Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 May;139(2):232-42.
doi: 10.1038/sj.bjp.0705287.

Pharmacology and direct visualisation of BODIPY-TMR-CGP: a long-acting fluorescent beta2-adrenoceptor agonist

Jillian G Baker  1 , Ian P Hall, Stephen J Hill
Affiliations

Pharmacology and direct visualisation of BODIPY-TMR-CGP: a long-acting fluorescent beta2-adrenoceptor agonist

Jillian G Baker et al. Br J Pharmacol. 2003 May.

Abstract

1 Fluorescence techniques offer a way to circumvent several problems associated with many radioligand binding and functional assays and the need for large numbers of cells. Fluorescent ligands also offer the advantage of allowing real time direct visualisation of ligand - receptors interactions. A fluorescent analogue of CGP 12177 (BODIPY-TMR-CGP) has thus been evaluated as a beta(2)-adrenoceptor ligand in CHO-K1 cells expressing the human beta(2)-adrenoceptor. 2 Studies of (3)H-cAMP accumulation showed that BODIPY-TMR-CGP stimulated an increase in cAMP accumulation and cyclic AMP response element (CRE)-mediated gene transcription with an EC(50) of 21-28 nM. Both of these responses were antagonised by the selective beta(2)-adrenoceptor antagonist ICI 118551. 3 Binding studies with (3)H-CGP 12177, and functional studies of CRE-regulated gene transcription showed that the BODIPY-TMR-CGP interaction with the human beta(2)-adrenoceptor is of very long duration. 4 Visualisation of the binding of BODIPY-TMR-CGP to single living mammalian cells was clearly demonstrated by confocal microscopy and showed that this ligand was able to selectively label cell surface beta(2)-adrenoceptors in living CHO-K1 cells transfected with the human beta(2)-adrenoceptor with an apparent K(D) of 27 nM. Studies with cells expressing a beta(2)-adrenoceptor-green fluorescent protein (GFP) fusion protein provided further strong evidence that BODIPY-TMR-CGP was binding to the beta(2)-adrenoceptor. 5 BODIPY-TMR-CGP is therefore a long-acting fluorescent beta(2)-adrenoceptor agonist that can be used to label beta(2)-adrenoceptors in the plasma membrane of living cells.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Structure of BODIPY-TMR-CGP.
Figure 2
Figure 2
(a) CRE-mediated SPAP production in response to isoprenaline and BODIPY-TMR-CGP obtained from CHO-β2 cells. Data points are mean±s.e.m. (triplicate determinations) from a single experiment and are representative of 15 separate experiments. Bar represents the SPAP production from unstimulated cells. (b) SPAP production induced by BODIPY-TMR-CGP in the presence and absence of 30 nM ICI 118551. Points represent mean±s.e.m. of triplicate determinations. The bars show basal SPAP production and that in response to 30 nM ICI 118551 and this single experiment is representative of nine separate determinations. (c) SPAP production induced by isoprenaline in the presence and absence of 100 nM BODIPY-TMR-CGP. Bars represent basal response and SPAP production in the presence of 100 nM BODIPY-TMR-CGP. Data points are mean±s.e.m. (triplicate determinations) and this single experiment is representative of six separate determinations.
Figure 3
Figure 3
SPAP production induced by BODIPY-TMR-CGP in the presence and absence of 100 nM isoprenaline. Bars represent basal response and SPAP production in the presence of 100 nM isoprenaline. Data points are mean±s.e.m. (triplicate determinations) and this single experiment is representative of four separate experiments.
Figure 4
Figure 4
(a) Specific binding of 3H-CGP 12177 to CHO-β2 cells in control conditions (absence of BODIPY-TMR-CGP; 2 h incubation, filled circles), in the presence of 100 nM BODIPY-TMR-CGP (BOD-CGP, open circles) added 1 h before 3H-CGP 12177 (2 h subsequent incubation in the presence of both ligands), and following wash out of the BODIPY-TMR-CGP before addition of 3H-CGP 12177 (triangles). In this latter case, BODIPY-TMR-CGP was incubated for 1 h, then each well washed twice with 200 μl phosphate-buffered saline. The cells were then incubated in media for a further hour in order to give time for any BODIPY-TMR-CGP to dissociate from the receptor. The cells were then washed twice again before the 3H-CGP 12177 was added for a 2 h incubation. Nonspecific binding was determined in the presence of 100 nM ICI 118551. Data points represent mean±s.e.m. for triplicate values and this single experiment is representative of four separate experiments. Curves were fitted by nonlinear regression as described under Methods. Fitted parameters in this experiment for BMAX and KD were: (filled circles) 784.7 fmol mg protein−1, 0.23 nM; (open circles) 315.9 fmol mg protein−1, 4.8 nM; (triangles) 313.1 fmol mg protein−1, 0.36 nM. (b) Inhibition of the specific binding of 0.3 nM 3H-CGP 12177 by BODIPY-TMR-CGP (BOD-CGP). Closed symbols show BODIPY-TMR-CGP and 3H-CGP 12177 after 2 h incubation. Open symbols are where the BODIPY-TMR-CGP was washed out as described in (a) before incubation with radioligand. Data points represent mean±s.e.m. for triplicate determinations in a single experiment and is representative of four experiments.
Figure 5
Figure 5
Confocal images and quantification of the binding of increasing concentrations (3–300 nM) of BODIPY-TMR-CGP (10 min exposure) to CHO-β2 cells. Data are from a single experiment and are representative of four separate experiments. The final panel shows the quantification of these data. Average pixel intensities were calculated from the grey-scale images as described under Methods. Values represent the mean of up to four separate images obtained in a single experiment. Similar data were obtained in three other experiments. Cells were imaged following washout of all ligands.
Figure 6
Figure 6
Confocal images showing the effect of increasing concentrations of 30 min preincubation with (a) ICI 118551 or (b) CGP 12177 on the binding of 50 nM BODIPY-TMR-CGP to CHO-β2 cells (10 min) in the continued presence of ICI 118551 or CGP 12177. Images shown are from a single experiment. The final panel shows the quantification of these data. Average pixel intensities were calculated from the grey-scale images as described under Methods. Values represent the mean of up to four separate images obtained in a single experiment. Similar data were obtained in (a) three and (b) two other experiments. Cells were imaged following washout of all ligands.
Figure 7
Figure 7
Confocal images showing the effects of 50–300 nM concentrations of BODIPY-TMR-CGP (10 min exposure) in CHO-β2 cells and control CHO-K1 cells. In CHO-β2 cells experiments were performed with and without 30 min pre-incubation with 100 nM ICI 118551. Inset images show a close up of a group of cells (from the centre of each image) at four times the magnification of the rest of the image. Images are from a single experiment that was representative of 155 cells in 10 different experiments. Cells were imaged following washout of all ligands.
Figure 8
Figure 8
Binding of BODIPY-TMR-CGP to CHO-β2-GFP cells. In (b) the fluorescence monitored in the green GFP channel (488 nm) is shown. (a) Shows the binding of 50 nM BODIPY-TMR-CGP to the same cells as in (b) in the red channel (543 nm). (d) Shows an overlay of the two images (a and b) with colocalisation shown by the yellow pixels. (c) Shows the phase contrast image of these cells. In this case, the BODIPY-TMR-CGP has not been washed out before imaging.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. ARUNLAKSHANA O., SCHILD H.O. Some quantitative uses of drug antagonists. Br. J. Pharmacol. Chemother. 1959;14:48–58. - PMC - PubMed
    1. BAKER J.G., HALL I.P., HILL S.J.Pharmacology and direct visualization of the fluorescent β2-adrenoceptor ligand BODIPY-CGP 12177 in CHO cells transfected with the human β2-adrenoceptor Br. J. Pharmacol. 200113450p
    1. BAKER J.G., HALL I.P., HILL S.J. Agonist and Antagonist effects of CGP 12177 on β2-adrenoceptor-stimulated gene transcription in CHO cells transfected with the human β2-adrenoceptor. Br. J. Pharmacol. 2002;137:400–408. - PMC - PubMed
    1. COLEMAN R.A., JOHNSON M., NIALS A.T., VARDEY C.J. Exosites: their current status, and their relevance to the duration of action of long-acting beta 2-adrenoceptor agonists. Trends Pharmacol. Sci. 1996;17:324–330. - PubMed
    1. D'ALLAIRE F., ATGIE C., MAURIEGE P., SIMARD P.M., BUKOWIECKI L.J. Characterization of beta 1- and beta 3-adrenoceptors in intact brown adipocytes of the rat. Br. J. Pharmacol. 1995;114:275–282. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources