RNA Interference-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 (AEA) occurs has been the source of much debate. In the current study, we show that neuronal differentiated CAD (dCAD) cells accumulate anandamide by a process that is inhibited in a dose-dependent manner by N-(4-hydroxyphenyl)arachidonylamide (AM404). We also show that dCAD cells express functional fatty acid amide hydrolase, 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, RNA interference (RNAi)-mediated knockdown of dynamin 2, a protein involved in endocytosis, blocked the internalization of the fluorescently labeled anandamide analog SKM 4-45-1 ([3Ј,6Ј-bis(acetyloxy)-. RNAi-mediated knockdown of the ␤2 subunit of the clathrin-associated activator protein 2 complex had no effect on SKM 4-45-1 internalization. We were surprised to find that dynamin 2 knockdown in dCAD cells did not affect [ 3 H]AEA uptake. However, dynamin 2 knockdown caused a significant increase in the overall levels of intact [3 H]AEA associated with the cells, suggesting that trafficking of [ 3 H]AEA to FAAH had been disrupted. This finding may be the result of an accumulation of the anandamide carrier protein in detergent-resistant membranes after 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.The endocannabinoid anandamide (AEA) is an agonist of the cannabinoid 1 and 2 receptors (Matsuda et al., 1990;Munro et al., 1993) and some vanilloid type ion channels (Di Marzo et al., 2001;Voets and Nilius, 2003). AEA is internalized by most cell types and is thought to be metabolized primarily by the intracellular enzyme fatty acid amide hydrolase (FAAH) (Deutsch and Chin, 1993;Di Marzo et al., 1994;Cravatt et al., 1996). Previous studies from our laboratory have indicated that AEA uptake potentially occurs by a caveolae-related, or clathrin-independent, endocytic process (McFarland et al., 2004). Interestingly, neurons are not thought to express caveolin-1 but do exhibit lipid raft-related endocytic processes (Cameron et al., 1997). Thus, our studies seek to validate a neuronal-like cell line as a useful model in the study of AEA uptake and to show that AEA uptake can be disrupted by using molecular inhibitors that are specifically targeted to endocytic processes.Cath.a cells display neuronal properties and express panneuronal markers but lack classic neuronal morphology (Suri et al., 1993;Lazaroff, 1996). Cath.a differentiated (CAD) cells are derived from the Cath.a cell line and are chara...

A nucleic acid-hydrolyzing antibody penetrates into cells via caveolae-mediated endocytosis, localizes in the cytosol and exhibits cytotoxicity

“…Small interfering RNA (siRNA) oligonucleotides were synthesized at Bioneer Co. (Daejeon, Korea). The mRNA sequences targeted by caveolin-1, clathrin, and dynamin-2 siRNA were 5 -AGA CGA GCU GAG CGA GAA GCA -3 [18,19], 5 -AAC CUG CGG UCU GGA GUC AAC -3 [20], and 5 -GUG GAC CUG GUU AUC CAG GAG CUA A -3 [21] respectively. An unrelated (scramble) siRNA with sequence of 5 -CCU ACG CCA CCA AUU UCG U -3 was employed as a control.…”

Oxidation of the Endogenous Cannabinoid Arachidonoyl Ethanolamide by the Cytochrome P450 Monooxygenases: Physiological and Pharmacological Implications

“…These observations are not cell-type specific because they are also present when using the neuronal CAD cells. Furthermore, down-regulation of dynamin 2, a small GTPase involved in endocytic internalization, in CAD cells leads to a decrease in the uptake of a fluorescently labeled anandamide analog and disrupts the trafficking of radiolabeled anandamide to FAAH (McFarland et al, 2008).…”

Inactivation and Biotransformation of the Endogenous Cannabinoids Anandamide and 2-Arachidonoylglycerol

“…1D) (Rakhshan et al, 2000;McFarland et al, , 2008. We demonstrated that inhibition of caveolae-related endocytosis or prevention of caveolae formation led to a significant decrease in cellular AEA accumulation, thus implicating a role for these membrane microdomains in the AEA uptake process (McFarland et al, , 2008.…”

“…We demonstrated that inhibition of caveolae-related endocytosis or prevention of caveolae formation led to a significant decrease in cellular AEA accumulation, thus implicating a role for these membrane microdomains in the AEA uptake process (McFarland et al, , 2008. We propose that extracellular AEA binds a carrier protein located within caveolae, and that subsequently, caveolae-derived vesicle formation and endocytosis of the membrane-packaged endocannabinoid are induced McFarland et al, , 2008. The subsequent delivery of internalized AEA to FAAH may be a critical step in freeing the carrier protein for additional AEA transport events.…”

“…Several different models have been proposed for endocannabinoid uptake that fit under the general heading of a protein-facilitated event: 1) transmembrane carrier (Beltramo et al, 1997;Hillard and Jarrahian, 2000); 2) intracellular sequestration (Hillard and Jarrahian, 2003;Hillard et al, 2007); 3) passive diffusion driven by fatty acid amide hydrolase (FAAH) (Glaser et al, 2003); and 4) carrier-mediated caveolae-related endocytosis (Rakhshan et al, 2000;McFarland et al, , 2008. The majority of these models have been suggested as a result of experiments studying AEA transport only.…”