Pharmacological evaluation of endocannabinoid synthesis and release in rat cortical slices

Sarmad, Sarir (2006) Pharmacological evaluation of endocannabinoid synthesis and release in rat cortical slices. In: BPS 75th anniversary meeting, December 2006, Oxford UK.

Pharmacological evaluation of endocannabinoid synthesis and release in rat cortical slices
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Item Type:Conference or Workshop contribution (Poster)
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Endocannabinoids (ECs) are proposed to be extracellular retrograde messengers that regulate the excitability of neurons in the brain by CB1 receptor-dependent inhibition of neurotransmitter release. The archetypal EC, anandamide (AEA), can also have excitatory effects by activating TRPV1 receptors at the intracellular face of neuronal plasma membranes. It is thus important to understand the factors regulating intracellular and extracellular EC concentrations. EC levels are governed by the balance between synthesis, metabolism and trans-membrane transport and we have, therefore, examined the effects of different excitatory stimuli and of URB597, an inhibitor of the major AEA catabolic enzyme {fatty acid amide hydrolase (FAAH)}, on intra- and extracellular ECs in a brain slice preparation.

Mini-prisms (350x350μm) were prepared from the cerebral cortices of male Lister Hooded rats (>250g). Following a 60 min incubation in Krebs Henseleit buffer (KHB, pH 7, 37oC) 50μl aliquots of slices were transferred to 5ml insert vials containing KHB with 1% BSA , final volume 500 μl, and exposed to drugs for 20 min, after which the slices were separated from the medium by rapid filtration under vacuum. Medium and slices were rapidly frozen in liquid nitrogen and stored at -80oC prior to assay. Following organic solvent and solid phase extraction, EC levels were measured by liquid chromatography-tandem mass spectrometry (Richardson et al., in press). All drugs except for carbachol and glutamate (KHB) were dissolved in ethanol to a stock concentration of 10-2M with subsequent dilution in KHB. Statistical analysis (Student’s t-test) compared basal levels with those following drug exposure.

Depolarizing levels of KCl (50 mM) stimulated anandamide (AEA) synthesis in the tissue (basal level 6.0±1.4 pmol/g, n=4) by 2.7±0.2 fold (n=4, P =0.001; mean ± s.e.m.), oleoylethanolamide (OEA) by 2.03 ±0.16 fold (n=3, P=0.0079) whilst no changes were observed for 2-arachidonoyl glycerol (2-AG ) or palmitoyl-ethanolamide (PEA). Stimulation of excitatory amino acid receptors by glutamate (10 mM) enhanced AEA synthesis non-significantly by 1.9±0.6 fold (n=3, P=0.2309) with no effect on other ECs. The cholinergic receptor agonist carbachol (1mM) slightly increased AEA synthesis by 1.2 fold (n=9, P=0.3), but had no effect on 2-AG, OEA or PEA. The TRPV1 agonist capsaicin (1μM) had no effect. Blockade of FAAH by URB597 (1μM) significantly increased anandamide levels in the slices (3.2±0.3 fold, n=33 P<0.0001), OEA by 2.0±0.2 fold (n=30, P=0.0001) and PEA by 1.5±0.1 fold (n=25, P=0.0227) but had no effect on 2-AG. The increased EC levels due to URB597 were not altered in the presence of the CB1 receptor antagonist (SR141617A, 1μM) or the TRPV1 antagonist, capsazepine (1μM). Basal EC levels detected in the medium were; AEA (0.5±0.1), 2-AG (2.9±0.8), OEA (39.2±8.31) and PEA (243.1±37.4) pmol/ml. These were enhanced by URB597; AEA by 4.3±0.6 fold (n=5, P=0.0190), OEA 1.9±0.2 fold (n=5, P=0.0087) but PEA 1.3±0.07 fold (n=5, P=0.39) and 2-AG 1.3±0.2 fold (n=5, P=0.4801) non-significantly. The effects of the other drugs on extracellular ECs mirrored tissue changes with the exception that no increased release was observed with K+ (50 mM) or carbachol.

The data presented indicate differential effects of excitatory stimuli on EC levels in brain slices and provide no evidence for tonic regulation of synthesis by CB1 or TRPV1 receptors. The effects of URB597 suggest a high turnover rate of AEA, PEA and OEA in this preparation and that 2-AG is not subject to catabolism by FAAH. The release of ECs from cortical slices appears, in the main, to be passively driven by the trans-membrane concentration gradient. The cortical slice preparation provides, therefore, a suitable in vitro model for the investigation of EC synthesis, metabolism and release in the brain.

Keywords:Endocannabinoids, Mass spectrometry, FAAH inhibitor
Subjects:B Subjects allied to Medicine > B140 Neuroscience
Divisions:College of Science > School of Chemistry
ID Code:31327
Deposited On:20 Oct 2018 20:28

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