Direct effects of adenylyl 5'-(beta,gamma-methylene)diphosphonate, a stable ATP analogue, on relaxant P1-purinoceptors in smooth muscle

Abstract

1. Previous results obtained with the rat colon muscularis mucosae, which contracts in response to adenosine and adenosine 5'-triphosphate (ATP), had suggested that adenylyl 5'-(beta,gamma-methylene)diphosphonate (AMPPCP), a stable ATP analogue, acted on P1-purinoceptors rather than, as expected, on P2-purinoceptors. This possibility has been examined in two tissues in which adenosine and ATP both cause relaxation, the guinea-pig taenia caeci and the rat duodenum. 2. ATP, 2-methylthio-ATP (2-MeSATP), AMPPCP, adenosine 5'-(alpha,beta-methylene)triphosphonate (AMPCPP) and adenosine each relaxed the taenia caeci and the duodenum, and the order of potency of the nucleotides in each tissue was 2-MeSATP greater than ATP greater than AMPCPP greater than AMPPCP, indicating that these effects were mediated by P2Y-purinoceptors. 3. The P1 antagonist 8-(p-sulphophenyl)theophylline (8-SPT) (100 microM) did not affect the responses to ATP, 2-MeSATP or AMPCPP in either tissue, but inhibited the responses of adenosine and of AMPPCP in both tissues. In the duodenum a lower concentration of 8-SPT caused a parallel shift to the right of the concentration-response curve to adenosine and to AMPPCP but to different extents, with AMPPCP being inhibited more powerfully than adenosine. A dose-ratio of around 5 was observed for adenosine and AMPPCP at concentrations of 8-SPT of 20 microM and 2 microM respectively, but Schild analysis resulted in plots with slopes greater than unity. In the taenia caeci, however, 8-SPT inhibited adenosine more powerfully than AMPPCP, and a range of concentrations (10-20 microM) only caused a two fold shift in the concentration-response curve for AMPPCP, although the concentration-response curve to adenosine was shifted in a concentration-dependent manner and Schild analysis gave a pA2 value of 5.13 with a slope of 0.90.4. As has been shown in other tissues, including the guinea-pig taenia caeci, ATP (100 microM) was rapidly dephosphorylated by enzymes present in the rat duodenum, with less than 10% remaining after 20min incubation, whereas AMPPCP (100 microM) was resistant to degradation, with greater than 90% remaining at the same time point.5. AMPPCP therefore has pronounced but variable agonist actions on P,-purinoceptors, and appears to act entirely via these receptors on the rat duodenum although in the guinea-pig taenia caeci this action is less important and it acts largely via P2y-purinoceptors. These Pl-purinoceptor effects of AMPPCP are direct and are not due to its degradation to adenosine.