Brain monoglyceride lipase participating in endocannabinoid inactivation

T Dinh

D Carpenter

F Leslie

T Freund

Departments of ^{Pharmacology and}^{Neurology, University of California, Irvine, CA 92697-4625;}^{Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest 1450, Hungary; and}^{Department of Neurology, University G. d'Annunzio, Chieti 66013, Italy}

^{To whom reprint requests should be addressed. E-mail:}ude.icu@illemoip.

Communicated by James L. McGaugh, University of California, Irvine, CA

Received 2002 Feb 22; Accepted 2002 Jun 4.

Abstract

The endogenous cannabinoids (endocannabinoids) are lipid molecules that may mediate retrograde signaling at central synapses and other forms of short-range neuronal communication. The monoglyceride 2-arachidonoylglycerol (2-AG) meets several criteria of an endocannabinoid substance: (i) it activates cannabinoid receptors; (ii) it is produced by neurons in an activity-dependent manner; and (iii) it is rapidly eliminated. 2-AG inactivation is only partially understood, but it may occur by transport into cells and enzymatic hydrolysis. Here we tested the hypothesis that monoglyceride lipase (MGL), a serine hydrolase that converts monoglycerides to fatty acid and glycerol, participates in 2-AG inactivation. We cloned MGL by homology from a rat brain cDNA library. Its cDNA sequence encoded for a 303-aa protein with a calculated molecular weight of 33,367 daltons. Northern blot and in situ hybridization analyses revealed that MGL mRNA is heterogeneously expressed in the rat brain, with highest levels in regions where CB^{cannabinoid receptors are also present (hippocampus, cortex, anterior thalamus, and cerebellum). Immunohistochemical studies in the hippocampus showed that MGL distribution has striking laminar specificity, suggesting a presynaptic localization of the enzyme. Adenovirus-mediated transfer of MGL cDNA into rat cortical neurons increased MGL expression and attenuated}N-methyl-D-aspartate/carbachol-induced 2-AG accumulation in these cells. No such effect was observed on the accumulation of anandamide, another endocannabinoid lipid. The results suggest that hydrolysis by means of MGL is a primary mechanism for 2-AG inactivation in intact neurons.

Abstract

Cannabinoid receptors, the molecular target for Δ^{-tetrahydrocannabinol in marijuana, are activated by endogenous lipids that include the fatty ethanolamide, anandamide, and the monoglyceride, 2-arachidonoylglycerol (2-AG) (}1–3). These compounds, collectively called endocannabinoids, are generated by cells on demand through stimulus-dependent cleavage of membrane phospholipid precursors and, after release, undergo rapid biological deactivation (4, 5). Because of their nonsynaptic production and fast elimination, the endocannabinoids are thought to act as short-range modulators of cell and synapse activity rather than classical hormones or neurotransmitters (for review, see ref. 6). In the brain striatum, for example, locally released anandamide may participate in an inhibitory feedback loop countering dopamine-induced facilitation of psychomotor activity (7, 8). Furthermore, in hippocampus and cerebellum, endogenously released 2-AG or anandamide produced from depolarized neurons may serve as a transsynaptic messenger, regulating neurotransmitter release (9–11) and influencing neuronal network activity (12, 13).

Anandamide and 2-AG are eliminated through a two-step process consisting of carrier-mediated transport into cells and subsequent enzymatic hydrolysis (for review, see ref. 14). In the brain, anandamide breakdown to arachidonic acid and ethanolamine is mediated by fatty acid amide hydrolase (FAAH), a member of the “amidase-signature family” of enzymes (15, 16). FAAH can function as a general hydrolytic enzyme not only for anandamide and other fatty ethanolamides but also for fatty esters such as 2-AG. This unusually broad substrate selectivity, which was demonstrated in experiments with membrane and purified enzyme (17–19), has led researchers to suggest that FAAH may terminate the biological actions of both anandamide and 2-AG. Two findings, however, argue against this idea. First, a 2-AG hydrolase activity distinct from FAAH has been partially purified from porcine brain (20). Second, in intact astrocytoma cells, inhibition of FAAH activity prevents the hydrolysis of anandamide but not that of 2-AG (21). Thus, although 2-AG can be hydrolyzed by FAAH in vitro, different enzyme(s) may be responsible for its degradation in vivo. A possible candidate for this role is monoglyceride lipase (MGL), a serine hydrolase that cleaves 2- and 1-monoglycerides into fatty acid and glycerol (22). To test this hypothesis, we cloned rat brain MGL, determined its anatomical distribution, and used adenovirus-mediated gene transfer to investigate its role in neuronal 2-AG inactivation.

Acknowledgments

We thank Dr. Cecilia Holm (Lund University, Lund, Sweden) for the generous gift of mouse adipocyte MGL cDNA; Drs. M. L. Solbrig and A. Giuffrida for critical reading of the manuscript; Drs. F. Désarnaud, J. Fu, and C. Sanchez; and Y. Chen, S. Rao, D. Ton-That, and J. Yoo for experimental assistance. This work was supported by National Institute on Drug Abuse Grants 12447 and 3412 (to D.P.) and by the Howard Hughes Medical Institute and Orszgos Tudomnyos Kutatsi Alap (to T.F.F.). T.P.D. was supported by National Institute of Aging Fellowship AG00096, and S.L.S. was supported by National Institute of Aging Grant AG00919.

Acknowledgments

Abbreviations

2-AG, 2-arachidonoylglycerol
MGL, monoglyceride lipase
FAAH, fatty acid amide hydrolase
NMDA, N-methyl-d-aspartate

Abbreviations

Notes

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AY081195).

Notes

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AY081195).

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