Differential heterologous and homologous desensitization of two receptors for ATP (P2y purinoceptors and nucleotide receptors) coexisting on endothelial cells

Bovine aortic endothelial cells in culture contain two coexisting phosphoinositidase C-linked receptors for ATP, the P2y-purinoceptors [for which 2-methylthio-ATP (2MeSATP) is a selective agonist] and the nucleotide (or P2u) receptors (for which UTP is a selective agonist). Here we have investigated the occurrence of homologous and heterologous desensitization of these two receptors and the involvement of protein kinase C-dependent mechanisms. Measuring total [3H]inositol (poly)phosphate accumulation in the presence of lithium, we showed that with long (15-min) stimulations with UTP or 2MeSATP desensitization occurred to a maximum of 40% within several minutes of preexposure to either agonist, i.e., with this procedure there is no difference between the heterologous and the homologous experimental design. In the remainder of the experiments reported we measured inositol-1,4,5-trisphosphate mass levels, using a protocol of 5-min preincubation, 2-min wash, and 5-sec stimulation. We found that preincubation with either agonist led to desensitization of the response to the same agonist of about 40%. However, whereas preincubation with 2MeSATP did not affect the subsequent response to UTP, preincubation with UTP did attenuate the 2MeSATP response. These results demonstrate that homologous desensitization occurs with both P2Y and nucleotide receptors but that heterologous desensitization follows only from activation of the nucleotide receptors. Preincubation with the protein kinase C inhibitor Ro 31-8220 enhanced the subsequent inositol-1,4,5-trisphosphate response to 2MeSATP but did not affect the desensitization of this response by preincubation with the same agonist. However, whereas the response to UTP was not enhanced by preincubation with the protein kinase C inhibitor, the desensitization caused by preincubation with UTP was partially inhibited by Ro 31-8220. These results show that multiple desensitizing events occur during the first few minutes of receptor activation and that these events are different for each of the receptors for ATP.