158 V. Marzo et al.
- Substances that inhibit the EMTenhanceresponses to exogenous AEA that are
elicited at extracellular sites (i.e. at CB 1 receptors) andinhibitthose that are
elicited at intra-cellular targets (i.e. TRPV1 receptors, see De Petrocellis et al.
2001)—if these compounds were simply acting by inhibiting FAAH, they should
enhance AEA effects in both cases. - A selective EMT inhibitor can modify the distribution of de novo biosynthesized
AEA between the intracellular and extracellular milieu, without altering its total
amounts (Ligresti et al. 2004). - 2-AGE and NADA, two endocannabinoids that are resistant and refractory to
enzymatic hydrolysis, respectively, are still taken up by cells in a temperature-
dependent way; their uptake is inhibited competitively by AEA (Huang et al.
2002; Fezza et al. 2002), although none of the specific EMT inhibitors mentioned
above has ever been tested on the cellular uptake of these compounds. - Lipopolysaccharide inhibits FAAH expression without affecting AEA cellular
uptake (Maccarrone et al. 2001); conversely, nitric oxide, peroxynitrite and
superoxide anions stimulate AEA cellular re-uptake (Maccarrone et al. 2000a),
while acute or chronic ethanol inhibits this process (Basavarajappa et al. 2003),
without affecting FAAH activity.
These data suggest that, although intracellular hydrolysis does greatly influence
the rate of AEA facilitated diffusion, the uptake process is likely to be mediated
by a mechanism subject to regulation and distinct from the one catalysing AEA
hydrolysis.
3.2
Enzymatic Hydrolysis
3.2.1
Anandamide Hydrolysis
The hydrolysis of AEA is catalysed by FAAH, an enzyme originally purified and
cloned from rat liver microsomes (Cravatt et al. 1996), that also catalyses the
hydrolysis of other long-chain NAEs and, in vitro, of 2-AG. Since the hydrolysis
products do not activate cannabinoid receptors, this reaction represents a true
inactivationmechanism.FAAHisprobablythesameenzymeidentifiedinthe1970s
and 1980s as a NAE-hydrolysing enzyme (see Natarajan et al. 1984, for an example).
It also catalyses the hydrolysis of arachidonoyl methyl ester, and hence it is possible
that virodhamine is also a substrate, although this possibility has not been tested
yet. Finally, FAAH also catalyses the hydrolysis of long-chain primary fatty acid
amides, such as the putative sleep-inducing factor oleamide (Maurelli et al. 1995;
Cravatt et al. 1996). The structural and kinetic properties of FAAH have been
widely reviewed in the literature (Bisogno et al. 2002; Cravatt and Lichtman 2003,
forrecentreviews)andwillbedescribedinmoredetailinotherchaptersofthis
volume.Inbrief,theenzymehasanalkalineoptimalpHandisfoundinintracellular