[H]BQ-123 binding to native endothelin ET(A) receptors in human astrocytoma 1321N1 cells and screening of potential ligands

Abstract

Endothelin ET(A) receptors on the human astrocytoma 1321N1 cell line were characterized in radioligand binding studies using the ET(A)-selective antagonist radioligand [(3)H]BQ-123. A membrane preparation of the cell line showed a high expression level of ET(A) receptors (B(max) = 128 fmol/mg protein). The K(D) value for [(3)H]BQ-123 (K(D) = 2.29 nmol/l) was comparable to that previously determined at the human neuroblastoma cell line SK-N-MC. The specific labeling of ET(A) receptors on astrocytoma 1321N1 cells by [(3)H]BQ-123 was confirmed by determining the rank order of potency of a series of agonists and antagonists. A competition assay for the screening of potential ET(A) ligands was established and a series of new disubstituted imidazo[1,2-c]pyrimidine and pyrimido[1,6-a]pyrimidine derivatives was investigated. Moderate ET(A) affinity was detected for several derivatives [30-40% inhibition of [(3)H]BQ-123 binding (3 nmol/l) at a concentration of 10 micromol/l]. Astrocytoma 1321N1 cells natively express relatively high levels of ET(A) receptors. They are useful for the screening of potential ET(A) ligands in radioligand binding assays with [(3)H]BQ-123.