CALCIUM-INDEPENDENT FORMATION OF ENDOCANNABINOIDS IN RAT BRAIN SLICES.

Sarir Sarmad, Annie Patel, David Barrett, Charles Marsden, Stephen Alexander, David Kendall
1School of Biomedical Sciences (University of Nottingham), Nottingham, United Kingdom, 2School of Pharmacy, Nottingham, United Kingdom

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 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) and removal of Ca2+ from the medium on EC levels in rat cortical slices.

Male Lister hooded rats (>250 g) decapitated and brain slices 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) and monoacylglycerol lipase (MAGL) activity were measured (n=3) in rat brain soluble fraction and rat liver microsomes, using [3H]-2-oleoylglycerol (2 μM) and oleamide (100 μM) as respective substrates and methylarachidonoylfluorophosphonate (1 μM) and URB597 (10 μM) as respective inhibitors (Patel et al., 2007; Vandevoorde et al., 2005). 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 changed AEA levels (non- significantly) 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.

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