Anandamide uptake by synaptosomes from human, mouse and rat brain: inhibition by glutamine and glutamate
© Battista et al; licensee BioMed Central Ltd. 2002
Received: 1 August 2002
Accepted: 3 September 2002
Published: 3 September 2002
Anandamide (N-arachidonoylethanolamine, AEA) belongs to an emerging class of endogenous lipids, called "endocannabinoids". A specific AEA membrane transporter (AMT) allows the import of this lipid and its degradation by the intracellular enzyme AEA hydrolase. Here, we show that synaptosomes from human, mouse and rat brain might be an ideal ex vivo system for the study of: i) the accumulation of AEA in brain, and ii) the pharmacological properties of AMT inhibitors. Using this ex vivo system, we demonstrate for the first time that glutamine and glutamate act as non-competitive inhibitors of AEA uptake by human, mouse and rat brain AMT.
Endocannabinoids are an emerging class of lipid mediators, which activate cannabinoid receptors and regulate sleep induction, interact with GABAergic, serotonergic and dopaminergic neurotransmission, and are also formed during glutamate-induced neurotoxicity [1, 2]. The biological activity of anandamide (N-arachidonoylethanolamine, AEA), a major endocannabinoid, is controlled by cellular uptake through a specific AEA membrane transporter (AMT) , followed by intracellular degradation by anandamide hydrolase (fatty acid amide hydrolase, FAAH) . The critical role of AMT in controlling AEA action, and the potential use of AMT inhibitors in the treatment of human pathologies such as multiple sclerosis, Parkinson's disease and Huntington's disease [1, 5, 6], prompted us to investigate whether brain synaptosomes might be used as a new ex vivo system, able to accumulate AEA through a selective transporter with the features of AMT.
Human brain specimens were obtained from twelve different male patients (aged 73–77), undergoing surgical operation to remove meningioma tumors from the pterional area. Brain tissues, removed and donated by Dr. G. De Caro (Neurosurgery Division, University of Rome "Tor Vergata", Sant'Eugenio Hospital, Rome, Italy), were kept on ice until processed (within 30 min). In each case the perilesional white matter (150 mg of fresh tissue) surrounding the tumor area (300 mg) was removed and used as healthy control . Clearance of the local Ethnics Committee was obtained to use human brain biopsies. Swiss mice weighing 20–25 g were from Charles River (Calco, Italy), and Wistar rats weighing 250–280 g were from Morini (San Paolo D'Anza, Italy). All animal procedures met the guidelines of the U.S. National Institutes of Health, detailed in the Guide for the Care and Use of Laboratory Animals, and the European Community directives regulating animal research. The experimental procedures were also approved by the local Animal Care Committee. To prepare synaptosomes from human, mouse or rat brain, fresh tissues were resuspended in ice-cold 0.32 M sucrose, 5 mM Tris.HCl buffer (pH 7.4) and were gently disrupted by 10 up-and-down strokes in a Teflon-glass homogenizer (weight/volume ratio = 1:10). The homogenate was centrifuged at 1000 × g for 5 min, at 4°C, the supernatant was then centrifuged again at 17000 × g for 15 min, at 4°C. The final pellet was resuspended in 136 mM NaCl, 5 mM KCl, 0.16 mM CaCl2, 0.1 mM EGTA, 1.3 mM MgCl2, 10 mM glucose, 10 mM Tris.HCl buffer (pH 7.4), at a protein concentration of 1 mg/ml . The activity of AMT and its kinetic properties were determined as described . Synaptosomes (100 μl/test) were incubated for different time intervals, at 37°C or 4°C to discriminate carrier-mediated from non-carrier-mediated transport of AEA through cell membranes, with 200 nM [3H]AEA (223 Ci/mmol; NEN Dupont de Nemours, Köln, Germany). Alternatively, they were incubated for 15 min with different concentrations of [3H]AEA, in the range 0–1000 nM (also in this case, the uptake at 4°C was subtracted from that at 37°C). The kinetic constants of AMT, i.e. apparent Michaelis-Menten constant (Km) and maximum velocity (Vmax), and the inhibition constant (Ki), were calculated by non-linear regression analysis of the experimental points, using a Prism 3 program (GraphPAD Software for Science, San Diego, CA). Q10 value was calculated as the ratio of AEA uptake at 30°C and 20°C . The effect of various compounds (AEA, arachidonic acid, ethanolamine, D/L-glutamine, D/L-glutamate, phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid, N-ethylmaleimide, sodium nitroprusside (SNP), all from Sigma Chemical Co., St. Louis, MO; 2-arachidonoylglycerol (2-AG) and N-(4-hydroxyphenyl)-arachidonoylamide (AM404), from Research Biochemicals International, Natick, MA; VDM11, from Tocris-Cookson, Bristol, UK; leukotriene B4 and prostaglandin E2, from Cayman Chemical Company, Ann Arbor, MI; 3-morpholinosydnonimine (SIN-1), from Alexis Corporation, Läufelfingen, Switzerland) on AMT activity was determined by adding directly each substance to the assay buffer, at the indicated concentrations, and incubating for 15 min at 37°C . Data are reported as mean (± S.D.) of at least four independent determinations, each performed in duplicate. Statistical analysis was performed by the non-parametric Mann-Whitney test, elaborating experimental data through the InStat 3 program (GraphPAD Software for Science).
Kinetic Constants of the AEA Membrane Transporter (AMT) in Brain Synaptosomes and Inhibition by Glutamine and Glutamate
793 ± 128
660 ± 80
483 ± 52
Vmax (pmol/min per mg protein)
219 ± 19
370 ± 23
357 ± 18
Ki of L-Glutamine (nM)a
1100 ± 90
970 ± 80
850 ± 80
Ki of D-Glutamine (nM)a
525 ± 50
480 ± 50
430 ± 45
Ki of L-Glutamate (nM)a
4000 ± 370
3580 ± 300
3300 ± 310
Ki of D-Glutamate (nM)a
1230 ± 110
1100 ± 90
850 ± 80
In summary, this study describes how to prepare synaptosomes suitable to investigate presence and kinetic properties of the anandamide membrane transporter in human, mouse and rat brain. This system allows to extend ex vivo the model of AEA transport already described in vitro with cell suspensions, and has the potential to provide insights on the regulation and function of AMT in the context of mature brain tissue, where control mechanisms lost in immortalized cells might be retained. In this line, the finding that AMT in synaptosomes is enhanced by nitric oxide-donor SNP, and even more by peroxynitrite-donor SIN-1, speaks in favour of a physiological meaning for the nitric oxide-mediated link between AEA receptor and AEA degradation, previously demonstrated in vitro. Moreover, the inhibition of AMT in human, mouse and rat brain synaptosomes by glutamine and glutamate seems relevant to better understand the role of AEA in glutamate-induced neurotoxity. In conclusion, brain synaptosomes appear to be a good tool for studies aimed at discovering new inhibitors of AMT of potential therapeutic value.
We are grateful to Dr. G. De Caro for kindly donating human brain specimens (Neurosurgery Division, University of Rome "Tor Vergata", Sant'Eugenio Hospital, Rome, Italy) and to Dr. M. Di Rienzo for skilfull assistance. This study was supported by Consiglio Nazionale delle Ricerche, Progetto strategico "Neuroscienze".
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