Cellular localization and pharmacological characterization of functioning alpha-1 adrenoceptors by fluorescent ligand binding and image analysis reveals identical binding properties of clustered and diffuse populations of receptors.
A fluorescent quinazoline derivative was shown to retain high affinity for, and act as a competitive antagonist at, alpha-1 adrenoceptors. This allowed it to be used in live cells to localize receptors and to quantify receptor binding characteristics. The technique was demonstrated and validated on fibrobasts transfected with a recombinant alpha-1d adrenoceptor. Using confocal laser scanning microscopy and image analysis methods both diffuse and clustered binding sites were found: their binding characteristics were assessed and found comparable to radioligand binding on membrane preparations. This approach should have widespread applicability in nonradioactive assays determining the location, quantity and binding properties of receptors and other biological molecules on live tissue.
Daly
C J
CJ
Clinical Research Initiative and Division of Neuroscience and Biomedical Systems, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland.
Milligan
C M
CM
Milligan
G
G
Mackenzie
J F
JF
Mcgrath
J C
JC
D000317:Adrenergic alpha-Antagonists / Q000493:pharmacokinetics; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D018613:Microscopy, Confocal; D011224:Prazosin / Q000493:pharmacokinetics; D011869:Radioligand Assay; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism* / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure
D000317:Adrenergic alpha-Antagonists; D005456:Fluorescent Dyes; D007295:Inositol Phosphates; D018340:Receptors, Adrenergic, alpha-1; D011224:Prazosin