We pharmacologically studied the a,-adrenoceptor (AR) subtype(s) involved in receptormediated 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 (K, value) of 0.4 nM and a maximum number of binding .sites (Bmm) of lOOfmolmg"1 protein. Catecholamines competed for [125I]-HEAT binding stereospecifically and with the characteristic oi-AR potency series. 3 Displacement curves for BMY-7378 and KMD-3213 best fitted a one-site model with a pKj value (-logio (equilibrium inhibition constant)) of 6.06 and 7.07, respectively. 4 Reverse transcription-polymerase chain reaction (RT-PCR) assay detected m- and aiD-AR, but not IA-AR transcript. 5 Chlorethylclonidine (CEC) treatment nearly abolished (-)noradrenaline (NA) (10 /(M)-induced inositol[l,4,5]trisphosphate (IP3) production, and BMY-7378 inhibited the response with a K, value of 0.3 nM, which value was similar to that obtained in the cells expressing 1D-AR. In both AC01 cells and cells expressing ociD-AR, BMY-7378 protected i-ARs from CEC alkylation while it had little protective effect on CEC alkylation and NA-induced IP3 production in cells expressing a,B-AR. 6 The results indicate that AC01 cells contain predominantly a]B-ARs and a small population of a1D-ARs; however, phosphoinositide (PI)/Ca2 + signalling is mainly mediated through the minor population of a)D-ARs, rather than the iB-ARs.
- Vascular smooth muscle cells
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