Murphy P M, Tiffany H L
Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1990 Jul 15;265(20):11615-21.
Stimulation of phagocytic cells with micromolar concentrations of extracellular ATP primes the production of toxic oxygen metabolites in response to chemoattractants and independently activates a secretory response in vitro. It is hypothesized that extracellular ATP derived from platelet storage granules and damaged endothelium at sites of localized tissue damage or infection may potentiate the pro-inflammatory effects of phagocytic cells in vivo. ATP-dependent functional responses in the phagocyte appear to be due to stimulation of putative P2 purinoreceptors that are coupled to the activation of a phospholipase C via a pertussis toxin-sensitive G-protein. The existence in nature of at least four subtypes of P2 purinoreceptors has been proposed based on the rank order of potency of nucleotide analogs of ATP studied in a variety of cell types. However, no studies involving the structural identification and characterization of the putative receptors have been reported. We have used the Xenopus oocyte expression system to demonstrate acquired adenosine 5'-(thio) triphosphate (ATP gamma S) responsiveness in oocytes injected with mRNA from the promyelocytic leukemia cell line HL60 by measuring the accelerated efflux of intracellular calcium. Two peaks of ATP gamma S responsiveness (Peak I and Peak II) were detected in sucrose gradient fractionated RNA that corresponded to transcript sizes of 4 and 6 kilobases and that were distinct from a third peak previously shown to be enriched in formyl peptide chemoattractant receptor activity. Peak I and Peak II RNA endowed receptor activity in the oocyte that was pharmacologically indistinguishable: ADP and AMP were inactive whereas UTP and ITP exhibited activity that was similar in potency to that of ATP gamma S. Both Peak I and Peak II ATP gamma S-dependent activity was inhibited by pertussis toxin. These data strongly support the concept of phagocytic cell receptors for extracellular nucleotide triphosphates whose ligand specificity is distinct from all other previously described P2 purinoreceptor subtypes, with the exception of the P2 receptor described in Ehrlich ascites tumor cells, by virtue of the ineffectiveness of ADP as a stimulus. These receptors are most likely composed of a single polypeptide chain that can be expressed in the Xenopus oocyte in a functional form regulated by a pertussis toxin-sensitive G-protein.
