Nevertheless, in the same models, AM404 increases the responses elicited by exogenous anandamide, and this potentiation is reversed by the CB1 antagonist SR141716A . Despite the absence of overt cannabimimetic properties, AM404 resembles anandamide and other cannabinoid receptor agonists in certain respects. For example, when administered alone, AM404 causes a reduction in motor activity, which is prevented by the CB1 antagonist SR141716A . Furthermore, AM404 reduces the yawning evoked by low doses of the mixed D1/D2 dopamine agonist apomorphine and inhibits the hyperactivity elicited by the selective D2 agonist quinpirole . AM404 also decreases the levels of circulating prolactin, but the role of CB1 receptors in this response is unknown . Can the effects of AM404 be explained by its in vitro affinity for vanilloid receptors ? The fact that SR141716A, a selective CB1 antagonist, blocks the motor inhibitory effects produced by AM404 argues against this possibility. Furthermore, vanilloid agonists such as capsaicin have very different, in some cases even opposite, effects. For example, capsaicin causes hyperkinesia and pain , whereas AM404 elicits hypokinesia and enhances anandamide’s analgesic properties . Therefore, a more plausible interpretation of the available data is that, by inhibiting anandamide clearance, AM404 may cause this lipid to accumulate outside cells and activate local cannabinoid receptors. In further support of this possibility,planting racks the systemic administration of AM404 in rats was found to cause a time-dependent increase in circulating anandamide levels . Finally, it is important to point out that several anandamide responses are not affected by AM404.
One example is the inhibition of intestinal motility, which anandamide may produce in rodents by activating CB1 receptors on the surface of enteric neurons . This effect is not enhanced by AM404, suggesting that the predominant pathway of endocannabinoid inactivation in the intestine may be through enzymatic hydrolysis, not transport .Alternatively, anandamide transport may occur in the intestine through transport mechanisms that are insensitive to AM404.Long before the discovery of anandamide, Schmid and coworkers identified in rat liver an amidohydrolase activity, which catalyzes the hydrolysis of fatty acid ethanolamides to free fatty acid and ethanolamine . That anandamide may serve as a substrate for this activity was first suggested on the basis of biochemical evidence and then demonstrated by molecular cloning and heterologous expression of the enzyme involved . AAH is an intracellular membrane-bound protein whose primary structure displays significant similarities with a group of enzymes known as “amidase signature family” . AAH may act as a general hydrolytic enzyme not only for fatty acid ethanolamides but also primary amides and even esters . Site-directed mutagenesis experiments indicate that this unusually wide substrate preference may be underpinned by 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 may also 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 .
Like other hydrolase enzymes, AAH may act in reverse, catalyzing the synthesis of anandamide from free arachidonate and ethanolamine . The high KM values reported for anandamide synthase activity suggest, however, that under normal circumstances AAH acts predominantly as a hydrolase. One exception is represented by the rat uterus, where substrate concentrations in the micromolar range are required for the synthase reaction to occur, implying that in this tissue AAH could contribute to anandamide biosynthesis . In addition to AAH, other ill-characterized enzyme activities may participate in the breakdown of anandamide and2-AG. A fatty acid ethanolamide-hydrolyzing activity catalytically distinct from AAH was described in rat brain membranes and human megakaryoblastic cells . Furthermore, evidence indicates that 2-AG degradation may be predominantly catalyzed by an enzyme different from AAH, possibly a monoacylglycerol lipase .Modifications in three potential pharmacophores have helped define several general requisites for endocannabinoid hydrolysis by AAH. First, reducing the number of double bonds in the hydrophobic carbon chain causes a gradual increase in metabolic stability . Thus, [3 H]anandamide hydrolysis is inhibited by fatty acid ethanolamides in the 20 carbon atom series with the following rank order of potency: 20:4 _x0005_ 20:3 _x0005_ 20:2 _x0005_ 20:1 _x0005_ 20:0 no effect . Second, replacing the ethanolamine moiety with a primary amide leads to good AAH substrates. For example, the rate of hydrolysis of arachidonylamide is approximately twice that of anandamide . Third, anandamide congeners containing a tertiary nitrogen in the ethanolamine moiety are poor AAH substrates . Fourth, introduction of a methyl group at the C2, C1_x0007_ , or C2_x0007_ positions of anandamide yields analogs that are resistant to hydrolysis, likely as a result of increased steric hindrance around the carbonyl group . Fifth, substrate recognition at the AAH active site is stereoselective, at least with fatty acid ethanolamide congeners containing a methyl group in the C1_x0007_ or C2_x0007_ positions . Finally, as a result of AAH’s remarkable “directed nonspecificity” , fatty acid esters also serve as substrates for this enzyme. Thus, 2-AG is hydrolyzed by AAH at a rate that is about 4 times faster than anandamide is .
AAH is widely distributed in the brain, with particularly high levels in cortex, hippocampus, cerebellum, amygdala, thalamus, and pontine nuclei . Immunohistochemical studies suggest that neurons, not glia, are the predominant cell type expressing AAH ,sub irrigation cannabis although astrocytes in primary culture have been shown to contain AHH activity . CB1 cannabinoid receptors are present in various brain regions that also express AAH, but there appears to be no direct correlation between the concentrations of these two proteins . This discrepancy may reflect the participation of AAH in the degradation of noncannabinoid lipid amides, such as oleamide and OEA.AAH mRNA and enzyme activity have been measured in a variety of nonneural cells lines, including lung carcinoma , human breast carcinoma , leukemia basophils , human monocytic leukemia , rat renal endothelial and mesangial cells , rat macrophages , human platelets , and human lymphocytes . Furthermore, high AAH levels have been found in rat liver, testis, kidney, lung, spleen, uterus, small intestine, and stomach; whereas lower levels were observed in heart and skeletal muscle . The distribution of AAH in human tissues is somewhat different from the rat, with expression levels that are reportedly higher in pancreas, brain, kidney, and skeletal muscle than in liver .The armamentarium of AAH inhibitors available to the experimentalist has been recently enriched by two important groups of molecules. The first are fatty acid sulfonyl fluorides, such as the compound AM374 . AM374 irreversibly inhibits AAH activity with an IC50 value in the low nanomolar range and displays a 50-fold preference for AAH inhibition versus CB1 cannabinoid receptor binding . In superfused hippocampal slices, AM374 augments anandamide’s ability to inhibit [3 H]acetylcholine release, although it does not affect release when it is applied alone . The second group of AAH inhibitors is represented by a series of substituted -keto-oxazolopyridines , which are reversible and extremely potent . Little information is as yet available on the pharmacological selectivity and in vivo properties of these interesting compounds.Systemic administration of the potent AAH inhibitor AM374 does not produce clear cannabimimetic effects in rats but enhances the operant lever pressing response evoked by anandamide administration . These results suggest that AM374 protects exogenous anandamide from degradation but does not cause a significant accumulation of endogenously generated anandamide. This idea is consistent with the finding that, in contrast to the transport inhibitor AM404 , AM374 does not increase circulating anandamide levels in rats . Further studies will be required to fully evaluate the behavioral impact of AAH inhibitors and to assess the biological availability and pharmacokinetics of these molecules.What place will inhibitors of endocannabinoid clearance occupy in medicine, if any, will largely depend on the answers to two key questions. The first is whether endogenously produced anandamide and 2-AG participate in the modulation of specific disease states. Drugs that block endocannabinoid inactivation should magnify this adaptive function in the same way as serotonin reuptake or monoaminooxidase inhibitors heighten the mood-regulating actions of endogenous biogenic amines. The second question is whether inhibiting endocannabinoid clearance provides a therapeutic advantage over direct activation of cannabinoid receptors with agonist drugs.
The latter approach has been generally favored thus far, and several classes of sub-type-selective cannabinoid agonists are already available for preclinical use . Thus, demonstrating that inhibitors of endocannabinoid inactivation possess a unique pharmacological profile is essential to justify the substantial efforts associated with the development of a new class of drugs. In the following sections, we illustrate with some examples the endocannabinoids’ rolein pathology and discuss the potential therapeutic value of drugs that target endocannabinoid inactivation. Pain. Considerable evidence indicates that the endocannabinoid system plays an essential role in pain regulation . For example, in vivo microdialysis experiments have shown that peripheral injections of the chemical irritant formalin are accompanied by increases in anandamide outflow within the PAG, a brain region intimately involved in pain processing . Since activation of CB1 receptors in the PAG causes profound analgesia, it has been argued that inhibitors of anandamide inactivation “may form the basis of a modern pharmacotherapy of pain, particularly in instances where opiates are ineffective” . The fact that the endocannabinoid transport inhibitor AM404 has no antinociceptive effect in models of acute pain seems to contradict this possibility . It should be noted, however, that neither AM404 nor any other inhibitor of anandamide clearance has yet been tested in animal models that are directly relevant to pathological pain states in humans. In models that mimic such states , the CB1 receptor antagonist SR141716A exacerbates pain when administered alone, suggesting that inflammation and nerve injury may be associated with compensatory increases in cannabinergic activity . If this hypothesis is correct, one would expect endocannabinoid inactivation inhibitors to alleviate inflammatory or neuropathic pain. This possibility has not yet been tested, however. Hypotensive Shock. During hemorrhagic and septic shock, anandamide and 2-AG may be released from macrophages and platelets, activate CB1-type receptors on the surface of vascular smooth muscle cells, and produce vasodilatation . The physiological significance of this response is still unclear. Nevertheless, the fact that a CB1 antagonist reduces survival time in “shocked” rats suggests that activation of the endocannabinoid system may have beneficial effects, possibly by redistributing cardiac output to or improving microcirculation in vital organs such as the kidneys . If this is true, inhibitors of endocannabinoid inactivation that do not appear to exert direct vasoactive effects could be used to prolong life expectancy in hemorrhagic and septic shock. Disorders of Dopamine Transmission. Functional interactions between dopamine and endocannabinoids are well documented. CB1 receptors are highly expressed in CNS regions that are innervated by dopamine-releasing neurons . In one of these regions, the striatum, anandamide release is stimulated by activation of dopamine D2-family receptors . Furthermore, the CB1 antagonist SR141716A, which has no effect on motor activity when administered alone, enhances the motor hyperactivity elicited by D2-family agonists . These findings suggest that one role of the endocannabinoid system in the CNS may be to act as an inhibitory feedback mechanism countering dopamine-induced facilitation of psychomotor activity . A corollary of this idea is that drugs that prevent endocannabinoid clearance should antagonize dopamine-mediated responses. As a test of this hypothesis, the endocannabinoid transport inhibitor AM404 was injected into the cerebral ventricles of rats that were then systemically treated with the mixed D1/D2 dopamine agonist apomorphine or the selective D2-family agonist quinpirole. AM404 blocked the yawning evoked by apomorphine and reduced the motor stimulation elicited by quinpirole. By contrast, when administered alone, AM404 produced only a mild hypokinesia, not other cannabinoid actions such as catalepsy . The effects of AM404 were also studied in juvenile spontaneously hypertensive rats . Juvenile SHR are not yet hypertensive but are hyperactive and show a number of attention deficits, which have been linked to alterations in mesocorticolimbic dopamine transmission and dopamine receptor expression .