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RJ Murrin and MR Boarder
Department of Pharmacology and Therapeutics, University of Leicester, UK.
We have investigated the characteristics of the receptor for ATP on neuronal cells and the involvement of phospholipase C and phospholipase D in the effector mechanisms, using PC12 rat phaeochromocytoma cells in culture. We show that the cells respond, with generation of total inositol phosphates, to ATP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) but not to 2-methylthioadenosine5'-triphosphate (2MeSATP), beta,gamma-methylene ATP, or adenosine 5'-O-(2- thiodiphosphate) (ADP beta S). The largest response to ATP gamma S was mainly independent of extracellular calcium, had an EC50 of 7.93 +/- 0.76 microM, and was competitively inhibited by the nonspecific antagonist suramin. The pyrimidine nucleotide UTP also elicited a response in these cells. Measurement of [3H]inositol triphosphate showed a rapid rise to maximum (10-15 sec) in response to both ATP gamma S and UTP but no response to 2MeSATP. Cells prelabeled with 32Pi and stimulated in the presence of 50 mM butanol responded to ATP gamma S, ATP, and UTP with enhanced formation of [32P]phosphatidylbutanol as well as [32P]phosphatidic acid, indicating that agonist-stimulated phosphatidic acid occurs by both phospholipase D and phospholipase C activity. The stimulation of phospholipase D was inhibited by the presence of a protein kinase C inhibitor, Ro 31-8220. The dose-response curve for the stimulation by ATP gamma S of phospholipase C was shifted to the right by the presence of UTP, indicating that both compounds act on the same receptors. The data provide the first evidence for the existence of a nucleotide receptor on neuronal cells (insensitive to both purines and pyrimidines) and show that this receptor is linked to both phospholipase C and phospholipase D.
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