Multiple P2X and P2Y receptor subtypes in mouse J774, spleen and peritoneal macrophages

We investigated P2 receptor expression and function in macrophages from mouse, and in the J774 cell line, and revealed a larger spectrum of P2 receptor subtypes than previously recognised. The nucleotides adenosine triphosphate (ATP), adenosine diphosphate, uridine triphosphate and uridine diphosphate evoked an increase in intracellular calcium and the activation of a potassium current. The sensitivity of these responses to the antagonists suramin, PPADS, MRS 2179 and Cibacron blue suggest the presence of at least three functional P2Y receptor subtypes, most probably P2Y₂, P2Y₄ and P2Y ₆. ATP also activated P2X receptors, giving rise to a rapidly activating cation conductance. This response was insensitive to the antagonists suramin and Cibacron blue, was potentiated by Zn²⁺ and inhibited by acidification suggesting involvement of P2X₄ receptors. In low divalent cation solution, responses to ATP became larger, and dibenzoyl-ATP became more potent than ATP, indicating the presence of P2X₇ receptors. Immunofluorescence, flow cytometry, Western blots and RT-PCR show that P2X₄ and P2X₇ receptors are the most prominent in both macrophage types, while the expression of the other P2X subunits is variable and sometimes weak or undetectable. These techniques also demonstrated the presence of mRNA for P2Y₁, P2Y₂, P2Y₄ and P2Y₆ receptors along with protein expression for the three subtypes we investigated, namely, P2Y₁, P2Y₂ and P2Y₄.