ATP-induced Ca2+ response mediated by P(2U) and P(2Y) purinoceptors in human macrophages: Signalling from dying cells to macrophages

Abstract

The activation of macrophages by various stimuli leading to chemotactic migration and phagocytosis is known to be mediated by an increase in intracellular Ca2+ concentration ([Ca2+](i)). We measured changes in [Ca2+](i) using a Ca2+ imaging method in individual human macrophages differentiated from freshly prepared peripheral blood monocytes during culture of 1-2 days. A transient rise in [Ca2+](i) (duration 3-4 min) occurred in 10-15 macrophages in the vicinity of a single tumour cell that was attacked and permeabilized by a natural killer cell in a dish. Similar Ca2+ transients were produced in 90% of macrophages by application of supernatant obtained after inducing the lysis of tumour cells with hypo- osmotic treatment. Ca2+ transients were also evoked by ATP in a dose- dependent manner between 0.1 and 100 μM. The ATP-induced [Ca2+](i) rise was reduced to less than one-quarter in Ca2+-free medium, indicating that it is mainly due to Ca2+ entry and partly due to intracellular Ca2+ release. UTP (P(2U) purinoceptor agonist) was more potent than ATP or 2- chloro-ATP (P(2Y) agonist). Oxidized ATP (P(2Z) antagonist) had no inhibitory effect. Both cell lysate- and ATP-induced Ca2+ responses were inhibited by Reactive Blue 2 (P(2Y) and P(2U) antagonist) to the same extent, but were not affected by PPADS (P(2X) antagonist). Sequential stimuli by cell lysate and ATP underwent long-lasting desensitization in the Ca2+ response to the second stimulation. The present study supports the view that macrophages respond to signal messengers discharged from damaged or dying cells to be ingested, and ATP is at least one of the messengers and causes a [Ca2+](i) rise via P(2U) and P(2Y) receptors.