Nerve injury is accompanied by a liberation of diverse nucleotides, some of which act as ‘find/eat-me’ signals in mediating neuron-glial interplay. Intercellular Ca2+ wave (ICW) communication is the main approach by which glial cells interact and coordinate with each other to execute immune defense. However, the detailed mechanisms on how these nucleotides participate in ICW communication remain largely unclear. In the present work, we employed a mechanical stimulus to an individual BV-2 microglia to simulate localized injury. Remarkable ICW propagation was observed no matter whether calcium was in the environment or not. Apyrase (ATP/ADP-hydrolyzing enzyme), suramin (broad-spectrum P2 receptor antagonist), 2-APB (IP3 receptor blocker) and thapsigargin (endoplasmic reticulum calcium pump inhibitor) potently inhibited these ICWs, respectively, indicating the dependence of nucleotide signals and P2Y receptors. Then, we detected the involvement of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose) by desensitizing receptors. Results showed that desensitization with ATP and ADP could block ICW propagation in a dose-dependent manner, whereas other nucleotides had little effect. Meanwhile, the expression of P2Y receptors in BV-2 microglia was identified and their contributions were analyzed, from which we suggested P2Y12/13 receptors activation mostly contributed to ICWs. Besides, we estimated that extracellular ATP and ADP concentration sensed by BV-2 microglia was about 0.3 μM during ICWs by analyzing calcium dynamic characteristics. Taken together, these results demonstrated that the nucleotides ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICW communication through acting on P2Y12/13 receptors in BV-2 microglia.
Citation: Jiang P, Xing F, Guo B, Yang J, Li Z, Wei W, et al. (2017) Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y12/13 receptors among BV-2 microglia. PLoS ONE 12(8): e0183114. https://doi.org/10.1371/journal.pone.0183114
Editor: Vincenzo Lionetti, Scuola Superiore Sant'Anna, ITALY
Received: May 4, 2017; Accepted: July 28, 2017; Published: August 11, 2017
Copyright: © 2017 Jiang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the National Natural Science Foundation of China (no. 11574165), the PCSIRT (Program for Changjiang Scholars and Innovative Research Team in University) (no. IRT_13R29), the 111 Project (no. B07013). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The intracellular roles of nucleotides (ATP, ADP, UTP, etc.) have long been recognized as energy currency or building blocks for DNA and RNA. However, in the extracellular compartment, mounting evidences reveal that they function as key signal molecules to participate in various processes including inflammation, infection and tissue damage through activation of membrane P2 nucleotide receptors [1, 2]. For instance, during nerve injury, amount of ATP, ADP and UTP liberated from apoptotic neurons and acted as ‘find-me’ signal via activating P2Y receptors, resulting in oriented migration of microglia [3, 4]. The ‘eat-me’ signal such as UDP will promote phagocytic clearance of dead cells and tissue debris by microglia through activating P2Y6 receptor [5, 6]. Moreover, extracellular nucleotide signaling by P1 and P2 receptors was found to regulate numerous inflammatory responses, which contributed to maintain the pro/anti-inflammatory balance of the central nervous system (CNS) [1, 7].
In the CNS, microglia acted as the first and main immune defense against infectious and pathological events . They could promptly survey changes of the surroundings and sense extracellular messengers via cell surface receptors , subsequently congregate together rapidly to build a protective barrier between normal and damaged tissues . Intercellular Ca2+ wave (ICW) communication was the principal approach by which microglia [11, 12], astrocytes , and neurons  coordinate and synchronize with each other to execute immune defense and maintain homeostasis of CNS. ICWs could prefigure and guide the migration of microglia in response to neuronal damage .
Many reports showed that extracellular nucleotide ATP played as an important signal transmitter in ICWs [16–18]. However, it remains unclear whether and how other types of extracellular nucleotides contribute to ICW communication in microglia. In the present study, we evoked ICWs in an immortalized murine microglial cell line (BV-2) by localized micro-stimulation on single cell using glass microelectrodes. We investigated the involvement of potential intercellular messengers and corresponding receptors. Results provided compelling evidences that both ATP and ADP played pivotal roles in ICW communication via P2Y12/13 receptors in BV-2 microglia, rather than other types of nucleotides.
Materials and methods
BV-2 cell is an immortalized mouse microglial cell line that exhibits the morphological and functional characteristics of microglia . The BV-2 cells, a generous gift from Dr. Linhua Jiang (Institute of Membrane and System Biology, Faculty of Biological Sciences, University of Leeds, Leeds, U.K.), were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Biological Industries, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, USA) at 37°C under 5% CO2. Before test, cells were isolated with 0.25% trypsin + 0.04% EDTA (Gibco, USA) and plated on glass coverslips at a density of 2×104 cells/cm2 to be adherent for 18 h.
Single-cell level mechanical stimulation
Local mechanical stimulus was performed using glass microelectrodes (1 μm tip diameter), drawn by flaming/brown micropipette puller (Model P-97-6368, Sutter Instrument Co., USA). The glass microelectrodes were mounted on a Three-axis Hanging Joystick Oil Hydraulic Micromanipulator (MMO-202ND, Narishige, Japan), which was fixed on the Axio observer D1 inverted fluorescent microscope (Carl Zeiss, Germany). During fluorescence image acquisition, the tip of the microelectrode was controlled to provoke an acute, short-lasting mechanical stimulation on target cells.
Measurements of cytosolic calcium concentration ([Ca2+]c) in microglia was performed as described previously . Briefly, BV-2 microglial cells were incubated with 3 μM calcium-sensitive Fluo-4 AM (Invitrogen, USA) for 40 min at 37°C in Hanks balanced salt solution (HBSS, 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2·6H2O, 10 mM glucose, 10 mM HEPES, pH 7.4) in the presence of 10% pluronic F-127 (Biotium, USA), subsequently washed for 10 min with HBSS for de-esterification. Ca2+ dynamics was observed by the Axio observer D1 inverted fluorescent microscope. Fluo-4 was excited by a mercury with a 485/20 nm excitation filter, and fluorescence was collected by a fluar 40×/1.30 oil objective with 540/50 nm emission filter. Images were acquired by an electron multiplying charge coupled device (EMCCD) (DU-897D-CS0-BV, Andor, U.K.), which was controlled by MetaMorph software (Universal Imaging Corp., USA). Each fluorescence image was acquired for 50 ms with a 1 s interval between frames. The obtained data were quantitatively analyzed for changes of fluorescence intensities within the region of interest (ROI). The [Ca2+]c level over time was presented as relative fluorescence intensity (F/F0, intensity after stimulation/basal intensity before stimulation).
Total RNA from BV-2 microglial cells was isolated using RNAprep pure Cell/Bacteria Kit (Tiangen, Beijing). Then, the RNA (1 μg) was subjected to reverse transcription (RT) using a RT system (Promega, USA) in a total volume of 20 μl that contained MgCl2 (25 mM, 4 μl), reverse transcription 10× buffer (2 μl), dNTP mixture (10 mM, 2 μl), RNase inhibitor (0.6 μl), AMV reverse transcriptase (1 μl) and Oligo(dT) primer (1 μl). The reaction mixtures were incubated at 45°C for 30 min, 99°C for 5 min to inactivate the enzyme, and then chilled on ice for 5 min. Subsequently, the product of RT reaction (1 μl) was amplified using a GoTaq Green Master Mix (Promega, USA) in a total volume of 50 μl PCR buffer containing Green Master Mix, 2× (25 μl), sense primer (0.5 μl) and antisense primer (0.5 μl). The reaction mixtures were preheated to 95°C for 2 min followed by 40 thermal cycles in a PCR machine (MJMiniTM, BIO-RAD, USA). For each cycle, denaturation was at 95°C for 30 s, annealing at 63.5°C for 30 s, and extension at 72°C for 1 min. Sequences of gene-specific primers (S1 Table) were designed using the Primer 5 (Premier Biosoft, USA) and evaluated with Oligo 7 (Molecular Biology Insights, USA).
BV-2 microglia were seeded in a 6-well plate and lysed in 100 μL RIPA solution (Beyotime, China). The total protein concentration was determined by BCA assay. Total protein lysates (~80 μl) were denatured at 95°C for 10 min. Proteins (20 μg/lane) were subjected to 8% SDS-PAGE by 80 V at room temperature for 2 h. The resolved proteins were transferred onto nitrocellulose membranes by 80 V for 1.5 h. Upon blocking non-specific binding sites with 3% BSA for 1 h at 4°C, membranes were incubated using the following primary antibodies overnight at 4°C: rabbit anti-P2Y12 (1:500; 75 kDa; Alomone, Isreal) and mouse anti-β-actin (1:1000; 42 kDa; Beyotime, China). After washing, membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit (for P2Y12; 1:1000; Alomone, Isreal) and goat anti-mouse (for β-actin; 1:1000; Beyotime, China) secondary antibodies for 1h. Finally, bands were visualized with the enhanced chemiluminescence (ECL; Tanon, China) and the Tanon 5200 MultiImage System (Tanon, China).
BV-2 microglial cells were prepared as described above and seeded on a glass cover slip in a 6-well plate (Corning, USA) for 24 h. For living cell immunostaining, cells were incubated with anti-P2Y12 (extracellular) rabbit antibody (1:500; Alomone, Israel) resolved in DMEM at 37°C for 1h, followed by washing with HBSS for three times. Then, cells were stained with Alexa-488 goat anti-rabbit IgG (1:500; Abcam, UK) at 37°C for 1h. After washing with HBSS for three times, images were obtained by 535 nm emission filter based on an Olympus IX51 inverted fluorescence microscope with 40× oil objective.
A cell was defined as responsive to mechanical stimulation if it showed a calcium transient with a peak magnitude not less than 1.1 times of baseline. The response rate was defined as the number of responsive neighboring cells (except the stimulated cell) divided by the total number of neighboring cells in each group. All data were analyzed using the IBM software SPSS (version 22). For multiple comparisons, the data were analyzed using unpaired Student’s t-test and analysis of variance (ANOVA) followed by the Scheffé post hoc test. P < 0.05 was considered statistically significant.
Mechanical stimulation on a single BV-2 microglia initiates intercellular Ca2+ wave propagations
To simulate local injury in the CNS, we mechanically stimulated BV-2 microglia using glass microelectrodes (Fig 1A). Before stimulation treatment, all cells should be in resting states indicated by stable basal [Ca2+]c (F/F0 = 1 for Fig 1B). In addition, we paid attention to keeping the integrity of plasma membrane after treatment with mechanical stress tested by propidium iodide staining (S1 Fig). Fig 1B displayed the Ca2+ traces of the stimulated cell and five representative neighboring cells. Fig 1C and S1 Movie showed a typical process of ICW propagations among BV-2 microglia in response to mechanical stimulus. It can be found that mechanical stimulation triggered a rapid rise of [Ca2+]c that originated from the point of the stimulation and then spread throughout the whole cell body (Fig 1C, 1s and 2s). Upon reaching the cell boundaries, the Ca2+ transient propagated to the neighboring cells in a wave-like manner, namely, intercellular calcium waves (ICWs). The velocity of these ICWs was 10.43 ± 2.66 μm/s.
MS indicates the mechanical stimulation event. Pentagram indicates the stimulated cell. (A) Representative image of BV-2 microglia and glass microelectrode visualized by differential interference contrast (DIC) microscope. (B) The Ca2+ traces of the stimulated cell (Cell 1) and five neighboring cells (Cell 2–6). (C) A sequence of pseudocolor images that show mechanical stimulation elicited-propagation of ICWs among isolated BV-2 microglia. The white arrow indicates the application of mechanical stimulation on the selected microglia. Relative changes in [Ca2+]c are depicted on a pseudocolor scale with white representing the highest and black the lowest values. Scale Bar = 50 μm.
ICWs are mainly due to Ca2+ release from intracellular stores
To verify the contribution of external Ca2+, we calculated response rate and [Ca2+]c peak value by dividing neighboring cells into four groups with different distances, that was, 0–25 μm group, 25–50 μm group, 50–75 μm group and 75–100 μm group. For Ca2+-containing condition, the response rate of ICW was 81.7 ± 13.7% (0–25 μm group), 72.4 ± 20.8% (25–50 μm), 41.5 ± 27.9% (50–75 μm) and 33.0 ± 17.4% (75–100 μm) (blue color, Fig 2A). For Ca2+-free buffer, it was 94.3 ± 9.0% (0–25 μm), 72.4 ± 13.3% (25–50 μm), 51.7 ± 19.1% (50–75 μm) and 28.3 ± 12.3% (75–100 μm) (green color, Fig 2A). There was no statistically significant difference in the response rate between the Ca2+-containing and the Ca2+-free groups. For peak value, the mean of 0–25 μm group was taken as 1, and other groups were normalized to 0–25 μm group. It was 1 ± 0.16 (0–25 μm), 0.86 ± 0.19 (25–50 μm), 0.78 ± 0.26 (50–75 μm) and 0.70 ± 0.24 (75–100 μm) in Ca2+-containing buffer (Fig 2B). Similarly, the peak value was 1 ± 0.23 (0–25 μm), 0.78 ± 0.28 (25–50 μm), 0.68 ± 0.28 (50–75 μm), and 0.66 ± 0.21 (75–100 μm) in Ca2+-free medium (Fig 2C). Together, we believed that ICWs resulted from mechanical stimulation did not rely on external Ca2+. To further address the calcium source of ICWs, cells were treated with 2-aminoethyl diphenylborate (2-APB, potent IP3 receptor blocker) and thapsigargin (TG, specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) prior to mechanical stimulus, respectively. Data showed that mechanical stimulus did not induce ICW propagation in the presence of 2-APB (50 μM for 15 min) (Fig 2D) or TG (2 μM for 5 min) (Fig 2E). The statistical response rate of ICWs by 75 μm was 55.4 ± 11.6% (Ca2+-containing), 61.3 ± 10.4% (Ca2+-free), 2.0 ± 4.5% (2-APB), and 0 ± 0% (TG), respectively (Fig 2F). In brief, these results indicated that Ca2+ waves propagation evoked by mechanical stimulus depended on IP3-sensitive calcium store release in BV-2 microglia.
MS indicates the mechanical stimulation event. Pentagram indicates the stimulated cell. (A) The response rate of ICWs in the presence and absence of extracellular Ca2+ (n ≥ 20 cells for each distance from five independent experiments). All values are expressed as mean ± SD. Data are statistically analyzed by the unpaired Student’s t-test. N.S., no significant difference. (B and C) The distribution of ICW peak values (n ≥ 25 cells for each distance from six independent experiments) is shown in box plot with and without extracellular Ca2+. The mean peak value of 0–25 μm group are taken as 1, and other groups are normalized to 0–25 μm group. (D) Inhibition of ICWs by pretreatment with 2-APB (50 μM for 15 min). (E) Pretreatment with 2 μM TG for 5 min inhibits ICW propagation. (F) Statistical data of the response rate within 75 μm for all experimental groups (n ≥ 90 cells for each condition from at least three independent experiments). Data are statistically analyzed by one-way ANOVA followed by Scheffé post hoc test. ***P < 0.001.
ICW propagation is mediated by ATP-dependent P2Y purinergic signaling
Our data showed that the 1st treatment with ATP/ADP could not only strongly suppress Ca2+ mobilization strength in response to the 2nd challenge, but also significantly delay response time (S2 Fig), indicating that purinoceptors could be desensitized in BV-2 cells with applications of nucleotides. Thus, to investigate whether ICWs were linked to ATP release, we applied different doses of exogenous ATP (0.3, 1 and 10 μM) prior to mechanical stimuli to desensitize corresponding purinoceptors. Results showed that ICWs were largely blocked by 0.3 μM ATP (Fig 3A), and completely suppressed by 1 and 10 μM ATP (Fig 3B and 3C). Besides, preincubation with apyrase (10 U/mL for 30 min), an ATP-hydrolyzing enzyme, evidently blocked the ICWs (Fig 3D), further demonstrating the importance of ATP during ICWs.
MS indicates the mechanical stimulation event. Pentagram indicates the stimulated cell. (A-C) Application of exogenous ATP (A: 0.3 μM; B: 1 μM; C: 10 μM) blocks mechanical stimulation-induced ICWs in a dose-dependent manner. (D) Preincubation with 10 U/mL apyrase for 15 min suppresses calcium waves. (E) Pretreatment with 100 μM suramin for 15 min abolishes ICW propagation. (F) Statistical data of the response rate within 75 μm for all experimental groups (n ≥ 90 cells for each condition from at least three independent experiments). All values are expressed as mean ± SD. Data are statistically analyzed by one-way ANOVA followed by Scheffé post hoc test. ***P < 0.001.
Because of the relevance between ATP and P2 purinergic receptors, we determined the involvement of P2 receptors by pretreatment of suramin (a broad-spectrum P2 receptor antagonist). Results showed that ICWs were almost abolished by 100 μM suramin (Fig 3E). As summarized in Fig 3F, the response rate of ICWs (0–75 μm) was 55.4 ± 11.6% for control, 5.4 ± 11.2% for 0.3 μM ATP group, 0 ± 0% for 1 and 10 μM ATP, 10.2 ± 11.8% for apyrase and 2.9 ± 6.4% for suramin, respectively. It was known that P2 receptors could be classified into two families: ionotropic P2X ion channels, which mediate calcium influx, and metabotropic G protein-coupled P2Y receptors that initiate intracellular calcium release via IP3-sensitive calcium store . Thus, considering that the ICWs were due to intracellular calcium release (Fig 2), the calcium response was mostly associated with P2Y receptors. These results together demonstrated that the mechanical stimulation-induced ICWs were mediated by extracellular nucleotides ATP via activation of P2Y subtypes in BV-2 microglia.
The key roles of ADP and P2Y12/13 receptors in microglial ICWs
We further tested the effects of other four naturally occurring nucleotides (ADP, UTP, UDP and UDP-glucose) on mechanical stimulation-evoked ICWs. Data showed that ADP could abolish ICW propagation in a dose-dependent manner (Fig 4A and 4G). In contrast, UTP (Fig 4B), UDP (Fig 4C) and UDP-glucose (Fig 4D) had no effect on these ICWs. These results suggested that ADP was also a significant signal molecule during mechanical stimulation-induced ICWs. To investigate the roles of various purinoceptors in ICW propagations, we subsequently examined the expression of different subtypes of P2Y receptors in BV-2 microglia using RT-PCR. Data showed high transcripts of P2Y12 receptors, as well as significant expression P2Y2,6,13,14 receptors (Fig 4H). The detailed expression rate of mRNA was 40.3 ± 11.3% for P2Y2, 40.6 ± 20.2% for P2Y6, 90.7 ± 4.0% for P2Y12, 51.4 ± 2.1% for P2Y13, and 53.6 ± 2.9% for P2Y14 (Fig 4I; as the positive control, the expression of GAPDH was taken as 100%). Excluding P2Y2,4 (activated by UTP), P2Y6 (UDP) and P2Y14 receptors (UDP-glucose), therefore, the P2Y12/13 receptors, which can be activated and desensitized by ATP/ADP [21, 22], should contribute to ICWs. In order to verify the key roles of P2Y12/13 receptors, we performed inhibition experiments utilizing their specific antagonists, MRS2395 (for P2Y12R) and MRS2211 (for P2Y13R). Results showed that application of 10 μM MRS2395 (15 min) could partially suppress the ICWs (Fig 4E). Meanwhile, 10 μM MRS2211 (15 min) significantly blocked these processes (Fig 4F). As summarized in Fig 4G, the response rate of ICWs (0–75 μm) was 55.4 ± 11.6% for control, 16.4 ± 22.2% for 0.3 μM ADP, 14.2 ± 4.2% for 1 μM ADP, 2.5 ± 5.0% for 10 μM ADP, 60.6 ± 21.9% for 10 μM UTP, 54.6 ± 24.4% for 10 μM UDP, 66.8 ± 11.8% for 10 μM UDP-glucose, 26.5 ± 25.8% for 10 μM MRS2395 and 3.6 ± 3.7% for 10 μM MRS2211, respectively. In addition, we typically confirmed the presence of P2Y12 receptor in BV-2 microglia using western-blot assay (Fig 4J) and immunofluorescence (Fig 4K).
MS indicates the mechanical stimulation event. Pentagram indicates the stimulated cell. (A) Application of ADP prior to mechanical stimulation blocks ICWs. (B-D) Application of 10 μM UTP (B), 10 μM UDP (C) and 10 μM UDP-glucose (D) have no inhibitory effects on ICW communication. (E) Pretreatment with 10 μM for 15 min MRS2395 partially inhibits ICWs. (F) Preincubation with 10 μM MRS2211 for 15 min suppresses ICW propagation. (G) Summary inhibitory effects of nucleotides on ICWs within 75 μm (n ≥ 88 cells for each condition from at least three independent experiments). All values are expressed as mean ± SD. *P < 0.05 and ***P < 0.001, one-way ANOVA, Scheffé post hoc test. N.S., no significant difference. (H) Gene expression of P2Y receptors in BV-2 microglia at mRNA level detected by RT-PCR. The ten lanes in the gel are as follows: P2Y1,2,4,6,12,13,14 (experimental group with primers directed towards the P2YR mRNA); nuclease-free water (negative control); GAPDH (positive control) and marker (with a list of standardized DNA sequences from 100 bp to 800 bp). (I) Quantitative statistical results of RT-PCR normalized to GAPDH (n = 3). All values are expressed as mean ± SD. (J) Western-blot assay indicates expression of P2Y12 receptor in BV-2 microglia. (K) Immunolocalization of P2Y12 receptor on the membrane of BV-2 cells. Scale bar = 30 μm.
Quantitative estimation of extracellular ATP/ADP concentrations sensed by neighboring BV-2 microglia during Ca2+ wave propagation
As a critical mechanism of [Ca2+]c regulation, store operated calcium entry (SOCE) can be triggered by a depletion of intracellular calcium stores. Mechanical stimulus-induced ICWs were found to depend on IP3 receptor-sensitive stores release in BV-2 microglia (Fig 2). However, readdition of 2 mM external calcium did not lead to a second rising of [Ca2+]cin non-mechanically stimulated BV-2 microglia (cell 2–4 in Fig 5A), indicating the independence of SOCE. Thus, we used different concentration of exogenous ATP and ADP to mimic the pattern of calcium mobilization during ICWs. Data showed that application of 0.1 μM ATP induced a sustained and regular calcium oscillation in Ca2+-free HBSS (Fig 5B). 0.1 μM ADP only resulted in weak Ca2+ responses (data not shown). Interestingly, similar to that of mechanical stimulation, application of 0.3 μM ATP (Fig 5C) and 0.3 μM ADP (Fig 5E) induced Ca2+ mobilization without SOCE. In contrast, 1 μM ATP (Fig 5D) and 1 μM ADP (Fig 5F) could evoke evident SOCE. Taken together, we suggested that the concentration of ATP and ADP sensed by neighboring BV-2 microglia was about 0.3 μM during mechanical stimulation-induced ICWs.
MS indicates the mechanical stimulation event. Pentagram indicates the stimulated cell. (A) Mechanical stimulus is used to evoke ICWs in the absence of extracellular Ca2+, followed by re-addition of 2 mM external Ca2+. (B) Application of 0.1 μM ATP without extracellular Ca2+. (C and D) Application of ATP (0.3 and 1μM) in the absence of extracellular Ca2+ followed by replenishment of 2 mM external Ca2+. (E and F) Addition of ADP (0.3 and 1μM) in Ca2+-free buffer followed by replenishment of 2 mM external Ca2+. Dashed rectangle indicates SOCE.
ICWs appear to be a widespread phenomenon by which various cell types communicate with each other to coordinate and synchronize their activities. For instance, ICWs prefigure and guide the migration of microglia in response to neuronal damage , as well as regulate the functions of glial cells in inflammation and immunity . In the present study, we employed mechanical stimulation to an individual BV-2 microglia, and observed remarkable ICWs among adjacent cells. Subsequently, we determined the involvement of several naturally occurring nucleotides in these ICWs. Data showed that ATP and ADP, rather than UTP, UDP and UDP-glucose, were the predominant signal messengers for ICW communication. Furthermore, we indicated that calcium waves were mostly mediated by P2Y12/13 receptors.
As significant paracrine messengers in the CNS, nucleotides were known to be released in response to biochemical or physical stimuli, such as oxygen radicals, mechanical forces and virus infection [24, 25]. Nucleotides could be released from the stimulated cells through exocytosis pathway or conductive mechanisms (including pannexin channels, P2X7 receptors etc.) [26–28]. The released nucleotides gradually diffused to adjacent cells, which subsequently evoked ICW propagation by activating membrane-bound P2 receptors . In this study, we found that mechanical stimulation triggered remarkable ICWs among BV-2 microglia (Fig 1). Subsequently, we investigated the effects of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose) on the ICWs. It has been well established that prior challenge with agonists could result in desensitization of P2 receptors, especially the G protein-coupled P2Y receptors to a second challenge with the same agonists [30, 31]. Our results showed that desensitization with ATP (Fig 3A to 3C) and ADP (Fig 4A) significantly blocked mechanical stimulation-induced Ca2+ waves. In contrast, application of UTP (Fig 4B), UDP (Fig 4C) and UDP-glucose (Fig 4E) did not abolish ICW propagation. Therefore, these results indicated that ATP and ADP were predominant transmitters during mechanical stimulation-induced ICW communication in BV-2 microglia.
Furthermore, according to our results, the ICWs were independent on extracellular Ca2+ (Fig 2A to 2C) and relied on IP3-sensitive calcium store release (Fig 2D and 2E). Thus, it should be attributed to metabotropic G protein-coupled P2Y receptors, rather than ionotropic P2X channels . Among P2Y subtypes expressed in BV-2 microglia (P2Y2, 6, 12, 13, 14; Fig 4H and 4I), UTP could activate P2Y2 receptors, UDP activated P2Y6 and UDP-glucose targeted for P2Y14 [21, 22]. In view of the negative effects for these three nucleotides in ICWs (Fig 4B to 4D), P2Y2,6,14 could be excluded from the candidate receptors mediating ICWs. On the other hand, the predominantly expressed P2Y12/13 receptors, could be activated by both ATP and ADP [21, 22]. Our data showed that desensitizing by ATP/ADP and application of MRS2395/MRS2211 could all eliminate ICWs. Therefore, it determined the pivotal roles of P2Y12/13 receptors in ICW communication between BV-2 cells. In addition, although ATP cannot activate P2Y12/13 receptors directly [32, 33], membrane-localized ecto-NTPase usually catalyzes hydrolysis of ATP and generates ADP that predominantly activates P2Y12/13 receptors [32, 34–36]. Thus, inhibition of ecto-NTPase with ARL 67156 suppressed the ICW propagation in BV-2 microglia (S3 Fig). We summarized an ATP/ADP-P2Y12/13R-PLC-IP3-Ca2+ signaling pathway for mechanical stimulation-induced ICWs by a diagram (Fig 6).
① A range of nucleotides, including ATP, ADP and UDP etc., are released into extracellular space in response to mechanical stimulus. ② P2Y12/13 receptors localized on the membrane of neighboring cells can sense ATP/ADP released from the stimulated cell, then activate PLC/IP3/Ca2+ signaling. ③ Upon sensing and responding to released ATP/ADP, Ca2+ mobilization sequentially occur in neighboring cells, thus perform as intercellular calcium waves. ④ ATP cannot activate P2Y12/13 receptors directly. Ecto-NTPase located on the plasma membrane could catalyze hydrolysis of ATP and generates ADP that predominantly activates P2Y12/13 receptors.
This mechanism we proposed was differ from previous studies, such as P2Y1,2 receptors mediated ICWs in rat astrocytes , P2Y11 receptors contributed to ICWs in rat cardiac myofibroblasts  and N-methyl-D-aspartic acid (NMDA) receptor was involved in zebrafish microglial ICWs . Importantly, in addition to participate in ICWs, extracellular nucleotides were associated with a wide array of physiological effects of microglia through activation of specific P2 receptors . For instance, P2X7 receptors are relevant to membrane pore formation, microvesicle shedding and cell apoptosis [40, 41]. P2X4 as well as P2Y12 receptors mediated microglial chemotaxis toward injured sites [42–44], and P2Y6 receptor was critical player in promoting microglial phagocytosis resulted from Ca2+ mobilization .
Metabolic G protein-coupled P2Y receptors are divided into two classes: Gq protein-coupled receptors, mainly P2Y1-11 receptors, and Gi-coupled P2Y12-14 receptors. Activation of Gq-protein could stimulate phospholipase C β (PLCβ), then cleave PIP2 into two types of second messengers IP3 and DAG, and triggered Ca2+ mobilization . In contrast, activation of Gi-coupled P2Y12/13 receptors inhibited the adenylate cyclase (AC)/cAMP-dependent pathway . Meanwhile, Gi-coupled P2Y12/13 receptors resulted in activation of the phosphatidylinositol 3’-kinase (PI3K) pathway, which was known to be a crucial enzyme in the regulation of chemotaxis . Nevertheless, some studies reported that activation of P2Y12/13 receptor could also mediate PLC/IP3/Ca2+ pathway [47, 48]. Our study indicated the correlation between PLC/IP3/Ca2+ signaling and P2Y12/13 receptors, which were consistent with de Simone’s  and Zeng’s work .
Subsequently, we estimated the concentrations of paracrine ATP and ADP sensed by BV-2 microglia during the ICW. The traditional method detecting concentration of ATP was luciferin-luciferase assay. However, this method was mostly used for the measurements of the overall level of ATP concentration and not applicable to ADP. In this work, we found that mechanical stimulation-evoked ICWs was due to calcium release rather than SOCE (Fig 5A). Therefore, we observed whether the [Ca2+]c pattern in response to exogenous ATP and ADP at different concentration were in agreement with mechanical stimulation-induced ICW. Data showed that different concentration of nucleotides could induce different patterns of Ca2+ transient, which was similar to that of Visentin’s work . Only application of 0.3 μM ATP or ADP exhibited a similar characteristic behavior to that of mechanical stimulation-evoked ICWs (Fig 5C and 5E), indicating that the concentration of ATP and ADP sensed by purinergic receptors of BV-2 microglia was about 0.3 μM. These findings might be significant in the field of drug analysis and development and could provide the basis for pharmacological experiments.
One of the main limitation on this study was the application of BV-2 microglia. Although BV-2 has been known as an immortalized mouse microglial cell line that exhibits the morphological and functional characteristics of primary microglia [19, 51], there is no denying that it cannot fully represent the real physiological features of primary microglia in situ or in vivo . Therefore, we plan to study and understand the procedure of primary microglia isolation. Much work will be performed on primary microglia that makes our research more interesting and significant.
In summary, our study demonstrated that ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICWs through P2Y12/13 receptors activation in BV-2 microglia. The concentration of ATP/ADP sensed by BV-2 microglia was about 0.3 μM. Our research may bring new insights in the mechanism of cell-to-cell communication of microglia to investigation of its immune defense in the CNS.
S1 Movie. Intercellular calcium wave triggered by mechanical stimulus in BV-2 microglia.
S1 Fig. BV-2 microglia maintain or lose plasma membrane integrity after different mechanical stimulations.
S2 Fig. Desensitization of purinoceptors by application of ATP and ADP.
S3 Fig. Ectonucleotidase inhibitor ARL 67156 significantly blocks the ICW propagations in BV-2 microglia.
This work was supported by the National Natural Science Foundation of China (No. 11574165), Program for Changjiang Scholars and Innovative Research Team in University (No. IRT_13R29), the 111 Project (No. B07013).
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