The effect of differentiation on adenosine receptor expression in SH-SY5Y human neuroblastoma cells

Nouh M. H. Aljarari & Stephen P.H. Alexander, Institute of Neuroscience and School of Biomedical Sciences, University of Nottingham Medical School, Nottingham NG7 2UH, ENGLAND.

Adenosine ( ADO) is a ubiquitous signalling molecule, the majority of the effects of which are mediated through four cell-surface receptors (A1, A2A, A2B & A3, Alexander et al. 2006). SH-SY5Y cells are a commonly-employed human-derived neuroblastoma cell line, which have been previously described to express adenosine receptors (Peterfreund et al. 1997). We have, therefore, examined this cell line to investigate the expression of adenosine receptors at gene, protein and functional levels, before & after differentiation.

RT-PCR using human-specific primers was employed to assess expression of mRNA encoding adenosine receptors, while immuno-blot analysis was performed using specific antibodies directed against human A2A and A2B receptors. Functional receptors were investigated through estimating changes in [3H]-cyclic AMP accumulation in [3H]-adenine pre-labelled cells. Differentiation of SH-SY5Y cells was conducted over 6 days using 10µM retinoic acid (RA) or 10nM tetradecanoyl phorbol acetate (TPA). Data presented were obtained from 3 separate passages (29-39) of cells and were analysed for statistical significance using one-way ANOVA with post-hoc Dunnett’s comparison test.

RT-PCR analysis (30 cycles) allowed the detection of mRNA encoding A2A and A2B receptors, while A1 receptor expression was only detected at 52 cycles. In contrast, there was no evidence for A3 receptor expression up to 52 cycles. Following 6-day exposure to RA or TPA, an apparently selective increase in A2A receptor mRNA was observed, as assessed visually. Immuno-blot analysis revealed that RA- and TPA-exposed cells showed significantly enhanced levels of Bcl-2 immunoreactivity, from 22 ± 3 to 45 ± 3 (P<0.001) and 91 ± 5 (P<0.001) % relative to β-actin immunoreactivity. This was accompanied by a significant increase of A 2A–like immunoreactivity compared to control (RA from 18 ± 1 to 45 ± 3, P<0.01; and TPA from 54 ± 3 to 115 ± 8, P<0.001). In contrast, A2B receptor immunoreactivity exhibited a small but significant decrease from 166 ± 9 to 135 ± 12 (P<0.001) after RA exposure, with no concomitant change following TPA (254 ± 11 to 261 ± 14). Undifferentiated SH-SY5Y cells exhibited a maximal cyclic AMP accumulation over basal of 0.70 ± 0.17 % conversion from [3H]-adenine nucleotides in response to the non-selective adenosine receptor agonist 5’-N-ethylcarboxamidoadenosine (NECA). Responses to 10µM NECA were antagonised by the A 2A – selective antagonist SCH58261 (1µM, 27 ± 5 % control, P<0.01), but not significantly by the A2B-selective MRS1754 (1µM, 77 ± 2). Following differentiation with RA and TPA, R max values were increased to 0.85 ± 0.17 % (P<0.05) and 1.73 ± 0.27 % (P<0.05), respectively. The potency (pEC50 values) of NECA-evoked cyclic AMP responses increased from 5.7 ± 0.2 to 7.4 ± 0.4 (P<0.05) and 6.9 ± 0.4 (P<0.05), in RA and TPA studies, respectively.

In summary, we observe selective expression of A2A and A2B receptors in SH-SY5Y cells, with a significant up-regulation of A2A receptors in both RA- and TPA-differentiated cells.

Alexander, S.P.H. et al. (2006). Br. J. Pharmacol., 147, S1-S183.
Peterfreund, R.A. et al. (1997). Eur. J. Pharmacol. , 336, 71-80.

 We thank the Libyan Government for financial support.