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Endothelin-1 receptor binding assay for high throughput chemical screening
M J Cain1, R K Garlick, P M Sweetman
1E. I. Du Pont de Nemours Company, Biotechnology Systems R&D, NEN Research Products, Billerica, Massachusetts 01862.
Journal of Cardiovascular Pharmacology
|January 1, 1991
Summary
A new assay for endothelin-1 (ET-1) receptors in A10 cells enables high-throughput screening. This reproducible method reduces radioactive waste by 90% compared to traditional assays.
Area of Science:
- Pharmacology
- Biochemistry
- Cell Biology
Background:
- Endothelin-1 (ET-1) plays a crucial role in cardiovascular function.
- High-affinity ET-1 receptors are present on rat aortic smooth muscle cells (A10 cell line).
- Efficient screening assays are needed for identifying ET-1 receptor modulators.
Purpose of the Study:
- To develop a high-throughput, microtiter-based assay for endothelin-1 (ET-1) receptor binding.
- To characterize the ET-1 receptor population on the A10 cell line.
- To assess the assay's suitability for automation and its efficiency in reducing radioactive waste.
Main Methods:
- Development of a 96-well microtiter filtration plate assay using [125I]Tyr13-ET-1.
- Saturation binding assays and Scatchard analysis to determine receptor affinity (Kd) and density.
- Determination of inhibition constants (Ki) for various ET receptor ligands.
Main Results:
- The A10 cell line expresses a single, high-affinity population of ET-1 receptors (Kd = 0.12 nM, ~40,000 receptors/cell).
- Specific Ki values were determined for ET-1, ET-2, sarafotoxin S6b, VIC, ET-3, and big human ET.
- ET receptors on A10 cells remained stable for at least 2 months when stored at -20°C.
Conclusions:
- A robust and reproducible microtiter-based ET-1 receptor binding assay has been established.
- The assay is suitable for high-throughput screening and automation.
- This method significantly reduces radioactive waste (90%) compared to conventional assays.