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

Briones, A. M., Daly, C. J. , Jimenez-Altayo, F., Martinez-Revelles, S., Gonzalez, J. M., McGrath, J. C. and Vila, E. (2005) Direct demonstration of β1- and evidence against β2- and β3-adrenoceptors, in smooth muscle cells of rat small mesenteric arteries. British Journal of Pharmacology, 146(5), pp. 679-691. (doi: 10.1038/sj.bjp.0706369)

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Publisher's URL: http://dx.doi.org/10.1038/sj.bjp.0706369

Abstract

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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:McGrath, Professor John and Daly, Professor Craig
Authors: Briones, A. M., Daly, C. J., Jimenez-Altayo, F., Martinez-Revelles, S., Gonzalez, J. M., McGrath, J. C., and Vila, E.
College/School:College of Medical Veterinary and Life Sciences > School of Life Sciences
Journal Name:British Journal of Pharmacology
Publisher:Wiley
ISSN:0007-1188
ISSN (Online):1476-5381

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