The antagonist effect of antipsychotic drugs on human α1A1 -adrenoceptors stably expressed in a HEK-293 cell line

Z. Nourian, T. Kristensen 1, M.J. Mulvany. Departments of Pharmacology and 1Molecular Biology, University of Aarhus, Aarhus, Denmark

The α1 -adrenoceptor ( α1 -AR) family consists of the α1A , α1B and α1D subtypes. According to the recently sequenced human genome, there are at least 16 splice variants transcribed from the α1A -AR gene. Five of them, ADRA1A_v1-5, encode functional adrenoceptors that differ from each other only in the C-terminal ends and share similar pharmacology in terms of ligand binding and noradrenaline-induced [Ca2+]i responses (Hawrylyshyn, K.A. et al., 2004). We have investigated α1A -AR, the dominant subtype in the resistance vasculature, with a view to determining the basis for the orthostatic hypertensive effects of antipsychotic drugs. This study was aimed at developing a cell line which would stably express human α1A -AR, and to determine the functional response of these to the adrenoceptor agonist phenyephrine.

To determine the relevant splice variant in human resistance vessels, we took subcutaneous vessels from six different subjects (investigation approved by the Local Ethics Committee). Expression level of α1A-AR isoforms were quantified by real-time PCR. The statistical analysis showed the higher expression of α1A1 than α1A3 (1.6 times) and α1A4 (5.8 times) in human subcutaneous arteries. α1A2 and α1A5 have not been included as it has not yet been possible to obtain primers of sufficient specificity. Therefore we have expressed α1A1 -AR in Flp-In 293 cells (HEK293 cells prepared with the Flp-In system).

Total RNA for cDNA synthesis was isolated from human liver. The blunt-end a αA1 -AR cDNA was ligated into the pCRII-TOPO vector. Single colonies were isolated after transformation in TOP10 cells. After confirmation of the sequence, the α1A1 -AR cDNA was transferred to the pcDNA5/FRT expression vector. Flp-In 293 cells (Invitrogen) were transfected with the recombinant expression vector by lipofection. Selection for stably integrated cDNA in the Flp-In 293 cells was performed using hygromycine and a beta-galactosidase activity test. Successful integration was confirmed by RT-PCR and Western blotting. The developed cell line was stable. Positive functional response of the α1A1-AR was determined by measuring the elevation of [Ca2+]i in the presence of phenylephrine using a Fura-2 assay (Victor 3, Perkin-Elmer).

Cumulative concentration-response curves to phenylephrine (1 nM to 10μM, 2-4 measurements per experiment, 4-6 experiments per point) in the absence and presence of prazosin (30nM) as positive control, risperidone (3, 10, 30 nM) and haloperidol (100 nM) were constructed for the Flp-In 293 cells. Analysis showed: EC50=2.02×10-8 (control), 6.85×10-7 (30 nM prazosin), giving pA2=9.02 for prazosin: EC50=3.05×10-8 (control), 3.80×10-7 (30 nM risperidone), giving pA2=8.57 for risperidone; EC50=2.63×10-8 (control), 1.56×10-7 (100 nM haloperidol), giving pA2=7.69 for haloperidol. These values are consistent with the higher adrenoceptor antagonistic potency of risperidone than haloperidol.

In summary, we have established a stable transfected Flp-In 293 cell line expressing human α1A1 -AR that could be a useful model for future research on this subtype of α1 -AR.

Hawrylyshyn, K.A. et al. (2004) Trends in Pharmacol. Sci.25, 449-455.