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

• Published in: British journal of pharmacology 1999-06, Vol.127 (3), p.756-762
• Main Authors: Ohmi, K, Shinoura, H, Nakayama, Y, Goda, N, Tsujimoto, G
• Format: Article
• Language: English
• Subjects: Alkylation; Animals; Aorta; Binding Sites - drug effects; Cell Line; Cells, Cultured; CHO Cells; Clone Cells; Clonidine - analogs & derivatives; Clonidine - pharmacology; Cricetinae; Humans; Inositol 1,4,5-Trisphosphate - biosynthesis; Mice; Mice, Knockout; Muscle, Smooth, Vascular - metabolism; Muscle, Smooth, Vascular - physiology; Piperazines - pharmacology; Receptors, Adrenergic, alpha-1 - biosynthesis; Receptors, Adrenergic, alpha-1 - genetics; Receptors, Adrenergic, alpha-1 - metabolism; Receptors, Adrenergic, alpha-1 - physiology; RNA, Messenger - biosynthesis; Tumor Suppressor Protein p53 - deficiency; Tumor Suppressor Protein p53 - genetics
• ISSN: 0007-1188

1. We pharmacologically studied the alpha 1-adrenoceptor (AR) subtype(s) involved in receptor-mediated signalling in a novel vascular smooth muscle cell line cloned from p53 knockout mice, P53LMAC01 (AC01) cells. 2. Radioligand binding studies with [125I]-HEAT showed the existence of a homogeneous population of binding site with an affinity (Kd value) of 0.4 nM and a maximum number of binding sites (Bmax) of 100 fmol mg-1 protein. Catecholamines competed for [125I]-HEAT binding stereospecifically and with the characteristic alpha 1-AR potency series. 3. Displacement curves for BMY-7378 and KMD-3213 best fitted a one-site model with a pKi value (-log10 (equilibrium inhibition constant)) of 6.06 and 7.07, respectively. 4. Reverse transcription-polymerase chain reaction (RT-PCR) assay detected alpha 1B- and alpha 1D-AR, but not alpha 1A-AR transcript. 5. Chlorethylclonidine (CEC) treatment nearly abolished (-)noradrenaline (NA) (10 microM)-induced inositol[1,4,5]trisphosphate (IP3) production, and BMY-7378 inhibited the response with a Ki value of 0.3 nM, which value was similar to that obtained in the cells expressing alpha 1D-AR. In both AC01 cells 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-ARs, rather than the alpha 1B-ARs.