Vol. 40, No. 5, November 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

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1 Vol. 40, No. 5, November 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages ENZYMATIC SYNTHESIS OF OLEA~DE (cls-9,10-octadecenoa~tide), AN ENDOGENOUS SLk~P-INDUCING LIPID, BY RAT BRAINMICROSOMES Takaynki Suglura I*, Sachlko Kondo 1, Tomoko Kodaka I, Takashi Tonegawa I, Shinji Nakane 1, Atsushi Yamashita 1, Yoshio Ishlma 2 and Keizo Waku 1 1Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa , Japan, and ~Ishlma Institute for Neurosclences, Kunitachi, Tokyo 186, Japan RcceivedJuly13,1996 Summary: The enzymatic synthesis of a novel sleep-lnduclng lipid, oleamide (cis-9,10-octadecenoamide), was studied using rat brain subcellular fractions as enzyme sources. We found that oleamide was formed from olelc acid and ammonia on incubation with a brain homogenate. The enzyme activity catalyzing the formation of oleamlde from olelc acid and ammonia was highest in the microsomal fraction among the subcellular fractions. Boiled microsomes did not exhibit appreciable enzyme activity, These results strongly suggest that oleamlde can be synthesized enzymatically in the brains of stimulated animals. Key words: oleamide (cis-9,10-octadecenoamide); sleep-induclng lipid; fatty acid amlde; enzymatic synthesis; brain mlcrosomes. There is increasing evidence that several sledp-inducing substances play important roles in physiological sleep in mammals (i), though their mechanisms of action have not been fully elucidated yet. Recently, Lerner and co-workers (2) reported that pmol of an unusual lipid was obtained from i00 # I of cerebrosplnal fluid from a sleep-deprlved cat, but not from that of normal animals. The chemical structure of this substance was then proved to be that of oleamlde (cls-9,10-octadecenoamlde) by TLC, FAB-MS, GC-MS, MS-MS and NMR analy- ses in 1995 (3). They found that oleamlde induces sleep in rats upon both intraventrlcular and intraperltoneal injection (3). Importantly, the sleep induced by oleamide in rats resembles the physiological sleep observed in the same animals, on the basis of several criteria. The above observations strongly suggest that oleamide, a novel type of bloactlve lipid, is one of the Important physiological modulators of sleep, at least in several mammalian species. Despite its possible physiological importance, however, little is known so far about the biosynthesis of oleamide in mammals. It will be very important to investigate thls precisely to better understand the physlologlcal meaning of *To whom correspondence should be addressed. FAX: /96/ /0 Copyright O 1996 by Academic Press Australia. All rights of reproduction in any form reserved.

2 oleamlde In mammalian tissues, especially in nervous tissues. In this study, we Investigated the biosynthesis of oleamlde in detail uslng rat brain subcellular fractions as enzyme sources. MATERIALS AND METHODS Chemicals and animals: [14C]Olelc acid {50 mai/mmo~}, [14C]palmitic acid (57 mcl/mmol}, [14C]linoleic acid (53 mcl/mmol), and [14C]arachldonlc acid (55 mci/mmo!) were purchased from Du Pont-New England Nuclear {Boston, MA, U.S.A.). Unlabeled fatty acids, essentially fatty acid-free bovlne serum albumin (BSA), L-glutamlc acid and CoA were obtained from Sigma (St. Louis, MO, U.S.A.}. ATP, dithlothreltol (DTT}, oleamlde, L-glutamlne and an aqueous 25 ~ ammonia solution were from Wako Pure Chem. Ind. {Osaka, Japan}. The ph of the aqueous 100 mm ammonia solution was usually adjusted to 8.0 wlth conc. HCl before use. Wistar rats {male, g body weight} were obtained from Sankyo Lab. Service {Tokyo, Japan}. Subcellular fractions: Rat brains were homogenized in 10 mm Hepes-NaOH buffered 0.25 M sucrose {ph 7.4) using a glass-teflon homogenizer. The mlcrosomal ( x g pellet fraction}, mitochondrla-rich {7000 x g pellet fraction}, and cytosolic { x g supernatant fraction) fractions were prepared as described elsewhere (4). The synaptosomal fraction was prepared by the sucrosedensity gradient centrlfugation method (5). The protein content was estimated according to the method of Lowry et al. (6). Enzyme assays: The standard assay conditions were as follows. The$~t brain mlcrosomal fraction (250 ~g protein} was incubated with 200 zm [x~c]oleic acid (5 mcl/mmol} and 10 mm ammonia in 250 ~ 1 of 20 mm Hepes-NaOH buffer (ph 8.0) containing 2 mm DTT and 2 mm EGTA at 37 C for 30 min. In some cases, the incubation was carried out on a double scale. In the experiments In which a rat brain homogenate was employed as the enzyme source, the homogenate (4 mg protein} was incubated with 200 ~M [14C]olelc acid and SO mm ammonia, 50 mm glutamine or 50 mm glutamlc acid plus 5 mm NAD In 500 # 1 of 20 mm Hepes-NaOH buffer containing 2 mm DTT and 2 mm EGTA. The incubation was stopped by the addition of chloroform:methanol (1:2, v/v), and total lipids were extracted by the method of Bligh and Dyer (7). Individual liplds were separated by twodimensional TLC, being developed first wlth petroleum ether:dlethyl ether:acetlc acld {20:80:1, v/v} and then wlth chloroform:methanol:25 ~ ammonia {80:20:2, v/v). The Rf value of oleamide on the first development was 0.27, and that on the second development was The area co-mlgrating with standard oleamlde was scraped off the TLC plate into a counting vial. The radioactivity was determined wlth a liquid scintlllatlon counter (Aloka LSC 3000, Japan). RESULTS AND DISCUSSION First, we investigated whether or not [14C]oleamide is formed from free [14C]olelc acid and ammonia on incubation with a rat brain homogenate. As shown in Flg. I, we found that [14C]oleamlde was formed when free [14C]oleic acld was incubated wlth ammonia. We also found that a small amount of [14C]oleamlde was formed when the homogenate was incubated with [14C]olelc acld and glutamlne. The boiled homogenate lacked appreciable activity in both cases, indicating that the reaction Is an enzyme-catalyzed one. Then, we examined the distribution of the enzyme activity forming [14C]oleamlde from free [14C]olelc acid and ammonia among the subcellular fractions. As shown in Flg. 2, the mlcrosomal fraction exhibited the highest 932

3 (A) ~ (S) (c) (D) (E) (F) (G) (H) Activity(pmol/30 min/mg protein) Fig. 1. Formation of [14C]oleamide from [14C]oleic acid by a rat brain homogenate. The rat brain bomogenate (4 mg protein) or bolled (lo0 C for 5 mln) homogenate {4 mg protein) was incubated with 200 #M [14C]oleic acid (5 mai/mmol} and 50 mm ammonia ((A) and (B)), 50 mm glutamlne {(C) and {D)), 50 mm glutamlc acid ((E) and (F)), or 50 mm glutamic acid plus 5 mm NAD ((G) and (H)) in 500 # 1 of 20 mm Hepes-NaOH buffer (ph 8.0) containing 2 mm DTT and 2 mm EGTA at 37 C for 30 min. ((A), {C), {E), (G)) homogenate; {(B), (D), (F), (H)) boiled bomogenate. The data are the means + SD of four determinations. I (A) (B) (C) (D) -q q (E) (F) I (G) (H) --q o t'o 5o Activity (pmol / 30 min / mg protein) Fie. 2. Distribution of the enzyme activity catalyzing the formation of i~ 14 [ C]oleamide from [ C]oleic acid and ammonia.1~at brain subcellular fractions (500 # g protein) were incubated with 200 #M [±~C]oleic acid (5 mcl/mmol) and i0 mm ammonia in 500 pl of 20 mm Bepes-NaOH buffer (ph 8.0) containing 2 mm DTT and 2mM EGTA at 37 C for 30 min. ((A) and (B)) microsomal fraction; ((C) and (D)) mitochondrla-rich fraction; ((E) and (F)) synaptosomal fraction; ((G) and (H)) cytosollc fraction. ((A), (C), (E), (G)) subcellular fractions; ((B), (D), (F), (H)) boiled subcellular fractions. The data are the means + SD of four determinations. 933

4 enzyme activity among the fractions. Some enzyme activity was detected In the mltochondrlal and synaptosomal fractions as well, whereas negllglble activity was observed in the cytosollc fraction. Fig. 3 shows the kinetics of the enzymatic formation of [14C]oleamlde from free [14C]olelc acld and ammonia by the microsomal fraction. The formation of [14C]oleamlde increased wlth time (Flg.3 (A)), with increasing amounts of mlcrosomal proteln (Fig. 3 (B)), and wlth increasing concentrations of the substrates (Fig. 3 (C) and (D)). The reaction followed the Mlchaells-Menten equation. The apparent Km value for olelc acid (estimated in the presence of 75 mm ammonia) was 208 #M, and that for ammonia was 65 mm (estimated In the presence of 200 #M olelc acid). The optimal ph appeared to be above 9.0 (Fig. 3 (E)). The boiled mlcrosomal fraction dld not exhibit apparent enzyme activity except at phs above 9.0. We further confirmed that the product Is oleamlde using different solvent systems: chloroform:methanol:water (85:15:2, v/v)(rf=0.82), ethyl acetate:hexane:acetlc acld (55:45:1, v/v)(rf=0.37), and petroleum ether:dlethyl ether:acetone:acetic acid (30:40:20:1, v/v)(rf=0.76). The fatty acld specificity of the enzymatic formation of [14C]fatty acid amides is shown In Fig. 4. Several types of fatty acids with different chain lengths and different degrees of unsaturatlon were shown to serve as acyl donors in the formation of fatty acld amides. Finally, we examined the effects of the addition of cofactors such as CoA and ATP-Mg 2+ on the enzymatic formation of oleamide. The addition of ATP-Mg 2 and CoA rather reduced the overall enzyme reaction (data not shown), suggesting that free olelc acld Itself, but not oleoyl-coa, acts as an acceptor of ammonia in thls enzyme reaction. In the 1960s, Udenfrlend and co-workers (8,9) reported that fatty acld amides of ethanolamlne such as N-palmltoylethanolamlne were produced enzymatlcally from free fatty acids and ethanolamlne by rat liver mlcrosomes. The enzymatic formation of an endogenous cannablnold receptor ligand, N- arachldonoylethanolamine (anandamlde), from free arachldonlc acld and ethanolamlne has also been described by several investigators (10-14). Bachur and Udenfrlend {9) showed that not only ethanolamlne but also various types of amines, such as histamine, tyramlne, phenethylamine and tryptamine, acted as acceptors for fatty acld substrates. The enzymatic formation of N-acyl phenylalanine has also been described by Fukul and Axelrod (15). However, llttle is known so far about the enzymatic synthesis of ammonia derivatives of fatty acld amides such as oleamlde In mammallan tissues, although Bachur and 934

5 , o < -- _ c 150 ~ ~. ->' ~ so "~ < 0]~o o ~.. o~'~ ~o --' ~o ~o ~'? 0 ~ ~.o,.o "~ Time (min).-. Protein (mg) <C NH3 (mm) c m L,h 14o. ~= E,~ E 80, c 50. c E t=60 o o c,) ~'),., 2o. ~_ ~ 40 v,o. v~,o 0 r-- m 0 -- o..... "~ o o O ~ 5, < < 18:1 (FM) ph Fig. 3. Kinetics of the formation of [14C]oleamide from [14C]oleic acid and ammonia by brain microsomes. The standard conditions were as follows. Rat brain mlcrosomes (250 ~ g protein) were incubated wlth 200 ~M [14C]oleic acid (5 mci/mmol) and 10 ~4 ammonia in 250 ~ 1 of 20 ~Hepes-HaOH buffer (ph 8.0) containing 2 mm DTT and 2 mm EGTA at 37 C for 30 min. (A) Time course; (B) effect of the content of microsomal protein; (C) effect of the amount of ammonia; (D) effect of the amount of oleic acid; (E) effect of ph. Closed circles, microsomes; open circles, boiled microsomes. The data are the means of four to five determinations. 0 I" 0.< -4 m z z I"

6 (A) ~ (B) (C) (D) (E) (F) (G) i (H) p o 1o to 80 Activity (pmol/3omin/mg protein) Fig. 4. Formation of [14C]fatty acld amides from [14C]fatty acids and ammonia by brainm~crosomes. Rat brain mlcrosomes (250 # g protein) were incubated with 200 #M [14C]fatty acids (5 mcl/mmol) and i0 mm ammonia in 250 ~ 1 of 20 mm Hepes-NaOH buffer (ph 8.0) containing 2 mm DTT and 2 m~ EGTA at 37 o C for 30 min. (~) and (B)) [14C]palmitlc acid; (~) and (D)) [1~C]olelc acid; ((E)and (F)) [±~C]llnoleic acid; ((G) and (H)) [±~C]arachidonic acid. ((A), (C), (E), (G)) mlcrosomes; ((B), (D), (F), (H)) boiled microsomes. The data are the means + SD of four determinations. I Udenfrlend (9) observed that i00 mm ammonia reduced the formation of N- palmltoylethanolamlne from free palmltlc acid (I mm) and ethanolamine (12 mm) by rat liver mlcrosomes and the conversion of olelc acld to oleamlde in Bacillus me~aterlum was described recently (16). The results of the present investigation clearly indicate that oleamlde can be formed enzymatlcally from oleic acid and ammonia, the enzyme activity itself being rather low even In the presence of a relatively hlgh concentration of ammonia (50 mm)(20 pmol/30 mln/mg protein bomogenate; 200 pmol/30 mln/mg protein mlcrosomes). The tissue level of ammonia in the brain is usually low (0.36 #mol/g tissue (17); 0.28 mg per cent (18)). However, thls does not imply that the formation of substantial amounts of ammonia does not occur In brain tissues; the turnover of ammonia Is very rapid in such tissue. Richter and Dawson (18) reported that the level of ammonia Is increased in the brains of stimulated animals such as electric shock rats, plcrotoxin-administered rats, decapitated rats and rats exposed to anoxlc conditions. Explosive generation of ammonia may occur in these rat brains. Ammonia can be formed from several types of amino acids, such as glutamlc acid and glutamlne, in brain. The ammonia released is usually taken up into other molecules very rapidly, the level of ammonia itself thereby being kept relatively low. Contrary to in the case of 936

7 stimulated animals, the level of ammonia was very low in rats anesthetized with Nembutal (17). Thus, it seems possible that a portion of the ammonia generated in the brains of animals, receiving various stimuli, is incorporated into fatty acids to yield fatty acld amides. It Is noteworthy that oleamlde was detected in sleep-deprlved animals, but not in resting animals (18). Furthermore, In addition to the change In the level of ammonia, it has been shown that the levels of free fatty acids are also dramatically Increased In Ischemlc and electric shock rat brains (19). A small amount of oleamide was formed when a brain homogenate was incubated wlth glutamlne (Fig. i). The mechanism underlying the formation of oleamlde from oleic acld and glutamlne is not yet fully understood. There are two possibilities. One is the formation of oleamide from oleic acid and ammonia released from glutamlne through the action of glutamlnase. The other possibility is the formation of oleamide through the direct transfer of an amlde group. The question of how oleamlde is formed from oleic acid and glutamlne remains to be answered in the future. Whatever the exact mechanism, it is noticeable that brain tissues are enriched In glutamine ( #mol/g tissue for rat brain) besides glutamlc acld and aspartic acid (20). Recently, DI Marzo and co-workers (21) suggested that oleamlde is hydrolyzed by anandamide amidohydrolase. Concerning this enzyme, Ueda et al. (13) found that the same enzyme Protein is responsible for the degradation and synthesis of anandamlde, using a partially purified enzyme. It seems possible, therefore, that such an amldohydrolase is also involved in the synthesis of oleamlde, a novel sleep-lnduclng lipid, from olelc acld and ammonia. Further detailed studies are necessary for a full understanding of the mechanism underlying the biosynthesis of oleamide in nervous tissues. REFERENCES i. Inoue, S. (1989)Biology of Sleep Substances, CRC Press, Boca Raton. 2. Lerner, R.A., Siuzdak, G., Prospero-Garcla, 0., Henrlksen, S.J., Boger, D.L. and Cravatt, B.F. {1994) Proc. Natl. Acad. Scl. USA 91, Cravatt, B.F., Prospero-Garcla, 0., Sluzdak, G., Gllula, N.B., Henriksen, S.J., Boger, D.L. and Lerner, R.A. {1995) Science 268, Sugiura, T., Masuzawa, Y. and Waku, K. (1988) J. Biol. Chem. 263, Sugiura, T., Kondo, S., Sukagawa, A., Nakane, S., Shinoda, A., Itoh, K., Yamashlta, A. and Waku, K. (1995) Biochem. Biophys. Res. Commun. 215, Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, Bllgb, E.G. and Dyer, W.J. (1959) Can. J. Blochem. Physiol. 37, Colodzln, M., Bachur, N~R., Welssbach, H. and Udenfriend, S. (1963) Blochem. Blopbys. Res. Commun. 10,

8 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL 9. Bachur, N.R. and Udenfriend, S. (1966) J. Biol. Chem. 241, Deutsch, D.G. and Chin, S.A. (1993) Blochem. Pharmacol. 46, Kruszka, E.K. and Gross, R.W. (1994) J. Biol. Chem. 269, Devane, W.A. and Axelrod, J. (1994) Proc. Natl. Acad. Sci. USA 91, Ueda, N., Kurahashl, Y., Yamamoto, S. and Tokunaga, T. (1995) J. Biol. Chem. 270, Sugiura, T., Kondo, S., Sukagawa, A., Tonegawa, T., Nakane, S., Yamashita, A., Ishlma, Y. and Waku, K. (1996) Eur. J. Blochem. in press 15. Fukul, T. and Axelrod, B. (1961) J. Biol. Chem. 236, Kaneshiro, T., Vesonder, R.F., Peterson, R.E., Welsleder, D. and Bagby, M.O. (1994) J. Am. Oil Chem. Soe. 71, Tsukada, Y., Takagakl, G., Sugimoto, S. and Hirano, S. (1958) J. Neurochem. 2, Richter, D. and Dawson, R.M.C. (1948) J. Biol. Chem. 176, Bazan, N.G. (1970) Biochlm. Biophys. Acta 218, Tallan, H.H. (1962) In: Amino Acid Pools (Holden, J.T., ed), pp , Elsevier, Amsterdam. 21. Maurelli, S., Blsogno, T., De Petrocellis, L., Di Luccia, A., Marino, G. and Di Marzo, V. (1995) FEBS Lett