ReviewA role for monoglyceride lipase in 2-arachidonoylglycerol inactivation
Introduction
The life cycles of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide are similar in that they are both produced by neurons when need arises, act near their site of synthesis, and are rapidly eliminated to terminate their biological actions (Di Marzo et al., 1994, Stella et al., 1997, Piomelli et al., 1999, Beltramo and Piomelli, 2000). These properties, which distinguish 2-AG and anandamide from classical or peptide neurotransmitters, reflect the suggested role of these bioactive lipids as activity-dependent, short-range modulators of synaptic function. Neurochemical and electrophysiological data directly support such a role. For example, microdialysis experiments in the rat striatum indicate that locally released anandamide may serve as a negative feedback signal regulating dopaminergic activity (Giuffrida et al., 1999, Beltramo et al., 2000). In addition, electrophysiological experiments suggest that anandamide or 2-AG may act as transsynaptic messengers to modulate neurotransmitter release (Katona et al., 1999, Kreitzer and Regehr, 2001, Ohno-Shosaku et al., 2001, Wilson and Nicoll, 2001) and synaptic plasticity (Carlson et al., 2002, Gerdeman et al., 2002, Marsicano et al., 2002, Robbe et al., 2002) in neurons.
Despite these broad analogies, the specific routes by which 2-AG and anandamide are produced and inactivated appear to be quite different. Anandamide is thought to be generated from the hydrolysis of an N-acylated species of phosphatidylethanolamine (PE), N-arachidonoyl-PE, which requires the activity of an unknown phospholipase D (Di Marzo et al., 1994, Cadas et al., 1996, Sugiura et al., 1996, Cadas et al., 1997). By contrast, 2-AG synthesis may involve the same enzymatic cascade that catalyzes the formation of the second messengers inositol-(1,4,5)-trisphosphate and 1,2-diacylglycerol (DAG). Phospholipase C (PLC) acting on membrane phosphoinositides generates DAG, which is then converted to 2-AG by a DAG-lipase activity (Stella et al., 1997).
After release, anandamide may be accumulated back into neurons and glial cells by means of an energy- and Na+-independent transport system (Di Marzo et al., 1994, Beltramo et al., 1997), and may be broken down intracellularly to arachidonic acid and ethanolamine by fatty acid amide hydrolase (FAAH) or other amidase enzymes (Schmid et al., 1985, Cravatt et al., 1996, Ueda et al., 2001). There is evidence suggesting that 2-AG may be transported into cells through a mechanism similar to that of anandamide. For example, in human astrocytoma and other cell types, [3H]anandamide and [3H]2-AG transport have similar kinetic properties (Piomelli et al., 1999, Bisogno et al., 2001). Moreover, anandamide and 2-AG can prevent each other's transport (Beltramo and Piomelli, 2000, Bisogno et al., 2001). Finally, the accumulation of both endocannabinoids is blocked by the anandamide analog 4-(hydroxyphenyl)-arachidonamide (AM404) (Beltramo and Piomelli, 2000, Bisogno et al., 2001). Yet, significant differences between anandamide and 2-AG transport also have been documented. [3H]2-AG uptake by astrocytoma cells is inhibited by arachidonic acid, whereas [3H]anandamide accumulation is not (Beltramo and Piomelli, 2000). This discrepancy may be explained in two alternative ways. Arachidonic acid may directly interfere with a 2-AG carrier distinct from anandamide's; or the fatty acid may indirectly prevent the facilitated diffusion of 2-AG by inhibiting its enzymatic conversion to arachidonic acid. If the latter explanation is correct, agents that interfere with the incorporation of arachidonic acid into phospholipids, such as triacsin C (an inhibitor of acyl–coenzyme A synthesis), also should decrease [3H]2-AG uptake. This is indeed the case in astrocytoma cells (Beltramo and Piomelli, 2000). Thus, while anandamide and 2-AG may be internalized through similar transport mechanisms, or even share a common one, they appear to differ in how their intracellular breakdown can affect the rate of transport into cells.
The fact that FAAH catalyzes the hydrolysis of both 2-AG and anandamide in vitro has led to the suggestion that this enzyme may be responsible for the elimination of both endocannabinoids. This hypothesis is contradicted, however, by several observations. Synthetic 2-AG is rapidly degraded in mouse blood whereas anandamide is stable under the same conditions (Jarai et al., 2000). In addition, inhibitors of FAAH activity have no effect on 2-AG hydrolysis at concentrations that completely block anandamide degradation (Beltramo and Piomelli, 2000). Furthermore, 2-AG hydrolysis is preserved in mutant FAAH−/− mice, which cannot dispose of either endogenous or exogenous anandamide (Lichtman et al., 2002). In agreement with these results, a 2-AG-hydrolase activity distinct from FAAH has been partially purified from porcine brain (Goparaju et al., 1999b). This activity may correspond to monoacylglycerol lipase (MGL), a cytosolic serine hydrolase that converts 2- and 1-monoglycerides to fatty acid and glycerol (Karlsson et al., 1997). To test this hypothesis, we have cloned and characterized rat brain MGL (Dinh et al., 2002).
Section snippets
Cloning of rat brain MGL
We used a 1 kilobase (kb) fragment of mouse adipocyte MGL cDNA to screen a rat brain cDNA library by low-stringency hybridization. Upon initial screening of 2.5×105 phage plaques, we identified 40 positive clones, which we purified and subjected to secondary and tertiary screenings to ensure homogeneity. After phage purification, we transformed plasmids into competent bacteria and conducted restriction analysis to identify positive clones. We selected for sequencing five random inserts that
Discussion
Two findings of this study are relevant to a role of MGL in 2-AG inactivation. The first is that adenovirus-mediated overexpression of MGL in cortical neurons attenuated the receptor-dependent accumulation of endogenous 2-AG, but had no effect on either 2-AG synthesis or anandamide hydrolysis. A plausible interpretation of these results is that hydrolysis by means of MGL may be a primary route of 2-AG elimination in intact neurons. The second finding is that, unlike FAAH (Thomas et al., 1997,
Acknowledgements
We thank Dr C. Holm for the generous gift of mouse adipocyte MGL cDNA; Dr F.M. Leslie and Dr D. Carpenter for help with experiments; Dr M.-L. Solbrig and Dr A. Giuffrida for critical reading of the manuscript; Dr C. Gall for discussion. This work was supported by the National Institute on Drug Abuse Grants 12447 and 3412 (to D. Piomelli) and by the Howard Hughes Medical Institute and OTKA (to T.F. Freund). T.P. Dinh was supported by National Institute of Aging fellowship AG00096.
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