Abstract
Following its photolysis in the presence of the isolated guinea-pig vas deferens, the ATP photoaffinity label ANAPP 3 produces a specific antagonism of adenine nucleotide-induced contractile responses which are mediated by P 2-purinergic receptors. To characterize the site of covalent photoincorporation of ANAPP 3, intact vasa deferentia were treated with [ 3H]ANAPP 3 and samples of homogenate, cytosol and a crude membrane fraction were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Photolysis of [ 3H]ANAPP 3 (10 −5 M; 3.0 μCi/ml) resulted in the incorporation of radioactivity into cellular components with apparent molecular weights of 54–66 and 43–57 kilodaltons. The photoincorporation of [ 3H]ANAPP 3 was associated with the crude membrane fraction and not the cytosol, was reduced in the presence of ATP in an ATP-concentration-dependent manner, was lessened following pretreatment of the tissues with photolyzed nonradiolabeled ANAPP 3, and was unaffected by the nucleoside transport inhibitor, dipyridamole. In tension studies on the same tissues the presence of ATP resulted in a concentration-dependent reduction in the initial contractile response to [ 3H]ANAPP 3 the response to 3H was antagonized in tissues which had been pretreated with nonradiolabeled ANAPP 3, and dipyridamole had no effect on the contractile response to [ 3H]ANAPP 3. According to several criteria these findings indicate that the antagonism by photolyzed ANAPP 3 of adenine nucleotide-induced responses is a direct result of the covalent insertion at or near the recognition site of cell-surface P 2-purinergic receptors.
Published Version
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