An endothelial cell-line contains functional vasoactive intestinal polypeptide receptors : they control inwardly rectifying K+ channels

Abstract Bovine endothelial cells cultured from pulmonary artery (ATCC cell line No. 209) were found to contain a high density of 125 I-VIP (vasoactive intestinal polypeptide) binding sites. These were found to be saturable and to be fit by a single binding site model (K d 1.8 nM; B max 534 fmol/mg protein). Studies of association and dissociation of 125 I-VIP to this site revealed that binding was fully reversible and yielded a K d value similar to that from equilibrium binding. However competition studies showed that VIP competed for binding at two sites (K i1 1.2 × 10 −11 M, K i2 4.7 × 10 −9 M; N 1 =21%, N 2 =77%; K i a dissociation constant for inhibitor; N percentage of occupied receptors). [Phe 1 ]VIP also competed at two sites, but VIP-(10–28), PHM, [4-Cl-D-Phe 6 , Leu 17 ]VIP and [D-Ala 4 ]VIP displaced all specific VIP binding in a simple competitive manner. These VIP binding sites were shown to be functional. In patch clamp studies VIP 10 −8 –10 −7 M inhibited opening of inwardly rectifying K + channels on hyperpolarization. These channels were affected appropriately by alteration in the K + -gradient and by Ba 2+ or Cs + . The VIP antagonist [4-Cl-D-Phe 6 , Leu 17 ]VIP prevented or reversed the effects of VIP. These results show that functional VIP receptors are present in high density in a endothelial cell line and provide a possible model for analysis of the molecular biology of these receptors.