Direct demonstration of β1‐ and evidence against β2‐ and β3‐adrenoceptors, in smooth muscle cells of rat small mesenteric arteries

• Published in: British journal of pharmacology 2005-11, Vol.146 (5), p.679-691
• Main Authors: Briones, Ana M, Daly, Craig J, Jimenez‐Altayo, Francesc, Martinez‐Revelles, Sonia, Gonzalez, Jose M, McGrath, John C, Vila, Elisabet
• Format: Article
• Language: English
• Subjects: Adrenoceptors; Biological and medical sciences; fluorescent ligands; imaging; Medical sciences; Pharmacology. Drug treatments; vascular smooth muscle, mesenteric resistance artery; β‐adrenoceptors
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0706369

Recent evidence supports additional subtypes of vasodilator β‐adrenoceptor (β‐AR) besides the ‘classical’ β2. 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 (pEC50: 7.70±0.1) (β1 and β2). Salbutamol (β2), BRL 37344 (β3) and CGP 12177 (atypical β) caused relaxation but were 144, 100 and 263 times less potent than isoprenaline; the ‘β3‐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 α1‐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 β1‐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 μM) 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 β1‐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