Key words: Cannabinoid, Lipid rafts/microdomains, Fluorescence techniques, RNA/siRNA