nystatin-a1 and anandamide

The mechanisms of endogenous cannabinoid biosynthesis are not completely understood. We hypothesized that anandamide could be recycled by the cell to form new endocannabinoid molecules and released into the extracellular space. We determined that new endocannabinoids derived from exogenous anandamide or arachidonic acid were synthesized and released from RBL-2H3 cells in response to ionomycin. Treatment of RBL-2H3 cells with nystatin and progesterone, agents that disrupt organization of lipid raft/caveolae, resulted in the attenuation of anandamide and 2-arachidonyl glycerol synthesis and/or release in response to stimulation with ionomycin suggesting a role for these membrane microdomains in endocannabinoid biosynthesis. Furthermore, anandamide synthesis may be independent of N-acyl phosphatidylethanolamine phospholipase D as expression of the enzyme was not detected in RBL-2H3 cells. We also established that extracellular calcium is necessary for endocannabinoid biosynthesis because release of intracellular calcium stores alone does not promote endocannabinoid biosynthesis. Next, we examined the role of calcium as a 'switch' to activate the synthesis of anandamide and simultaneously reduce uptake. Indeed, [(3)H] anandamide uptake was reduced in the presence of calcium. Our findings suggest a mechanism indicative of calcium-modulated activation of anandamide synthesis and simultaneous termination of uptake.

Topics: Animals; Arachidonic Acids; Biological Transport; Calcium; Caveolae; Cell Line, Transformed; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Glycerides; Ionomycin; Ionophores; Lactones; Nystatin; Phospholipase D; Polyunsaturated Alkamides; Progesterone; Progestins; Rats; Thapsigargin; Time Factors; Tritium

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.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cell Differentiation; Cells, Cultured; Dogs; Dose-Response Relationship, Drug; Dynamin II; Endocannabinoids; Endocytosis; Fluorescent Dyes; Kinetics; Lactones; Neurons; Nystatin; Polyunsaturated Alkamides; Progesterone; RNA Interference; RNA, Small Interfering; Transfection

The cellular uptake of anandamide is reduced by inhibitors of fatty acid amide hydrolase (FAAH) and by agents disrupting endocytotic mechanisms. However, it is not clear if these events occur over the same time frame and if they occur to the same extent in different cells. We have therefore investigated the effects of such compounds in three cell lines of different origins using different assay incubation times and temperatures.. FAAH activity and cellular uptake of anandamide was measured using anandamide, radio-labelled either in the ethanolamine or arachidonoyl part of the molecule.. The FAAH inhibitor URB597 inhibited the uptake of anandamide into C6 glioma, RBL2H3 basophilic leukaemia cells and P19 embryonic carcinoma cells at incubation time 4 min. However, a time-dependent and temperature-sensitive residual uptake remained after URB597 treatment. The combination of progesterone and nystatin reduced the uptake, but also decreased the amount of anandamide retained by the wells. Genistein inhibited anandamide uptake in a manner that was not additive to that of URB597. However, genistein was a potent competitive inhibitor of FAAH (K(i) value 8 microM).. The reduction of anandamide uptake by genistein can be explained by its ability to inhibit FAAH with a potency which overlaps that for inhibition of tyrosine kinase. The FAAH- resistant but time-dependent uptake of anandamide is seen in all three cell lines studied and is thus presumably a generally occurring process.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Brain; Carbamates; Cell Line, Tumor; Endocannabinoids; Genistein; Hydrolysis; Mice; Nystatin; Polyunsaturated Alkamides; Progesterone; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Rats