TY - JOUR
T1 - Direct demonstration of β 1- and evidence against β 2- and β 3-adrenoceptors, in smooth muscle cells of rat small mesenteric arteries
AU - Briones, Ana M.
AU - Daly, Craig J.
AU - Martinez-Revelles, Sonia
AU - Gonzalez, Jose M.
AU - McGrath, John C.
AU - Vila, Elisabet
PY - 2005/11/1
Y1 - 2005/11/1
N2 - 1. 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. 2. 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. 3. 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. 4. 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. 5. 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. 6. 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. © 2005 Nature Publishing Group All rights reserved.
AB - 1. 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. 2. 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. 3. 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. 4. 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. 5. 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. 6. 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. © 2005 Nature Publishing Group All rights reserved.
KW - Adrenoceptors
KW - Fluorescent ligands
KW - Imaging
KW - Mesenteric resistance artery
KW - Vascular smooth muscle
KW - β-adrenoceptors
U2 - https://doi.org/10.1038/sj.bjp.0706369
DO - https://doi.org/10.1038/sj.bjp.0706369
M3 - Article
VL - 146
SP - 679
EP - 691
JO - British Journal of Pharmacology
JF - British Journal of Pharmacology
SN - 0007-1188
ER -