P 2-Purinergic receptors activate a guanine nucleotide-dependent phospholipase C in membranes from HL-60 cells

Abstract

We have previously determined that human neutrophils and monocytes, as well as neutrophil/monocyte progenitor cells, express a subtype of P 2-purinergic receptors (for ATP) which activate the inositol phospholipid signalling system. In the present study, membranes prepared from HL-60 promyelocytic leukemia cells were used to examine the mechanism by which these ATP receptors activate phosphatidylinositol-specific phospholipase C (PI-PLC) under defined in vitro conditions. Micromolar concentrations of the receptor agonists ATP, UTP, and ATPγS stimulated the GTP-dependent formation of inositol bisphosphate (IP 2) and inositol trisphosphate (IP 3) in washed membranes prepared from undifferentiated HL-60 cells prelabeled with [ 3H]inositol. The stimulatory effects of these nucleotides on PI-PLC appeared to be mediated through a GTP binding protein since minimal inositol polyphosphate accumulation was observed in the absence of guanine nucleotides. The increased inositol polyphosphate formation triggered by these nucleotide receptor agonists did not result from inhibition of GTP breakdown. Neither was it a consequence of increased [ 3H]polyphosphatidylinositol levels resulting from enhanced activity of membrane-associated PI- or PIP-kinases. Instead, the stimulated phospholipase activity was apparently receptor-mediated. The rank order of potency observed in these in vitro membrane assays (ATP = UTP > ATP γS ⪢ TTP > CTP ⪢ β, γ-CH-ATP) was similar to that observed with intact HL-60 cells. This order of potency appears to distinguish the P 2-purinergic receptors expressed by human phagocytic leukocytes from the P 2y-purinergic receptors which activate PI-PLC in turkey erythrocyte membranes.