(Received for publication, November 21, 1994; and in revised form, January 17, 1995)
From the
An amidohydrolase activity present in rat brain microsomes
catalyzes the hydrolysis of N-arachidonoyl[H]ethanolamine
([
H]anandamide), an endogenous cannabimimetic
substance, forming [
H]ethanolamine and
arachidonic acid. Amidohydrolase activity is maximal at pH 6 and 8, is
independent of divalent cations, has an apparent K
for [
H]anandamide of 12.7 ± 1.8
µM, and has a V
of 5630 ±
200 pmol/min/mg of protein. Phenylmethylsulfonyl fluoride, a serine
protease inhibitor, and p-bromophenacyl bromide, a
histidine-alkylating reagent, inhibit the activity, whereas N-ethylmaleimide and various nonselective peptidase inhibitors
(EDTA, o-phenanthroline, bacitracin) have no effect. Brain
amidohydrolase activity exhibits high substrate specificity for
[
H]anandamide; N-
-linolenoyl-, N-homo-
-linolenoyl-, and N-11,14-eicosadienoyl-
are hydrolyzed at markedly slower rates. Moreover, N-11-eicosaenoyl- and N-palmitoyl-[
H]ethanolamine are not
hydrolyzed. [
H]Anandamide hydrolysis is inhibited
competitively by nonradioactive anandamide and by other N-acylethanolamines with the following rank order of potency:
anandamide > N-linoleoyl- = N-cis-linolenoyl- = N-
-linolenoyl- = N-homo-
-linolenoyl-
> N-11,14-eicosadienoyl- > N-oleoyl- > N-docosahexaenoyl- > N-docosatetraenoyl > N-linoelaidoyl- > N-eicosaenoyl- > N-palmitoyl
N-elaidoyl- = N-eicosanoyl-ethanolamine = no effect. Amidohydrolase
activity is high in liver and brain and low in heart, kidney,
intestine, stomach, lung, spleen, and skeletal muscle. Within the
central nervous system, highest activity is found in globus pallidus
and hippocampus, two regions rich in cannabinoid receptors, and lowest
activity is found in brainstem and medulla, where cannabinoid receptors
are sparse. The results, showing that brain amidohydrolase activity is
selective for anandamide and enriched in areas of the central nervous
system with high density of cannabinoid receptors, suggest that this
activity may participate in the inactivation of anandamide at its sites
of action.
The discovery of a G protein-coupled membrane receptor that
recognizes -tetrahydrocannabinol, the major
psychoactive principle of Cannabis sativa, has prompted the
search for an endogenous substance with cannabimimetic properties (for
review, see Howlett et al.(1990)). This search has recently
led to the isolation from porcine brain of a cannabimimetic lipid
derivative, identified as N-arachidonoylethanolamine and named
anandamide (Devane et al., 1992). Like
-tetrahydrocannabinol, anandamide binds with high
affinity to brain cannabinoid receptors, reduces electrically evoked
contractions in mouse vas deferens, and modulates the activities of
adenylyl cyclase and voltage-dependent Ca
channels in
neuroblastoma cell lines (Devane et al., 1992; Vogel et
al., 1993; Felder et al., 1993; Mackie et al.,
1993). Moreover, anandamide produces, in vivo, a series of
behavioral responses typical of cannabinoid drug administration,
including catalepsy, hypothermia, analgesia, and activation of the
hypothalamo-pituitary axis (Smith et al., 1994; Weidenfeld et al., 1994).
Recently, two other cannabimimetic N-acylethanolamines (N-homo--linolenoyl- and N-docosatetraenoyl ethanolamine) have been identified in brain
tissue (Hanuset al., 1993; Pertwee et al., 1994), suggesting that anandamide may belong to a
family of lipid mediators serving as endogenous cannabimimetic
messengers in the central nervous system. Biochemical studies lend
further support to this hypothesis. Rat brain preparations were shown
to catalyze the synthesis of anandamide via energy-independent
condensation of arachidonic acid with ethanolamine (Kruszka and Gross,
1994; Devane and Axelrod, 1994). Furthermore, studies in our laboratory
have shown that rat brain neurons in primary culture produce and
release anandamide and other N-acylethanolamines when they are
stimulated with membrane-depolarizing agents or Ca
ionophores. This reaction, which is both
Ca
-dependent and neuron-specific, involves the
phosphodiesterase-mediated cleavage of a membrane phospholipid
precursor, N-acylphosphatidylethanolamine (Di Marzo et
al., 1994). Together, these results suggest that multiple pathways
of anandamide formation may coexist in nervous tissue.
Despite these
important advances, the fate of endogenous anandamide in the central
nervous system is still poorly documented. Before the discovery of
anandamide, the pioneering studies of Schmid and co-workers have
demonstrated that ethanolamides of saturated fatty acids are hydrolyzed
in tissues by an amidohydrolase (amidase) activity with broad substrate
specificity (Natarajan et al., 1984; Schmid et al.,
1985, 1990). That anandamide may be a substrate for amidohydrolase
activity was suggested by recent reports showing hydrolytic cleavage of
exogenous [H]anandamide by rat brain homogenates
(Deutsch and Chin, 1993) or by intact brain neurons in primary culture
(Di Marzo et al., 1994).
We report now that rat brain
microsomes contain an amidohydrolase activity that acts with high
selectivity on [H]anandamide and other
polyunsaturated N-acylethanolamines. This ``anandamide
amidohydrolase'' activity is discretely distributed in rat central
nervous system and is abundant in regions where cannabinoid receptors
are densely expressed. Our results suggest therefore that anandamide
amidohydrolase activity may participate in the degradation of
endogenous anandamide at synaptic sites.
Figure 1:
Hydrolysis of
[H]anandamide by rat brain microsomes. A, time dependence. Microsomes (0.2 mg of protein/ml) were
incubated at 37 °C in 5 ml of Tris buffer (50 mM, pH 7.5)
containing [
H]anandamide (14 µM,
10,000 dpm). At the indicated times, samples of the incubation mixture
were collected and diluted with methanol, and
[
H]ethanolamine was measured as described under
``Experimental Procedures.'' B, protein dependence.
Amidohydrolase activity was determined by measuring the
[
H]ethanolamine formed in assay mixtures
containing varying quantities of microsome protein and incubated under
standard conditions (see ``Experimental Procedures''). C, pH dependence. Standard amidohydrolase assays were carried
out in the following buffer solutions: sodium acetate (100
mM), pH 4-6; Tris (100 mM), pH 6.5-9;
sodium borate (100 mM), pH
9-10.
Mouse liver microsomes incubated in the presence of
NADPH convert anandamide into several oxygenated products, possibly
formed by cytochrome P-450-dependent monooxygenation (Bornheim et
al., 1993). Under our experimental conditions, oxidative
metabolism does not appear, however, to participate in
[H]anandamide degradation. When samples from
amidohydrolase assays (10-min incubation) were analyzed by
reversed-phase HPLC, no evidence was found for the formation of polar
[
H]anandamide metabolites (data not shown).
Amidohydrolase activity in microsomes was dependent on the
concentration of protein (Fig. 1B) and was optimal at
pH 6 and 8 (Fig. 1C). Moreover, the activity was
dependent on the concentration of [H]anandamide,
with an apparent K
of 12.7 ± 1.8 µM and a V
of 5630 ± 200 pmol/min/mg of
protein (n = 3). Divalent cations were neither
necessary for nor stimulatory on amidohydrolase activity; adding EGTA,
EDTA, CaCl
, or MgCl
(each at 10 mM)
had little or no effect (Table 2).
The serine protease inhibitor, phenylmethylsulfonyl fluoride, was shown to prevent the degradation of anandamide in brain homogenates (Deutsch and Chin, 1993) and to improve its metabolic stability in binding assays carried out on rat brain membranes (Abadji et al., 1994; Childers et al., 1994). In our experiments, phenylmethylsulfonyl fluoride inhibited amidohydrolase activity in a concentration-dependent manner (Table 2). The effects of other protein-alkylating reagents (N-ethylmaleimide and p-bromophenacyl bromide), as well as of nonselective peptidase inhibitors (EDTA, o-phenanthroline, bacitracin) are also shown in Table 2. Among these, only p-bromophenacyl bromide, which alkylates histidine residues on proteins, produced a significant inhibition of the activity.
Figure 2:
Substrate specificity of brain microsome
amidohydrolase activity. Relative rates of hydrolysis of various N-acyl-[H]ethanolamines are shown. The N-acyl-[
H]ethanolamines depicted in the
figure, synthesized as described under ``Experimental
Procedures,'' were incubated (28 µM, 20,000 dpm/ml)
for 5 min at 37 °C with 0.2 mg/ml of microsome protein (specific
radioactivities are listed under ``Experimental
Procedures''). Results are from one experiment, representative of
three, carried out in quintuplicate.
In a second series of
experiments, [H]anandamide hydrolysis was
measured in the presence of various nonradioactive N-acylethanolamines (Fig. 3). The concentration of
[
H]anandamide in these assays was 14
µM, and that of the competing products was 200
µM. The results show that
[
H]anandamide hydrolysis was best inhibited by
unsaturated ethanolamides with a fatty acyl chain containing
18-20 carbon atoms and a number of cis double bonds
comprised between two and four (Fig. 3). Structural
modifications that resulted in reduced inhibitory efficacy included the
following: 1) elongating the fatty acyl chain above 20 carbon atoms; 2)
replacing cis double bonds with trans double bonds;
3) decreasing the number of double bonds to one or eliminating them (Fig. 3).
Figure 3:
Substrate specificity of brain microsome
amidohydrolase activity. Effects of various N-acylethanolamines on [H]anandamide
hydrolysis are shown. Standard assays were in the presence of
[
H]anandamide (14 µM, 10,000 dpm)
and the N-acylethanolamines shown in the figure (200
µM). Results are from one experiment, representative of
three, carried out in quintuplicate.
Although our experiments do not provide a complete characterization of the structural requirements of brain microsome amidohydrolase activity, which may be best accomplished on a purified enzyme preparation, they do suggest that this activity is highly selective for anandamide. That other unsaturated N-acylethanolamines may be also substrates for this activity is in agreement with studies showing that more than one anandamide may be produced and released by stimulated neurons (Hanus et al., 1993; Pertwee et al., 1994; Di Marzo et al., 1994).
Figure 4:
Distribution of amidohydrolase activity (A) in microsomes from various tissues and (B) in
homogenates of various brain regions of the rat. Assays were carried
out on microsomes under standard conditions (A), or on
freshly-prepared homogenates of various brain regions (1 mg/ml of
protein, 10 min), with 22 µM [H]anandamide (20,000 dpm/ml) (B).