Advertisement

Anandamide Induces Apoptosis in Human Cells via Vanilloid Receptors

EVIDENCE FOR A PROTECTIVE ROLE OF CANNABINOID RECEPTORS*
  • Mauro Maccarrone
    Affiliations
    Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via di Tor Vergata 135, I-00133 Rome, Italy
    Search for articles by this author
  • Tatiana Lorenzon
    Affiliations
    Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via di Tor Vergata 135, I-00133 Rome, Italy
    Search for articles by this author
  • Monica Bari
    Affiliations
    Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via di Tor Vergata 135, I-00133 Rome, Italy
    Search for articles by this author
  • Gerry Melino
    Affiliations
    Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via di Tor Vergata 135, I-00133 Rome, Italy
    Search for articles by this author
  • Alessandro Finazzi-Agrò
    Correspondence
    To whom correspondence should be addressed. Tel./Fax: 39-06-72596468
    Affiliations
    Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via di Tor Vergata 135, I-00133 Rome, Italy
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by Istituto Superiore di Sanità (III AIDS Program), by Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Rome (to A.F.A.), and by Telethon Grant E872 (to G. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:October 13, 2000DOI:https://doi.org/10.1074/jbc.M005722200
      The endocannabinoid anandamide (AEA) is shown to induce apoptotic bodies formation and DNA fragmentation, hallmarks of programmed cell death, in human neuroblastoma CHP100 and lymphoma U937 cells. RNA and protein synthesis inhibitors like actinomycin D and cycloheximide reduced to one-fifth the number of apoptotic bodies induced by AEA, whereas the AEA transporter inhibitor AM404 or the AEA hydrolase inhibitor ATFMK significantly increased the number of dying cells. Furthermore, specific antagonists of cannabinoid or vanilloid receptors potentiated or inhibited cell death induced by AEA, respectively. Other endocannabinoids such as 2-arachidonoylglycerol, linoleoylethanolamide, oleoylethanolamide, and palmitoylethanolamide did not promote cell death under the same experimental conditions. The formation of apoptotic bodies induced by AEA was paralleled by increases in intracellular calcium (3-fold over the controls), mitochondrial uncoupling (6-fold), and cytochrome c release (3-fold). The intracellular calcium chelator EGTA-AM reduced the number of apoptotic bodies to 40% of the controls, and electrotransferred anti-cytochrome c monoclonal antibodies fully prevented apoptosis induced by AEA. Moreover, 5-lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid and MK886, cyclooxygenase inhibitor indomethacin, caspase-3 and caspase-9 inhibitors Z-DEVD-FMK and Z-LEHD-FMK, but not nitric oxide synthase inhibitorNω-nitro-l-arginine methyl ester, significantly reduced the cell death-inducing effect of AEA. The data presented indicate a protective role of cannabinoid receptors against apoptosis induced by AEA via vanilloid receptors.
      AEA
      anandamide (arachidonoylethanolamide)
      2-AG
      2-arachidonoylglycerol
      AM404
      N-(4-hydroxyphenyl)arachidonoylamide
      ATFMK
      arachidonoyl-trifluoromethyl ketone
      Caps
      capsazepine
      CBD
      cannabidiol
      CB1/2R
      type 1/2 cannabinoid receptor
      AM
      acetoxymethyl ester
      ELISA
      enzyme-linked immunosorbent assay
      ETYA
      5,8,11,14-eicosatetraynoic acid
      FAAH
      fatty acid amide hydrolase
      LEA
      linoleoylethanolamide
      l-NAME
      Nω-nitro-l-arginine methyl ester
      OEA
      oleoylethanolamide
      PBS
      phosphate-buffered saline
      PEA
      palmitoylethanolamide
      VR
      vanilloid receptor
      Z-DEVD-FMK
      Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fluoromethyl ketone
      Z-LEHD-FMK
      Z-Leu-Glu(OCH3)-His-Asp (OCH3)-fluoromethyl ketone
      GAM-AP
      goat anti-mouse alkaline phosphatase
      PCD
      programmed cell death
      Anandamide (arachidonoylethanolamide, AEA)1 belongs to an emerging class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids and collectively indicated as “endocannabinoids” (
      • Pop E.
      ). In fact, AEA has been isolated and characterized as an endogenous ligand for cannabinoid receptors in the central nervous system (CB1 subtype) and peripheral immune cells (CB2 subtype). AEA is released from depolarized neurons, endothelial cells and macrophages (
      • Di Marzo V.
      • Bisogno T.
      • De Petrocellis L.
      • Melck D.
      • Orlando P.
      • Wagner J.A.
      • Kunos G.
      ), and mimics the pharmacological effects of Δ9-tetrahydrocannabinol, the active principle of hashish and marijuana (
      • Pertwee R.G.
      ). Recently, attention has been focused on the possible role of AEA and other endocannabinoids in regulating cell growth and differentiation, which might account for some pathophysiological effects of these lipids. An anti-proliferative action of AEA has been reported in human breast carcinoma cells, due to a CB1-like receptor-mediated inhibition of the action of endogenous prolactin at its receptor (
      • De Petrocellis L.
      • Melck D.
      • Palmisano A.
      • Bisogno T.
      • Laezza C.
      • Bifulco M.
      • Di Marzo V.
      ). An activation of cell proliferation by AEA has been reported instead in hematopoietic cell lines (
      • Derocq J.-M.
      • Bouaboula M.
      • Marchand J.
      • Rinaldi-Carmona M.
      • Ségui M.
      • Casellas P.
      ). Moreover, preliminary evidence that the immunosuppressive effects of AEA might be associated with inhibition of lymphocyte proliferation and induction of programmed cell death (PCD or apoptosis) has been reported (
      • Schwarz H.
      • Blanco F.J.
      • Lotz M.
      ), and growing evidence is being collected that suggests that AEA might have pro-apoptotic activity, both in vitro (
      • Sarker K.P.
      • Obara S.
      • Nakata M.
      • Kitajima I.
      • Maruyama I.
      ) and in vivo (
      • Galve-Roperh I.
      • Sànchez C.
      • Cortes M.L.
      • del Pulgar T.G.
      • Izquierdo M.
      • Guzman M.
      ). This would extend to endocannabinoids previous observations on Δ9-tetrahydrocannabinol, shown to induce PCD in glioma tumors (
      • Galve-Roperh I.
      • Sànchez C.
      • Cortes M.L.
      • del Pulgar T.G.
      • Izquierdo M.
      • Guzman M.
      ), glioma cells (
      • Sànchez C.
      • Galve-Roperh I.
      • Canova C.
      • Brachet P.
      • Guzmàn M.
      ), primary neurons (
      • Chan G.C.-K.
      • Hinds T.R.
      • Impey S.
      • Storm D.R.
      ), hippocampal slices (
      • Chan G.C.-K.
      • Hinds T.R.
      • Impey S.
      • Storm D.R.
      ), and prostate cells (
      • Ruiz L.
      • Miguel A.
      • Diaz-Laviada I.
      ). However, the mechanism of AEA-induced PCD is unknown. The various effects of AEA in the central nervous system and in immune system (reviewed in Refs.
      • Pop E.
      ,
      • Di Marzo V.
      • Bisogno T.
      • De Petrocellis L.
      • Melck D.
      • Orlando P.
      • Wagner J.A.
      • Kunos G.
      ,
      • Pertwee R.G.
      ), as well as its ability to reduce the emerging pain signals at sites of tissue injury (
      • Walker J.M.
      • Huang S.M.
      • Strangman N.M.
      • Tsou K.
      • Sañudo-Peña M.C.
      ), are terminated by a rapid and selective carrier-mediated uptake of AEA into cells (
      • Maccarrone M.
      • Bari M.
      • Lorenzon T.
      • Bisogno T.
      • Di Marzo V.
      • Finazzi-Agrò A.
      ), followed by its degradation to ethanolamine and arachidonic acid by the enzyme fatty acid amide hydrolase (FAAH) (
      • Maccarrone M.
      • Valensise H.
      • Bari M.
      • Lazzarin N.
      • Romanini C.
      • Finazzi-Agrò A.
      ). Recently, we showed that human neuroblastoma CHP100 cells and human lymphoma U937 cells do have these tools to eliminate AEA (
      • Maccarrone M.
      • van der Stelt M.
      • Rossi A.
      • Veldink G.A.
      • Vliegenthart J.F.G.
      • Finazzi-Agrò A.
      ). Therefore, these cell lines were chosen to investigate how AEA and related endocannabinoids induce apoptosis and how the removal and degradation of AEA are related to this process. The existence of a neuroimmune axis appears to be confirmed by the finding that endocannabinoids elicit common responses in these two cell types.

      DISCUSSION

      We have shown that AEA can induce apoptotic body formation and DNA fragmentation, hallmarks of PCD, in human neuronal and immune cells through a pathway involving rise in intracellular calcium, mitochondrial uncoupling, and cytochrome c release. Activation of the arachidonate cascade and of the caspase cascade are critical steps in the death program. The pro-apoptotic activity of AEA was observed at physiological concentrations of this compound (
      • Di Marzo V.
      • Sepe N.
      • De Petrocellis L.
      • Berger A.
      • Crozier G.
      • Fride E.
      • Mechoulam R.
      ). Unlike AEA, other structurally related and biologically active endocannabinoids, such as 2-AG, LEA, OEA, and PEA (
      • Pop E.
      ,
      • Di Marzo V.
      • Bisogno T.
      • De Petrocellis L.
      • Melck D.
      • Orlando P.
      • Wagner J.A.
      • Kunos G.
      ,
      • Pertwee R.G.
      ), were unable to force cells into PCD under the same experimental conditions (TableI), ruling out the possibility that the observed effects of AEA were due to unspecific cell poisoning. Since 2-AG may release arachidonate through FAAH activity faster than AEA (
      • Goparaju S.K.
      • Ueda N.
      • Yamaguchi H.
      • Yamamoto S.
      ), the lack of pro-apoptotic activity of this compound rules out the possibility that AEA-induced PCD might be due to arachidonate, as reported for U937 cells (
      • Vanags D.M.
      • Larsson P.
      • Feltenmark S.
      • Jakobsson P.-J.
      • Orrenius S.
      • Claesson H.-E.
      • Aguilar-Santelises M.
      ). Consistently, inhibition of FAAH by ATFMK potentiated, instead of reducing, the apoptotic activity of AEA (Table II). Also inhibition of AEA degradation by blocking its uptake enhanced AEA-induced PCD (TableII). Since a slower degradation leads to an increased concentration of AEA in the extracellular matrix, these findings suggest that the pro-apoptotic activity of AEA is mediated by a target molecule on the cell surface. Indeed, [3H]AEA binds to CHP100 and U937 cell membranes (Fig. 3 A). However, in these cell lines a different binding site must be involved because the “classical” CB1 or CB2 receptors are not present (Fig. 2). Previous reports have shown that AEA can bind and modulate receptors other than CB1R and CB2R (
      • Wagner J.A.
      • Varga C.
      • Jàrai Z.
      • Kunos G.
      ), and recently a CB receptor for AEA, distinct from type 1 or type 2, has been described in endothelial cells (
      • Járai Z.
      • Wagner J.
      • Varga K.
      • Lake K.D.
      • Compton D.R.
      • Martin B.R.
      • Zimmer A.M.
      • Bonner T.I.
      • Buckley N.E.
      • Mezey E.
      • Razdan R.K.
      • Zimmer A.
      • Kunos G.
      ). However, this new CB receptor was not expressed in CHP100 or U937 cells, because its selective antagonist CBD (
      • Járai Z.
      • Wagner J.
      • Varga K.
      • Lake K.D.
      • Compton D.R.
      • Martin B.R.
      • Zimmer A.M.
      • Bonner T.I.
      • Buckley N.E.
      • Mezey E.
      • Razdan R.K.
      • Zimmer A.
      • Kunos G.
      ) was ineffective on [3H]AEA binding (Fig. 3 B) and on AEA-induced PCD (Table II). On the other hand, it is becoming increasingly evident that AEA behaves as a full agonist at human vanilloid receptors (
      • Zygmunt P.M.
      • Petersson J.
      • Andersson D.A.
      • Chuang H.-H.
      • Sorgård M.
      • Di Marzo V.
      • Julius D.
      • Högestätt E.D.
      ,
      • Smart D.
      • Jerman J.C.
      ), whose activation can induce apoptosis in neuronal (
      • Sugimoto T.
      • Takeyama A.
      • Xiao C.
      • Takano-Yamamoto T.
      • Ichikawa H.
      ) and immune (
      • Macho A.
      • Calzado M.A.
      • Munoz-Blanco J.
      • Gomez-Diaz C.
      • Gajate C.
      • Mollinedo F.
      • Navas P.
      • Munoz E.
      ) cells. Therefore, the possibility that the pro-apoptotic activity of AEA might occur through this receptor was investigated. Indeed, it was found that capsazepine, a selective antagonist of VR (
      • Zygmunt P.M.
      • Petersson J.
      • Andersson D.A.
      • Chuang H.-H.
      • Sorgård M.
      • Di Marzo V.
      • Julius D.
      • Högestätt E.D.
      ), prevented [3H]AEA binding to CHP100 or U937 cells (Fig. 3 B) and inhibited AEA-induced PCD (Table II), whereas the VR agonist capsaicin (
      • Zygmunt P.M.
      • Petersson J.
      • Andersson D.A.
      • Chuang H.-H.
      • Sorgård M.
      • Di Marzo V.
      • Julius D.
      • Högestätt E.D.
      ) mimicked the pro-apoptotic activity of AEA in these cells. Altogether, these findings suggest that AEA-induced PCD was mediated by vanilloid receptors. It should be stressed that this hypothesis is consistent with the observation that 2-AG and the other endocannabinoids did not promote PCD (Table I), because these compounds do not activate vanilloid receptors (
      • Zygmunt P.M.
      • Petersson J.
      • Andersson D.A.
      • Chuang H.-H.
      • Sorgård M.
      • Di Marzo V.
      • Julius D.
      • Högestätt E.D.
      ) or have a much lower potency than AEA (
      • Smart D.
      • Gunthorpe M.J.
      • Jerman J.C.
      • Nasir S.
      • Gray J.
      • Muir A.I.
      • Chambers J.K.
      • Randall A.D.
      • Davis J.B.
      ). In this context, it seems noteworthy that AM404 alone was ineffective on PCD, mitochondrial uncoupling, intracellular calcium concentration, or cytochrome c release from cells, although it did potentiate the effect of AEA (Tables Table II, Table III, Table IV and Fig. 4 B). These findings suggest that AM404 was unable to activate directly human VR, at variance with a previous report suggesting that it is an agonist for rat VR (
      • Smart D.
      • Jerman J.C.
      ).
      A major finding of this investigation is that CB1R or CB2R antagonists, SR141716 or SR144528, were ineffective in CHP100 or U937 cells, which lack cannabinoid receptors (Fig. 2), but they did potentiate AEA-induced PCD in C6 or DAUDI cells (Table III). In fact, these cells express functional CB1 or CB2 receptors, respectively (Fig. 2), and were able to bind larger amounts of [3H]AEA than CHP100 or U937 cells. Capsazepine displaced approximately 30% [3H]AEA from C6 or DAUDI cells, suggesting that the remaining 70% was bound to CB receptors. Remarkably, capsazepine prevented AEA-induced PCD in these cells in a way fully analogous to that observed in CHP100 or U937 cells (Table III), suggesting that vanilloid receptors mediate the pro-apoptotic activity of AEA also in C6 and DAUDI cells. As a matter of fact, specific vanilloid responses have been described in C6 cells (
      • Bı́ró T.
      • Brodie C.
      • Modarres S.
      • Lewin N.E.
      • Ács P.
      • Blumberg P.M.
      ). Like in CHP100 or U937 cells, cannabidiol was ineffective on the pro-apoptotic activity of AEA in C6 or DAUDI cells, ruling out that the new “endothelial” CBR (
      • Járai Z.
      • Wagner J.
      • Varga K.
      • Lake K.D.
      • Compton D.R.
      • Martin B.R.
      • Zimmer A.M.
      • Bonner T.I.
      • Buckley N.E.
      • Mezey E.
      • Razdan R.K.
      • Zimmer A.
      • Kunos G.
      ) might be involved. On the other hand, it seems noteworthy that the ability of C6 or DAUDI cells to degrade AEA through intracellular uptake and degradation by FAAH was similar to that of CHP100 or U937 cells, respectively. Therefore, it is tempting to speculate that cells bearing functional CB1 or CB2 receptors on their surface are protected against the toxic effects of physiological concentrations of AEA. In C6 or DAUDI cells, the effects on PCD of co-administration of the transporter inhibitor AM404, which increases extracellular concentration of AEA, or of CBR antagonists SR141716 and SR144528, which prevent CBR activation (Table III), support this concept. These findings can be interpreted by suggesting a regulatory loop between CB receptors and the AEA transporter, which has been recently demonstrated in human endothelial cells (
      • Maccarrone M.
      • Bari M.
      • Lorenzon T.
      • Bisogno T.
      • Di Marzo V.
      • Finazzi-Agrò A.
      ). In this loop, the binding of AEA to CB receptors triggers the activation of AEA uptake by cells, followed by intracellular degradation of AEA by FAAH. Elimination of AEA from the extracellular space might terminate its activity at vanilloid receptors, thus inhibiting the induction of apoptosis. Scheme FSI summarizes the main features of this model.
      Figure thumbnail grfsi
      Figure FSIRole of vanilloid receptor and cannabinoid receptor in AEA-induced programmed cell death. Binding of extracellular AEA to VR triggers a sequence of events starting with a rise in intracellular calcium and followed by activation of cyclooxygenase and lipoxygenase, drop in mitochondrial membrane potential (Δψ), release of cytochrome c, and activation of caspases, ultimately leading to programmed cell death (apoptosis). Binding of AEA to cannabinoid receptors (CBR) activates transporter (T)-mediated uptake of AEA and its subsequent cleavage to arachidonic acid and ethanolamine by membrane-bound FAAH. These latter events inhibit the pro-apoptotic activity of AEA.
      PCD of CHP100 or U937 cells induced by AEA was executed through a series of events common to several types of unrelated apoptotic stimuli (
      • Wallace D.C.
      ). It involved the following: (i) rise in cytosolic calcium concentration (within 6 min), (ii) uncoupling of mitochondria (within 6 h), and (iii) release of cytochrome c (within 8 h). These events required gene expression of proteins necessary for apoptosis, as shown by the protective effect of actinomycin D and cycloheximide (Table II). Consistently with the data on apoptotic body formation and [3H]AEA binding to cell membranes, (i) capsazepine inhibited the events triggered by AEA, (ii) AM404 or ATFMK potentiated them, and (iii) SR141716, SR144528, or CBD were ineffective (Table IV and Fig. 4 B). At variance with other types of PCD (
      • Ghafourifar P.
      • Klein S.D.
      • Schucht O.
      • Schenk U.
      • Pruschy M.
      • Rocha S.
      • Richter C.
      ), calcium rise induced by AEA was not acting through activation of nitric-oxide synthase, because the nitric-oxide synthase inhibitorl-NAME was ineffective in protecting cells against AEA. Instead, arachidonate degradation by 5-lipoxygenase and cyclooxygenase activities, which might be enhanced as a consequence of a rise in intracellular Ca2+ (
      • Ford-Hutchinson A.W.
      • Gresser M.
      • Young R.N.
      ,
      • Datta K.
      • Biswal S.S.
      • Xu J.
      • Towndrow K.M.
      • Feng X.
      • Kehrer J.P.
      ,
      • Ara G.
      • Teicher B.A.
      ), had a role in the process, because the inhibitors ETYA and MK886 significantly inhibited AEA-induced PCD (Table II). It must be mentioned that MK886 can exert lipoxygenase-unrelated effects on mammalian cells (
      • Datta K.
      • Biswal S.S.
      • Xu J.
      • Towndrow K.M.
      • Feng X.
      • Kehrer J.P.
      ). However, the observation that ETYA and MK886 yielded the same inhibition of apoptosis seems to rule out the involvement of lipoxygenase-independent pathways. This seems interesting, because formation of arachidonate products unbalances the intracellular redox level and has been implicated in apoptotic death of several cell types (
      • Sarker K.P.
      • Obara S.
      • Nakata M.
      • Kitajima I.
      • Maruyama I.
      ,
      • Maccarrone M.
      • Catani M.V.
      • Finazzi-Agrò A.
      • Melino G.
      ,
      • Datta K.
      • Biswal S.S.
      • Xu J.
      • Towndrow K.M.
      • Feng X.
      • Kehrer J.P.
      ,
      • Vanags D.M.
      • Larsson P.
      • Feltenmark S.
      • Jakobsson P.-J.
      • Orrenius S.
      • Claesson H.-E.
      • Aguilar-Santelises M.
      ). In particular, it should be stressed that a function for lipoxygenase in programmed organelle degradation has been recently demonstrated, showing that the enzyme can make pore-like structures in the lipid bilayer (
      • Van Leyen K.
      • Duvoisin R.M.
      • Engelhardt H.
      • Wiedmann M.
      ). This activity might contribute to uncouple directly the mitochondria (Table IV). However, opening of the mitochondrial permeability transition pore (
      • Andreyev A.
      • Fiskum G.
      ) did not contribute to AEA-induced PCD, as suggested by the lack of effect of cyclosporin A (Table II). On the other hand, an unbalanced redox level in the cell has been associated to release of cytochrome c, a converging point in apoptosis induced by different stimuli in various cell types (
      • Ushmorov A.
      • Ratter F.
      • Lehmann V.
      • Dröge W.
      • Schirrmacher V.
      • Umansky V.
      ,
      • Garrido C.
      • Bruey J.-M.
      • Fromentin A.
      • Hammann A.
      • Arrigo A.P.
      • Solary E.
      ,
      • Wallace D.C.
      ,
      • Ghafourifar P.
      • Klein S.D.
      • Schucht O.
      • Schenk U.
      • Pruschy M.
      • Rocha S.
      • Richter C.
      ). Cytochrome c release was observed also in AEA-induced PCD (Fig. 4 B), and it was essential for apoptosis, because sequestering cytochrome c within intact U937 cells by electrotransferred anti-cytochrome cmonoclonal antibodies was able to prevent AEA-induced PCD (
      • Neame S.J.
      • Rubin L.L.
      • Philpott K.L.
      ). Cytochrome c release in the cell cytosol is usually followed by activation of a caspase cascade, initiated by caspase-3 and caspase-9 which are the most proximal members of the proteolytic chain (
      • Ushmorov A.
      • Ratter F.
      • Lehmann V.
      • Dröge W.
      • Schirrmacher V.
      • Umansky V.
      ,
      • Garrido C.
      • Bruey J.-M.
      • Fromentin A.
      • Hammann A.
      • Arrigo A.P.
      • Solary E.
      ,
      • Neame S.J.
      • Rubin L.L.
      • Philpott K.L.
      ). Caspases are thought to form a proteolytic machinery within the cell, resulting in the breakdown of key enzymes and cellular structures, and to activate DNases responsible for chromatin degradation seen in apoptosis (
      • Neame S.J.
      • Rubin L.L.
      • Philpott K.L.
      ). Also AEA-induced PCD seemed to be executed through this series of events, because caspase-3 or caspase-9 inhibitors reduced apoptotic body formation to approximately 20–30% of the controls (Table II). Altogether, these results suggest that PCD induced by AEA occurs through an apoptotic pathway based on calcium rise, mitochondrial uncoupling, and cytochrome c release. Upstream activation of the arachidonate cascade leads to redox unbalance and organelle disruption, which both favor cytochromec release, then caspases act as downstream executioners of the death program. In this context, it seems noteworthy that also capsaicin-induced PCD occurs through intracellular calcium rise, imbalance of the redox level, and drop in mitochondrial membrane potential (
      • Macho A.
      • Calzado M.A.
      • Munoz-Blanco J.
      • Gomez-Diaz C.
      • Gajate C.
      • Mollinedo F.
      • Navas P.
      • Munoz E.
      ), further strengthening the hypothesis that AEA is acting through vanilloid receptors. Scheme FSI summarizes the series of events responsible for AEA-induced cell death. It seems noteworthy that these findings might be relevant also for neuronal apoptosis induced by alcohols (
      • Saito M.
      • Saito M.
      • Berg M.J.
      • Guidotti A.
      • Marks N.
      ), where an increase in AEA concentration has been reported (
      • Basavarajappa B.S.
      • Hungund B.L.
      ). Moreover, they demonstrate major differences in the cytotoxicity of the different endocannabinoids, which might be relevant for understanding their pathophysiological roles (
      • Pop E.
      ,
      • Di Marzo V.
      • Bisogno T.
      • De Petrocellis L.
      • Melck D.
      • Orlando P.
      • Wagner J.A.
      • Kunos G.
      ,
      • Pertwee R.G.
      ). Finally, this study shows that endocannabinoids exert similar actions in neuronal and immune cells, perhaps (and significantly) through common signals.

      Acknowledgements

      We thank Dr. Dale G. Deutsch (Department of Biochemistry and Cell Biology, State University of New York, Stony Brook) for the kind gift of C6 glioma cells, Drs. Marco Ranalli and Rita Agostinetto for their skillful assistance with cytofluorimetric analysis and cell culture, and Dr. Francesca Bernassola for helpful discussions.

      REFERENCES

        • Pop E.
        Curr. Opin. Chem. Biol. 1999; 3: 418-425
        • Di Marzo V.
        • Bisogno T.
        • De Petrocellis L.
        • Melck D.
        • Orlando P.
        • Wagner J.A.
        • Kunos G.
        Eur. J. Biochem. 1999; 264: 258-267
        • Pertwee R.G.
        Pharmacol. Ther. 1997; 74: 129-180
        • De Petrocellis L.
        • Melck D.
        • Palmisano A.
        • Bisogno T.
        • Laezza C.
        • Bifulco M.
        • Di Marzo V.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8375-8380
        • Derocq J.-M.
        • Bouaboula M.
        • Marchand J.
        • Rinaldi-Carmona M.
        • Ségui M.
        • Casellas P.
        FEBS Lett. 1998; 425: 419-425
        • Schwarz H.
        • Blanco F.J.
        • Lotz M.
        J. Neuroimmunol. 1994; 55: 107-115
        • Sarker K.P.
        • Obara S.
        • Nakata M.
        • Kitajima I.
        • Maruyama I.
        FEBS Lett. 2000; 472: 39-44
        • Galve-Roperh I.
        • Sànchez C.
        • Cortes M.L.
        • del Pulgar T.G.
        • Izquierdo M.
        • Guzman M.
        Nat. Med. 2000; 6: 313-316
        • Sànchez C.
        • Galve-Roperh I.
        • Canova C.
        • Brachet P.
        • Guzmàn M.
        FEBS Lett. 1998; 436: 6-10
        • Chan G.C.-K.
        • Hinds T.R.
        • Impey S.
        • Storm D.R.
        J. Neurosci. 1998; 18: 5322-5332
        • Ruiz L.
        • Miguel A.
        • Diaz-Laviada I.
        FEBS Lett. 1999; 458: 400-404
        • Walker J.M.
        • Huang S.M.
        • Strangman N.M.
        • Tsou K.
        • Sañudo-Peña M.C.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12198-12203
        • Maccarrone M.
        • Bari M.
        • Lorenzon T.
        • Bisogno T.
        • Di Marzo V.
        • Finazzi-Agrò A.
        J. Biol. Chem. 2000; 275: 13484-13492
        • Maccarrone M.
        • Valensise H.
        • Bari M.
        • Lazzarin N.
        • Romanini C.
        • Finazzi-Agrò A.
        Lancet. 2000; 355: 1326-1329
        • Maccarrone M.
        • van der Stelt M.
        • Rossi A.
        • Veldink G.A.
        • Vliegenthart J.F.G.
        • Finazzi-Agrò A.
        J. Biol. Chem. 1998; 273: 32332-32339
        • Maccarrone M.
        • Veldink G.A.
        • Vliegenthart J.F.G.
        Eur. J. Biochem. 1992; 205: 995-1001
        • Maccarrone M.
        • Catani M.V.
        • Finazzi-Agrò A.
        • Melino G.
        Cell Death Differ. 1997; 4: 396-402
        • Maccarrone M.
        • Nieuwenhuizen W.F.
        • Dullens H.F.J.
        • Catani M.V.
        • Melino G.
        • Veldink G.A.
        • Vliegenthart J.F.G.
        • Finazzi-Agrò A.
        Eur. J. Biochem. 1996; 241: 297-302
        • Maccarrone M.
        • Fiorucci L.
        • Erba F.
        • Bari M.
        • Finazzi-Agrò A.
        • Ascoli F.
        FEBS Lett. 2000; 468: 176-180
        • Deutsch D.G.
        • Goligorsky M.S.
        • Schmid P.C.
        • Krebsbach R.J.
        • Schmid H.H.O.
        • Das S.K.
        • Dey S.K.
        • Arreaza G.
        • Thorup C.
        • Stefano G.
        • Moore L.C.
        J. Clin. Invest. 1997; 100: 1538-1546
        • Smiley S.T.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3671-3675
        • Vandenberghe P.A.
        • Ceuppens J.L.
        J. Immunol. Methods. 1990; 127: 197-205
        • Ushmorov A.
        • Ratter F.
        • Lehmann V.
        • Dröge W.
        • Schirrmacher V.
        • Umansky V.
        Blood. 1999; 93: 2342-2352
        • Di Marzo V.
        • Sepe N.
        • De Petrocellis L.
        • Berger A.
        • Crozier G.
        • Fride E.
        • Mechoulam R.
        Nature. 1998; 396: 636
        • Piomelli D.
        • Beltramo M.
        • Glasnapp S.
        • Lin S.Y.
        • Goutopoulos A.
        • Xie X.Q.
        • Makriyannis A.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5802-5807
        • Koutek B.
        • Prestwich G.D.
        • Howlett A.C.
        • Chin S.A.
        • Salehani D.
        • Akhavan N.
        • Deutsch D.G.
        J. Biol. Chem. 1994; 269: 22937-22940
        • Járai Z.
        • Wagner J.
        • Varga K.
        • Lake K.D.
        • Compton D.R.
        • Martin B.R.
        • Zimmer A.M.
        • Bonner T.I.
        • Buckley N.E.
        • Mezey E.
        • Razdan R.K.
        • Zimmer A.
        • Kunos G.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14136-14141
        • Zygmunt P.M.
        • Petersson J.
        • Andersson D.A.
        • Chuang H.-H.
        • Sorgård M.
        • Di Marzo V.
        • Julius D.
        • Högestätt E.D.
        Nature. 1999; 400: 452-457
        • Deutsch D.G.
        • Chin S.A.
        Biochem. Pharmacol. 1993; 46: 791-796
        • Galiègue S.
        • Mary S.
        • Marchand J.
        • Dussossoy D.
        • Carrière D.
        • Carayon P.
        • Bouaboula M.
        • Shire D.
        • Le Fur G.
        • Casellas P.
        Eur. J. Biochem. 1995; 232: 54-61
        • Maki A.
        • Berezesky I.K.
        • Fargnoli J.
        • Holbrook N.J.
        • Trump B.F.
        FASEB J. 1992; 6: 919-924
        • Ford-Hutchinson A.W.
        • Gresser M.
        • Young R.N.
        Annu. Rev. Biochem. 1994; 63: 383-417
        • Datta K.
        • Biswal S.S.
        • Xu J.
        • Towndrow K.M.
        • Feng X.
        • Kehrer J.P.
        J. Biol. Chem. 1998; 273: 28163-28169
        • Ara G.
        • Teicher B.A.
        Prostaglandins Leukot. Essent. Fatty Acids. 1996; 54: 3-16
        • Andreyev A.
        • Fiskum G.
        Cell Death Differ. 1999; 6: 825-832
        • Garrido C.
        • Bruey J.-M.
        • Fromentin A.
        • Hammann A.
        • Arrigo A.P.
        • Solary E.
        FASEB J. 1999; 13: 2061-2070
        • Neame S.J.
        • Rubin L.L.
        • Philpott K.L.
        J. Cell Biol. 1998; 142: 1583-1593
        • Goparaju S.K.
        • Ueda N.
        • Yamaguchi H.
        • Yamamoto S.
        FEBS Lett. 1998; 422: 69-73
        • Vanags D.M.
        • Larsson P.
        • Feltenmark S.
        • Jakobsson P.-J.
        • Orrenius S.
        • Claesson H.-E.
        • Aguilar-Santelises M.
        Cell Death Differ. 1997; 4: 479-486
        • Wagner J.A.
        • Varga C.
        • Jàrai Z.
        • Kunos G.
        Hypertension. 1999; 33: 429-434
        • Smart D.
        • Jerman J.C.
        Trends Pharmacol. Sci. 2000; 21: 134
        • Sugimoto T.
        • Takeyama A.
        • Xiao C.
        • Takano-Yamamoto T.
        • Ichikawa H.
        Brain Res. 1999; 818: 147-152
        • Macho A.
        • Calzado M.A.
        • Munoz-Blanco J.
        • Gomez-Diaz C.
        • Gajate C.
        • Mollinedo F.
        • Navas P.
        • Munoz E.
        Cell Death Differ. 1999; 6: 155-165
        • Smart D.
        • Gunthorpe M.J.
        • Jerman J.C.
        • Nasir S.
        • Gray J.
        • Muir A.I.
        • Chambers J.K.
        • Randall A.D.
        • Davis J.B.
        Br. J. Pharmacol. 2000; 129: 227-230
        • Bı́ró T.
        • Brodie C.
        • Modarres S.
        • Lewin N.E.
        • Ács P.
        • Blumberg P.M.
        Mol. Brain Res. 1998; 56: 89-98
        • Wallace D.C.
        Science. 1999; 283: 1482-1493
        • Ghafourifar P.
        • Klein S.D.
        • Schucht O.
        • Schenk U.
        • Pruschy M.
        • Rocha S.
        • Richter C.
        J. Biol. Chem. 1999; 274: 6080-6084
        • Van Leyen K.
        • Duvoisin R.M.
        • Engelhardt H.
        • Wiedmann M.
        Nature. 1998; 395: 392-395
        • Saito M.
        • Saito M.
        • Berg M.J.
        • Guidotti A.
        • Marks N.
        Neurochem. Res. 1999; 24: 1107-1115
        • Basavarajappa B.S.
        • Hungund B.L.
        J. Neurochem. 1999; 72: 522-528