Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation

Abstract

Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca2+-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca2+ entry (ROCE). Although store-operated Ca2+ entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca2+ imaging. After depleting internal Ca2+ stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca2+]i whereas lower concentrations (≤100 μM) also induced Ca2+ oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca2+ oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca2+ signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca2+ oscillations (P2X4) and sustained [Ca2+]i increases. We demonstrate specific drug effects on purinergic Ca2+ pathways and provide new pharmacological insights into Ca2+ oscillations in BV2 cells. For example, minocycline inhibits both P2X7- and P2X4-mediated Ca2+-responses, and this may explain its anti-inflammatory action in neuroinflammatory disease. As a technical result, our novel automated bio-screening approach provides a biomedical engineering platform to allow high-content drug library screens to study neuro-inflammation in vitro.

Simulation of P2X‐mediated calcium signalling in microglia

“…It is reported, however, that in some cells lines like BV2 P2Y effects on intracellular Ca 2+ responses are small, especially compared to those mediated by P2X channels (Gilbert et al . ). For this reason, our initial model of microglia Ca 2+ ‐dependent function may be particularly applicable to P2X‐specific signalling in commonly used cell lines such as BV2 that have insignificant P2Y activity.…”

“…Our computational model of P2X-mediated microglial signalling portrayed in Fig. 1 assumes: (1) ATP-dependent activation of P2X receptors assuming minimal contributions from P2Y receptors is minor as is reported in BV2 microglial cell lines (Gilbert et al 2016); (2) Ca 2+ -dependent activation of signalling proteins calmodulin (CaM), CN, NFAT, as well as Ca 2+ extrusion and uptake mechanisms; and (3) TNFα transcription, translation and exocytosis. For clarity, we numbered these reactions and list their supporting literature in Table S1.…”

“…It is expected that including both P2X-and P2Y-mediated responses, as well as a realistic model of ectonucleotidase activity (Sandefur et al 2017) and associated regulation of synaptic nucleotide pools, would constitute a significant step toward modelling in vivo microglia activity. It is reported, however, that in some cells lines like BV2 P2Y effects on intracellular Ca 2+ responses are small, especially compared to those mediated by P2X channels (Gilbert et al 2016). For this reason, our initial model of microglia Ca 2+ -dependent function may be particularly applicable to P2X-specific signalling in commonly used cell lines such as BV2 that have insignificant P2Y activity.…”

Chloroquine is a potent pulmonary vasodilator that attenuates hypoxia‐induced pulmonary hypertension

“…In our study, ATP‐mediated activation of P2Y receptors, a family of GPCRs, increased [Ca 2+ ] cyt by causing SOCE (due to IP 3 ‐mediated active store depletion and the opening of SOCC) and ROCE (due to the DAG‐mediated opening of ROCC). ATP‐mediated activation of P2X receptors, a family of cation‐permeable ligand‐gated ion channels, increases [Ca 2+ ] cyt by directly inducing Ca 2+ influx through the channels and/or indirectly inducing Ca 2+ influx through VDCC via membrane depolarization (Gilbert et al ., ). In HEK293 cells traniently transfected with the gene encoding TRPC6 channels, chloroquine attenuated the ATP‐mediated increase in [Ca 2+ ] cyt (Supporting Information Figure S1), indicating that chloroquine may partly block TRPC6 channels, which contribute to forming ROCC and SOCC (Thebault et al ., ; Albarran et al ., ), and thus induce PA relaxation.…”

“…Rats were given an s.c. injection of 20 mg·kg À1 SU5416 or vehicle, followed by hypoxia (SuHx) for 3 weeks (10% O 2 ) and normoxia for an additional 2 weeks to elicit severe PH. After 3 weeks of hypoxia, the SuHx rats were divided into three groups: hypoxia with no intervention (SuHx3w), vehicle treatment (SuHx3w + Nor2w) and treatment with chloroquine (CHQ; 50 mg·kg À1 ·day À1 ) (SuHx3w + CHQ2w depolarization (Gilbert et al, 2016). In HEK293 cells traniently transfected with the gene encoding TRPC6 channels, chloroquine attenuated the ATP-mediated increase in [Ca 2+ ] cyt (Supporting Information Figure S1), indicating that chloroquine may partly block TRPC6 channels, which contribute to forming ROCC and SOCC (Thebault et al, 2005;Albarran et al, 2014), and thus induce PA relaxation.…”