We studied the localization of N-acyl phosphatidylethanolamine (NAPE), a putative cannabinoid precursor, in primary cultures of striatal and cortical neurons from the rat brain. We probed intact neurons with various exogenous phospholipases, including S. chromofuscus phospholipase D (PLD). S. chromofuscus PLD does not penetrate into neurons (as demonstrated by a lack of internalization of 125I-labeled PLD), and does not cause gross damage to the neuronal membrane (as demonstrated by a lack of effect of PLD on [3H]gamma-aminobutyric acid release). When neurons, labeled to isotopic equilibrium with [3H]ethanolamine, were incubated for 10 min with S. chromofuscus PLD, approximately 50% of neuronal NAPE was hydrolysed. This hydrolysis was accompanied by the release of a family of N-acyl ethanolamines (NAE) (assessed by high performance liquid chromatography), which included the cannabinoid receptor agonist, anandamide. Exogenous phospholipase A2 (PLA2) (Apis mellifera) and PLC (B. cereus) mobilized [3H]arachidonate and [3H]diacylglycerol, respectively, but had no effect on NAE formation under these conditions. These experiments indicate that approximately 50% of neuronal NAPE is localized in a compartment that is easily accessible to extracellular PLD, possibly the plasmalemma, where it would also be easily hydrolyzed upon stimulation to produce NAE

Cadas, H

Piomelli, D

Schinelli, S

H. CADAS

D. PIOMELLI

SCHINELLI, SERGIO

Archivio Istituzionale della Ricerca - Università degli Studi di Pavia

Membrane localization of N-acylphosphatidylethanolamine in central neurons: studies with exogenous phospholipases.

Cadas, Hugues

Schinelli, Sergio

Piomelli, Daniele

eScholarship - University of California

UC IrvineUC Irvine Previously Published WorksTitleMembrane localization of N-acylphosphatidylethanolamine in central neurons: Studies with exogenous phospholipasesPermalinkhttps://escholarship.org/uc/item/0nr4p6s2JournalJournal of Lipid Mediators and Cell Signalling, 14(1-3)ISSN0929-7855AuthorsCadas, HuguesSchinelli, SergioPiomelli, DanielePublication Date1996-09-01DOI10.1016/0929-7855(96)00510-xCopyright InformationThis work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaE L S E V I E R  J. Lipid Mediators Cell Signalling 14 (1996) 63-70 JotntNAt Or LIPID MEDIATORS A N D  CILL SIGNALI.ING Membrane localization of N-acylphosphatidylethanolamine in central neurons: studies with exogenous phospholipases Hugues Cadas, Sergio Schinelli l, Daniele Piomelli* The Neurosciences Institute, 10640 J.J. Hopkins Drive, San Diego, CA 92121, USA Abstract We studied the localization of N-acyl phosphatidylethanolamine (NAPE), a putative cannabinoid precursor, in primary cultures of striatal and cortical neurons from the rat brain. We probed intact neurons with various exogenous phospholipases, including S. chromofuscus phospholipase D (PLD). S. chromofuscus PLD does not penetrate into neurons (as demonstrated by a lack of internalization of 125I-labeled PLD), and does not cause gross damage to the neuronal membrane (as demonstrated by a lack of effect of PLD on [3H]7-aminobutyric acid release). When neurons, labeled to isotopic equilibrium with [3H]ethanolamine, were incubated for 10 min with S. chromofuscus PLD, approximately 50% of neuronal NAPE was hydrolysed. This hydrolysis was accompanied by the release of a family of N-acyl ethanolamines (NAE) (assessed by high performance liquid chromatogra- phy), which included the cannabinoid receptor agonist, anandamide. Exogenous phospholi- pase A 2 (PLA2) (Apis mellifera) and PLC (B. cereus) mobilized [3H]arachidonate and [3H]diacylglycerol, respectively, but had no effect on NAE formation under these conditions. These experiments indicate that ~ 50% of neuronal NAPE is localized in a compartment that is easily accessible to extracellular PLD, possibly the plasmalemma, where it would also be easily hydrolyzed upon stimulation to produce NAE. Keywords: Cannabinoid; N-acylethanolamine; Anandamide; Phospholipase D; Neuromodu- lation * Corresponding author. Tel.: + 1 619 6262170; fax: + 1 619 6262199; e-mail: piomelli@nsi.edu l Current address: Department of Pharmacology, School of Pharmacy, University of Pavia, Italy. 0929-7855/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved Pll S0929-7855(96)00510-X 64 H. Cadas et al. / J. Lipid Mediators Cell Signalling 14 (1996) 63 70 1. Introduction Since the pioneering studies of Schmid and coworkers, it has been recognized that mammalian tissues have the ability to produce small quantities of saturated and unsaturated fatty acid ethanolamides (N-acylethanolamines, NAEs) (reviewed in Schmid et al., 1990). The biological roles of these lipids have remained elusive, however, until the isolation of N-arachidonoylethanolamine (anandamide), and the discovery that this naturally-occurring NAE binds to and activates CBl-type cannabinoid receptors in the brain (Devane et al., 1993; reviewed in Mechoulam et al., 1994). That anandamide may be the long-sought endogenous cannabinoid substance is indicated not only by its remarkable pharmacological properties, largely overlapping with those of plant-derived or synthetic cannabinoid receptor agonists, but also by a growing body of biochemical evidence. Anandamide is produced in and released from central neurons, but not from astrocytes, in a calcium ion-dependent manner (Di Marzo et al., 1994). Also, anandamide is rapidly and selectively taken up into neurons and astrocytes (Di Marzo et al., 1994), and hydrolyzed to ethanolamine and arachidonate by a selective anandamide amidohydrolase activity (Desarnaud et al., 1995). These properties (neuronal synthesis, calcium-dependent release, local inacti- vation) are characteristic of brain signaling molecules, supporting the hypothesis that anandamide may serve such a function in the mammalian central nervous system. In an important sequel to these findings, it has now been suggested that the saturated congener of anandamide, N-palmitoylethanolamine, may exert a parallel action on non-neuronal cells, by binding selectively to CB2-type cannabinoid receptors, which have been identified thus far in mastocytes and other immune cells (Facci et al., 1995; Munro et al., 1993). While the case for a signaling role of anandamide and of related NAEs is gaining support, the molecular mechanisms underlying the generation of these lipids in tissues remain the object of debate. Two mechanisms have been proposed: the energy-inde- pendent condensation of ethanolamine and arachidonate, catalysed by anandamide synthetase activity (Deutsch and Chin, 1993; Devane and Axelrod, 1994; Kruszka and Gross, 1994); and the phospholipase D (PLD)-mediated cleavage of a membrane precursor phospholipid, N-acyl phosphatidylethanolamine (NAPE) (Di Marzo et al., 1994; Cadas et al., 1996). Although these two mechanisms need not be mutually exclusive (multiple paths of biosynthesis exist for many biologically active molecules, including several lipids) it would certainly be desirable to define their respective physiological roles in the formation of cannabimimetic NAEs. In primary cultures of rat brain neurons, NAPE may serve as the precursor for anandamide, N-palmitoylethanolamine and other NAEs. Evidence supporting this possibility includes the existence of an enzymatic activity that catalyses the formation of NAEs from exogenous NAPE, as well as the occurrence of endogenous NAPE in unstimulated neurons (Di Marzo et al., 1994). To serve as a precursor for extracellular messengers such as anandamide and N-palmitoylethanolamine, NAPE is expected to be located in a membrane compartment from which newly-formed NAEs can readily access the extracellular space. In the present study, we have investigated the cellular localization of NAPE in intact neurons in culture by probing their plasma membrane composition with exogenous phospholipases. H. Cadas et al. / J. Lipid Mediators Cell Signalling 14 (1996) 63 70 65 2. Experimental procedures Primary cultures of rat brain striatal neurons were prepared, kept in culture and labeled with [3H]ethanolamine or [3H]arachidonic acid as previously described (Di Marzo et al., 1994). The neurons (plated at a density of 2.5 x 1 0  7 cells/dish in 90 mm dishes) were used after 10 days in vitro. The labeled neurons were washed with Dulbecco Modified Eagle's Medium (DMEM) and incubated for 10 min in DMEM containing one of the following phospholipases: phospholipase D (PLD), from Streptomyces  chromofuscus (12.5 U/ml, Boehringer Mannheim or Sigma), phospho- lipase A2 (PLA2), from Apis mellifera (6.8 U/ml, Sigma) and phospholipase C (PLC), from Bacillus cereus (40 U/ml, Boehringer Mannheim). Incubations were stopped by adding methanol, immediately followed by chloroform extraction. Analyses of the [3H]NAEs present in the lipid extracts were carried out as described (Fontana et al., 1995) [3H]NAPE was isolated by thin-layer chromatography (TLC), using plastic- backed silica gel G plates eluted with a solvent system of chloroform/methanol/am- monia (80/20/1, v/v/v). Lipids were visualized with phosphomolybdic acid, and radioactivity measured by liquid scintillation counting after cutting the plates. [3H]Ethanolamine was determined in the aqueous phase of neuronal extracts by fractionation on polydivinylbenzene minicolumns, as described (Desarnaud et al., 1995). In some experiments, we measured [3H]arachidonic acid and [3H]diacylglycerol in the extracts by using standard TLC methods (Hamilton and Hamilton, 1992). To measure [3H]7-aminobutyric acid (GABA) release, the neurons were incubated with [3H]GABA (Amersham) for 30 min at 37°C, washed 5 times with DMEM, and then incubated in DMEM containing PLD (10 U/ml) for 10 min at 37°C. 3. Results and discussion Our main objective in carrying out the present experiments was to determine whether the plasma membrane of brain neurons contains NAPE. Interest in this question lies in the potential role of NAPE as precursor for anandamide and N-palmitoylethanolamine, two NAEs that are thought to be released from neurons (Di Marzo et al., 1994; Cadas et al., 1996) and to act extracellularly by binding to cannabinoid receptors on neighbouring neuronal or non-neuronal cells (Mechoulam et al., 1994; Facci et al., 1995). Because cellular fractionation methods require substantial quantities of biological material and are therefore poorly applicable to neurons in primary culture, we took an alternative course. By applying an approach extensively used with circulating blood cells (reviewed in Waite, 1987), we incubated prelabeled rat brain striatal neurons in culture with purified PLD (from S. chromofuscus)  and determined the effect of this treatment on the levels of [3H]NAPE and on the formation of [3H]NAEs. We found that incubation with PLD resulted in the formation of large amounts of [3H]NAEs, which we identified by a combination of TLC and normal-phase HPLC (Fig. la and Fig. ld). Analysis of these [3H]NAEs by two sequential reversed-phase HPLC steps revealed a family of saturated and unsaturated congeners (Fig. ld and 66 H. Cadas et al. / J. Lipid Mediators Cell Signalling 14 (1996) 63-70 a Striatal neurons: PLD -1 1 Anandamldc Ethanolamine Anandamide .., 6 ~ 3  2000" / ~  O] 0 | | C PLD " 1000" 0 0 5 l0 15 Anandamide DAG ~ ~ooo]... ~ . . . . . . .  - ,  . . . . . . . . .  ~ ~ c 3  ~" o 0 i 0 i~- 1000 C PLC C PLC o 0 5 10 15 £ PLA 2 1ooo o o 5 - , 10 15 migration (cm) Anandamldc AA  = ~ J 0 i D t~ 0 C PLA~ C PLA 2 d PLD 1000] Anandamide 3 14 • ' z ; u  \ i i 6 ~- 1'o 15 2o 25 500 E. 250 IE e,z Fig. 1. H. Cadas et al./ J. Lipid Mediators Cell Signalling 14 (1996) 63-70 67 Table l Compositions of the [3H]NAEs produced by striatal neurons in primary culture, incubated in the presence of exogenous PLD. Analyses were carried out by two sequential reversed-phase HPLC steps, as described (Fontana et al., 1995). Data are expressed as percent of total [3H]NAEs recovered (% of total) N-arachidonoylethanolamine N- 7-1inolenoylethanolamine N-linoleoylethanolamine N -oleoylethanolamine N-stearoylethanolamine N -palmitoylethanolamine 0.2 5.0 0.2 27 20 52 Table  1). These were similar in quali tat ive composi t ion  to those produced by s t imula t ion  of striatal neurons  with membrane-depola r iz ing  agents or by P LD  t rea tment  of purified NAPE,  bu t  showed a greater percentage of  saturated species (Di Marzo  et al., 1994). This discrepancy may  be explained by the fact that,  unl ike sa turated NAEs,  a n a n d a m i d e  and  other po lyunsa tu ra ted  N A E s  are good substrates for a n a n d a m i d e  amidohydrolase  activity, and  are subject to rapid hydrolytic degradat ion  (Desarnaud  et al., 1995). As expected, [3H]NAE format ion  was accom- panied by release of  [3H]ethanolamine, a product  of  P LD  hydrolysis, f rom the neurons.  By contrast ,  fo rmat ion  of [3H]arachidonate or [3H]diacylglycerol was no t  observed (Fig. la). The effect of  P L D  on [3H]NAE format ion  did no t  result f rom m e m b r a n e  damage.  In suppor t  of  this conclusion,  we found  that  t reat ing the neurons  with P L D  had no effect on either exclusion of the vital dye, T rypan  blue (data not  shown), or release of the striatal neurot ransmi t te r ,  [3H]GABA. Extracellularly released [3H]GABA was 0.283 _+ 0.03% of total  [3H]GABA in control  neurons ,  and  0.276 + 0.09% in neurons  with P L D  (mean + SEM, n = 5). Also, incuba t ion  with P LD  was not  accompanied  by in ternal iza t ion  of  this phospholipase.  When  the neurons  were incubated  for 10 Fig. t. Effect of exogenous phospholipases on [3H]NAE formation in rat brain striatal neurons. PLD stimulates the formation of NAEs. Representative TLC fractionation of samples from the 10-min incubation of neurons (one dish): a, with PLD (S. chromoJuscus, 12.5 U/ml); b, with PLC (B. Cereus, 40 U/ml); c, with PLA 2 (A. melliJera, 6.8 U/ml). Bars indicate the mobility of synthetic anandamide. Radioactivity associated with the TLC origin is composed of [3H]ethanolamine-labeled phospholipids, which are expected to be different after treatment with PLD, PLC or PLA 2. Bar graphs on the right show the mean + SEM of five experiments in which we measured the radioactive material in NAE TLC fractions. The graph also shows the effects of PLD, PLC and PLA 2 on the formation of products characteristic of their respective enzymatic activities, using neurons labeled with either [3H]ethanolamine or [3H]arachidonic acid: a, free [3H]ethanolamine and related water-soluble products (for PLD); b, [3H]diacylglycerol (DAG, for PLC); c, [3H]arachidonic acid (AA, for PLA2); d, identification of the [3H]NAEs produced by PLD treatment. Representative normal-phase HPLC (np-HPLC) and reversed- phase HPLC (rp-HPLC) fractionations of samples from the incubation of striatal neurons with PLD. Numbers indicate the retention times of the following synthetic NAEs: 1, N-7-1inolenoylethanolamine; 2, N-linoleoylethanolamine plus N-arachidonoylethanolamine (anandamide); 3, N-palmitoyl- ethanolamine; 4, N-oleoylethanolamine; 5, N-stearoylethanolamine. Further purification of component 2 was obtained by an additional rp-HPLC step (Fontana et al., 1995). 1 0 0 0 0 .  H. Cadas et al. / J. Lipid Mediators Cell Signalling 14 (1996) 63-70 min with [125I]PLD (3.6 x 106 dpm/dish), little radioactivity above background was recovered in association with the cells (2000 _+ 560 dpm/dish, or 0.06% of  total, n = 5). Finally, the effect of  PLD on [3H]NAE formation was specific. Exogenous PLA2 (from A. mellifera) and PLC (from B. Cereus) were potent in producing [3H]arachidonate and [3H]diacylglycerol, respectively, but had no effect on the formation of  [3H]NAEs (Fig. lb and Fig. lc). To determine whether NAPE served as substrate for exogenous PLD under our experimental conditions, we measured the levels of  [3H]NAPE by TLC. We found that incubating the neurons with PLD for 10 min resulted in a marked decrease in the quantity of [3H]NAPE recovered (Fig. 2). When the neurons were disrupted and NAPE c o n t r o l  u n  7500 ,  y 500o. c~ 2500 - 0"  1oooo o b. 7500 - 5000-  2500 - 68 L5 ; 7'.5 ~'o P L D  NAPE 0-  i i ~) 21.5 5 7,5 i1(i m i g r a t i o n  (cm)  Fig. 2. Effect of  exogenous PLD on [3H]NAPE levels in striatal neurons: PLD catalyses the hydrolysis of endogenous [3H]NAPE. Representative TLC fractionation of samples from the 10-min incubation of neurons (one dish): a, without PLD; b, with PLD. Bars indicate the mobilities of  synthetic N A P E  and NAEs,  as well as that of  an unknown [3H]ethanolamine-containing lipid component.  H. Cadas et al. / J. Lipid Mediators Cell Signalling 14 (1996) 63 70 69 endogenous  [3H]NAPE was purified and  subjected to digestion with PLD,  [3H]NAPE was hydrolysed quant i ta t ively  (data not  shown). The f inding that  S. chromofuscus  P L D  does no t  penetrate  neurons  and  yet is able to catalyse the hydrolysis of  endogenous  [3H]NAPE, indicates that  [3H]NAPE is present  in the p lasma m e m b r a n e  of striatal neurons  in culture. We estimate from our  results that  up to ~ 50% of  neurona l  [3H]NAPE is sensitive to P L D  treatment ,  and  may  be localized therefore in this m e m b r a n e  compar tment .  However,  because the extent to which S. chromofuscus  P LD  penetrates  the lipid bilayer in these neurons  is u n k n o w n ,  we canno t  conclude that  [3H]NAPE is restricted to the outer  leaflet of  this membrane .  While further experiments  will be necessary to address this issue (linked to that  of  the subcellular  localizat ion of  NAPE-metabo l i z ing  enzymes), the presence of  N A P E  in neu rona l  p lasma membranes  lends suppor t  to the proposed role of this phosphol ip id  as a cellular precursor  for anandamide ,  N-  pa lmi toy le thano lamine  and  other N A E s  that  may act as intercellular  messenger molecules. T rea tmen t  with P L D  revealed also that  release of [3H]NAEs can take place independent ly  of release of [3H]GABA, an inhibi tory  neuro t ransmi t t e r  re- leased by the major i ty  of  striatal neurons  both  in vivo and  in vitro. Our  results thus underscore  the dis t inct ion between cannab imimet ic  signaling via cleavage of precur- sor N A P E  and  vesicular release of classical neurot ransmit ters .  Acknowledgements We thank  Dr. Claude Gros  for [125I]iodination of P LD  and  Dr. Jean-Char les  Schwartz for discussion. 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Membrane localization of N-acylphosphatidylethanolamine in central neurons: Studies with exogenous phospholipases

Membrane localization of N-acylphosphatidylethanolamine in central neurons: studies with exogenous phospholipases.

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