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The lipid bilayer of eukaryotic cells’ plasma membrane is almost impermeable to small ions and large polar molecules, but its miniscule basal permeability in intact cells is poorly characterized. This report describes the intrinsic membrane permeability of A549 cells toward the charged molecules propidium (Pr(2+)) and ATP(4−). Under isotonic conditions, we detected with quantitative fluorescence microscopy, a continuous low-rate uptake of Pr (∼150 × 10(−21) moles (zmol)/h/cell, [Pr](o) = 150 μM, 32°C). It was stimulated transiently but strongly by 66% hypotonic cell swelling reaching an influx amplitude of ∼1500 (zmol/h)/cell. The progressive Pr uptake with increasing [Pr](o) (30, 150, and 750 μM) suggested a permeation mechanism by simple diffusion. We quantified separately ATP release with custom wide-field-of-view chemiluminescence imaging. The strong proportionality between ATP efflux and Pr(2+) influx during hypotonic challenge, and the absence of stimulation of transmembrane transport following 300% hypertonic shock, indicated that ATP and Pr travel the same conductive pathway. The fluorescence images revealed a homogeneously distributed intracellular uptake of Pr not consistent with high-conductance channels expressed at low density on the plasma membrane. We hypothesized that the pathway consists of transiently formed water pores evenly spread across the plasma membrane. The abolition of cell swelling-induced Pr uptake with 500 μM gadolinium, a known modulator of membrane fluidity, supported the involvement of water pores whose formation depends on the membrane fluidity. Our study suggests an alternative model of a direct permeation of ATP (and other molecules) through the phospholipid bilayer, which may have important physiological implications.

Subcutaneous mast cells (MCs) are vulnerable to mechanical stimulation from external environment. Thus, MCs immune function could be modulated by their mechanosensitivity. This property has been identified as the trigger mechanism of needling acupuncture, a traditional oriental therapy. Previously we have demonstrated the release of adenosine triphosphate (ATP), a stress-responsive signalling molecule, from mechanical-perturbed MCs. The current work explores its underlying mechanisms. We noticed that propagation of intracellular free Ca2+ occurred among HMC-1 cells in response to 50% hypotonic shock. Additionally, amplifying cascade of ATP-induced ATP release was observed in RBL-2H3 cells stimulated by medium displacement, which could be mimicked by exogenous ATP (exoATP). Secondary ATP liberation induced by low level (50 nmol/L) of exoATP was reduced by inhibiting ecto-ATPase-dependent ADP production with ARL67156, or blocking P2 receptors with suramin or PPADS, or with specific P2Y13 receptor antagonist MRS2211, or siRNA. Secondary ATP release induced by higher dose (200 μmol/L) of exoATP, sufficient to stimulate P2X7 receptor, was attenuated by suramin, PPADS or specific P2X7 receptor antagonist BBG, or siRNA. Finally, RT-PCR confirmed mRNA expression of P2Y13 and P2X7 in RBL-2H3 cells. Additionally, such secondary ATP release was attenuated by DPCPX, specific antagonist of adenosine A1 receptor, but not by MRS2179, specific inhibitor of P2Y1 receptor. In summary, mechanosensitive ATP release from MCs is facilitated by paracrine/autocrine stimulation of P2Y13 and P2X7 receptors. This multi-receptor combination could mediate transmission of information from a local site to distal areas, enabling communication with multiple surrounding cells to coordinate and synchronize their function.

Background/aims Prolonged hyperosmotic shrinkage evokes expression of osmoprotective genes via nuclear factor NFAT5-mediated pathway and activates Na+ influx via hypertonicity-induced cation channels (HICC). In human umbilical vein endothelial cells (HUVEC) elevation of intracellular sodium concentration ([Na+]i) triggers transcription of dozens of early response genes (ERG). This study examined the role of monovalent cations in the expression of Na+i-sensitive ERGs in iso- and hyperosmotically shrunken HUVEC. Methods Cell volume was measured by 3D reconstruction of cell shape and as 14C-urea available space. Intracellular Na+ and K+ content was measured by flame atomic absorption spectrometry. ERG transcription was estimated by RT-PCR. Results Elevation of medium osmolality by 150 mM mannitol or cell transfer from hypo- to isosmotic medium decreased cell volume by 40-50%. Hyperosmotic medium increased [Na+]i by 2-fold whereas isosmotic shrinkage had no impact on this parameter. Hyperosmotic but not isosmotic shrinkage increased up-to 5-fold the content of EGR1, FOS, ATF3, ZFP36 and JUN mRNAs. Expression of these ERGs triggered by hyperosmotic shrinkage and Na+,K+-ATPase inhibition by 0.1 µM ouabain exhibited positive correlation (R2=0.9383, p=0.0005). Isosmotic substitution of NaCl by N-methyl-D-glucamine abolished an increment of [Na+]i and ERG expression triggered by mannitol addition. Conclusion Augmented expression of ERGs in hyperosmotically shrunken HUVEC is mediated by elevation of [Na+]i.

The lytic release of ATP due to cell and tissue injury constitutes an important source of extracellular nucleotides and may have physiological and pathophysiological roles by triggering purinergic signalling pathways. In the lungs, extracellular ATP can have protective effects by stimulating surfactant and mucus secretion. However, excessive extracellular ATP levels, such as observed in ventilator-induced lung injury, act as a danger-associated signal that activates NLRP3 inflammasome contributing to lung damage. Here, we discuss examples of lytic release that we have identified in our studies using real-time luciferin-luciferase luminescence imaging of extracellular ATP. In alveolar A549 cells, hypotonic shock-induced ATP release shows rapid lytic and slow-rising non-lytic components. Lytic release originates from the lysis of single fragile cells that could be seen as distinct spikes of ATP-dependent luminescence, but under physiological conditions, its contribution is minimal

Pancreatic β-cells continuously sense blood glucose levels and secrete insulin to maintain normoglycemia. In β-cells, ATP generated by glycolysis promotes the closure of ATP-sensitive K+ channels, thereby increasing intracellular calcium and ultimately insulin secretion. 14-3-3 proteins, and in particular 14-3-3ζ, have been found to regulate ATP synthase and mitochondrial respiration, suggesting that members of this family of scaffold proteins in β-cells may influence glucose-stimulated insulin secretion (GSIS) and glucose homeostasis. To date, we have identified critical contributions of 14-3-3 proteins to GSIS. In mouse and human islets, pan-inhibition of 14-3-3 proteins with cell-permeable inhibitors potentiated ex-vivo GSIS. This was associated with increased mitochondrial function, as oxygen consumption and ATP synthesis rates were significantly enhanced. Moreover, increased ATP production was confirmed with luciferase-based assays. Of the seven isoforms, we previously reported critical metabolic roles of 14-3-3ζ in glucose homeostasis, and to understand its role in β-cells, β-cell-specific knockout mice (Ins1CreThor:14-3-3ζfl/fl, β-KO) were generated. When compared to control mice, no differences in body weights or glucose and insulin tolerance were observed, but β-KO mice displayed significantly enhanced insulin secretion following i.p. glucose (2 g/kg), and ex vivo islet perifusion studies revealed enhanced 2nd-phase secretion of GSIS from β-KO islets. Similar to pan-14-3-3 inhibition, increased mitochondrial activity and ATP synthesis were detected in islets of β-KO mice. In summary, these results demonstrate critical functions of 14-3-3ζ and its related proteins in mitochondrial activity in β-cells and the regulation of GSIS. These data also suggest that 14-3-3ζ inhibition may represent a promising target to enhance pancreatic β-cell function in the context of diabetes. Disclosure Y. Mugabo: None. M. Galipeau: None. J. Tan: None. E. Fadzeyeva: None. R. Grygorczyk: None. G.E. Lim: None. Funding Canadian Institutes of Health Research; Canada Research Chairs Program (PJT-153144)

This study examines the action of hypoxia on integrity, fluidity and protein composition of red blood cell (RBC) membrane. Twenty-min exposure to oxygen-free environment decreases rat RBC integrity documented by 3-fold elevation of hemoglobin release without any action on the membrane fluidity estimated by electron magnetic resonance spectroscopy of spin-labeled stearic acid analogues. The proteomics technology in combination with relative label free quantification analysis revealed a dozen of membrane-bound proteins, including elevated content of hemoglobin, reproducibly affected by hypoxia. Mapping the identified proteins in the KEGG pathway database we found that the proteins of multi subunit Cullin-Rbx E3 ubiquitin ligase complex are presented in normoxic RBC ghosts but not in the hypoxic samples. Our results suggest that Cullin-Rbx E3 complex, associated with RBC membrane in normoxia, provides detection and deletion of membrane proteins damaged by reactive oxygen species. In hypoxic conditions, deoxy-Hb binds to band 3 protein, resulting in dissociation of Cullin-Rbx E3 complex from RBC membrane and impaired clearance of damaged cytoskeleton proteins. These rearrangements of membrane proteins might be involved in attenuated membrane integrity revealed in hypoxic RBC. Significance This study demonstrate that sustained deoxygenation of rat erythrocytes alters the composition of membrane-bound proteins including elevation of the content of hemoglobin without any changes in the viscosity of erythrocyte membrane lipid bilayer. These results suggest that the changes the composition of membrane proteins result in attenuated membrane integrity and contribute to augment release of hemoglobin seen in hypoxic conditions.

A method to simultaneously assess the changes in intracellular calcium concentration and cell volume in single cells was developed using the Ca2+-sensitive fluorescent probe Fura-2 and a three-dimensional image-surface reconstruction technique, respectively. Studies with this method showed that Fura-2 loading had no significant effect on the kinetics of A549 human epithelial cell swelling in a hypotonic solution, as well as the volume restoration kinetics. Significant changes in intracellular Ca2+ concentration were not observed in the examined volume modulation range. The results suggest that Ca2+-mediated signaling pathways are not involved in the autoregulation of the cell volume in A549 cells exposed to hypotonic conditions.

Red blood cells are involved not only in transportation of oxygen and carbon dioxide but also in autoregulation of vascular tone by ATP release in hypoxic conditions. Molecular mechanisms of the ATP release from red blood cells in response to a decrease in partial oxygen pressure still remain to be elucidated. In this work we have studied effects of hypoxia on red blood cell hemolysis in humans and rats and compared the effects of inhibitors of ecto-ATPase and pannexin on the release of ATP and hemoglobin from rat erythrocytes. The 20-min hypoxia at 37°C increased hemolysis of red blood cells in humans and rats 1.5- and 2.5-fold, respectively. In rat erythrocytes a significant increase in hypoxia-induced extracellular ATP level was found only in the presence of ecto-ATPase inhibitor ARL 67156. In these conditions we observed a positive correlation (R2 = 0.5003) between the increase in free hemoglobin concentration and the ATP release. Neither carbenoxolon nor probenecid, the inhibitors of low-selectivity pannexin channels, altered the hypoxia-induced ATP release from rat erythrocytes. The obtained results indicate a key role of hemolysis in the ATP release from red blood cells.

Abnormalities of mucus viscosity play a critical role in the pathogenesis of several respiratory diseases, including cystic fibrosis. Currently, there are no approaches to assess the rheological properties of mucin granule matrices in live cells. This is the first example of the use of a molecular rotor, a BODIPY dye, to quantitatively visualize the viscosity of intragranular mucin matrices in a large population of individual granules in differentiated primary bronchial epithelial cells using fluorescence lifetime imaging microscopy.

An upregulation of Egr-1 expression has been reported in models of atherosclerosis and intimal hyperplasia and, various vasoactive peptides and growth promoting stimuli have been shown to induce the expression of Egr-1 in vascular smooth muscle cells (VSMC). Angiotensin-II (Ang-II) is a key vasoactive peptide that has been implicated in the pathogenesis of vascular diseases. Ang-II elevates intracellular Ca2+ through activation of the store-operated calcium entry (SOCE) involving an inositol-3-phosphate receptor (IP3R)-coupled depletion of endoplasmic reticular Ca2+ and a subsequent activation of the stromal interaction molecule 1 (STIM-1) /Orai-1 complex. However, the involvement of IP3R/STIM-1/Orai-1-Ca2+-dependent signaling in Egr-1 expression in VSMC remains unexplored. Therefore, in the present studies, we have examined the role of Ca2+ signaling in Ang-II-induced Egr-1 expression in VSMC and investigated the contribution of STIM-1 or Orai-1 in mediating this response. 2-aminoethoxydiphenyl borate (2-APB), a dual non-competitive antagonist of IP3R and inhibitor of SOCE, decreased Ang-II-induced Ca2+ release and attenuated Ang-II-induced enhanced expression of Egr-1 protein and mRNA levels. Egr-1 upregulation was also suppressed following blockade of calmodulin and CaMKII. Furthermore, RNA interference-mediated depletion of STIM-1 or Orai-1 attenuated Ang-II-induced Egr-1 expression as well as Ang-II-induced phosphorylation of ERK1/2 and CREB. In addition, siRNA-induced silencing of CREB resulted in a reduction in the expression of Egr-1 stimulated by Ang-II. In summary, our data demonstrate that Ang-II-induced Egr-1 expression is mediated by STIM-1/Orai-1/Ca2+-dependent signaling pathways in A-10 VSMC. This article is protected by copyright. All rights reserved

Subcutaneous mast cells (MCs) are vulnerable to mechanical stimulation from external environment. Thus, MCs immune function could be modulated by their mechanosensitivity. This property has been identified as the trigger mechanism of needling acupuncture, a traditional oriental therapy. Previously we have demonstrated the release of adenosine triphosphate (ATP), a stress-responsive signalling molecule, from mechanical-perturbed MCs. The current work explores its underlying mechanisms. We noticed that propagation of intracellular free Ca

Extracellular ATP and its metabolites are potent paracrine modulators of lung alveolar cell function, including surfactant secretion and fluid transport, but the sources and mechanism of intra-alveolar ATP release remain unclear. To determine the contribution of gas-exchanging alveolar type 1 (AT1) and surfactant-secreting type 2 (AT2) cells to stretch-induced ATP release, we used quantitative real-time luminescence ATP imaging and rat primary alveolar cells cultured on silicon substrate for 2–7 days. When cultured on solid support, primary AT2 cells progressively transdifferentiated into AT1-like cells with ~20% of cells showing AT1 phenotype by day 2–3 (AT2:AT1 ≈ 4:1), while on day 7, the AT2:AT1 cell ratio was reversed with up to 80% of the cells displaying characteristics of AT1 cells. Stretch (1 s, 5–35%) induced ATP release from AT2/AT1 cell cultures, and it was highest on days 2 and 3 but declined in older cultures. ATP release tightly correlated with the number of remaining AT2 cells in culture, consistent with ~10-fold lower ATP release by AT1 than AT2 cells. ATP release was unaffected by inhibitors of putative ATP channels carbenoxolone and probenecid but was significantly diminished in cells loaded with calcium chelator BAPTA. These pharmacological modulators had similar effects on stretch-induced intracellular Ca2+ responses measured by Fura2 fluorescence. The study revealed that AT2 cells are the primary source of stretch-induced ATP release in heterocellular AT2/AT1 cell cultures, suggesting similar contribution in intact alveoli. Our results support a role for calcium-regulated mechanism but not ATP-conducting channels in ATP release by alveolar epithelial cells.

Although several mechanical stressors promote ATP secretion from eukaryotic cells, few mechanosensitive pathways for ATP release have been precisely characterized and none have been clearly identified. To facilitate progress, we report here a wide field of view (∼20 × 20 mm sample area) imaging technique paired with a quantitative image analysis to accurately map the dynamics of ATP release from a cell population. The approach has been tested on A549 cells stretched at high initial strain rate (2–5 s−1) or swelled by hypotonic shock. The amount of ATP secreted in response to a series of five graded stretch pulses (5–37% linear deformation, 1-s duration at 25°C) changed nonmonotonically with respect to strain amplitude and was inhomogeneous across the cell monolayer. In a typical experiment, extracellular ATP density averaged 250 fmol/mm2, but the area of detectable signal covered only ∼40% of the cells. In some areas, ATP accumulation peaked around 900 fmol/mm2, which corresponded to an estimated concentration of 4.5 µM. The total amount of ATP released from the combined stretch pulses reached 384 ± 224 pmol/million cells ( n = 4). Compared with stretch, hypotonic shock (50%, 30°C) elicited a more homogeneous ATP secretion from the entire cell population but at a lower yield totaling 28 ± 12 pmol/million cells ( n = 4). The quantitative extracellular ATP mapping of several thousand cells at once, with this wide field of view imaging system, will help identify ATP release pathways by providing unique insights on the dynamics and inhomogeneities of the cellular ATP secretion that are otherwise difficult to assess within the smaller field of view of a microscope.

Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that stimulate purinergic receptors and regulate diverse processes in the normal lungs. They are also associated with pathogenesis of a number of respiratory diseases and clinical complications including acute respiratory distress syndrome and ventilator induced lung injury. Mechanical forces are major stimuli for cellular ATP release but precise mechanisms responsible for this release are still debated. The present review intends to provide the current state of knowledge of the mechanisms of ATP release in the lung. Putative pathways of the release, including the contribution of cell membrane injury and cell lysis are discussed addressing their strength, weaknesses and missing evidence that requires future study. We also provide an overview of the recent technical advances in studying cellular ATP release in vitro and ex vivo. Special attention is given to new insights into lung ATP release obtained with the real-time luminescence ATP imaging. This includes recent data on stretch-induced mechanosensitive ATP release in a model and primary cells of lung alveoli in vitro as well as inflation-induced ATP release in airspaces and pulmonary blood vessels of lungs, ex vivo.

Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that stimulate purinergic receptors and regulate diverse processes in the normal lungs. They are also associated with pathogenesis of a number of respiratory diseases and clinical complications including acute respiratory distress syndrome and ventilator induced lung injury. Mechanical forces are major stimuli for cellular ATP release but precise mechanisms responsible for this release are still debated. The present review intends to provide the current state of knowledge of the mechanisms of ATP release in the lung. Putative pathways of the release, including the contribution of cell membrane injury and cell lysis are discussed addressing their strength, weaknesses and missing evidence that requires future study. We also provide an overview of the recent technical advances in studying cellular ATP release in vitro and ex vivo. Special attention is given to new insights into lung ATP release obtained with the real-time luminescence ATP imaging. This includes recent data on stretch-induced mechanosensitive ATP release in a model and primary cells of lung alveoli in vitro as well as inflation-induced ATP release in airspaces and pulmonary blood vessels of lungs, ex vivo.

Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that regulate diverse processes critical for lung function, including mucociliary clearance, surfactant secretion, and local blood flow. Cellular ATP release is mechanosensitive; however, the impact of physical stimuli on ATP release during breathing has never been tested in intact lungs in real time and remains elusive. In this pilot study, we investigated inflation-induced ATP release in rat lungs ex vivo by real-time luciferin-luciferase (LL) bioluminescence imaging coupled with simultaneous infrared tissue imaging to identify ATP-releasing sites. With LL solution introduced into air spaces, brief inflation of such edematous lung (1 s, ∼20 cmH2O) induced transient (−6 M, relevant for autocrine/paracrine signaling, but it remained spatially restricted to single alveolar sacs or their clusters. ATP release was stimulus dependent: prolonged (100 s) inflation evoked long-lasting ATP release that terminated upon alveoli deflation/derecruitment while cyclic inflation/suction produced cyclic ATP release. With LL introduced into blood vessels, inflation induced transient ATP release in many small patchlike areas the size of alveolar sacs. Findings suggest that inflation induces ATP release in both alveoli and the surrounding blood capillary network; the functional units of ATP release presumably consist of alveolar sacs or their clusters. Our study demonstrates the feasibility of real-time ATP release imaging in ex vivo lungs and provides the first direct evidence of inflation-induced ATP release in lung air spaces and in pulmonary blood capillaries, highlighting the importance of purinergic signaling in lung function.

The plasma membrane plays a prominent role in the regulation of cell volume by mediating selective transport of extra- and intracellular osmolytes. Recent studies show that upstream sensors of cell volume changes are mainly located within the cytoplasm that displays properties of a hydrogel and not in the plasma membrane. Cell volume changes occurring in anisosmotic medium as well as in isosmotic environment affect properties of cytoplasmic hydrogel that, in turn, trigger rapid regulatory volume increase and decrease (RVI and RVD). The downstream signaling pathways include reorganization of 2D cytoskeleton and altered composition of polyphosphoinositides located on the inner surface of the plasma membrane. In addition to its action on physico-chemical properties of cytoplasmic hydrogel, cell volume changes in anisosmotic conditions affect the ionic strength of the cytoplasm and the [Na

The plasma membrane plays a prominent role in the regulation of cell volume by mediating selective transport of extra- and intracellular osmolytes. Recent studies show that upstream sensors of cell volume changes are mainly located within the cytoplasm that displays properties of a hydrogel and not in the plasma membrane. Cell volume changes occurring in anisosmotic medium as well as in isosmotic environment affect properties of cytoplasmic hydrogel that, in turn, trigger rapid regulatory volume increase and decrease (RVI and RVD). The downstream signaling pathways include reorganization of 2D cytoskeleton and altered composition of polyphosphoinositides located on the inner surface of the plasma membrane. In addition to its action on physico-chemical properties of cytoplasmic hydrogel, cell volume changes in anisosmotic conditions affect the ionic strength of the cytoplasm and the [Na+]i/[K+]i ratio. Elevated intracellular ionic strength evoked by long term exposure of cells to hypertonic environment resulted in the activation of TonEBP and augmented expression of genes controlling intracellular organic osmolyte levels. The role of Na+i/K+i -sensitive, Ca2+i -mediated and Ca2+i-independent mechanisms of excitation-transcription coupling in cell volume-adjustment remains unknown.

Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~ 10-fold elevation of furosemide-resistant 86Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2 +. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2 +-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel.

Objective To investigate the role of adenosine triphosphate (ATP) purinergic signaling in mast cells (MCs) modulated by heat to further understand the molecular mechanisms of moxibustion. Methods Skin temperatures induced by monkshood cake moxibustion were evaluated by measuring the Neiguan acupoint (PC 6) from 31 participants with a digital thermocouple thermometer. Temperatures of 43 °C and 52 °C were applied to cultured human leukemia mast cell line HMC-1 in vitro. Calcium fluorescence was applied to detect intracellular Ca2+ ([Ca2+]i). Extracellular ATP contents were measured by luciferin-luciferase assay. Results Maximum skin temperatures mostly ranged from 40–45 °C, but some reached up to 50 °C. Both 43 °C and 52 °C induced MC degranulation, which was accompanied by an increase in [Ca2+]i and ATP release. Complexing extracellular Ca2+ with 5 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited the noxious heat-induced elevation of [Ca2+]i and prevented the enhanced ATP secretion by those cells at 52 °C, but not 43 °C. Conclusion Monkshood cake moxibustion can generate heat sufficient to trigger cellular events of MCs, including degranulation, [Ca2+]i elevation, and ATP release, suggesting that purinergic signals originating from MCs are possibly the initiating response of acupoints to moxibustion.

The hypothesis that regulated ATP release from red blood cells (RBCs) contributes to nitric oxide-dependent control of local blood flow has sparked much interest in underlying release mechanisms. Several stimuli, including shear stress and hypoxia, have been found to induce significant RBC ATP release attributed to activation of ATP-conducting channels. In the present study, we first evaluated different experimental approaches investigating stimulated RBC ATP release and quantifying hemolysis. We then measured ATP and free hemoglobin in each and every RBC supernatant sample to directly assess the contribution of hemolysis to ATP release. Hypotonic shock, shear stress, and hypoxia, but not cyclic adenosine monophosphate agonists, significantly enhanced ATP release. It tightly correlated, however, with free hemoglobin in RBC supernatants, indicating that lysis was responsible for most, if not all, ATP release. Luminescence ATP imaging combined with simultaneous infrared cell imaging showed that ATP was released exclusively from lysing cells with no contribution from intact cells. In summary, with all stimuli tested, we found no evidence of regulated ATP release from intact RBCs other than by cell lysis. Such a release mechanism might be physiologically relevant in vivo, eg, during exercise and hypoxia where intravascular hemolysis, predominantly of senescent cells, is augmented.

Extracellular ATP and other purines are ubiquitous mediators of local intercellular signaling within the body. While the last two decades have witnessed enormous progress in uncovering and characterizing purinergic receptors and extracellular enzymes controlling purinergic signals, our understanding of the initiating step in this cascade, i.e., ATP release, is still obscure. Imaging of extracellular ATP by luciferin–luciferase bioluminescence offers the advantage of studying ATP release and distribution dynamics in real time. However, low-light signal generated by bioluminescence reactions remains the major obstacle to imaging such rapid processes, imposing substantial constraints on its spatial and temporal resolution. We have developed an improved microscopy system for real-time ATP imaging, which detects ATP-dependent luciferin–luciferase luminescence at ∼10 frames/s, sufficient to follow rapid ATP release with sensitivity of ∼10 nM and dynamic range up to 100 μM. In addition, simultaneous differential interference contrast cell images are acquired with infra-red optics. Our imaging method: (1) identifies ATP-releasing cells or sites, (2) determines absolute ATP concentration and its spreading manner at release sites, and (3) permits analysis of ATP release kinetics from single cells. We provide instrumental details of our approach and give several examples of ATP-release imaging at cellular and tissue levels, to illustrate its potential utility.

Cell death is accompanied by the dissipation of electrochemical gradients of monovalent ions across the plasma membrane that, in turn, affects cell volume via modulation of intracellular osmolyte content. In numerous cell types, apoptotic and necrotic stimuli caused cell shrinkage and swelling, respectively. Thermodynamics predicts a cell type-specific rather than an ubiquitous impact of monovalent ion transporters on volume perturbations in dying cells, suggesting their diverse roles in the cell death machinery. Indeed, recent data showed that apoptotic collapse may occur in the absence of cell volume changes and even follow cell swelling rather than shrinkage. Moreover, side-by-side with cell volume adjustment, monovalent ion transporters contribute to cell death machinery engagement independently of volume regulation via cell type-specific signaling pathways. Thus, inhibition of Na+-K+-ATPase by cardiotonic steroids (CTS) rescues rat vascular smooth muscle cells from apoptosis via a novel Na+i-K+i-mediated, Ca2+i-independent mechanism of excitation-transcription coupling. In contrast, CTS kill renal epithelial cells independently of Na+-K+-ATPase inhibition and increased [Na+]i/[K+]iratio. The molecular origin of [Na+]i/[K+]isensors involved in the inhibition of apoptosis as well as upstream intermediates of Na+i/K+i-independent death signaling triggered by CTS remain unknown.

Maintenance of cell volume is a fundamental housekeeping function in eukaryotic cells. Acute cell swelling activates a regulatory volume decrease (RVD) process with poorly defined volume sensing and intermediate signaling mechanisms. Here, we analyzed the putative role of Ca2+ signaling in RVD in single substrate-adherent human lung epithelial A549 cells. Acute cell swelling was induced by perfusion of the flow-through imaging chamber with 50 % hypotonic solution at a defined fluid turnover rate. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and cell volume were monitored simultaneously with ratiometric Fura-2 fluorescence and 3D reconstruction of stereoscopic single-cell images, respectively. Hypotonic challenge caused a progressive swelling peaking at ∼20 min and followed, during the next 20 min, by RVD of 60 ± 7 % of the peak volume increase. However, at the rate of swelling used in our experiments, these processes were not accompanied by a measurable increment of [Ca2+]i. Loading with intracellular Ca2+ chelator BAPTA slightly delayed peak of swelling but did not prevent RVD in 82 % of cells. Further, electrophysiology whole-cell patch-clamp experiments showed that BAPTA did not block activation of volume-regulated anion channel (VRAC) measured as swelling-induced outwardly rectifying 5-nitro-2-(3-phenylpropyl-amino) benzoic acid sensitive current. Together, our data suggest that intracellular Ca2+-mediated signaling is not essential for VRAC activation and subsequent volume restoration in A549 cells.

Background/Aims: ATP release from erythrocyte plays a key role in hypoxia-induced elevation of blood flow in systematic circulation. We have previously shown that hemolysis contributes to erythrocyte ATP release triggered by several stimuli, including hypoxia, but the molecular mechanisms of hypoxia-increased membrane fragility remain unknown. Methods: In this study, we compared the action of hypoxia on hemolysis, ATP release and the composition of membrane-bound proteins in human erythrocytes. Results: Twenty minutes incubation of human erythrocytes in the oxygen-free environment increased the content of extracellular hemoglobin by ∼1.5 fold. Paired measurements of hemoglobin and ATP content in the same samples, showed a positive correlation between hemolysis and ATP release. Comparative analysis of SDS-PAGE electrophoresis of erythrocyte ghosts obtained under control and deoxygenated conditions revealed a ∼2-fold elevation of the content of membrane-bound protein with Mr of ∼60 kDa. Conclusion: Deoxygenation of human erythrocytes affects composition of membrane-bound proteins. Additional experiments should be performed to identify the molecular origin of 60 kDa protein and its role in the attenuation of erythrocyte integrity and ATP release in hypoxic conditions.

In the present work, we compared the outcome of hyperosmotic and isosmotic shrinkage on ion transport and protein phosphorylation in C11-MDCK cells resembling intercalated cells from collecting ducts and in vascular smooth muscle cells (VSMC) from the rat aorta. Hyperosmotic shrinkage was triggered by cell exposure to hypertonic medium, whereas isosmotic shrinkage was evoked by cell transfer from an hypoosmotic to an isosmotic environment. Despite a similar cell volume decrease of 40%–50%, the consequences of hyperosmotic and isosmotic shrinkage on cellular functions were sharply different. In C11-MDCK and VSMC, hyperosmotic shrinkage completely inhibited Na+,K+-ATPase and Na+,Picotransport. In contrast, in both types of cells isosmotic shrinkage slightly increased rather than suppressed Na+,K+-ATPase and did not change Na+,Picotransport. In C11-MDCK cells, phosphorylation of JNK1/2 and Erk1/2 mitogen-activated protein kinases was augmented in hyperosmotically shrunken cells by ∼7- and 2-fold, respectively, but was not affected in cells subjected to isosmotic shrinkage. These results demonstrate that the data obtained in cells subjected to hyperosmotic shrinkage cannot be considered as sufficient proof implicating cell volume perturbations in the regulation of cellular functions under isosmotic conditions.

Contrasting cell volume behaviours (swelling vs. shrinkage) are considered as criteria to distinguish necrosis from apoptosis. In this study, we employed a time-lapse, dual-image surface reconstruction technique to assess the volume of single vascular smooth muscle cells transfected with E1A-adenoviral protein (E1A-VSMC) and undergoing rapid apoptosis in the absence of growth factors or in the presence of staurosporine. After 30- to 60-min lag-phase, serum-deprived E1A-VSMC volume was increased by ~40%, which preceded maximal increments of caspase-3 activity and chromatin cleavage. Swollen cells underwent rapid apoptotic collapse, documented by plasma membrane budding, and terminated in 10-15 min by the formation of numerous apoptotic bodies. Suppression of apoptosis by inhibition of Na(+),K(+)-ATPase and activation of cAMP signalling with ouabain and forskolin, respectively, completely abolished the swelling of serum-deprived E1A-VSMC. In contrast to serum deprivation, apoptotic collapse of staurosporine-treated E1A-VSMC preceded attenuation of their volume by ~30%. Neither transient hyposmotic swelling nor isosmtotic shrinkage triggered apoptosis. Our results show that cell shrinkage can not be considered as ubiquitous hallmark of apoptosis. The involvement of stimulus-specific cell volume perturbations in initiation and progression of apoptosis in vascular smooth muscle cells should be examined further.

Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca(2+)-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells.

Extracellular nucleotides regulate mucociliary clearance in the airways and surfactant secretion in alveoli. Their release is exquisitely mechanosensitive and may be induced by stretch as well as airflow shear stress acting on lung epithelia. We hypothesized that, in addition, tension forces at the air-liquid interface (ALI) may contribute to mechanosensitive ATP release in the lungs. Local depletion of airway surface liquid, mucins, and surfactants, which normally protect epithelial surfaces, facilitate such release and trigger compensatory mucin and fluid secretion processes. In this study, human bronchial epithelial 16HBE14o−and alveolar A549 cells were subjected to tension forces at the ALI by passing an air bubble over the cell monolayer in a flow-through chamber, or by air exposure while tilting the cell culture dish. Such stimulation induced significant ATP release not involving cell lysis, as verified by ethidium bromide staining. Confocal fluorescence microscopy disclosed reversible cell deformation in the monolayer part in contact with the ALI. Fura 2 fluorescence imaging revealed transient intracellular Ca2+elevation evoked by the ALI, which did not entail nonspecific Ca2+influx from the extracellular space. ATP release was reduced by ∼40 to ∼90% from cells loaded with the Ca2+chelator BAPTA-AM and was completely abolished by N-ethylmalemide (1 mM). These experiments demonstrate that in close proximity to the ALI, surface tension forces are transmitted directly on cells, causing their mechanical deformation and Ca2+-dependent exocytotic ATP release. Such a signaling mechanism may contribute to the detection of local deficiency of airway surface liquid and surfactants on the lung surface.

Pseudomonas aeruginosa is a gram-negative bacterium that causes chronic infection in cystic fibrosis patients. We reported recently that P. aeruginosa modulates epithelial Na+ channel (ENaC) expression in experimental chronic pneumonia models. For this reason, we tested whether LPS from P. aeruginosa alters ENaC expression and activity in alveolar epithelial cells. We found that LPS induces a ∼60% decrease of ENaC apical current without significant changes in intracellular ENaC or surface protein expression. Because a growing body of evidence reports a key role for extracellular nucleotides in regulation of ion channels, we evaluated the possibility that modulation of ENaC activity by LPS involves extracellular ATP signaling. We found that alveolar epithelial cells release ATP upon LPS stimulation and that pretreatment with suramin, a P2Y2 purinergic receptor antagonist, inhibited the effect of LPS on ENaC. Furthermore, ET-18-OCH3, a PLC inhibitor, and Go-6976, a PKC inhibitor, were able to partially prevent ENaC inhibition by LPS, suggesting that the actions of LPS on ENaC current were mediated, in part, by the PKC and PLC pathways. Together, these findings demonstrate an important role of extracellular ATP signaling in the response of epithelial cells to LPS.

The expression of the epithelial Na + channel (ENaC) is tissue-specific and dependent on a variety of mediators and interacting proteins. Here we examined the role of intracellular Na + ((Na + ) i ) as a modulator of the expression of rat ENaC in Xenopus laevis oocytes. We manipulated (Na + ) i of ENaC-expressing oocytes in the range of 0-20 mM by incubating in extracellular solutions of different (Na + ) o . Electrophysiological, protein biochemical and fluorescence optical methods were used to determine the effects of different (Na + ) i on ENaC expression and membrane abundance. In voltage-clamp experiments we found that amiloride- sensitive ENaC current (I ami ) and conductance (G ami ) peak at a (Na + ) i of ~10 mM Na + , but were significantly reduced in 5 mM and 20 mM (Na + ) i . Fluorescence

Extracellular ATP is a potent surfactant secretagogue but its origin in the alveolus, its mechanism(s) of release and its regulatory pathways remain unknown. Previously, we showed that hypotonic swelling of alveolar A549 cells induces Ca(2+)-dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. In this study, we examined sources of Ca(2+) for the elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) evoked by acute 50% hypotonic stress and the role of autocrine purinergic signalling in Ca(2+)-dependent ATP release. We found that ATP release does not directly involve Ca(2+) influx from extracellular spaces, but depends entirely on Ca(2+) mobilization from intracellular stores. The [Ca(2+)](i) response consisted of slowly rising elevation, representing mobilization from thapsigargin (TG)-insensitive stores and a superimposed rapid spike due to Ca(2+) release from TG-sensitive endoplasmic reticulum (ER) Ca(2+) stores. The latter could be abolished by hydrolysis of extracellular triphospho- and diphosphonucleotides with apyrase; blocking P2Y(2)/P2Y(6) receptors of A549 cells with suramin; blocking UDP receptors (P2Y(6)) with pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS); emptying TG-sensitive stores downstream with TG or caffeine in Ca(2+)-free extracellular solution; or blocking the Ca(2+)-release inositol 1,4,5-triphosphate receptor channel of the ER with 2-aminoethyldiphenylborinate. These data demonstrate that the rapid [Ca(2+)](i) spike results from the autocrine stimulation of IP(3)/Ca(2+)-coupled P2Y, predominantly P2Y(6), receptors, accounting for approximately 70% of total Ca(2+)-dependent ATP release evoked by hypotonic shock. Our study reveals a novel paradigm in which stress-induced ATP release from alveolar cells is amplified by the synergistic autocrine/paracrine action of coreleased uridine and adenosine nucleotides. We suggest that a similar mechanism of purinergic signal propagation operates in other cell types.

Extracellular nucleotides play an important role in lung defense, but the release mechanism and relative abundance of different nucleotide species secreted by lung epithelia are not well defined. In this study, to minimize cell surface hydrolysis, we used a low-volume, flow-through chamber and examined adenosine and uridine nucleotide concentrations in perfusate aliquots of human lung A549 cells challenged by 50% hypotonic shock. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (Ado) were quantified in high-performance liquid chromatography (HPLC) analysis of fluorescent etheno derivatives, and uridine triphosphate (UTP) and uridine diphosphate (UDP) were measured using HPLC-coupled radioenzymatic assays. After the onset of hypotonic shock, ATP, ADP, UTP, and UDP in the perfusates increased markedly and peaked at approximately 2.5 min, followed by a gradual decay in the next 15-20 min; peak changes in Ado and AMP were relatively minor. The peak concentrations and fold increment (in parentheses) were: 34 +/- 13 nM ATP (5.6), 11 +/- 5 nM ADP (3.7), 3.3 +/- 1.2 nM AMP (1.4), 23 +/- 7 nM Ado (2.1), 21 nM UTP (7), and 11 nM UDP (27). Nucleotide release was almost completely abolished from cells loaded with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Under isotonic conditions, elevation of intracellular calcium with the calcium ionophore ionomycin (5 muM, 3 min) also released nucleotides with kinetics and relative abundance as above, albeit less robust. ADP:ATP (1:3) and UDP:UTP (1:2) ratios in perfusates from stimulated cells were markedly higher than the cytosolic ratios of these species, suggesting that a nucleotide diphosphate (NDP)-rich compartment, e.g., the secretory pathway, contributed to nucleotide release. Laser confocal microscopy experiments illustrated increased FM1-43 uptake into the plasma membrane upon hypotonic shock or ionomycin treatment, consistent with enhanced vesicular exocytosis under these conditions. In summary, our results strongly suggest that calcium-dependent exocytosis is responsible, at least in most part, for adenosine and uridine nucleotide release from A549 cells.

Despite the existence of a functional arginine vasopressin (AVP) system in the adult heart and evidence that AVP induces myogenesis, its significance in cardiomyogenesis is currently unknown. In the present study, we hypothesized a role for AVP in cardiac differentiation of D3 and lineage-specific embryonic stem (ES) cells expressing green fluorescent protein under the control of atrial natriuretic peptide (Anp) or myosin light chain-2V (Mlc-2V) promoters. Furthermore, we investigated the nitric oxide (NO) involvement in AVP-mediated pathways. AVP exposure increased the number of beating embryoid bodies, fluorescent cells, and expression of Gata-4 and other cardiac genes. V1a and V2 receptors (V1aR and V2R) differentially mediated these effects in transgenic ES cells, and exhibited a distinct developmentally regulated mRNA expression pattern. A NO synthase inhibitor, L-NAME, powerfully antagonized the AVP-induced effects on cardiogenic differentiation, implicating NO signaling in AVP-mediated pathways. Indeed, AVP elevated the mRNA and protein levels of endothelial NO synthase (eNOS) through V2R stimulation. Remarkably, increased beating activity was found in AVP-treated ES cells with down-regulated eNOS expression, indicating the significant involvement of additional pathways in cardiomyogenic effects of AVP. Finally, patch clamp recordings revealed specific AVP-induced changes of action potentials and increased L-type Ca2+ (ICa,L) current densities in differentiated ventricular phenotypes. Thus, AVP promotes cardiomyocyte differentiation of ES cells and involves Gata-4 and NO signaling. AVP-induced action potential prolongation appears likely to be linked to the increased ICa,L current in ventricular cells. In conclusion, this report provides new evidence for the essential role of the AVP system in ES cell-derived cardiomyogenesis.

In this issue of Blood , 2 independent reports by [Rapetti-Mauss et al][1] and [Glogowska et al][2] describe the first example of a human disease: a novel type of hemolytic hereditary xerocytosis (HX) that is caused by a genetic defect in the Gardos channel, a Ca2+-sensitive K+ channel critical for

Background Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K+ channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before. Results Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair. Conclusion Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.

We appreciate the willingness of Kirby and colleagues to discuss methodological concerns in red blood cell (RBC) adenosine triphosphate (ATP) release studies. In our investigations, before moving to mechanism-probing experiments, we spent most of the time evaluating the different methods that have been used in the field to assess RBC ATP release and the contribution of hemolysis to that release. Although we began with the hypothesis that regulated ATP release mechanisms do operate in RBCs, after extensively verifying each experimental approach, we came to the conclusion expressed in the title: ie, that “Hemolysis is a primary ATP release mechanism in human erythrocytes.”1 In the course of the study, while attempting to reproduce previously published findings, we identified a number of methodological pitfalls (the major ones are discussed in the supplemental Methods available on Blood Web site), which likely precluded a proper evaluation of ATP release and also the contribution of hemolysis in the earlier studies2-10 cited by Kirby et al. We agree that “liberation of ATP secondary to hemolysis is not mutually exclusive of regulated export.”11 Our point is that without the controls described in our paper, a confounding effect of hemolysis cannot be excluded. We determined that the most reliable and direct evaluation of a hemolysis contribution to RBC ATP release requires the following. (1) The use of RBC supernatants for extracellular ATP (ATPec) evaluation rather than RBC suspensions is necessary. We verified that centrifugation (500g, 10 minutes, 4°C), even when repeated 5 times, does not elevate ATPec above the low basal level seen after the first or second centrifugation. (2) The measurement of free hemoglobin (Hb) in an aliquot of the same supernatant used for ATPec determination is necessary. To the best of our knowledge, such paired measurements have been performed only for plasma ATP-Hb determinations by Gorman et al12 and Kirby et al2 or toxin-induced ATP release.13 When ATPec-Hb values of all samples were plotted to evaluate the correlation between these variables, they showed a linear relationship and exactly matched those obtained with freshly prepared RBC lysates (see Figure 1D in Sikora et al1). Any “upward” deflection in the linear ATP-Hb relationship would indicate a contribution of nonlytic ATP release. With these restrictions, we found that ATP and Hb release were tightly correlated for all stimuli tested and exactly matched expectations based on the number of lysed cells and independently determined intracellular ATP concentration. Our conclusions were fully confirmed by directly visualizing single-cell ATP release and cell lysis using luminescence ATP imaging and simultaneous infrared cell imaging, respectively. We encourage other investigators to use these criteria and to examine the correlation between ATP and Hb release in individual experiments, so that conclusions can be made concerning regulated, hemolysis-independent mechanisms.

Low-level-laser therapy (LLLT) is an effective complementary treatment, especially for anti-inflammation and wound healing in which dermis or mucus mast cells (MCs) are involved. In periphery, MCs crosstalk with neurons via purinergic signals and participate in various physiological and pathophysiological processes. Whether extracellular ATP, an important purine in purinergic signaling, of MCs and neurons could be modulated by irradiation remains unknown. In this study, effects of red-laser irradiation on extracellular ATP content of MCs and dorsal root ganglia (DRG) neurons were investigated and underlying mechanisms were exploredin vitro. Our results show that irradiation led to elevation of extracellular ATP level in the human mast cell line HMC-1 in a dose-dependent manner, which was accompanied by elevation of intracellular ATP content, an indicator for ATP synthesis, together with [Ca2+]ielevation, a trigger signal for exocytotic ATP release. In contrast to MCs, irradiation attenuated the extracellular ATP content of neurons, which could be abolished by ARL 67156, a nonspecific ecto-ATPases inhibitor. Our results suggest that irradiation potentiates extracellular ATP of MCs by promoting ATP synthesis and release and attenuates extracellular ATP of neurons by upregulating ecto-ATPase activity. The opposite responses of these two cell types indicate complex mechanisms underlying LLLT.