Characterization of the kinetics and distribution of N-arachidonylethanolamine (anandamide) hydrolysis by rat brain

et Biophysics &ta

ELSEVIER

Biochimica et Biophysics Acta 1257 (1995) 249-256

Characterization of the kinetics and distribution of iV-arachidonylethanolamine ( anandamide) hydrolysis by rat brain * Cecilia J. Hillard *, Douglas M. Wilkison, William S. Edgemond, William B. Campbell Department of Pharmacology and Toxicology, Medical College

ofWisconsin, Milwaukee, WI 53226, USA

Received 10 January 1995; accepted 6 April 1995

Abstract Arachidonoylethanolamide or ‘anandamide’ is a naturally occurring derivative of arachidonic acid that has been shown to activate cannabinoid receptors in the brain. Its metabolic inactivation by brain tissue has been investigated. Anandamide is hydrolyzed by the membrane fraction of rat brain homogenate to arachidonic acid and ethanolamine. The hydrolysis is temperature and pH- dependent (pH maximum at 8.5) and abolished by boiling. Anandamide hydrolysis is protein dependent in the range of 25-100 pg protein/ml; does not require calcium and is inhibited by phenylmethylsulfonylfluoride, diisopropylfluorophosphate, thimerosal and arachidonic acid. Hydrolysis of 10 PM anandamide by brain membranes follows first order kinetics; at 3O”C, the rate constant for anandamide catabolism is 0.34 min-’ mg protem -I. The K, for anandamide hydrolysis is 3.4 /IM, and the V,,, is 2.2 nmol/min per mg protein. Hydrolysis occurs in all subcellular fractions except cytosol with the highest specific activity in myelin and microsomes. The distribution of anandamide hydrolytic activity correlates with the distribution of cannabinoid receptor-binding sites; the hippocampus, cerebellum and cerebral cortex exhibit the highest metabolic activity, while activity is lowest in the striatum, brain stem and white matter. Keywords: Cannabinoid; N-acylethanolamine; (Marijuana); Phenylmethylsulfonyl fluoride; Amidohydrolase

1. Introduction A’-Tetrahydrocannabinol ( A9-THC) was isolated and identified from cannabis sativa 30 years ago [I]. A receptor has been characterized in neuroblastoma cells and in the brain that selectively binds both and synthetic cannabinoids [2-41. cannabinoid receptor in neuroblastoma

naturally occurring Activation of the cells results in inhi-

bition of adenylyl cyclase [5] via a pertussis G-protein [6] and a decrease in the influx

toxin sensitive of calcium via

N-type, voltage-operated calcium channels [7,8]. N-Arachidonylethanolamine (anandamide) has been recently identified as a brain constituent that binds to the cannabinoid receptor [9]. Anandamide mimics several of the physiological effects of d9-THC including production

* This work was supported in Grants, DA 4800 (CJH), HL 51055 Foundation of the Medical College * Corresponding author. Fax: +

part by National Institutes of Health and HL 37981 (WBC) and The Peters of Wisconsin (WSE). 1 (414) 266 8460.

0005.2760/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0005-2760(95)00087-9

of hypothermia and analgesia [lo] and has been found to inhibit adenylyl cyclase activity in cells that have been transfected with the cannabinoid receptor [ 11,121 and voltage-operated calcium channels in N18 cells [ 121. Taken together, these reports support the hypothesis that anandamide is an endogenous ligand of the cannabinoid receptor in brain. Studies by other investigators have shown that anandamide is degraded by membranes from brain tissue and that the serine protease/esterase inhibitor phenylmethylsulfonyl fluoride (PMSF) blocks the hydrolysis of anandamide [ 131. These observations have been extended in this report, and data is presented characterizing the kinetics, time course and inhibitor profile of the hydrolysis of anandamide by brain membranes. We conclude from these studies that the enzyme responsible for the hydrolysis of anandamide has many characteristics in common with N-acylethanolamine amidohydrolase, an enzyme previously identified in liver and dog brain that catalyzes the hydrolysis of N-acylethanolamides to ethanolamine and fatty acid [14-161.

250

2. Materials

C.J. Hillard et al. /Biochimiccr

et Biophysics Acta 1257 (19951 249-256

and methods

2.1. Materials 14C-Labeled anandamide was synthesized from arachidonyl chloride following published methods [9]. Briefly, uniformly labeled [ I4Clarachidonic acid (specific activity 866 mCi/mmol; DuPont) was mixed with unlabeled arachidonic acid and added to dry dichloromethane with 1.2 equivalents of oxalyl chloride in the presence of 1 equivalent of dimethylformamide at 0°C to form the acid chloride of arachidonic acid. After 15 min, the acid chloride was added to a IO-fold excess of ethanolamine and incubated for 60 min at 0°C. The reaction mixture was washed with water followed by removal of the solvent under N,. The [ 14C]anandamide was separated from [ I4Clarachidonic acid using thin-layer chromatography on silica-gel HL plates developed with hexane/ethyl acetate/methanol (60:40:5). The authenticity and purity of the synthesized anandamide was determined by GC-MS (as described below) and NMR. A Bruker AC 300 was used for both ‘H-and “C-NMR analysis of samples that were dissolved in 0.3 ml CDCl, with 0.03% TMS in 5 mm tubes. Arachidonic acid was purchased from NuChek Prep (Elysian, MN) and all other chemicals were purchased from Sigma Chemical or Aldrich Chemical. The final specific activity of the [14C]anandamide was 0.2-0.5 mCi/mmol. 2.2. Membrane

preparation

Male, Sprague-Dawley rats (250-350 g) were obtained from Harlan Sprague-Dawley (Madison, Wisconsin) and were maintained on a 12 h light/dark schedule before sacrifice. Food and water were available ad libitum. These studies were carried out in accordance with the Declaration of Helsinki and following the Guide for the Care and Use of Laboratory Animals. Forebrain membranes were prepared by homogenization in buffer (50 mM Tris-HCl, 1.0 mM EDTA and 3.0 mM MgCl,, pH 7.4) followed by centrifugation at 11 300 X g for 20 min at 4°C. The pellet was resuspended in buffer and incubated as described below. In studies of the distribution of the hydrolase activity in the brain, the method of Glowinski and Iversen [ 171 was used to prepare the brain regions. 2.3. Preparation

of subcellular fractions

Subcellular fractions of forebrain were prepared using the method of Gray and Whittaker [ 181. The brains were homogenized in ice cold 0.32 M sucrose using a Thomas C glass vessel with a Telfon pestle and the homogenate was centrifuged at 1000 X g for 10 min at 4°C. The supematant was recentrifuged at 13 000 X g for 20 min and the pellet from this fraction (P,) was either used without further

purification or was purified as outlined below. The supernatant from this centrifugation (S,) was recentrifuged at 100000 X g; the pellet from this centrifugation (P,) contains the microsomes, the supernatant is the cytosolic fraction (S). The P2 pellet was resuspended in cold 0.32 M sucrose and layered on top of discontinuous sucrose gradients (0.8 M, 1.2 M and 2.0 M). The gradients were centrifuged at 53 500 X g for 60 min. The myelin fraction was collected at the 0.32-0.8 M interface; the synaptosoma1 fraction was collected from the 0.8-1.2 M interface and the mitochondrial fraction from above the 2.0 M sucrose. Each fraction was diluted with distilled water to 0.32 M sucrose and was centrifuged at 37 000 X g for 20 min. The resultant pellets were resuspended in buffer and assayed for anandamide hydrolysis as described. Nuclei were prepared following the Percoll gradient procedure outlined in [19] through the first discontinuous gradient separation. The nuclei isolated in fraction I (neuronal nuclei) were used. Synaptosomal plasma membranes were prepared using Ficoll density and sucrose density centrifugation as described previously [20]. Succinate dehydrogenase was determined by the method of King [21], NaK ATPase by the method reported in [22] and inosine diphosphatase by the method of Plaut [23]. The protein concentration of each tissue preparation was determined using the dye binding method of Bradford [24] using reagent and protein standard I (bovine y-globulin) obtained from BioRad Laboratories (Richmond, CA). 2.4. Studies of 1 i4Clunandamide

cutubolism

Brain tissue was prepared as described above and was incubated in buffer containing 1 mg/ml fatty acid-free bovine serum albumin (BSA). [ ‘3C]Anandamide was added to the membrane suspension and incubated with shaking. In the studies with inhibitors, the inhibitor was added to the tissue 5 min prior to the addition of [ ‘“Clanandamide and the incubation continued. At the times indicated, 0.5 ml of the tissue suspension was removed and added to 2 ml of chloroform/methanol (1:2) and extracted following established methods [25]. This mixture was vortexed and allowed to stand at room temperature for 30 min; 0.6 ml water and 0.6 ml of chloroform were added with vortexing to induce clear phase separation. After 10 min, the extraction mixture was centrifuged to separate the layers, and the aqueous layer was removed. The organic phase was dryed under a stream of nitrogen and spotted onto channelled silica-gel H TLC plates (20 cm X 20 cm, 250 pm thickness, Whatman LKSD). The plates were developed in hexane/ethyl acetate/methanol (90:60:7.5). In most experiments, the radioactivity was visualized using a radioanalytical imaging system (AMBIS, San Diego, CA) using a scan time of 12 h. In the kinetic experiments, the anandamide and arachidonic acid spots were identified using iodine vapor and scraped. The silica-gel was extracted by vortexing in scintillation cocktail and, after the silica had

C.J. Hillard et al. / Biochimica et Biophysics Acta 1257 (1995) 249-256

settled, the radioactivity in the extracts using scintillation counting.

was determined

2.5. GC-MS identification acid

and arachidonic

of anandamide

branes from each of the brain regions studied (10 pg protein/incubate) were added to the wells containing 800 pM [3H]CP55,940. Incubations were carried out for 1 h at room temperature with shaking and were terminated by filtration. Filters were washed three times with BSA-containing buffer and counted. Nonspecific binding was determined in the presence of 10 PM A9-THC.

Membranes were incubated for 120 min at 30°C with 10 Following extraction and purificaPM [ l4 Clanandamide. tion by TLC as described above, the region of the plate containing radioactive material comigrating with arachidonic acid was scraped and eluted with chloroform/methanol (2: 1). The solvent was removed under a stream of nitrogen. The sample was dissolved in 50 ~1 of acetonitrile and treated with 5 ~1 of pentafluorobenzyl bromide (PFB) and 10 ~1 of N,N-diisopropylethylamine [26]. After incubating for 30 min at room temperature, the solvent was removed under nitrogen, and the sample was extracted twice with water and ethyl acetate. The ethyl acetate fractions were combined, dryed and resuspended in isooctane for analysis. The analysis utilized a Hewlett-Packard Engine Gas Chromatograph-Mass Spectrometer. Separation was carried out using a DB-5 capillary column (J and W Scientific) with helium as the carrier gas. The column temperature was 190°C and increased to 300°C at lO”C/min. Negative ion chemical ionization MS utilized methane as the ionization gas and an ionization Analysis of the synthetic potential of 70 eV. [ I4Clanandamide was performed on the undetivatized compound using the same conditions but in the positive ion chemical ionization mode. 2.6. Determination

of [3HjCP55,940-binding

3. Results Anandamide was hydrolyzed by membranes isolated from rat forebrain in a time dependent manner to a product that comigrates with arachidonic acid on TLC (Fig. 1). No other metabolic products were found and 103 & 4% of the added [ 14C] was accounted for as either unmetabolized anandamide or arachidonic acid (n = 20). The identity of arachidonic acid as the product of anandamide hydrolysis was confirmed by GC-MS (data not shown). The PFB derivative of the metabolite eluted as a single peak on GC that comigrated with the PFB ester of authentic arachidonic acid at a retention time of 12.5 min. The mass spectrum of this peak revealed a single major ion of 303 (m - 1) indicating a molecular weight of 304 for the parent acid. This mass spectrum was identical to that of the PFB ester of authentic arachidonic acid. The cytosolic fraction of rat forebrain failed to metabolize anandamide (Fig. 1 and Table 1). Anandamide was neither hydrolyzed in buffer alone nor by membranes that were boiled for 10 min (Table 1). Anandamide hydrolysis was not dependent upon the presence of calcium and was unaffected by the addition of EGTA. Anandamide hydrolysis was linearly related to temperature between 4°C and 37°C; the percent anandamide hydrolyzed during a 30 min incubation was 1.9, 13.5, 18.0, 26.9 and 36.5 at 4”C, 17°C 25°C 30°C and 37°C respectively. The hydrolysis of anandamide by brain membranes was also pH dependent, exhibiting a pH optimum at 8.5 (Fig. 2). No hydrolysis of

site density

Binding assays were performed using a Multiscreen Filtration System with Durapore 1.2 pm filters (Millipore, Bedford, MA). Incubations (total volume 0.2 ml) were carried out in the incubation buffer described above containing 1 mg/ml bovine serum albumin (BSA). Mem-

15 MEMBRANES

251

30

60

120

CYTOSOL

Fig. 1. [‘4C]Anandamide (AEA) is catabolized by brain membranes but not cytosol to a product that comigrates with arachidonic acid. Representative TLC autoradiogram of catabolism experiment. Protein concentration was 50 pg/ml for the membranes and 100 pg/ml for cytosol. Tissue was incubated in 5 ml with 3 PM [14C]anandamide at 37°C. At the times indicated, 0.5 ml of tissue suspension was removed to tubes containing chloroform/methanol (1:2) and the extractions were carried out as described in Section 2. This chromatograph is representative of 3 experiments.

C.J. Hillard et al. /Biochimica et Biophysics Acta 1257 (1995) 249-256

252 Table 1 Characterization branes Incubation

100

of

[ 14C]anandamide

hydrolysis

conditions

by rat forebrain

mem-

% Conversion to [ I4C]AA

Buffer alone Membranes (50 pg protein) Membranes + 5.0 mM EGTA Membranes + 5.0 mM CaCI, Boiled membranes Cytosolic fraction (100 /*g protein)

0 27.0 32.4 32.5 0 0.4

. Tissues were prepared from rat forebrain and were incubated with 3 PM [ 14C]anandamide for 30 min at 30°C. After extraction and separation on TLC, the percent conversion was calculated as [‘4C]arachidonate/([‘4C]anandamide + [‘4C]arachidonate) x 100. Each value is the mean of two experiments.

anandamide occurred at the temperatures or pH’s tested in the absence of membranes. Hydrolysis of anandamide occurs to the same extent in forebrain membranes taken from rats at 6 weeks of age and at 4 months of age (data not shown). Anandamide hydrolysis followed first order kinetics, and the rate of hydrolysis was dependent upon the tissue concentration (Fig. 3). The half-lives determined at 30°C were 133 mitt, 60 min and 26 min for incubations with 25, 50 and 100 pg/ml protein, respectively, resulting in K, values of 0.30, 0.33 and 0.39 min-’ mg protein-‘. The hydrolysis of anandamide varied with the substrate concentration and was saturable (Fig. 4). The K, of hydrolysis was 3.4 PM and the V,,, in the rat forebrain membrane preparation was 2.2 nmol/min per mg protein. The abilities of a series of inhibitors of proteases and amidases to block the hydrolysis of anandamide were tested (Table 2). PMSF, DFP and thimerosal were effec50

.

.

ti

.

.

. 0

i

1;0

Fig. 3. Anandamide hydrolysis by brain membranes follows first order kinetics and is tissue dependent, [14C]Anandamide (10 PM) was incubated with brain membranes at the protein concentrations noted in a total volume of 5 ml at 30°C. At the incubation times indicated, 0.5 ml of the tissue suspension was removed and extracted as indicated in Section 2. Anandamide remaining was calculated as the radioactivity of [“Clanandamide at the time indicated divided by the [ 14C]anandamide radioactivity at time zero (immediately after the addition of [ 14C]anandamide to the incubates). The lines were drawn using least squares linear regression. Each point is the mean of duplicate determinations, vertical lines represent the range of the duplicates.

tive blockers of anandamide hydrolysis, all having IC,, values in the low micromolar range when anandamide was present at 10 PM. Aprotinin (Trasylol) inhibited hydrolysis only at high micromolar concentrations. Chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), Ntosyl-L-lysine chloromethyl ketone (TICK) and 4-(2aminoethyl)benzene-sulfonylfluoride (AEBSF) were all ineffective inhibitors of anandamide hydrolysis at low concentrations. The hydrolysis of 3 PM [ 14C]anandamide was inhibited completely by 100 PM arachidonic acid (Fig. 5). The inhibition by arachidonic acid was not concentration so

40

40

30 0.3

5 ii ; z 0"

30

20

0.2 0 0.1

20

10

ac

0.0

/iis

-10

10

12

3

4

0 0

0

2

4

6

Anandamlde 5

6

7

6

9

10

8

10

12

(pY)

I

PB Fig. 2. Anandamide hydrolysis by brain membranes is pH dependent. [‘4C]Anandamide (3 PM) was incubated with brain membranes (50 pg/ml) for 60 min at the pH values indicated. Tris buffer was used to achieve pH values less than 8.5 and bicine was used to achieve higher pH values. Anandamide conversion to arachidonic acid was calculated as the ratio of the radioactivity as arachidonic acid to the total as anandamide plus arachidonic acid. Each value represents the mean of duplicate determinations, the range is shown by the vertical lines.

Fig. 4. Kinetic analysis of anandamide hydrolysis by brain membranes. [‘4C]Anandamide at the concentrations indicated was added to brain membranes (50 pg/ml) in a total volume of 2.5 ml and were incubated at 30°C. Aliquots were removed at 0, 5, 10 and 15 min after the addition of [‘4C]anandamide and were extracted as outlined in the Section 2. After development of the TLC plates, anandamide and arachidonic acid spots were identified under iodine vapor, scraped and extracted. The rate of anandamide hydrolysis was determined at each time point, and the values were averaged. Insert: Lineweaver-Burke transformation of data shown. Experiment shown is representative of 2 experiments.

C.J. Hillard et al. /Biochimica Table 2 Effect of inhibitors

on [14C]anandamide

hydrolysis

by rat forebrain

253

et Biophysics Acta 1257 (1995) 249-256

membranes

Inhibitor

Ga

Diisofluorophosphate (DFP) Phenylmethylsuifonyl fluoride (PMSF) Thimerosal Trasylol Chymostatin N-Tosyl-L-phenylalanine chloromethyl ketone N-p-Tosyl-L-lysine chloromethyl ketone 4-(2-aminoethyl)benzenesulfonylfluoride (AEBSF) Captopril E64

6.9 + 3.6 ILM 12.9 + 1.2 /.LM 17.0 + 1.3 PM )9CMlKIU/ml * )lmM )3mM )lmM )lmM )lmM ) 0.3 mM

Forebrain membranes were preincubated with the inhibitors for 5 min. followed by the addition of 3 pM [‘4C] anandamide and incubation for 30 min at 30°C. was determined using at least 5 concentrations. For each inhibitor, the percent inhibition of the conversion of [ 14C]anandamide to [‘4C]arachidonate Each value is the mean of 3 experiments, shown f S.E.M. * KIU refers to kallikrein inhibitory units; equivalent to approx. 300 PM.

dependent; concentrations less than 30 PM had minimal effect. The subcellular distribution of anandamide hydrolase activity in forebrain was determined. The homogenate was prepared and separated into four fractions, P, (crude nuclei), P2, P3 and the supematant obtained after 100 000 X g centrifugation (cytosol). Of these four fractions, the P3 (microsomes) removed fraction has the highest specific activity for anandamide hydrolysis, but all of the membrane fractions have a higher specific activity than homogenate (Table 3). The cytosolic fraction is essentially devoid of activity. The overall contribution of each of the four fractions can be estimated from the activity/g tissue; the P, fraction had the highest overall activity, accounting for nearly half of the total. The anandamide hydrolase

Table 3 Distribution

of anandamide

hydrolase

Fraction

Starting material (homogenate) PI p2 P3

Cytosol (S) Total (P, + P2 + P3 + S) Starting material (P2 ) Myelin (A) Synaptosomes (B) Mitochondria (C) Total (A + B + C) Purified nuclei SPM

activity in subfractions

activity was not enriched in purified nuclei compared to the P, preparation (Table 3). The P2 fraction was further fractionated into myelin, synaptosome and mitochondria enriched fractions; 65% of the P2 protein and 68% of the hydrolase activity were recovered following fractionation. The myelin fraction has the greatest hydrolase specific activity; however, the synaptosomes contain more than 60% of the total activity in the P2. The mitochondria and synaptic plasma membranes have fairly low activity. The relative enrichment of the subcellular fractions in succinate dehydrogenase (a mitochondrial enzyme), Na+,K+ ATPase, (plasma membrane enzyme), and inosine diphosphatase (an enzyme of the smooth endoplasmic reticulum) were determined (Table 4). Inosine diphosphatase was the only marker enzyme activity that correlated with the distri-

of rat forebrain

Protein (mg/g tissue)

Specific activity (activity/mg protein)

Activity (activity/ g tissue)

117(11) 35.7 (3) 24.6 (5) 10.70) 50.0 (4) 121 24.6 (5) 1.7 (0.3) 9.9 (1) 4.5 (1) 16.1 27.5 (4) 4.0 (.2)

12.0 (2) 17.9 (2) 18.5 (0.4) 21.9 (7) 2.0 (0.6)

1404 639 455 234 100 1428 455 46.6 192 69 308 451 56

18.5 27.4 19.4 15.3

(0.4) (6) (1) (1)

16.4 (2) 14.0 (1)

The fractions listed were prepared and assayed for anandamide hydrolysis. The tissue (50 pg protein/ml) was incubated with [ 14C]anandamide (3-10 PM) for 60 min at 30°C and the fraction [ “C]aracbidonic acid in a 0.5 ml aliquot was determined by TLC. ”Activity ” refers to the fractional conversion of [ I4Clanandamide to [ “C]arachidonic acid. Each point is the mean of 3-5 separate tissue preparations: S.E.M. are in parentheses.

Distribution (%o) 44.8 31.9 16.4 7.0 100 15.1 62.3 22.4 100

of [‘4C]anandamide

converted

to

254

C.J. Hillard et al. / Biochimica et Biophysics Acta 1257 (19951 249-256 Table 5 Distribution of anandamide brain regions

0

1

3

Arachidonic

10 acid

30

Brain region

%Conversion [t4C]~~

Cerebral cortex Cerebellum Brain stem Hypothalamus Hippocampus Striatum White matter

69.5 70.1 41.4 61.9 74.8 43.7 47.5

100

(pW)

Fig. 5. End product inhibition of [14C]anandamide hydrolysis by brain membranes. Brain membranes (50 pg/ml) were preincubated for 5 min with arachidonic acid in BSA containing buffer at the concentrations indicated. [‘4C]Anandamide (3 FM) was added and the incubation was continued for 60 min at 30°C. Anandamide conversion to arachidoinic acid was calculated as the ratio of the radioactivity as arachidonic acid to the total anandamide and arachidonic acid radioactivity. Control is the conversion of [‘4C]anandamide to [ “C]arachidonic acid in the absence of added arachidonic acid. Each value represents the mean of triplicate determinations, the S.E.M. is shown by the vertical lines.

hydrolysis to

and [‘H]CP55,940

binding

[ ‘H]CP55,940 binding (pmol/mg protein) 0.33 0.60 0.15 0.32 0.64 0.30 0.18

The brain regions were dissected and membranes were prepared as outlined in Section 2. Hydrolysis activity was determined by measurement of the conversion of [ ‘JC]anandamide (3 PM, final concentration) to [“Clarachidonic acid during a 60 min incubation at 30°C. The cannabinoid receptor density was determined in the same membrane fractions using [‘H]CP55,940 as the radioligand. Both assays were carried out using a final protein concentration of 50 pg/ml.

regions was significantly correlated with the binding density measured using [3H]CP55,940 (r* = 0.703). bution of anandamide hydrolase activity among the subcellular fractions. The distribution of the hydrolase activity among several brain regions was determined and compared to the density of cannabinoid receptors in the same preparation (Table 5). The amount of anandamide hydrolysis varied among brain regions; the highest activity was found in the cerebellum, hippocampus and the cerebral cortex while the lowest activity was found in the brain stem, striatum and white matter. The degree of hydrolysis of anandamide in these

Table 4 Relative specific forebrain

activities

of marker

enzymes

in subfractions

Fraction

Succinate dehydrogenase

Na+, K+ ATPase

Inosine diphosphatase

P, Pz P, (S) Cytosol Myelin (A) Synaptosomes (B) Mitochondria (C) Purified nuclei SPM r2 P

0.8 0.9 0.4 0.6 0.6 0.8 1.3 0.7 0.3 0.002 >O.l

0.8 1.0 0.5 0.05 2.7 1.4 0.8 I .2 4.4 0.035 >O.l

I .6

of rat

1.3 2.1 0.3 2.2 1.0 0.8 1.1 1.3 0.640 (0.01

The fractions were prepared and assayed for the activities indicated using methods as described. Activities reported are relative to the activity in the homogenate and have been corrected for protein. Each value is the mean of 2 separate tissue preparations. The correlation between the specific activity of each marker enzyme and the hydrolase activity in that tissue were determined using linear regression analysis and the P value was determined following conversion of the r’ to t value.

in rat

site

4. Discussion These studies indicate that membranes from rat forebrain hydrolyze anandamide to ethanolamine and arachidonic acid. Since arachidonic acid does not bind to the cannabinoid receptor or have cannabinoid-like activity, the process represents an inactivation of anandamide. The hydrolytic activity is abolished by boiling, follows Michaelis-Menton kinetics and is consistent with an enzymatic metabolic process. The enzyme is active over a wide pH range with an alkaline optimum and does not require calcium for activity. As was shown in [13], the hydrolase is inhibited by the nonselective esterase and amidase inhibitor PMSF. The IC,, for PMSF is in the micromolar range. In addition, we have found that the serine enzyme inhibitor DFP is an effective inhibitor of anandamide hydrolysis but that the water soluble serine esterase inhibitor AEBSF is without effect at micromolar concentrations. Furthermore, anandamide hydrolysis is inhibited by the sulfhydry-reactive agent thimerosal but not by E64, which is selective for cysteine residues that are part of the active site of the enzyme-removed [27]. This inhibitor profile suggests that serine is part of the active site and that disulfide bonding elsewhere in the protein is important for enzymatic activity. Anandamide hydrolysis does not occur via cathepsin G, chymotrypsin or trypsin based on the lack of inhibition by chymostatin, TPCK and TLCK, respectively [28-301. Several amidohydrolases have been identified and described that share some, but not all, of the characteristics of the hydrolytic activity described in the present study.

C.J. Hillard et al. /Biochimica

et Biophysics Acta 1257 (1995) 249-256

Ceramides, which are N-acyl derivatives of sphingosine, are metabolised by hydrolysis of an amide linkage between a fatty acid and sphingosine [3 11. Ceramidase is membrane associated and not dependent upon divalent cations [32]. Most ceramidase isoforms are active only at acidic pH; however, an alkaline-preferring ceramidase has been identified in human cerebellum [32]. There is no evidence that ceramidases with alkaline pH optima occur in rat brain [33]. Furthermore, N-oleoylethanolamide has been shown to inhibit the activity of some ceramidase isoforms [33] suggesting that N-acylethanolamines act as inhibitors rather than substrates for the ceramidases. Lipoyl-X hydrolase (LLH) is a membrane associated enzyme found in brain [34] and in serum [35] that hydrolyzes both amides and esters of lipoic acid. Like anandamide hydrolysis, LLH is inhibited by DFP, but it is not affected by PMSF and is also blocked by both TPCK and TLCK [35]. These inhibitor data argue against a major role for this enzyme in the hydrolysis of anandamide. The enzyme that hydrolyzes anandamide differs from deamidase (lysosomal protective protein), which hydrolyses peptides that contain hydrophobic amino acids in that deamidase is a cytosolic enzyme 1361. The data presented suggest that the enzyme that hydrolyzes anandamide is similar, if not identical, to Nacylethanolamide amidohydrolase, an enzyme identified in rat liver and dog brain [ 15,161. N-Acylethanolamide amidohydrolase hydrolyzes N-acylethanolamides to fatty acid and ethanolamine, is inhibited by fatty acids and is not dependent upon calcium. Furthermore, the liver amidohydrolase is found only in membrane fractions, is sensitive to sulfhydryl reactive agents and is active at high pH. The liver amidohydrolase is found in microsomal and mitochondrial membranes [15,37]. Our studies of the subcellular distribution of anandamide hydrolase in brain demonstrates that the specific activity of the microsomal fraction is high while relatively little activity is seen in the mitochondria. Bomheim and coworkers [38] have reported that anandamide is metabolized by cytochrome P-450 in liver microsomes to several oxygenated products when its catabolism by the amidase is inhibited by PMSF. While brain contains cytochrome P-450 activity [39], we failed to observe the formation of oxygenated metabolites in our studies. This discrepancy may be due to the absence in brain of the cytochrome P-450 isozyme that metabolizes anandamide or a difference in the in vitro conditions used. Although these studies have been carried out in vitro, the hydrolysis of anandamide could be involved in the inactivation of anandamide in vivo. Anandamide has a short duration of action in vivo, less that 30 min in most physiological and behavioral measures [10,40] which may be due to hydrolysis of anandamide in both liver and brain. In the context of the hypothesis that anandamide functions as a neuroactive agent in the brain, it is tempting to speculate that the catabolic process described here is im-

255

portant for the termination of action of anandamide. The K, of the hydrolase for anandamide (3 PM) is an order of magnitude above the Ki of anandamide for the cannabinoid receptor, which is 140 nM [41]. In addition, for the seven regions studied, the distribution of anandamide hydrolase activity is not homogeneous and correlates well with the distribution of cannabinoid receptor binding sites. It is interesting, however, that synaptic plasma membranes are not enriched in hydrolase activity while the microsomes and myelin are very active. This distribution of activity is not typical of other neurotransmitter termination systems; however, anandamide is rapidly taken up by neurons and glia which supports an intracellular site of inactivation [ 13,421. In summary, these studies indicate that anandamide is catabolized in rat forebrain to arachidonic acid and ethanolamine with a K, of 3 PM. The inactivation is inhibited by PMSF, DFP and thimerosal. This process may account for the short duration of action of anandamide.

Acknowledgements We thank Drs. Ervin G. Erdos, Randal A. Skidgel and H.H.O. Schmid for helpful discussions and Jody J. Pounds and Pamela J. Dumke for their technical assistance with these studies. The authors also thank Dr. Kasem Nithipatikom for his help with the GC-MS analysis.

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