Inhibitory effects of U73122 and U73343 on Ca 2+ influx and pore formation induced by the activation of P2X7 nucleotide receptors in mouse microglial cell line

Abstract

P2X7 receptors are ATP-gated ion channels and play important roles in microglial functions in the brain. Activation of P2X7 receptors by ATP or its agonist BzATP induces Ca 2+ influx from extracellular space, followed by the formation of non-selective membrane pores that is permeable to larger molecules, such as fluorescent dye. To determine whether phospholipase C (PLC) is involved in the activation of P2X7 receptors in microglial cells, U73122, a specific inhibitor of PLC, and its inactive analogue U73343 were examined on ATP and BzATP-induced channel and pore formation in an immortalized C57BL/6 mouse microglial cell line (MG6-1). ATP induced both a transient and a sustained increase in the intracellular Ca 2+ concentration ([Ca 2+] i) in MG6-1 cells, whereas BzATP evoked only a sustained increase. U73122, but not U73343, inhibited the transient [Ca 2+] i increase involving Ca 2+ release from intracellular stores through PLC activation. In contrast, both U73122 and U73343 inhibited the sustained [Ca 2+] i increase either prior or after the activation of P2X7 receptor channels by ATP and BzATP. In addition, these U-compounds inhibited the influx of ethidium bromide induced by ATP and BzATP, suggesting possible PLC-independent blockage of the process of P2X7-associated channel and pore formations by U-compounds in C57BL/6 mouse microglial cells.