Abstract
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.
Footnotes
-
Send reprint requests to: Dr. Craig J. Daly, Clinical Research Initiative, West Medical Building, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland.
-
↵1 This work was supported by the Medical Research Council, the Scottish Hospitals Endowments Research Trust and Pfizer.
- Abbreviations:
- CLSM
- confocal laser scanning microscope
- BODIPY
- borate-dipyrromethene
- QAPB
- quinazolinyl piperazine-BODIPY
- DMEM
- Dulbecco’s modified Eagle medium
- HBG
- Hanks buffered glucose
- Received January 26, 1998.
- Accepted April 14, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|