Interplay betweenCa ²⁺ and K ⁺ signaling in macrophages in response to ATP

Study Objective and Hypothesis: Macrophages mediate inflammatory reactions via assembly and activation of the NLRP3 inflammasome, leading to production and secretion of proinflammatory cytokines. In response the canonical activator ATP, increases in intracellular Ca 2+ and K + efflux have been recognized as critical signaling components leading to NLRP3 activation. However, the relationship between Ca 2+ signaling components and K + efflux and the influence of macrophage priming in these signals remain incompletely understood. We hypothesized that a direct, simultaneous measurement of these signaling events can reveal the interplay between them during the response to ATP. Methods: We devised a novel assay to simultaneously measure dynamic changes in intracellular Ca 2+ and K + concentrations using fura-2/AM and IPG-1/AM. Bone marrow-derived macrophages (BMDMs) were isolated from the femur and tibia of 16-week-old C57BL/6J male mice. Intracellular Ca 2+ and K + were simultaneously measured in response to micromolar (µM) to millimolar (mM) ATP concentrations in BMDMs pretreated with or without 100 nanograms/milliliter (ng/mL) LPS for 4 hrs. Magnitude of Ca 2+ signal was quantitated as the area under the curve of the increases in fractional fura-2 signal, whereas K + efflux was calculated as the rate of decay in IPG-1 fluorescence from unstimulated state. Results: Our dual Ca 2+ -K + measurement assay detects ATP-induced Ca 2+ and K + signals well within the sensitive ranges of fura-2 and IPG-1. In unprimed BMDMs, 5 mM and 100 µM ATP trigger rapid and sustained increases in Ca 2+ that are of similar in magnitude; however, only 5 mM ATP triggers K + efflux. In LPS-primed BMDMs, 100 µM ATP triggers ~6-fold larger Ca 2+ signal AUC than that induced by 5 mM ATP, but only 5 mM ATP stimulates K + efflux. Priming with LPS also doubles K + efflux induced by 5 mM ATP. To dissect the potential relationship between Ca 2+ and K + signaling, we tested the effects of ATP-induced Ca 2+ release from internal Ca 2+ stores and subsequent Ca 2+ entry on K + efflux in unprimed BMDMs. In extracellular Ca 2+ -free condition, 5 mM ATP triggers transient Ca 2+ signal and robust K + efflux. Addition of extracellular Ca 2+ triggers large increase in intracellular Ca 2+ yet no change in K + concentration. The Ca 2+ ionophore ionomycin triggers robust transient Ca 2+ release signal in Ca 2+ -free condition, but no change in K + concentration; subsequent addition of 5 mM ATP triggers no further release of Ca 2+ , but robust K + efflux signal. Inhibition of the IP3 receptor by pretreatment with 100 µM 2-APB completely prevents 5 mM ATP-induced Ca 2+ release and K + efflux, despite intact Ca 2+ store content. Conclusions: Our dual measurement of Ca 2+ and K + concentrations provides a novel approach to assess ionic components of the inflammatory response in macrophages. The mobilizability of IP 3 -sensitive Ca 2+ stores, rather than the release of stored Ca 2+ per se, regulates ATP-induced K + efflux. ATP-induced Ca 2+ entry does not affect K + efflux. Macrophage priming with LPS conditions for distinct Ca 2+ signaling pathways in response to ATP and enhances K + efflux in response to ATP. Funding: This study was supported in part by IOER grant 03-22-10 to EW and NIH grant HL173818 to Q-KT. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.