Comparison of the pharmacological profile of the native dopamine D1 receptor in SK-N-MC human neuroblastoma cells using cAMP and label-free determinations

Martyn Wood, Michel Gillard, Maggi Burton. UCB Pharma SA, Braine-l\'Alleud, Belgium

The human SK-N-MC neuroblastoma cell line endogenously expresses the dopamine (DA) D1 receptor, but not D2 or D5 receptors. They are therefore a useful tool to study the pharmacological profile of the D1 receptor under physiological expression levels and with the appropriate signaling machinery.D1 receptors in SK-N-MC cells induce changes in cellular dynamic mass redistribution (DMR; Peters & Scott, 2009). This response was mediated by Gi/Go proteins as it was pertussis-toxin (PTX) sensitive. Classically the D1 receptor activates Gs protein. We have compared the pharmacological profile of the D1 receptor in SK-N-MC cells using changes in cAMP and in DMR for Gs and Gi/o signalling.

SK-N-MC cells were cultured in complete medium with 10% v/v dialysed FBS and assays were run in HBSS with 20 mM HEPES (pH 7.4). Baseline readings in DMR (Corning Epic, Amsterdam, The Netherlands) in the presence or absence of antagonist were taken for 30 min, agonist or buffer added and the response followed for a further 120 min and peak response minus basal determined.). Cells (6,500 per well) were incubated in buffer containing IBMX (100 μM) for 1 hour at 37oC in the presence or absence of antagonist and/or agonist and cAMP levels determined (CisBio HTRF, Codolet, France). Data was analysed using 4-parameter logistic equation in GraphPad Prism (San Diego CA

Agonist induced changes in DMR and cAMP are shown below (potency as pEC50 and efficacy as the Emax compared to DA):

Data are mean + s.e.m. from (n) separate determinations

The DA response was antagonised by SCH23390 with pKb of 10.1 for the DMR response and 9.7 for the cAMP response.There was increase in functional potency and relative efficacy in the DMR signal compared to cAMP but the rank order was maintained. Apomorphine and rotigotine were partial agonists on the cAMP response but full agonists on the DMR response. This can be explained by a higher degree of receptor-response coupling.(higher receptor reserve) for the DMR signal.

In conclusion, the pharmacological profile of D1 mediated changes in cAMP and DMR are similar.in SK-N-MC cells. It would be interesting to confirm the PTX sensitivity, to look at changes in cAMP in the presence of forskolin (to measure the Gi response) and to test further compounds for biased signalling.

Peters M.E & Scott C.W., J Biomol Screening 14: 246, 2009