Dynamic regulation of cell volume and extracellular ATP of human erythrocytes

Introduction: The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe. Methods and Treatments: We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors. Results: In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40-50% and swelling by 40-60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%. Analysis and Discussion: Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop underlying ATP-induced ATP release of rbcs.Instituto de Física de Líquidos y Sistemas Biológico

Dynamic regulation of cell volume and extracellular ATP of human erythrocytes

Introduction: The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe. Methods and Treatments: We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors. Results: In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40-50% and swelling by 40-60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%. Analysis and Discussion: Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop underlying ATP-induced ATP release of rbcs.Instituto de Física de Líquidos y Sistemas Biológico

Effect of MST7 on hematocrit of rbcs.

<p>Rbcs were held in suspension in isosmotic medium at 20% hematocrit, and exposed 10 min to isosmotic medium in the absence and presence of POM1 (an ectoATPase inhibitor) followed by exposure to 10 μM MST7 for 2 min. Hematocrits were then determined, and expressed as percentage (%). Results are means ± SEM (N = 5, n = 46) (***, p < 0,001 versus Basal; **, p < 0,05 <i>versus</i> Basal + POM1; &: not significant). Numbers of determinations (n) from independent preparations (N) are indicated.</p

Effect of ATP release on the kinetics of Vr and [ATPe].

<p>(A) <i>Time course of Vr for rbcs exposed to MST7</i>. BCECF-loaded rbcs were incubated in 200 μL isosmotic medium in the absence or presence of 10 μM CBX (○), 10 μM PBC (), or 10 U/mL apyrase (Apy, ). After 20 min, 10 μM MST7 was added. Results are means ± SE of 20–30 rbcs for CBX (N = 4), PBC (N = 3) and apyrase (N = 3). The dashed arrow indicates addition of treatment and the full arrow indicates exposure to 10 μM MST7. Numbers of independent preparations (N) are indicated. (B) <i>Time course of [ATPe] for rbcs exposed to MST7</i>. Rbcs were incubated in 40 μL isosmotic medium in the absence or presence of 10 μM CBX (light grey line, N = 5, n = 8) or 10 μM PBC (dark grey line, N = 3, n = 5). After 20 min, 10 μM MST7 was added. The dashed arrow indicates addition of blockers and the full arrow indicates exposure to MST7. Numbers of determinations (n) from independent preparations (N) are indicated. (C) <i>Degree of swelling derived from results shown in A</i>. Results are given as Vr₂₀, i.e., the Vr value at 20 min post MST7 exposure. Results are means ± SEM (&, p < 0.001 <i>versus</i> CTROL, MST7 alone; #, p < 0.01 <i>versus</i> Apy; ^, p < 0.001 <i>versus</i> PBC10). (D) <i>Effect of apyrase on swelling</i>. BCECF-loaded rbcs were pre-incubated for 10 min with 10, 40 or 80 U/mL Apy, followed by exposure to 10 μM MST7 for 5 min. The degree of swelling was expressed as Vr₅, i.e., the Vr value at 5 min post MST7 exposure. Results are means ± SEM (&, p < 0.001 <i>versus</i> CTROL, MST7 alone; #, p < 0.001 among apyrase concentrations). CBX = carbenoxolone; PBC = probenecid, Apy = apyrase. Calibrations of Vr and [ATPe] were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158305#pone.0158305.g001" target="_blank">Fig 1</a>.</p

Hemolysis assessed for off-line ATPe determinations.

<p>Hemolysis assessed for off-line ATPe determinations.</p

ATPe concentration of a suspension of rbcs exposed to MST7.

<p>Rbcs were held in suspension in isosmotic medium (300 mosM) at 20% hematocrit and exposed to 10 μM MST7 for 2 and 6 min (T2 and T6 respectively). MST7 was added in the absence of additional treatments (MST7), or in the presence of hyperosmotic medium (Hyper, 345 mosM) or 10 μM probenecid (PBC). Controls were run in the absence of MST7, both in isosmotic (B.iso) as well as hyperosmotic (B. hyper) media. Assessment of hemolysis for each sample allowed to calculate the lytic contribution to ATPe concentration. Results are means of N = 3, n = 5. Numbers of determinations (n) from independent preparations (N) are indicated.</p

Model simulations of Vr kinetics in the absence and presence of blockers.

<p>Model dependent fit to experimental Vr kinetics in the absence of blockers (control condition), and in the presence of 10 μM CBX or 10 μM NF110. Fitting was performed simultaneously to the three experimental conditions. MST7 was added at t = 20 min. Black, grey and blue lines correspond to experimental data of Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158305#pone.0158305.g001" target="_blank">1A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158305#pone.0158305.g002" target="_blank">2A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158305#pone.0158305.g005" target="_blank">5A</a>, respectively. Red lines show the best fitting curves to experimental data. The dashed line indicates the critical value of Vr^c above which ATP release is triggered by swelling. Model for Vr kinetics is encoded in Eqs. A-B of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158305#pone.0158305.s001" target="_blank">S1 File</a>, with values of best fit for the parameters given in Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158305#pone.0158305.s001" target="_blank">S1 File</a>.</p

Mutual regulation of ATPe and Vr.

<p>The scheme summarizes the key features of this study. In rbcs, exposure to MST7 triggers activation of two ATP permeabilities (P_ATP¹ and P_ATP²), causing [ATPe] kinetics. In the absence of P2X activation, swelling is activated by ATPe, but the magnitude of volume increase is so small that it does not trigger ATP efflux. This is shown as the arrow leading to Vrc. Activation of a P2X receptor by ATPe leads to sodium influx, coupled to water influx and swelling. As Vr increases, it surpasses Vr^c (i.e., Vr>Vr^c), so that P_ATP is transiently activated, leading to ATP release. ATPi and ATPe denote intracellular and extracellular ATP, respectively. The chemical gradient refers to the difference between ATPi and ATPe at both sides of the plasma membrane.</p

Effect of P2X receptor and sodium on the kinetics of Vr and ATPe.

<p>(A) <i>Vr kinetics</i>. BCECF-loaded rbcs were incubated in isosmotic medium. After 10 min the medium was replaced by a sodium free isosmotic medium () or an isosmotic medium containing 10 μM NF110 (). Kinetics of Vr for rbcs exposed to MST7 in isosmotic (●) and hyperosmotic (○) media are shown for comparison. Calibration was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158305#pone.0158305.g001" target="_blank">Fig 1</a>. Results are means ± SE of 20–30 rbcs (isosmotic medium, N = 6; NF110, N = 4; Choline, N = 3) and 30–40 rbcs for hyperosmotic medium (N = 4). The dashed arrow indicates addition of blocker and the full arrow indicates addition of MST7. (B) <i>Degree of swelling obtained from A</i>. Values are expressed as Vr₂₀, i.e., the Vr value at 20 min post stimulus. Results are means ± SEM (***, p < 0.001 <i>versus</i> CTROL, MST7 alone). (C) <i>ATPe kinetics</i>. Rbcs were pre-incubated with 10 μM NF110 (blue line, N = 3, n = 5) for 10 min before exposure to 10 μM MST7. ATPe kinetics for MST7 in isosmotic (black line) and hyperosmotic (red line) media are shown for comparison. Results are expressed in nM for 3 10⁶ cells in 40 μL assay medium. The dashed arrow indicates addition of blocker and the full arrow indicates addition of MST7. (D) <i>Values of [ATPe] increase using data from C</i>. Values are expressed as ΔATP₂₀, i.e., the difference between [ATPe] at 20 min post stimulus and basal [ATPe]. Results are means ± SEM. (*, p < 0.05 <i>versus</i> CTROL, MST7 alone) and are expressed in nM for 3 10⁶ cells assayed in 40 μL of medium. Calibrations of Vr and [ATPe] were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158305#pone.0158305.g001" target="_blank">Fig 1</a>.</p