Characterization of endogenous cannabinoid systems in three human cell lines

Mauro Dionisi, Paul J. Millns, Yan Sun, Andrew J. Bennett, Stephen P.H. Alexander. University of Nottingham, Nottingham, United Kingdom.

The HEK293 human embryonic kidney fibroblast, SH-SY5Y human neuroblastoma and HeLa human cervical carcinoma cell lines are widely used host cells for heterologous expression studies and as models of cellular function. We have examined these cells for elements of the cannabinoid signalling system in order to ascertain their relative merits as tools for the study of cannabinoid action and metabolism.

Expression data from Taqman® analysis of cellular mRNA were normalized to the relative expression levels of the reference gene β-actin. Fatty acid amide hydrolase activity was assessed by monitoring liberation of [3H]-ethanolamine from labelled anandamide (Boldrup et al., 2004). cAMP accumulation was measured using the [3H]-adenine pre-labelling methodology (Alexander et al., 1996).

mRNA expression of CB1 cannabinoid receptors was greatest in HEK293 cells (0.35 arbitrary units relative to β-actin mRNA levels), and much lower in SH-SY5Y (0.034) and HeLa cells (2.7 x 10-4). A similar pattern of expression of CB2 receptor mRNA was observed: HEK293 (2.89) > SH-SY5Y (0.28) > HeLa (6.1 x 10-3). Fatty acid amide hydrolase mRNA levels, however, appeared similar in HEK293 (0.8) and SH-SY5Y cells (0.85), while HeLa expression levels were much lower (4.9 x 10-2). FAAH activity in cellular homogenates was unexpectedly higher in HEK293 cells compared to SH-SY5Y cells (128 ± 58 compared to 19 ± 6 pmol min-1 mg-1 protein, respectively; n=2). Incubation with the selective inhibitor of fatty acid amide hydrolase URB597 (1 μM) reduced activity markedly in both cell line homogenates (6 and 5 pmol min-1 mg-1 protein, respectively). As expected (Day et al., 2001), fatty acid amide hydrolase activity in HeLa cells was much lower (5.0 ± 0.1 pmol min-1 mg-1 protein) and unaffected by the presence of URB597 (5 pmol min-1 mg-1 protein). Preliminary studies in SH-SY5Y cells, demonstrated that forskolin (30 μM) evoked a significant enhancement of cAMP accumulation (basal 0.72 ± 0.12; forskolin 9.17 ± 0.47 % conversion from total [3H]-adenine nucleotides, n=4). However, CP55940 (1 μM) failed to alter the forskolin response (10.14 ± 0.31, n=2), while anandamide (10 μM) actually increased cAMP accumulation (12.18 ± 0.28, n=4, P<0.01 repeated measures ANOVA with Dunnetts’ multiple comparison).

These data indicate differential endogenous expression patterns of cannabinoid receptor and fatty acid amide hydrolase in human cell lines. The disparity between mRNA levels of fatty acid amide hydrolase and enzyme activity indicates that FAAH activity may be controlled by post-transcriptional mechanisms.

Alexander, SPH, et al. (1996). Br J Pharmacol 119:1286-1290

Boldrup, L, et al. (2004). J Biochem Biophys Methods 60:171-177

Day, TA, et al. (2001). Mol Pharmacol 59:1369-1375

We thank the MRC for a Studentship (MD).