Expression of the pore-forming P2Z purinoreceptor in Xenopus oocytes injected with poly (A) + RNA from murine macrophages
Nuttle, L.C.; el-Moatassim, C.; Dubyak, G.R.
Molecular Pharmacology 44(1): 93-101
1993
ISSN/ISBN: 0026-895X PMID: 7688070 Document Number: 417125
Extracellular ATP activates two distinct types of P-2 purinoreceptor-mediated signaling pathways in macrophages, 1) the rapid formation of nonselective pores/channels in the plasma membrane and 2) a guanine nucleotide-binding protein-dependent stimulation of phosphotidylinositol-specific phospholipase C, with subsequent mobilization of intracellular Ca-2+. Several studies have suggested that the pore-forming, or P-2z, purinoreceptor may be involved in the cytolytic effects of ATP on macrophages and other cell types. We have identified 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) and UTP as selective agonists for the P-2z purinoreceptor and Ca-2+-mobilizing nucleotide receptor, respectively, in BAC1.2F5 macrophages. In this paper we demonstrate that BzATP and ATP (but not UTP) activate membrane depolarization in BAC1.2F5 cells and also stimulate appropriate electrophysiological responses, consistent with the expression of the P-2z purinoreceptor, in Xenopus oocytes injected with poly(A)+ RNA derived from BAC1.2F5 cells. Micromolar concentrations of BzATP or millimolar concentrations of ATP induced a sustained increase in the membrane holding current in these voltage-clamped oocytes. This response was significantly potentiated in the absence of extracellular divalent cations, consistent with the specificity of the P-2z purinoreceptor for tetrabasic nucleotides. The sustained currents induced by BzATP or ATP were distinct from the transient and/or oscillating increases in Ca-2+-dependent Cl- currents that were stimulated by UTP but not BzATP. UTP-stimulated transient currents and nucleotide-dependent increases in aequorin luminescence in poly(A)+ RNA-injected oocytes were independent of extracellular Ca-2+ and were correlated with the mobilization of intracellular Ca-2+ stores. Sucrose fractionation of the poly(A)+ RNA from BAC1.2F5 cells resulted in the enrichment of mRNA species encoding the components of the P-2z purinoreceptor, as well as the Ca-2+-mobilizing nucleotide receptor, in fractions containing 2.5-4.0-kilobase species.