Article,

Characterization of adenosine A1 receptor in a cell line (28A) derived from rabbit collecting tubule

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Am J Physiol, 263 (2 Pt 1): C502-8 (August 1992)Spielman, W S Klotz, K N Arend, L J Olson, B A LeVier, D G Schwabe, U R01 DK-39654/DK/NIDDK NIH HHS/United States Research Support, U.S. Gov't, P.H.S. United states The American journal of physiology Am J Physiol. 1992 Aug;263(2 Pt 1):C502-8..

Abstract

We have previously reported that in several renal cell types, adenosine receptor agonists inhibit adenylyl cyclase and activate phospholipase C via a pertussis toxin-sensitive G protein. In the present study, in 28A cells, both of these adenosine receptor-mediated responses were inhibited by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a highly selective A1 adenosine receptor antagonist. The binding characteristics of the adenosine A1 receptor in the 28A renal cell line were studied using the radiolabeled antagonist 3HDPCPX to determine whether two separate binding sites could account for these responses. Saturation binding of 3HDPCPX to 28A cell membranes revealed a single class of A1 binding sites with an apparent Kd value of 1.4 nM and maximal binding capacity of 64 fmol/mg protein. Competition experiments with a variety of adenosine agonists gave biphasic displacement curves with a pharmacological profile characteristic of A1 receptors. Comparison of 3HDPCPX competition binding data from 28A cell membranes with rabbit brain membranes, a tissue with well-characterized A1 receptors, reveals that the A1 receptor population in 28A cells has similar agonist binding affinities to the receptor population in brain but has a considerably lower density. Addition of guanosine 5'-triphosphate (100 microM) to 28A cell membranes caused the competition curves to shift from biphasic to monophasic, indicating that the A1 receptors exist in two interconvertible affinity states because of their coupling to G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

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