Mechanisms for recycling and biosynthesis of endogenous cannabinoids anandamide and 2‐arachidonylglycerol

Abstract

Abbreviations used: 2-AG, 2-arachidonyl glycerol; D 9 -THC, delta-9-tetrahydrocannabinol; AEA, arachidonylethanolamine (anandamide); AM404, N-(4-hydroxyphenyl)-Arachidonylamide; ARA, arachidonic acid; BSA, bovine serum albumin; CAD, Cath.a differentiated; FAAH, fatty acid amide hydrolase; KRH, Kreb-Ringer Hepes; NAPE PLD, N-acyl phosphatidylethanolamine phospholipase D; SOCE, store-operated calcium entry. AbstractThe 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.

Activation of TRPC6 channels promotes endocannabinoid biosynthesis in neuronal CAD cells

“…To label the CAD cells, the tritiated precursor was added to the medium where it could be incorporated into the cells. Previously, we observed that the tritiated precursors (AEA or ArA) could be taken up into the cognate mast cell line RBL-2H3, metabolized, and then reincorporated into membrane phospholipids to be recycled into new endocannabinoids (AEA or 2-AG)(Placzek et al, 2008). The question was whether a similar phenomenon could be observed for the neuronal CAD cells.…”

“…Indeed, we have previously observed that these treatments deplete membrane cholesterol in CAD cells (McFarland et al, 2008). There is growing evidence linking endocannabinoid signaling and lipid rafts (Bari et al, 2005; Maccarrone et al, 2009; Rimmerman et al, 2008; Szoke et al ,2010) and specifically evidence supporting that endocannabinoid synthesis and release occurs at or near caveolae (Placzek et al, 2008; McFarland and Barker, 2005; McFarland et al, 2004; McFarland et al, 2008; Rimmerman et al, 2008). Therefore, the effects of the disruption of caveolae on OAG-induced endocannabinoid biosynthesis and release were assessed.…”

“…This method has proven useful for simple detection of endocannabinoid biosynthesis (Placzek et al, 2008). The TLC plate was subjected to 11:5 isooctane/ethylacetate mobile phase for one hour followed by one hour in an iodine chamber to visualize the location of standards.…”

“…2-AG is synthesized from the precursor diacylglycerol (DAG) through the calcium-dependent action of sn1-specific DAG lipase (Bisogno et al, 2003), although there is evidence for phospholipase C-independent mechanisms for 2-AG formation as well (Bisogno et al, 1999; Carrier et al, 2004). There is strong support for AEA being synthesized from a precursor, N-arachidonyl phosphatidylethanolamine (NAPE) a calcium-dependent enzyme NAPE phospholipase D (NAPE PLD) (Di Marzo et al, 1994; Okamoto et al, 2004; Placzek et al, 2008). Like 2-AG, calcium-independent pathways for AEA biosynthesis have been described (Vellani et al, 2008).…”

Sphingosine lysolipids in the CNS: Endogenous cannabinoid antagonists or a parallel pain modulatory system?

“…Nonetheless, eCBs and CB 1 Rs are co-localized in lipid rafts (Barnett-Norris et al 2005, Placzek et al 2008a), and sphingosine and sphinganine would be present in these microdomains where they could potentially act as endogenous CB 1 R antagonists. Moreover, S1P can regulate its own production from sphingosine through S1PR-induced increases in SphK activity (Meyer zu Heringdorf et al 2001).…”