Measuring the Number of β-adrenergic Receptors

In order to study the pathway of G-protein coupled receptors mediate, it is necessary to investigate the amount of β-adrenergic receptors in smooth muscle. First we must make four assumptions. We must assume: 1:1 binding between the ligand and the receptors, binding is specific, no interactions between receptors, and no internalization. I purpose utilizing an experiment that takes advantage of fluorescent tags to measure β-adrenergic receptors. In a study done by Briones et al., it was found that BODIPY-TMR-GCP (bordifluoropyrromethancetetramethylrhodamine-(+) was a long-acting fluorescent label used to label β-adrenergic receptors on the cell membrane of living cells. Moreover, any fluorescent or radiolabel can be used as long as it is selective for β-adrenergic receptors in smooth muscle. Now that a label was identified, we must take a tissue sample. The tissue sample should have the smooth muscle cells extracted into a cell suspension. There are various methods of doing this. Next, this cell suspension must be incubated for 1 hour at 4º C with increasing concentrations of the fluorescent tag. This low temperature will prevent endocytosis of the cellsurface receptors. To quantify the number of receptors labeled, the sample will be put through a flow cytometer. With appropriate thresholds set and gating, an accurate receptor count can be resolved. This quantification was for total binding (Briones et al, 2005). Since total binding was quantified, we need to find non-specific binding. A competing ligand in excess was tagged fluorescently and added 30 minutes prior to our original label. The original label may also be used in 100 fold excess if it was not labeled. Since molecules were fluorescently labeled (different emissions preferably), flow cytometry can be used to quantify only our original tag of interest, BODIPY-TMR-GCP (gating). Theoretically, BODIPY-TMRGCP should now be bound to only non-specific receptors. The total binding can be subtracted from the nonspecific binding and the specific binding is now obtained (Daly et al, 2010). References Briones, Ana M, Craig J. Daly, Francesc Jimenez-Altayo, Sonia Martinez-Revelles, Jose M. Gonzalez, John C. McGrath, and Elisabet Vila. "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 (2005): 679-691. Print. Daly, CJ, RA Ross, J Whyte, CM Henstridge, AJ Irving, and JC McGrath. "Themed Section: Imaging in Pharmacologyresearch Paper : Fluorescent Ligand Binding Reveals Heterogeneous Distribution of Adrenoceptors and ‘cannabinoid-Like’ Receptors in Small Arteries." British Journal of Pharmacology. 159.4 (2010): 787-796. Print.

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