Direct demonstration of β 1 ‐ and evidence against β 2 ‐ and β 3 ‐adrenoceptors, in smooth muscle cells of rat small mesenteric arteries

Recent evidence supports additional subtypes of vasodilator β ‐adrenoceptor ( β ‐AR) besides the ‘classical’ β 2 . The aim of this study was to investigate the distribution of β ‐ARs in the wall of rat mesenteric resistance artery (MRA), to establish the relative roles of β ‐ARs in smooth muscle and other cell types in mediating vasodilatation and to analyse this in relation to the functional pharmacology. We first examined the vasodilator β ‐AR subtype using ‘subtype‐selective’ agonists against the, commonly employed, phenylephrine‐induced tone. Concentration‐related relaxation was produced by isoprenaline (pEC 50 : 7.70±0.1) ( β 1 and β 2 ). Salbutamol ( β 2 ), BRL 37344 ( β 3 ) and CGP 12177 (atypical β ) caused relaxation but were 144, 100 and 263 times less potent than isoprenaline; the ‘ β 3 ‐adrenoceptor agonist’ CL 316243 was ineffective. In arteries precontracted with 5‐HT or U 46619, isoprenaline produced concentration‐related relaxation but salbutamol, BRL 37344, CGP 12177 and CL 316243 did not. SR 59230A, CGP 12177 and BRL 37344 caused a parallel rightward shift in the concentration–response curve to phenylephrine indicating competitive α 1 ‐AR antagonism, explaining the false‐positive ‘vasodilator’ action against phenylephrine‐induced tone. Endothelial denudation but not L ‐NAME slightly attenuated isoprenaline‐mediated vasodilatation in phenylephrine and U 46619 precontracted MRA. The β ‐AR fluorescent ligand BODIPY TMR‐CGP 12177 behaved as an irreversible β 1 ‐AR antagonist in MRA and bound to the surface and inside vascular smooth muscle cells in intact vascular wall. β ‐ARs in smooth muscle cells were observed in a perinuclear location, consistent with the location of Golgi and endoplasmic reticulum. Binding of BODIPY TMR‐CGP 12177 was inhibited by BAAM (1 μ M ) in all three vascular tunics, confirming the presence of β ‐ARs in adventitia, media and intima. Binding in adventitia was observed in both neuronal and non‐neuronal cell types. Lack of co‐localisation with a fluorescent ligand for α ‐ARs confirms the selectivity of BODIPY TMR‐CGP 12177 for β ‐ARs over α ‐ARs. Our results support the presence of functional vasodilator β 1 ‐ARs and show that they are mainly located in smooth muscle cells. Furthermore, we have demonstrated, for the first time, the usefulness of BODIPY TMR‐CGP 12177 for identifying β ‐AR distribution in the ‘living’ vascular wall. British Journal of Pharmacology (2005) 146 , 679–691. doi: 10.1038/sj.bjp.0706369