Calcium-independent formation of endocannabinoids in rat brain slices

Sarmad, Sarir (2008) Calcium-independent formation of endocannabinoids in rat brain slices. In: 18th symposium of international cannabinoids research society, June 2008, Aviemore, Scotland.

Calcium-independent formation of endocannabinoids in rat brain slices
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Item Type:Conference or Workshop contribution (Poster)
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Endocannabinoids (ECs) play physiologically essential roles in diverse biological systems. They are believed to be synthesized in cell membranes, on demand, via calcium–sensitive phospholipases, although the objective evidence for this is limited. The aim of the present study was to investigate whether EC synthesis and release in rat cerebral cortical slices is driven by increased intracellular Ca2+ concentration. We have, therefore, examined the effects of different excitatory stimuli KCl, Ca2+ ionophore (ionomycin), Ca2+-mobilising agents (glutamate and the cholinergic agonist carbachol), the Ca2+-channel blocker (verapamil), non-selective calcium channel blockers Lanthanum hydrochloride, manganese sulphate (MnSO4) and removal of Ca2+ from the medium on EC levels in rat cortical slices.
Brain slices from male Lister hooded rats (>250 g) were prepared as previously described (Sarmad et al., 2007) and EC levels measured by LC/tandem mass spectrometry. (Richardson et al., 2007). Fatty acid amide hydrolase (FAAH) activity were measured (n=3) in rat cortical slices and URB597 (1 µM), PMSF (100 µM), MAFP (10 µM) and AACOCF3 (10 µM) as respective inhibitors. Statistical analysis (one way ANOVA, Kruskal-Wallis or Dunnett’s multiple comparison test, compared basal levels with those following drug exposure.
Depolarising levels of KCl (50 mM) stimulated anandamide (AEA) synthesis significantly by 2.3±0.26 fold (mean±s.e.m) (n=6, P<0.05) and oleoylethanolamine (OEA) by 1.75±0.21fold (n=6, P<0.05) but had no effect on N-palmitoylethanolamine (PEA) and 2-arachidonoylglycerol (2-AG). Stimulation of excitatory amino acid receptors by glutamate (10 mM) and ionomycin slightly increased AEA levels but no changes were observed for OEA, PEA and 2-AG. The cholinergic receptor agonist carbachol (1mM) had no effect on ECs. Removal of Ca2+from the medium (±EGTA, 300µM) did not affect the ECs synthesis and release significantly. Verapamil (100 µM) significantly increased 2-arachidonoylglycerol (2-AG) levels in cortical slices (P<0.05, n=12) by 2.5±0.33 fold compared to basal but had no effect on other ECs. Verapamil decreased elevations of AEA, OEA and PEA due to the FAAH inhibitor URB597 (1μM) but significantly (P<0.001, n=12) enhanced URB597-elevated 2-AG by 3.69±0.74 fold.
In vitro analysis of FAAH activity indicated that verapamil up to 100 µM was without significant effect. MAGL activity was unaltered in the presence of 30 µM verapamil (92 ± 2 % control), but was inhibited slightly at 100 µM (84 ± 4 % control P<0.01).
In conclusion, we observed little evidence for Ca2+ driving EC formation in rat brain slices. Potentiation of 2-AG formation by verapamil is probably due to effects on a catabolic enzyme activity other than FAAH and MAGL.

Keywords:2-Arachidonyl glycerol, Endocannabinoids, LC-MS/MS
Subjects:B Subjects allied to Medicine > B140 Neuroscience
Divisions:College of Science > School of Chemistry
ID Code:31331
Deposited On:20 Oct 2018 20:31

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