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) 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.
Department of Pathology, National Children's Medical Research Center, Tokyo, Japan.
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Ohmi
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Shinoura
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Nakayama
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Goda
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Tsujimoto
D000478:Alkylation; D000818:Animals; D001011:Aorta; D001665:Binding Sites / Q000187:drug effects; D016466:CHO Cells; D002460:Cell Line; D002478:Cells, Cultured; D002999:Clone Cells; D003000:Clonidine / Q000031:analogs & derivatives / Q000494:pharmacology; D006224:Cricetinae; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000378:metabolism / Q000502:physiology*; D010879:Piperazines / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D018340:Receptors, Adrenergic, alpha-1 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency / Q000235:genetics
D010879:Piperazines; D012333:RNA, Messenger; D018340:Receptors, Adrenergic, alpha-1; D016159:Tumor Suppressor Protein p53; C051731:chlorethylclonidine; D015544:Inositol 1,4,5-Trisphosphate; C053428:BMY 7378; D003000:Clonidine