1: Biochem Pharmacol. 2003 Sep 1;66(5):757-67.  

Inhibition of C6 glioma cell proliferation by anandamide,
1-arachidonoylglycerol, and by a water soluble phosphate ester of anandamide:
variability in response and involvement of arachidonic acid.

Fowler CJ, Jonsson KO, Andersson A, Juntunen J, Jarvinen T, Vandevoorde S,
Lambert DM, Jerman JC, Smart D.

Department of Pharmacology and Clinical Neuroscience, Umea University, SE-90187
Umea, Sweden. cf@pharm.umu.se

It has previously been shown that the endocannabinoids anandamide and
2-arachidonoylglycerol (2-AG) inhibit the proliferation of C6 glioma cells in a
manner that can be prevented by a combination of capsazepine (Caps) and
cannabinoid (CB) receptor antagonists. It is not clear whether the effect of
2-AG is due to the compound itself, due to the rearrangement to form
1-arachidonoylglycerol (1-AG) or due to a metabolite. Here, it was found that
the effects of 2-AG can be mimicked with 1-AG, both in terms of its potency and
sensitivity to antagonism by Caps and CB receptor antagonists. In order to
determine whether the effect of Caps could be ascribed to actions upon vanilloid
receptors, the effect of a more selective vanilloid receptor antagonist,
SB366791 was investigated. This compound inhibited capsaicin-induced Ca(2+)
influx into rVR1-HEK293 cells with a pK(B) value of 6.8+/-0.3. The combination
of SB366791 and CB receptor antagonists reduced the antiproliferative effect of
1-AG, confirming a vanilloid receptor component in its action. 1-AG, however,
showed no direct effect on Ca(2+) influx into rVR1-HEK293 cells indicative of an
indirect effect upon vanilloid receptors. Identification of the mechanism
involved was hampered by a large inter-experimental variation in the sensitivity
of the cells to the antiproliferative effects of 1-AG. A variation was also seen
with anandamide, which was not a solubility issue, since its water soluble
phosphate ester showed the same variability. In contrast, the sensitivity to
methanandamide, which was not sensitive to antagonism by the combination of Caps
and CB receptor antagonists, but has similar physicochemical properties to
anandamide, did not vary between experiments. This variation greatly reduces the
utility of these cells as a model system for the study of the antiproliferative
effects of anandamide. Nevertheless, it was possible to conclude that the
antiproliferative effects of anandamide were not solely mediated by either its
hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of
phospholipase A(2) since palmitoyltrifluoromethyl ketone did not prevent the
response to anandamide. The same result was seen with the fatty acid amide
hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic
acid by administration of arachidonic acid methyl ester did not affect cell
proliferation, and the modest antiproliferative effect of umbelliferyl
arachidonate was not prevented by a combination of Caps and CB receptor
antagonists.

PMID: 12948856 [PubMed - indexed for MEDLINE]