P2X7-mediated ATP secretion is accompanied by depletion of cytosolic ATP
ATP and its metabolites are important extracellular signal transmitters acting on purinergic P2 and P1 receptors. Most cells can actively secrete ATP in response to a variety of external stimuli such as gating of the P2X7 receptor. We used Yac-1 murine lymphoma cells to study P2X7-mediated ATP release. These cells co-express P2X7 and ADP-ribosyltransferase ARTC2, permitting gating of P2X7 by NAD+-dependent ADP-ribosylation without the need to add exogenous ATP. Yac-1 cells released ATP into the extracellular space within minutes after stimulation with NAD+. This was blocked by pre-incubation with the inhibitory P2X7-specific nanobody 13A7. Gating of P2X7 for 3 h significantly decreased intracellular ATP levels in living cells, but these had returned to normal by 20 h. P2X7-mediated ATP release was dependent on a rise in cytosolic calcium and the depletion of intracellular potassium, but was not blocked by inhibitors of pannexins or connexins. We used genetically encoded FRET-based ATP sensors targeted to the cytosol to image P2X7-mediated changes in the distribution of ATP in 3T3 fibroblasts co-expressing P2X7 and ARTC2 and in Yac-1 cells. In response to NAD+, we observed a marked depletion of ATP in the cytosol. This study demonstrates the potential of ATP sensors as tools to study regulated ATP release by other cell types under other conditions.
KeywordsP2X7 Lymphoma ATP release FRET ARTC2 Nanobodies
Beyond its well-characterised intracellular role as the currency of energy metabolism, ATP is an important signalling molecule in the extracellular environment. Outside the cell, ATP either transmits signals directly by acting on P2 receptors, or is metabolised by the combined action of the ecto-nucleotidases CD39 and CD73 to adenosine, which acts on P1 receptors . The balance between extracellular ATP (eATP) and adenosine, and thus between P2 and P1 signalling, constitutes a key mechanism for the control of tissue inflammation and lymphocyte functions. ATP is a predominantly intracellular molecule; under steady-state conditions, its extracellular concentrations lie in the low nanomolar range . The appearance of ATP outside of cells is considered to be an immunological danger signal, alerting the immune system to an imminent threat. Thus, gating of the P2X7 receptor by eATP results in assembly of the NLRP3 inflammasome, initiating processing and secretion of the pro-inflammatory cytokine interleukin-1 beta (IL-1β) . Gating of P2X7 also induces activation of the ADAM17 metalloprotease that sheds the ectodomains of molecules such as TNF, CD62L and CD27 from the cell surface, a process known as ectodomain shedding [4, 5, 6]. Conversely, adenosine mediates mainly anti-inflammatory and suppressive effects by acting on the A2A and A2B P1 receptors, thereby negatively regulating effector functions of lymphocytes and antigen-presenting cells .
Because eATP constitutes the raw material for P2 and P1 signalling processes, the mechanisms by which ATP reaches the extracellular space have been the subject of intense study. While it was originally thought that eATP is primarily the product of leakage from dead or damaged cells, it is now generally agreed that most cells actively secrete ATP in a regulated fashion in response to external stimuli as a means of intercellular communication. Various stimuli that induce ATP secretion have been identified, among them mechanical and osmotic stress, activation of the T cell receptor (TCR) and gating of P2X7 itself [7, 8, 9]. Investigation of P2X7-mediated ATP release is difficult because the ligand required for gating of P2X7 is the same substance whose release is to be measured. The aim of this study was to characterise P2X7-mediated ATP release using a model that does not require the addition of exogenous ATP. To this end, we used the murine T lymphoma cell line Yac-1, which endogenously expresses both P2X7 and ADP-ribosyltransferase-C2 (ARTC2) and thus permits gating of P2X7 by covalent ADP-ribosylation in the presence of NAD+ . Importantly, Yac-1 cells do not express the classical ectonucleotidase CD39. Our results confirm that gating of P2X7 is a stimulus that induces cells to actively release ATP and shed light on the mechanism underlying this secretion. In addition, we used genetically encoded fluorescence resonance energy transfer (FRET)-based sensor proteins targeted to the cytosol to visualise P2X7-mediated changes in ATP distribution by live cell imaging. Besides conclusively identifying gating of P2X7 as a relevant stimulus for ATP release, our results show that this process is accompanied by depletion of ATP from the cytosol.
Materials and methods
Reagents and cell lines
Unless otherwise stated, chemicals were from Sigma-Aldrich (Munich, Germany). Yac-1 cells were originally kindly provided by Dr. Klaus Harbers, Hamburg, and maintained at the Institute of Immunology in RPMI 1640 medium supplemented with 10% foetal calf serum (FCS), 2 mM glutamine, 1 mM sodium pyruvate and 50 μM β-mercaptoethanol (all from Thermo Fisher Scientific, Darmstadt, Germany). Mouse 3T3 fibroblasts were originally kindly provided by Dr. Ingke Braren, Hamburg, and maintained at the Institute of Immunology in DMEM medium supplemented with 5% FCS, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer and 100 μM non-essential amino acids (all from Thermo Fisher Scientific). The P2X7-inhibitory nanobody 13A7 has been described elsewhere  and was used as a fusion protein containing the hinge and Fc domains of mouse IgG2c at a concentration of 1 μg/106 cells.
Flow cytometry was performed on FACS Canto2 and FACS Calibur instruments (Becton Dickinson, Heidelberg, Germany). Cells were stained for 30 min on ice with CD62L-APC (eBioscience) or CD27-PerCP-Cy5.5 (Biolegend, Koblenz, Germany) and washed once before analysis. For live/dead cell discrimination propidium iodide (PI) (Molecular Probes/Thermo Fisher Scientific) was added immediately before analysis. Analysis was performed using the FlowJo Software package (FlowJo, LLC, version 9.9.4). Flow cytometric monitoring of FRET signals was performed by generating the ratio of the 405-nm laser signals in the 450/50 and 510/50 nm channels using FlowJo.
Measurement of extracellular and intracellular ATP
Yac-1 cells were pre-incubated for 30 min at room temperature in the presence or absence of 13A7 (1 μg/106 cells), washed, and resuspended in ECS buffer (15 mM HEPES pH 7.4, 140 mM NaCl, 5 mM KCl, 10 mM D-glucose, 0.1% BSA) supplemented with 1 mM CaCl2 (ECS+). In some experiments, 1 mM MgCl2 was added additionally (ECS+/+). For the quantitative measurement of intra- and extracellular ATP, cells were suspended in 1 ml ECS+ or ECS+/20 μM NAD+ at a density of 2 × 105 cells/ml and incubated in FACS tubes for 3 h at 37 °C. The cells were centrifuged and the ATP content of the supernatant was measured in a Victor3 1430 photoluminometer (PerkinElmer, Rodgau, Germany) using the CellTiter Glo kit (Promega, Mannheim, Germany) according to the manufacturer’s instructions. The cell pellet was resuspended in ECS+ and one quarter of the cells was used for the measurement of intracellular ATP using the CellTiter Glo kit. Three quarters were subjected to flow cytometry to determine cell death and cell numbers. To this end, a suspension of fluorescent CaliBRITE microbeads (BD Biosciences, Heidelberg, Germany) in PBS was counted twice in a Neubauer chamber to determine the concentration of beads. Fifty microliters of this suspension containing 6.2 × 104 beads were added to 350 μl cell suspension. Flow cytometry was performed on a FACS Canto2, and acquisition was stopped when 2 × 104 beads were acquired. For other experiments, cells were incubated at a density of 1 × 106 cells/ml in 200 μl ECS+ buffer. In experiments involving 20 h of incubation, the cells were cultured in RPMI medium. For sequential measurements of extracellular ATP during the incubation period, the cells were seeded into wells of a white microtiter plate containing 5 μl of the CellTiter Glo luciferase/luciferin substrate dissolved in PBS instead of the lysis buffer. The plates were incubated at 37 °C in a Victor3 1430 photoluminometer and measured at the indicated time points.
Mechanisms of ATP secretion
Yac-1 cells were pre-incubated with carbenoxolone (10 min at room temperature); erioglaucine/Brilliant Blue FCF (20 min at 37 °C, 30 min at room temperature, and another 10 min at 37 °C); or BAPTA-AM (1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) and 30 min at 37 °C) before stimulation with NAD+. All chemicals were from Sigma-Aldrich. To impact ion fluxes across the P2X7 cation channel cells were either incubated in ECS buffer or K+ hi buffer (15 mM HEPES, pH 7.4, 145 mM KCl, 10 mM D-glucose, 0.1% BSA), supplemented with either 1 mM CaCl2 or 100 μM EDTA. Before measurement of eATP MgCl2 was added to concentration of 1 mM.
Induction of osmotic stress
To induce osmotic stress, 250 μl of cell suspension in ECS+/+ were diluted with 1750 μl of either water alone, 250 μl ECS+/+ + 1500-μl water, or 750 μl ECS+/+ + 1000 μl water, each containing luciferin/luciferase to reach reductions of osmolarity of 1:8, 1:4 or 1:2, respectively.
Construction of FRET sensors for ATP and transfection of cells
FRET sensors were constructed on the basis of the ATEAM sensors developed by Imamura and colleagues , with slight modifications introduced including alteration of restriction sites between the individual elements of the sensors. The sensors were assembled using published sequences for monomeric super-enhanced CFP (GenBank BAG31928.1) missing the terminal 11 amino acids (mseCFPd11), a circularly permutated  version of the Venus variant of YFP (GenBank CAO79590.1), and the epsilon subunits of ATP synthase from Bacillus subtilis (NCBI Reference Sequence WP_014478254.1) or Bacillus strain PS3 (SwissProt entry P07678.1). To create the ATP-non-binding RRKK variant, we replaced the arginine residues at positions 122 and 126 of the B. subtilis sequence by lysine residues. Sequences were assembled using the LaserGene Software package (DNAStar, Madison, WI, USA, version 8.1.2), and synthesised by GeneArt/Thermo Fisher (Regensburg, Germany) after codon optimisation for expression in human cells.
Live cell imaging
Live cell imaging was performed using an inverted microscope (Leica) with a CoolLED pE-100 light source (436 nm) and a dualview image splitter with 480/30 nm for CFP and 535/40 nm for YFP. 3T3 cells were seeded (4.5 × 105 cells per well) on a 6-well plate containing 25 mm cover slips coated with 0.1 mg/ml poly-L-lysine 24 h prior to measurement. Cover slips were mounted in an imaging chamber and washed once with 300 μl ECS+ buffer. Subsequently, 300 μl ECS buffer was added for measurement. Images were recorded using the Micromanger 1.4.5 software (ImageJ). A picture was taken every 5 s with an exposure time between 5 and 10 ms. After recording the baseline for 100 s, the same volume of ECS+ buffer containing a stimulus was added. Micromanager 1.4.5 software was used to create ROIs and to calculate CFP/YFP ratios. The ratio data were evaluated with Excel 2010 and Prism 7. Pseudocolour FRET Images were generated in FIJI (ImageJ2, ) according to the protocol of Kardash et al. .
Comparison of P2X7- and complement-mediated ATP release
Yac-1 cells stably transfected with the Bs.cyt or RRKK.cyt FRET sensors were suspended in 1 ml ECS+ and analysed on a FACS Canto2 flow cytometer (BD Biosciences) at 37 °C. After 60 s, cells were stimulated by adding either ATP to 500 μM, NAD to 20 μM, or 50 μl pooled human serum as a source of complement. Gates were set to identify morphologically intact cells (FSC/SSC) expressing the sensor (FITC channel). FRET was recorded as described above.
Human and animal rights
This article does not contain any studies with human or animal subjects performed by any of the authors.
NAD+-dependent ADP-ribosylation induces gating of P2X7 accompanied by rapid secretion of ATP
P2X7-mediated ATP secretion is accompanied by depletion of intracellular ATP in living cells
By contrast, the amount of intracellular ATP (iATP) recovered from the cell pellets was reduced in samples treated with NAD+ compared to untreated cells (268.3 ± 6.9 vs. 532.9 ± 21.9 pmol, Fig. 2g). However, when related to the total number of cells present in the culture, the amount of iATP/cell in NAD+-stimulated cells was also reduced (838 ± 36 amol (attomol)/cell vs. 1361 ± 124 amol/cell, Fig. 2h). Taking into account the higher death rate in the NAD+-treated samples and assuming that most of the intracellular ATP is contained in living cells, we calculated the amount of intracellular ATP per living cell. The results showed a mean ATP content of 919 ± 42 amol/living cell for NAD+-treated cells vs. 1406 ± 127 amol/living cell for untreated cells (Fig. 2i).
Continuous gating of P2X7 slows the proliferation of Yac-1 cells
A genetically encoded FRET-based ATP sensor reveals that gating of P2X7 is accompanied by a decrease in cytosolic ATP
To test whether these constructs could report cytosolic ATP levels, we transfected them into 3T3 fibroblasts. Besides visualisation by live imaging microscopy, the FRET signals of transfected cells could also be conveniently monitored by flow cytometry on a FACS Canto2 by forming the ratio of the signals in the 450 and 510 nm channels, enabling the simultaneous analysis of large populations of cells (Fig. 4a, b). Treatment with 10 μM of the mitochondrial uncoupling agent carbonyl cyanide m-chlorophenyl hydrazone (CCCP) caused a marked decrease in the FRET signals of 3T3 cells transfected with Bs.cyt, but not RRKK.cyt (Fig. 4a-c). To visualise the effects of P2X7-mediated ATP release on cytosolic ATP levels, we co-transfected 3T3 cells with Bs.cyt and P2X7. Treatment of these cells with 500 μM ATP resulted in a slow but steady decline in cytosolic ATP levels (Fig. 4d). In a similar fashion, Yac-1 cells transfected with Bs.cyt exhibited a slow and continuous decrease of FRET signals upon challenge with 500 μM ATP, an effect that was blocked by pre-treatment of the cells with the inhibitory nanobody 13A7, indicating that gating of P2X7 directly decreases cytosolic ATP levels in parallel with the export of ATP into the extracellular environment (Fig. 4e).
We used the Bs.cyt sensor to compare the kinetics of P2X7-mediated ATP release with that resulting from complement-mediated pore formation in the first minutes after stimulation. The results show that complement fixation leads to a rapid and almost complete loss of cytosolic ATP within the first 2 min after addition of serum, while gating of P2X7 induces a slow and continuous loss of ATP from the cytosol (Fig. 4f).
P2X7-induced ATP release is dependent on P2X7 ion channel activity
To investigate if P2X7-mediated ATP release depended on P2X7 ion channel activity, we gated P2X7 in buffers designed to minimise cation fluxes upon stimulation. Removal of extracellular calcium from the medium reduced ATP release by about 40% (Fig. 5c, e), and prevention of potassium efflux by gating in a high-potassium buffer diminished ATP release by about 75% (Fig. 5c). Interestingly, in contrast to ATP secretion, CD62L ectodomain shedding was not affected by the prevention of P2X7-associated cation fluxes (Fig. 5d). Since P2X7 affects cytosolic calcium levels not only by permitting the influx of extracellular calcium but also by stimulating the release of calcium from intracellular stores , we assessed the importance of cytosolic calcium for NAD+-mediated ATP release. Indeed, loading the cells with the calcium-chelating agent BAPTA before stimulation completely blocked the release of ATP (Fig. 5e).
Autocrine/paracrine gating of P2X7 by nucleotides released from Yac-1 cells by osmotic stress
However, osmotic stress also caused ectodomain shedding of CD62L and CD27, known indicators of P2X7 activation [6, 11] (Fig. 6c, e). This was completely abrogated by pre-treating the cells with 13A7. These results suggest that osmotic stress can induce Yac-1 cells to release ATP in amounts sufficient to gate P2X7 in a paracrine or autocrine manner. However, it should be noted that P2X7 on Yac-1 cells can also be gated via NAD+-dependent ADP-ribosylation, and that the release of NAD+ was not monitored in our experiment. It is therefore not possible to distinguish if the observed paracrine/autocrine activation of P2X7 consequent to osmotic stress was due to the release of ATP or NAD+.
The regulated release of ATP in response to external stimuli is an important mechanism for intercellular communication and a key event in the regulation of tissue inflammation. Interestingly, gating of P2X7 by ATP itself has been described as a stimulus for the regulated release of ATP . In this study, we used NAD+-dependent ADP-ribosylation as a model to investigate P2X7-mediated ATP secretion without needing to add exogenous ATP or a closely related compound. Treatment of Yac-1 cells with 20 μM NAD+ caused a rapid, P2X7-dependent increase of ATP in the extracellular space. Since gating of P2X7 is known to cause cell death, it is likely that a part of this increase in extracellular ATP was due to disintegration of dead or dying cells. However, the observation that the rise in extracellular ATP was accompanied by a corresponding decrease in the ATP content of living cells provided strong evidence that a substantial part of the increase was due to active ATP secretion by living cells.
Many tumour cells express P2X7, but the consequences of P2X7 signalling for tumour growth are ambivalent. On the one hand, expression of P2X7 positively affects the proliferation of many tumour cell lines by stimulating mitochondrial ATP production . On the other hand, strong and prolonged activation of P2X7 can cause cytolysis and thereby slow tumour growth. Our observations suggest a third possibility, namely that prolonged gating of P2X7 can slow the proliferation of living cells by depleting cytosolic ATP. The level of intracellular ATP in living cells was significantly decreased after 3 h of P2X7 gating, but returned to normal after 20 h. While the percentage of living cells remained constant over this time period (81% in cultures treated with NAD+ vs. 4–6% in untreated cultures), the proliferation index (calculated as the increase in total cell number from t = 3 h to t = 20 h, divided by the number of living cells at t = 3 h) was 1.19 for cells incubated with NAD+ compared to 1.86 for untreated cells. In cultures treated with 13A7, the proliferation rates were 1.90 (NAD+) and 1.71 (untreated), respectively. This observation suggests that the reduced cell number was not primarily due to increased cell death during the period of incubation but rather to reduced proliferation presumably resulting from the loss of cytosolic ATP. Quantification of intracellular ATP levels revealed a mean content of 1406 amol of ATP per living cell in untreated samples. Assuming Yac-1 to be perfect spheres of 10 μm diameter with uniform distribution of ATP inside the cell, this would correspond to an intracellular ATP concentration of 2.7 mM, a value within the range reported in the literature . Gating of P2X7 for 3 h decreased the mean ATP content of living cells to 919 amol (corresponding to 1.8 mM, assuming the size of the cells remained constant), a reduction by almost 35%.
ATP is a highly polar molecule that cannot traverse the lipid bilayer of the cell membrane under physiological conditions. Cells can release ATP either passively upon disintegration of cell membranes in the course of cell damage or death, or actively in response to a signal via a regulated transport mechanism. Regulated ATP release can occur either through exocytosis of ATP-containing vesicles  or through conductive pathways such as channels or pores . An important question was whether the increase in eATP observed following P2X7 activation was the result of P2X7-induced cell death. In our experiments, activation of P2X7 was always accompanied by an increase in the number of dead cells. Although cell death was never massive, ATP released from disintegrating dying cells might account for the increase in eATP. This is illustrated by the data reported in Online Resource 2. NAD+-stimulated cultures contained a mean of 12,556 dead cells and an eATP content of 24.9 pmol, implying a contribution of 890 amol of ATP from each dead cell, far below the total ATP content of 1400 amol calculated for unstimulated living cells. Thus, the amount of ATP originally contained in the dead cells would be more than sufficient to explain the increase in eATP, assuming that the dead cells released all of their ATP.
Although we cannot formally rule out that release of eATP comes from disintegrating or damaged cells, several observations point to a regulated mechanism of release. First of all, we found a notable difference in the amount of eATP that could be attributed to dead cells in untreated vs. NAD+-stimulated cultures (36 vs. 890 pmol). Under the assumption that all eATP was derived from dead cells, this would suggest that, even though they had died, P2X7-activated cells had released substantially more ATP than their unstimulated counterparts. More importantly, we also detected differences in the intracellular ATP content of living cells between untreated and NAD+-stimulated cells (1406 vs. 919 amol/living cell). This depletion of ATP in living cells had functional consequences for the cells, as the proliferation rate of these cells was also reduced (see Fig. 3). Furthermore, the dependence of ATP secretion on the cytosolic calcium and potassium efflux also suggest a regulated mechanism.
Taken together, gating of P2X7 for 3 h reduced the intracellular ATP content of Yac-1 cells to 50% of that of untreated control cells. The amount recovered from the supernatant of treated cells corresponded to an additional 5% of the total ATP of control cells. The fate of the remaining 45% is unclear. This ‘missing’ ATP may have been degraded by ectonucleotidases, although Yac-1 cells do not express the ectonucleotidase CD39. However, other ectoenzymes such as phosphatases may also degrade ATP, and ATP-metabolising enzymes may have been released from inside the cell in the course of cell death. It is equally possible, however, that part of the missing ATP might be due to alterations in intracellular metabolism as a consequence of sustained P2X7 signalling.
Our observations do not point to a specific mechanism. In fact, the distinction between ATP release resulting from cell death or regulated secretion is not clear-cut. For example, T cells undergoing apoptosis actively secrete ATP via pannexin-1 hemichannels that are activated by caspase-mediated cleavage of their C-terminal tails . It is therefore possible that the regulated secretion of ATP is associated with P2X7-mediated cell death. We observed a partial dependency of ATP on the presence of extracellular calcium, consistent with a requirement for the cation channel activity of P2X7. However, this dependency was not complete as long as elevations of cytosolic calcium from intracellular stores were not blocked. This phenomenon could be explained by the observation that P2X7 can induce the liberation of calcium from intracellular stores via the IP3 receptor in the absence of extracellular calcium . The finding that ATP secretion is associated with direct depletion of ATP in the cytosol seems to suggest a conductive mechanism. Pannexin-1 and connexin-43 hemichannels have been associated with the release of ATP from T lymphocytes. However, carbenoxolone, a broad-specificity inhibitor of pannexins and connexins did not block ATP export. On the other hand, the strong dependency of ATP release on P2X7-mediated K+ efflux suggests that, similar to IL-1β, ATP also may be released by a mechanism resembling unconventional protein secretion consequent to inflammasome assembly . Further investigations will be necessary to clarify this point.
The notion that activation of P2X7 by eATP induces the secretion of more eATP suggests that this may be part of a self-amplifying feed forward mechanism ensuring full activation of P2X7 once a signalling process has been started. This question was difficult to address in our system because manipulations that interfere with the ‘second messenger’ (released ATP) always also affect the measured outcome (released ATP). In addition, unlike gating of P2X7 via ATP, gating via NAD+-dependent ADP-ribosylation provides a covalently bound ligand that is difficult to titrate. Experiments using low doses of NAD+ near the activation threshold in the presence or absence of apyrase to hydrolyse released ATP failed to provide clear evidence for a synergistic feed-forward loop (data not shown). However, we did address the question of whether ATP released from cells in response to a physiological stimulus (osmotic stress) could activate P2X7 in an autocrine or paracrine fashion. Lowering the osmolarity of the medium to 25% induced Yac-1 cells to release ATP that in turn activated P2X7 on the same or neighbouring cells. This was evidenced by ecto-domain shedding of CD62L and CD27 that could be blocked by treating the cells with the P2X7-inhibitory nanobody 13A7. It is important to realise that this interaction took place in cell suspension where the ATP released from the cells is immediately diluted into the extracellular space, while most relevant scenarios for the activation of P2X receptors by secreted ATP are envisioned to take place in closed compartments such as immunological synapses [25, 26].
In this study, we used a genetically encoded FRET sensor to directly visualise the ATP content of the cytosol following the gating of P2X7. The results nicely complement the direct measurements of ATP content by the luciferase/luciferin method, and provide a convenient method to follow changes in cytosolic ATP either by live cell imaging or by flow cytometry. It will be interesting to use these tools in the context of more complex cellular interactions such as the interaction between T lymphocytes and antigen-presenting cells.
The authors thank G. Dubberke for technical assistance.
This study was supported by the Deutsche Forschungsgemeinschaft (SFB1328 and grant Ha2569/5).
Compliance with ethical standards
Conflicts of interest
BJ, KEK, SN, ES, JAN, RF, IMAW, AHG, and VON declare that they have no conflict of interest.
FKN and FH received royalties from the sales of antibodies developed in the laboratory via MediGate GmbH, a 100% subsidiary of the University Medical Center, Hamburg. FKN is also an inventor on patent applications on P2X7-specific nanobodies.
This article does not contain any studies with human participants or animals performed by any of the authors.
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