An endothelial cell-line contains functional vasoactive intestinal polypeptide receptors: they control inwardly rectifying K+ channels
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Bovine endothelial cells cultured from pulmonary artery (ATCC cell line No. 209) were found to contain a high density of 125I-VIP (vasoactive intestinal polypeptide) binding sites. These were found to be saturable and to be fit by a single binding site model (Kd 1.8 nM; Bmax 534 fmol/mg protein). Studies of association and dissociation of 125I-VIP to this site revealed that binding was fully reversible and yielded a Kd value similar to that from equilibrium binding. However competition studies showed that VIP competed for binding at two sites (Ki1 1.2 x 10(-11) M, Ki2 4.7 x 10(-9) M; N1 = 21%, N2 = 77%; Ki a dissociation constant for inhibitor; N percentage of occupied receptors). [Phe1]VIP also competed at two sites, but VIP-(10-28), PHM, [4-Cl-D-Phe6,Leu17]VIP and [D-Ala4]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 Ba2+ or Cs+. The VIP antagonist [4-Cl-D-Phe6, Leu17]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.
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