CELL-BASED ASSAY FOR FUNCTIONAL SCREENING OF COMPOUNDS ACTIVE AT THE HUMAN ENDOTHELIN A RECEPTOR

We have developed a cell based assay for the functional screening of c hemical libraries for novel chemical entities active at the human endo thelin A (ET(A)) receptor. The assay is relatively inexpensive, suitab le for dealing with large numbers of samples, and simple to operate; i t generates results quickly. We achieved this by expressing the cDNA f or the receptor in mammalian cell lines and determining whether coupli ng to pIA(A), occurred through the quantification of released radiolab eled arachidonic acid (AA) into the culture medium. Significant coupli ng was observed only when the receptor was expressed in the Chinese ha mster ovary (CHO) line DG44. The assay could distinguish between ET(A) (r) agonists and antagonists, and the IC,, (the concentration that inh ibits maximum response by 50%) values obtained were similar to those f rom other sources of receptor. The ET(B) receptor-selective agonist BQ 3020 did not stimulate AA release, indicating that the assay can also discriminate between receptor subtypes.