A role for detergent -resistant membrane microdomains in the uptake and recycling of the endogenous cannabinoid anandamide
The endocannabinoid anandamide is an endogenous agonist of the cannabinoid receptors and some members of the transient receptor potential, vanilloid type, family of cation channels. There have been many advances in our collective understanding of anandamide in the last decade and experimental evidence is mounting that pharmacological augmentation of endocannabinoid tone might have a significant therapeutic benefit in several disease states. However, the mechanisms responsible for the biosynthesis, cellular uptake, and intracellular processing of anandamide are not well understood and have been the source of much debate. The two aims of this work are to investigate how (1) pharmacological inhibitors of caveolar-related (clathrin-independent) endocytosis effect anandamide transport and recycling in the non-neuronal RBL-2H3 cell line, and (2) how molecular inhibitors targeted at specific endocytic processes effect anandamide uptake in the neuronal-like dCAD cell line. I propose that anandamide uptake may occur via a caveolae/lipid raft-related endocytic process. Inhibitors of caveolar-related endocytosis reduced anandamide transport by approximately 50% compared to control in RBL-2H3 cells, and fluorecein derived from fluorescently labeled anandamide colocalized with protein markers of caveolae at early time points following transport. In dCAD cells, molecular inhibitors of clatherin-dependant endocytosis had no effect on the internalization of fluorescently labeled anandamide while molecular inhibitors of caveolae-related endocytosis abolished internalization of fluorescently labeled anandamide. Following uptake of [ 3H]anandamide by RBL-2H3 cells, I found an accumulation of tritium in caveolin-rich membranes. Inhibitors of both anandamide uptake and metabohsm blocked the observed enrichment of tritium in the caveolin-rich membranes. Mass spectrometry of subcellular membrane fractions revealed that the tritium accumulation observed in the caveolin-rich membrane fraction was not representative of intact anandamide suggesting that following metabolism by the enzyme fatty acid amide hydrolase, anandamide metabolites are rapidly enriched in caveolae. Furthermore, Hela cells, which do not express high levels of fatty acid amide hydrolase, only showed an accumulation of tritium in the caveolin-rich membrane fraction when transfected with fatty acid amide hydrolase cDNA. Together, these data suggest that following uptake via caveolae/lipid raft-related endocytosis, anandamide is rapidly metabolized by fatty acid amide hydrolase with the metabolites efficiently recycled to caveolin-rich membrane domains.^
Eric L. Barker, Purdue University.
Health Sciences, Pharmacology