Organized trafficking of anandamide and related lipids.

N-arachidonylethanolamide (anandamide or AEA) is an endogenous long-chain fatty acid ethanolamide with activity at both the cannabinoid 1 (CB(1)) and cannabinoid 2 (CB(2)) receptors, as well as the transient receptor potential vanilloid 1 (TRPV1) receptor. Whereas the mechanisms for both AEA biosynthesis and metabolism are fairly well established, the manner by which AEA is accumulated into cells remains controversial. The overwhelming majority of scientific reports indicate that this lipid neuromodulator is taken into cells via a facilitated process. Some reports have suggested that AEA uptake occurs by facilitated diffusion. Recent evidence indicates that AEA uptake may occur via endocytosis, contesting the premise that passive diffusion is the mechanism by which AEA transverses the plasma membrane. This chapter serves as an introduction to the endocannabinoid field with an emphasis on the various proposed mechanisms for the cellular uptake of endocannabinoids and other related hydrophobic molecules.

Inactivation and biotransformation of the endogenous cannabinoids anandamide and 2-arachidonoylglycerol.

The cannabinoid field is currently an active research area. Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the most characterized endogenous cannabinoids (also known as endocannabinoids). These neuromodulators have been implicated in various physiologically relevant phenomena, including mood (Witkin et al., 2005), the immune response (Ashton, 2007), appetite (Kirkham and Tucci, 2006), reproduction (Wang et al., 2006), spasticity (Pertwee, 2002), and pain (Hohmann and Suplita, 2006). Pharmacological manipulation of AEA and 2-AG signaling should prove to have significant therapeutic applications in disorders linked to endocannabinoid signaling. One way to alter endocannabinoid signaling is to regulate the events responsible for termination of the endocannabinoid signal-cellular uptake and metabolism. However, to pharmacologically exploit AEA and/or 2-AG signaling in this way, we must first gain a better understanding of the proteins and mechanisms governing these processes. This review serves as an introduction to the endocannabinoid system with an emphasis on the proteins and events responsible for the termination of AEA and 2-AG signaling.

RNA interference-mediated knockdown of dynamin 2 reduces endocannabinoid uptake into neuronal dCAD cells.

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 ([3',6'-bis(acetyloxy)-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen-5-yl]-2-[[1-oxo-5Z,8Z,11Z,14Z-eicosatetraenyl]amino]ethyl ester carbamic acid). RNAi-mediated knockdown of the beta2 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 [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 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.

Cloning and expression of canine glucagon receptor and its use to evaluate glucagon receptor antagonists in vitro and in vivo.

Glucose homeostasis is maintained by the combined actions of insulin and glucagon. Hyperglucagonemia and/or elevation of glucagon/insulin ratio have been reported in diabetic patients and in animal models of diabetes. Therefore, antagonizing glucagon receptor function has long been considered a useful approach to lower hyperglycemia. Dogs serve as an excellent model for studying glycemic control and various aspects of glucagon biology in vivo; however, the amino acid sequence of the dog glucagon receptor has not been reported. To better understand the pharmacology of the dog glucagon receptor and to characterize glucagon receptor antagonists, we cloned a cDNA corresponding to the glucagon receptor from dog liver RNA. The dog glucagon receptor shares a significant (>75%) homology at both nucleotide and amino acid levels with the glucagon receptor from human, monkey, mouse, and rat. The protein is highly conserved among all species in areas corresponding to the 7 trans-membrane domains. However, it shows significant divergence at the carboxy terminus such that the receptor from dog has the longest cytoplasmic tail among all species examined. When expressed in chinese hamster ovary cells, the dog glucagon receptor bound [125I]Glucagon with a K(d) of 477+/-106 pM. Glucagon stimulated the rise of intracellular cAMP levels in these cells with an EC(50) of 9.6+/-1.7 nM and such effects could be blocked by known peptidyl and non-peptidyl small molecule antagonists. In addition we show that a small molecule glucagon receptor antagonist with significant activity in cell based assays also blocked the ability of glucagon to induce elevation in blood glucose in beagle dogs. These data demonstrate that the cloned cDNA encodes a functional dog glucagon receptor. The availability of the dog cDNA will facilitate the understanding of glucagon pharmacology and aid in the characterization of novel glucagon antagonists that may serve as anti-hyperglycemic treatment for type 2 diabetes mellitus.