The antagonistic effect of antipsychotic drugs on a HEK293 cell line stably expressing human α1A1-adrenoceptors

Antipsychotic drugs often cause orthostatic hypotension, probably through antagonist action on resistance vessel α1A-adrenoceptors. Here we have tested this possibility directly using cells transfected with a relevant human α1A-adrenoceptor splice variant. To determine a splice variant which was relevant, we used quantitative real-time polymerase chain reaction (qPCR) to determine the prevalence in human subcutaneous small arteries of three of the five splice variants ADRA1A_v1-5, which encode functional protein: α1A1-, α1A3-, α1A4-adrenoceptors. Our statistical analysis showed higher transcription levels of α1A1- than of α1A3- and α1A4-adrenoceptors (1.6 and 5.8 times, respectively). We therefore chose to study the α1A1-adrenoceptor, and the cDNA encoding it was transfected into the Flp-In-293 (modified from HEK-293) cell line to produce a cell line stably expressing a functional form of this splice variant. The expression of recombinant α1A1-adrenoceptor subtype was confirmed by Western immunoblot analysis, and its functionality demonstrated using a Fura-2 assay by a rise in intracellular calcium concentration ([Ca2+]i) when challenged with phenylephrine (EC50 = 1.61 × 10− 8 M). From Schild analysis, prazosin, sertindole, risperidone, and haloperidol caused a concentration-dependent, rightward shift of the cumulative concentration–response curves for phenylephrine in cells expressing human recombinant α1A1-adrenoceptors to yield pKB values of 8.40, 8.05, 8.26 and 7.38, respectively. In [7-methoxy-3H]-prazosin binding experiments, high expression was seen (Bmax = 48.5 ± 16.7 pmol/mg protein, ± S.E.M.) along with high affinity binding to a single site (Kd = 0.210 ± 0.034 nM). The pharmacological profiles of recombinant human α1A1-adrenoceptors in competition binding studies confirmed much higher antagonist affinity of sertindole and risperidone than haloperidol for these receptors. In summary, it can be concluded that there is an approximately 10-fold higher adrenoceptor affinity of risperidone and sertindole for human α1A1-adrenoceptors compared to haloperidol. These findings are consistent with the observation that risperidone and sertindole have a higher incidence of orthostatic hypotension than haloperidol.