Although separate enzymes cata lyze the syntheses of anandamide and NAPE, the two events are likely to occur simultaneously because Ca2+- stimulated anandamide production is often accompanied by de novo formationof NAPE .For example, the Ca2+ ionophore ionomycin stimulates [ 3 H]anandamide formation in cultures of rat striatal and cortical neurons labeled by incubation with [ 3 H]ethanolamine . In the same neurons, Ca2+-dependent [3 H]ana ndamide production may be elicited by the glutamate receptor agonist, kainate, by the K+ channel blocker 4- aminopyridine, and by membrane-depolarizing con centrations of K+ ions . Depolarizationof neural cells was also shown to evoke Ca2+-dependent anandamide release in vivo . Along with Ca2+ entry, activation of certain G protein-coupled receptors can also initiate anandamide generation. Administration of the dopamine D2-recep tor agonist quinpirole causes a profound stimulation of anandamide synthesis in the rat basal ganglia, which is prevented by the D2 antagonist raclopride . Importantly, cocaine elicits a similar response , suggesting a role for anandamide in the actions of these psychostimulant drugs. The ability of the anandamide transport inhibitor AM404 to reduce D2 agonist induced hyperactivity, discussed below,vertical farming market further sup ports this possibility .The biological elimination of anandamide proceeds through two successive steps of high-affinity transport into cells, followed by intracellular degradation . Brain neurons and astrocytes in culture internalize anandamide through a process that fulfills all key requirements of a carrier-mediated transport. Plots of the initial rates of [3 H]anandamide internalization in rat brain neurons and astrocytes in culture yield apparent Michaelis constants that are consistent with a saturable process and are comparable to the KM values of brain amine or amino-acid transporters .
Moreover, neurons and astrocytes in culture internalize [3 H]anandamide, along with a select group of structurally related compounds, in a stereoselective manner . Even further, [3 H]ana ndamide internalization can be inhibited by drugs that have no effect on the uptake of non-cannabinoid lipid mediators such as fatty acids and eicosanoids. Nevertheless, anandamide is internalized in a Na+- and energy-inde pendent manner , a feature that differentiates this lipid mediator from most conventional neurotransmitters.The prototype of this class of drugs, the arachido nate derivative AM404 , has provided important information on the properties of anandamide transport, not only aiding the in vitro characterization of this process, but also helping to reveal its possible functions in animals. Importantly, the partial cannabimetic profile exhibited by this agent in vivo suggests that anandamide transport might provide a useful tar get in disease conditions in which the endocannabinoid system is hypofunctional . Evidence indicates that one such condition could be opiate withdrawal, which is markedly reduced in rodents by administering AM404 . These theories have been hindered by the fact that the putative transport system responsible for anandamide internalization is still uncharacterized at the molecular level. In fact, the presence of such a system has been recently questioned, based on the observation that [ 3 H]anandamide uptake in certain cell lines is saturable at longer , but not at shorter incu bationtimes . This finding has been interpreted to suggest that fatty-acid amide hydrolase —a key enzyme of intracellular anandamide degradation, described in a subsequent section—may be responsible for the saturation of uptake noted at longer incubation times . However, the result may also be explained on purely technical grounds, as the high concentration of serum albumin used in the experiments of Glaser and collaborators was previously shown to prevent [3 H]anandamide internalization .
Consistent with this interpretation, recent studies have provided additional evidence for the existence of an anandamide transport system independent of FAAH . In particular, one of these studies has shown that cultures of cortical neurons isolated from the brain of FAAH null mice internalize anandamide as efficiently as do neurons that express normal levels of the enzyme. The same study also demonstrated that the transport inhibitor AM404 is equally effective at reducing anandamide internalization in neurons of FAAH-null and wild-type mice. These results indicate that FAAH does not pro vide the driving force for anandamide uptake or serve as a target for AM404. Invivo experiments further support this conclusion, showing that AM404 not only enhances the actions of exogenous anandamide in FAAH-null mice, but acts more effectively in this mutant strain than it does in control animals. This implies that AM404 is not in fact a FAAH inhibitor, as it has been proposed , but a FAAH substrate. In support of this idea, it was found that membranes prepared from the brains of normal mice rapidly hydrolyze AM404, whereas those prepared from mice that lack FAAH are unable to carry out this reaction.The fact that FAAH is not directly involved in anandamide internalization raises the question of what mechanism provides the driving force for this process. One possibility is that an intracellular protein may sequester anandamide at the membrane, driving its internalization and facilitating its movement to the mitochondria and the endoplasmic reticulum, where FAAH is primarily localized . If selective for anandamide, such a protein might participate in the transport process as well as serve as a target for transport inhibitors. This hypothetical model is consistent with fatty acid transport into cells, which is also thought to require the cooperation of membrane transporters and intracellular fatty-acid binding proteins .AM404 increases endogenous anandamide levels in brain tissue and peripheral blood of rats and mice . This effect is accompanied by a series of behavioral responses that, though blocked by the CB1 antagonist rimonabant , are clearly distinguishable from those of direct cannabinoid agonists. For example, administra tion of AM404 into the cerebral ventricles of rats decreases exploratory activity without producing cata lepsy and analgesia, two hallmarks of direct CB1 receptor activation.
Inaddition, AM404 reduces two characteristic effects caused by activationof D2 family receptors: the yawning response elicited in mice by low doses of the D1/D2-receptor agonist apomorphine; and the stimu lationof locomotor activity evoked inrats by the D2- receptor agonist quinpirole . These effects are observed at doses of AM404 that selectively target anandamide transport and produce only mild hypokinesia when the drug is administered alone . The results of this study, which have been confirmed in several subsequent reports , demarcate the pharmacological profile of AM404 from those of direct-acting cannabinoid drugs. This distinction may result from the ability of AM404 to enhance anandamide signaling in an activity-dependent manner, causing anandamide to accumulate in discrete regions of the brain and only when appropriate stimuli initiate its release. Pharmacological activation of D2 receptors may represent one such stimulus, suggesting that blockade of anandamide transport might offer an innovative strategy to correct abnormalities associated with dysfunction in dopaminergic transmission. Initial tests of this hypothesis have shown that systemic administration of AM404 normalizes movement in spontaneously hypertensive rats , an inbred line in which hyperactivity and attention deficits have been linked to a defective regulation of mesocorticolimbic dopamine pathways .FAAH was first identified as an amide hydrolase activity present in rat liver tissue, which catalyzes the hydrolysis of the fatty-acid ethanolamides palmitoy lethanolamide and oleoylethanolamide . That anandamide serves as a substrate for this activity was first suggested onthe basis of biochemical evidence and later demonstrated by molecular cloning,vertical farming pros and cons heterologous expression and generation of FAAH null mice by homologous recombination . FAAH belongs to a group of enzymes known as ‘amidase signature family’ and catalyzes the hydrolysis not only of anandamide and other fatty-acid ethanola mides, but also of primary amides such as oleamide and even of fatty-acid esters such as 2-AG . Elegant site-directed mutagenesis and X-ray diffraction studies have demonstrated that this unusually broad substrate preference is due to a novel catalytic mechanism involving the amino-acid residue lysine 142. This residue may act as a general acid catalyst, favoring the protonation and consequent detachment of reaction products from the enzyme’s active site . Three serine residues that are conserved in all amidase signature enzymes also may be essential for enzymatic activity: serine 241 may serve as the enzyme’s catalytic nucleophile, while serine 217 and 218 may modulate catalysis through an as-yet-unidentified mechanism . Electron microscopy experiments in the rat and mouse brain have shown that FAAH is predominantly, if not exclusively localized to intracellular membrane com partments, particularly to the endoplasmic reticulum and the mitochondria . Although FAAH appears to be the predominant route of anandamide hydrolysis in the brain, other enzymes are likely to participate in the breakdown of this endocannabinoid in peripheral tissues. An acid amide hydrolase activity catalytically distinct from FAAH has been characterized in human mega karyoblastic cells and shown to be highly expressed in the rat thymus, lungs and intestine .The search for small-molecule inhibitors of intra cellular FAAH activity has led to the emergence of several potent and selective agents, which include substituted sulfonyl fluorides , alpha-keto-oxazolopyridines an d carbamic acid esters .
The latter were identified during structure– activity relationship studies aimed at determining whether esters of carbamic acid such as the insecticide carbaryl inhibit FAAH activity. It was found that, although carbaryl is ineffective in this regard, variations in its template result in significant inhibitory potencies. Further structural optimizations yielded a group of highly potent inhibitors, a representative example of which is provided by the compound URB597 . Kinetic and dialysis experiments indicate that URB597 interacts non-competitively with FAAH, which is suggestive of anirreversible or slowly reversible association with the enzyme. Importantly, URB597 has no notable effect on CB1 or CB2 binding, anandamide transport, or rat brain monoglyceride lipase , a cytosolic serine hydrolase that catalyzes the hydrolysis of the second endocannabinoid, 2-arachido noylglycerol. Following administration to rats in vivo, URB597 produces profound, dose-dependent inhibition of brain FAAH activity. After injection of a maximal dose of compound , FAAH inhibitionis rapid , persistent and associated with a 3-fold increase in brain anandamide levels. Furthermore, the inhibitor intensifies and pro longs the effects produced by exogenous anandamide, yet it elicits no overt cannabinoid-like actions when administered alone; for example, it does not cause hypothermia, hot-plate analgesia, or hyperphagia .Although URB597 does not display a typical canna binoid profile in live animals, it exerts several pharma cological effects that might be therapeutically relevant. One such effect, the ability to reduce anxiety-like beha viorsinrats, was demonstrated in two distinct experimental models: the elevated ‘zero maze’ test, and the isolation-induced ultrasonic emission test . The ‘zero maze’ consists of an elevated annular platform with two open and two closed quad rants and is based on the conflict between an animal’s instinct to explore its environment and its fear of open spaces where it may be attacked by predators . Benzodiazepines and other clinically used anxiolytic drugs increase the proportion of time spent in, and the number of entries made into, the open com partments. In a similar fashion, URB597 elicits anxio lytic-like responses at a dose that corresponds to those required to inhibit brain FAAH activity. Moreover, these effects are prevented by the CB1-selective antagonist rimonabant. Analogous results were obtained in the ultrasonic vocalization emission test, which measures the number of stress-induced vocalizations emitted by rat pups removed from their nest . If confirmed in further behavioral models, these findings would suggest that inhibition of intracellular FAAH activity might offer an innovative target for the treatment of anxiety , which is also a feature of marijuana withdrawal .2-AG was identified as a second endocannabinoid substance in 1995 . The multiple roles of this lipid compound in cell metabolism and its high levels in brain tissue— about 200-fold higher than those of anandamide—suggest that much of cellular 2-AG may be involved in housekeeping functions. The diversity of roles played by this compound also complicates our efforts to establish biochemical route involved in its physiological formation. Nevertheless, one pathway has emerged as the most likely candidate . This pathway starts with the phospholipase-mediated generation of 1, 2- diacylglycerol . This serves as a substrate for two enzymes: DAG kinase, which catalyzes DAG phosphorylationto phosphatidic acid; and DAG lipase , which hydrolyzes DAG to monoacylglycerol . Pharmacological inhibition of phospholipase C and DGL prevent the Ca2+-dependent accumulation of 2-AG in rat cortical neurons, which suggests a key role of this pathway in2-AG generation . However, additional routes of 2-AG synthesis also may exist, including phospho lipase A1 , hormone-sensitive lipase or a lipid phosphatase . In neurons and glia, 2-AG synthesis may be initiated by a rise incytosolic Ca2+ levels. For example, incultures of rat cortical neurons, the Ca2+ ionophore ionomycin and the glutamate receptor agonist N-methyl d-aspartate stimulate 2-AG productionina Ca2+-dependent manner .