Characterization of alpha(1)-adrenoceptors expressed in a novel vascular smooth muscle cell line cloned from p53 knockout mice, P53LMAC01 (AC01) cells

1 We pharmacologically studied the alpha(1)-adrenoceptor (AR) subtype(s) in volved in receptor-mediated signalling in a novel vascular smooth muscle ce ll line cloned from p53 knockout mice, P53LMAC01 (AC01) cells. 2 Radioligand binding studies with [I-125]-HEAT showed the existence of a h omogeneous population of binding site with an affinity (K-d value) of 0.4 n hl and a maximum number of binding sites (B-max) of 100 fmol mg(-1) protein . Catecholamines competed for [I-125]-HEAT binding stereospecifically and w ith the characteristic alpha(1)-AR potency series. 3 Displacement curves for BMY-7378 and KMD-3213 best fitted a one-site mode l with a pK(1) value (-log(10) (equilibrium inhibition constant)) of 6.06 a nd 7.07, respectively. 4 Reverse transcription-polymerase chain reaction (RT-PCR) assay detected a lpha(1B)- and alpha(1D)-AR, but not alpha(1A)-AR transcript. 5 Chlorethylclonidine (CEC) treatment nearly abolished (-)noradrenaline (NA ) (10 mu M)-induced inositol[1,4,5]trisphosphate (IP3) production, and BMY- 7378 inhibited the response with a K-i value of 0.3 nM, which value was sim ilar to that obtained in the cells expressing alpha(1D)-AR. In both AC01 ce lls and cells expressing alpha(1D)-AR, BMY-7378 protected alpha(1)-ARs from CEC alkylation while it had little protective effect on CEC alkylation and NA-induced IP3 production in cells expressing alpha(1B)-AR 6 The results indicate that AC01 cells contain predominantly alpha(1B)-ARs and a small population of alpha(1D)-ARs; however, phosphoinositide (PI)/Ca2 + signalling is mainly mediated through the minor population of alpha(1D)-A Rs, rather than the alpha(1B)-ARs.