Influence of ER leak on resting cytoplasmic Ca2+ and receptor-mediated Ca2+ signalling in human macrophage

Abstract

Mechanisms controlling endoplasmic reticulum (ER) Ca²⁺ homeostasis are important regulators of resting cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto) and receptor-mediated Ca²⁺ signalling. Here we investigate channels responsible for ER Ca²⁺ leak in THP-1 macrophage and human primary macrophage. In the absence of extracellular Ca²⁺ we employ ionomycin action at the plasma membrane to stimulate ER Ca²⁺ leak. Under these conditions ionomycin elevates [Ca²⁺]_cyto revealing a Ca²⁺ leak response which is abolished by thapsigargin. IP₃ receptors (Xestospongin C, 2-APB), ryanodine receptors (dantrolene), and translocon (anisomycin) inhibition facilitated ER Ca²⁺ leak in model macrophage, with translocon inhibition also reducing resting [Ca²⁺]_cyto. In primary macrophage, translocon inhibition blocks Ca²⁺ leak but does not influence resting [Ca²⁺]_cyto. We identify a role for translocon-mediated ER Ca²⁺ leak in receptor-mediated Ca²⁺ signalling in both model and primary human macrophage, whereby the Ca²⁺ response to ADP (P2Y receptor agonist) is augmented following anisomycin treatment. In conclusion, we demonstrate a role of ER Ca²⁺ leak via the translocon in controlling resting cytoplasmic Ca²⁺ in model macrophage and receptor-mediated Ca²⁺ signalling in model macrophage and primary macrophage.

Keywords: Calcium leak; Endoplasmic reticulum; Macrophage; Purinergic; Translocon.