A novel method using fluorescence microscopy for real-time assessment of ATP release from individual cells

Many cell types release ATP in response to mechanical or biochemical stimulation. The mechanisms responsible for this release, however, are not well understood and may differ among different cell types. In addition, there are numerous difficulties associated with studying the dynamics of ATP release immediately outside the cell membrane. Here, we report a new method that allows the visualization and quantification of ATP release by fluorescence microscopy. Our method utilizes a two-enzyme system that generates NADPH when ATP is present. NADPH is a fluorescent molecule that can be visualized by fluorescence microscopy using an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The method is capable of detecting ATP concentrations <1 microM and has a dynamic range of up to 100 microM. Using this method, we visualized and quantified ATP release from human polymorphonuclear leukocytes and Jurkat T cells. We show that upon cell stimulation, the concentrations of ATP can reach levels of up to 80 microM immediately outside of the cell membrane. This new method should prove useful for the study of the mechanisms of release and functional role of ATP in various cell systems, including individual cells.     

Water induces autocrine stimulation of tumor cell killing through ATP release and P2 receptor binding

Although exposure of cells to extreme hypotonic stress appears to be a purely experimental set up, it has found an application in clinical routine. For years, surgeons have washed the abdominal cavity with distilled water to lyse isolated cancer cells left after surgery. No data are available supporting this practice or evaluating the potential mechanisms of cell injury under these circumstances. Recent evidence indicates that increases in cell volume stimulate release of adenosine triphosphate and autocrine stimulation of purinergic (P2) receptors in the plasma membrane of certain epithelial cell types. Under physiological conditions, purigenic stimulation can contribute to cell volume recovery through activation of solute efflux. In addition, adenosine triphosphate-P2 receptor binding might trigger other mechanisms affecting cell viability after profound hypotonic stress. This study demonstrates a novel pathway of cell death by apoptosis in human colon cancer cells following a short hypotonic stress. This pathway is induced by transitory cell swelling which leads to extracellular release of adenosine triphosphate (ATP) and specific binding of ATP to P2 receptors (probably P2X7). Extracellular ATP induced activation of caspases 3 and 8, annexin V, release of cytochrome c, and eventually cell death. The effect of ATP can be blocked by addition of (i) apyrase to hydrolyse extracellular ATP and (ii) suramin, a P2 receptor antagonist. Finally, (iii) gadolinium pretreatment, a blocker of ATP release, reduces sensitivity of the cells to hypotonic stress. The adenosine triphosphate-P2 receptor cell death pathway suggests that autocrine/paracrine signaling may contribute to regulation of viability in certain cancer cells disclosed with this pathway.     

Autocrine and paracrine actions of ATP in rat carotid body

Carotid bodies are peripheral chemoreceptors that detect lowering of arterial blood O(2) level. The carotid body comprises clusters of glomus (type I) cells surrounded by glial-like sustentacular (type II) cells. Hypoxia triggers depolarization and cytosolic [Ca(2+)] ([Ca(2+)](i)) elevation in glomus cells, resulting in the release of multiple transmitters, including ATP. While ATP has been shown to be an important excitatory transmitter in the stimulation of carotid sinus nerve, there is considerable evidence that ATP exerts autocrine and paracrine actions in carotid body. ATP acting via P2Y(1) receptors, causes hyperpolarization in glomus cells and inhibits the hypoxia-mediated [Ca(2+)](i) rise. In contrast, adenosine (an ATP metabolite) triggers depolarization and [Ca(2+)](i) rise in glomus cells via A(2A) receptors. We suggest that during prolonged hypoxia, the negative and positive feedback actions of ATP and adenosine may result in an oscillatory Ca(2+) signal in glomus cells. Such mechanisms may allow cyclic release of transmitters from glomus cells during prolonged hypoxia without causing cellular damage from a persistent [Ca(2+)](i) rise. ATP also stimulates intracellular Ca(2+) release in sustentacular cells via P2Y(2) receptors. The autocine and paracrine actions of ATP suggest that ATP has important roles in coordinating chemosensory transmission in the carotid body.     

Human Pannexin 1 channel: Insight in structure–function mechanism and its potential physiological roles

Pannexins, large non-gap junction super family exists in vertebrates, play multiple roles in different cellular functions through their ATP release. Panx1-mediated adenosine 5′-triphosphate (ATP) release plays a vital role in physiological and pathophysiological conditions and is known major extracellular molecule in purinergic signaling. To modulate their function in vivo, a proper regulation of channel is necessary. Post-translational modifications are considered to be some regulating mechanisms for PANX1, while PANX2, PANX3 have been uncharacterized to date. Through their significant evidences, PANXs exclude from gap junction and conduits ATP release and other cellular molecules from cells by various mechanisms. PANX1 is most extensive characterized and implicated in ATP signaling and inflammatory processes. Despite the constant advances, much significance of PANX1 in physiological processes remains elusive. Recently, various research groups along with our group have reported the Cryo-EM structure of Panx1 channel and uncovered the hidden functions in structure–function mechanism as well as to provide the clear understanding in physiological and pathophysiological roles. These research groups reported the novel heptameric structure with contains 4 transmembrane helices (TM), two extracellular loops and one intracellular loop with N and C terminus located at the intracellular side. In addition, the structure contains a large pore of which an inhibitor CBX act as a plug that blocking the passage of substrate. In this context, this review will present current mechanistic understanding in structure and function together with significant physiological roles particularly ATP release in health and disease. As such, this review emphasizes on recent functional properties associated with novel heptameric channel and demystifies channel-mediated ATP release function.

     

Autocrine/paracrine stimulation of purinergic receptors in osteoblasts: contribution of vesicular ATP release

Extracellular nucleotides such as ATP and UTP are released in response to mechanical stimulation in different cell systems. It is becoming increasingly evident that ATP release plays a role in autocrine and paracrine stimulation of osteoblasts. Mechanical stimulation, as shear stress, membrane stretch or hypo-osmotic swelling, as well as oscillatory fluid flow, stimulates ATP release from different osteoblastic cell lines. Human osteoblast-like initial transfectant (HOBIT) cells release ATP in response to mechanical stimulation. In the present study, we show that HOBIT cells are activated by nanomolar levels of extracellular ATP, concentrations that can be detected under resting conditions and increase following hypotonic shock. Cell activation by hypotonic medium induced intracellular Ca2+ oscillations, and Egr-1 synthesis and DNA-binding activity. Quinacrine staining of living, resting cells revealed a granular fluorescence, typical of ATP-storing vesicles. Monensin prevented quinacrine staining and considerably inhibited hypotonic-induced ATP release. Finally, elevated levels of cytosolic Ca2+ activated massive ATP release and a dose-dependent loss of quinacrine granules. The contribution of a vesicular mechanism for ATP release is proposed to sustain paracrine osteoblast activation.     

Involvement of SLC17A9-dependent Vesicular Exocytosis in the Mechanism of ATP Release during T Cell Activation

Recent reports have shown that T cell receptor (TCR)-dependent ATP release from T cells is involved in production of interleukin-2 (IL-2) through activation of P2 receptors. Stimulation of TCR induces ATP release from T cells through gap junction hemichannels and maxianion channels, at least in part. However, the mechanisms of ATP release from activated T cells are not fully understood. Here, we studied the mechanisms of ATP release during TCR-dependent T cell activation by investigating the effects of various inhibitors on TCR-dependent ATP release from murine T cells. We found that not only anion channel and gap junction hemichannel inhibitors, but also exocytosis inhibitors suppressed the ATP release. These results suggest that ATP release from murine T cells is regulated by various mechanisms, including exocytosis. An inhibitor of exocytosis, bafilomycin A, significantly blocked TCR signaling, such as Ca²⁺ elevation and IL-2 production. Furthermore, bafilomycin A, ectonucleotidase, and P2Y₆ receptor antagonist significantly inhibited production of pro-inflammatory cytokines from external antigen-restimulated splenocytes, indicating that vesicular exocytosis-mediated purinergic signaling has a significant role in TCR-dependent cytokine production. We also detected vesicular ATP in murine T cells and human T lymphoma Jurkat cells, both of which also expressed mRNA of SLC17A9, a vesicular nucleotide transporter. Knockdown of SLC17A9 in Jurkat cells markedly reduced ATP release and cytosolic Ca²⁺ elevation after TCR stimulation, suggesting involvement of SLC17A9-dependent vesicular exocytosis in ATP release and T cell activation. In conclusion, vesicular exocytosis of ATP appears to play a role in T cell activation and immune responses.     

Pharmacological dissection of the cellular mechanisms associated to the spontaneous and the mechanically stimulated ATP release by mesentery endothelial cells: roles of thrombin and TRPV

Endothelial cells participate in extracellular ATP release elicited by mechanosensors. To characterize the dynamic interactions between mechanical and chemical factors that modulate ATP secretion by the endothelium, we assessed and compared the mechanisms participating in the spontaneous (basal) and mechanically stimulated secretion using primary cultures of rat mesentery endothelial cells. ATP/metabolites were determined in the cell media prior to (basal) and after cell media displacement or a picospritzer buffer puff used as mechanical stimuli. Mechanical stimulation increased extracellular ATP that peaked within 1 min, and decayed to basal values in 10 min. Interruption of the vesicular transport route consistently blocked the spontaneous ATP secretion. Cells maintained in media lacking external Ca²⁺ elicited a spontaneous rise of extracellular ATP and adenosine, but failed to elicit a further extracellular ATP secretion following mechanical stimulation. 2-APB, a TRPV agonist, increased the spontaneous ATP secretion, but reduced the mechanical stimulation-induced nucleotide release. Pannexin1 or connexin blockers and gadolinium, a Piezo1 blocker, reduced the mechanically induced ATP release without altering spontaneous nucleotide levels. Moreover, thrombin or related agonists increased extracellular ATP secretion elicited by mechanical stimulation, without modifying spontaneous release. In sum, present results allow inferring that the spontaneous, extracellular nucleotide secretion is essentially mediated by ATP containing vesicles, while the mechanically induced secretion occurs essentially by connexin or pannexin1 hemichannel ATP transport, a finding fully supported by results from Panx1^?/? rodents. Only the latter component is modulated by thrombin and related receptor agonists, highlighting a novel endothelium-smooth muscle signaling role of this anticoagulant.     

5-Nitro-2-(3-phenylpropylamino)benzoic Acid (NPPB) Stimulates Cellular ATP Release through Exocytosis of ATP-enriched Vesicles

Cells release ATP in response to physiologic stimuli. Extracellular ATP regulates a broad range of important cellular functions by activation of the purinergic receptors in the plasma membrane. The purpose of these studies was to assess the role of vesicular exocytosis in cellular ATP release. FM1-43 fluorescence was used to measure exocytosis and bioluminescence to measure ATP release in HTC rat hepatoma and Mz-Cha-1 human cholangiocarcinoma cells. Exposure to a Cl⁻ channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (50–300 μm) stimulated a 5–100-fold increase in extracellular ATP levels within minutes of the exposure. This rapid response was not a result of changes in cell viability or Cl⁻ channel activity. NPPB also potently stimulated ATP release in HEK293 cells and HEK293 cells expressing a rat P2X7 receptor indicating that P2X7 receptors are not involved in stimulation of ATP release by NPPB. In all cells studied, NPPB rapidly stimulated vesicular exocytosis that persisted many minutes after the exposure. The kinetics of NPPB-evoked exocytosis and ATP release were similar. Furthermore, the magnitudes of NPPB-evoked exocytosis and ATP release were correlated (correlation coefficient 0.77), indicating that NPPB may stimulate exocytosis of a pool of ATP-enriched vesicles. These findings provide further support for the concept that vesicular exocytosis plays an important role in cellular ATP release and suggest that NPPB can be used as a biochemical tool to specifically stimulate ATP release through exocytic mechanisms.     

Astrocyte-derived ATP induces vesicle shedding and IL-1 beta release from microglia

ATP has been indicated as a primary factor in microglial response to brain injury and inflammation. By acting on different purinergic receptors 2, ATP is known to induce chemotaxis and stimulate the release of several cytokines from these cells. The activation of purinergic receptors 2 in microglia can be triggered either by ATP deriving from dying cells, at sites of brain injury or by ATP released from astrocytes, in the absence of cell damage. By the use of a biochemical approach integrated with video microscopy experiments, we investigated the functional consequences triggered in microglia by ATP released from mechanically stimulated astrocytes, in mixed glial cocultures. Astrocyte-derived ATP induced in nearby microglia the formation and the shedding of membrane vesicles. Vesicle formation was inhibited by the ATP-degrading enzyme apyrase or by P2X(7)R antagonists. Isolation of shed vesicles, followed by IL-1beta evaluation by a specific ELISA revealed the presence of the cytokine inside the vesicular organelles and its subsequent efflux into the extracellular medium. IL-1beta efflux from shed vesicles was enhanced by ATP stimulation and inhibited by pretreatment with the P2X(7) antagonist oxidized ATP, thus indicating a crucial involvement of the pore-forming P2X(7)R in the release of the cytokine. Our data identify astrocyte-derived ATP as the endogenous factor responsible for microvesicle shedding in microglia and reveal the mechanisms by which astrocyte-derived ATP triggers IL-1beta release from these cells.     

The effect of anodal/cathodal biphasic electrical stimulation on insulin release

We studied the effects of electrical stimulation on insulin release from rat insulinoma (INS-1) cells. The anodal/cathodal biphasic stimulation (ACBPS) electrical waveform resulted in a voltage- and stimulation duration-dependent increase in insulin release. ACBPS elicited insulin release both in the presence and absence of glucose. Basal and ACBPS-induced insulin secretion could be inhibited by mitochondrial poisons and calcium channel blockers, indicating that insulin release was dependent on adenosine triphosphate (ATP) and the influx of calcium. ACBPS parameters that released insulin caused no detectable plasma membrane damage or cytotoxicity, although temporary morphological changes could be observed immediately after ACBPS. ACBPS did not alter the plasma membrane transmembrane potential but did cause pronounced uptake of MitoTracker Red into the mitochondrial membrane, indicating an increased mitochondrial membrane potential. While the ATP:ADP ratio after ACBPS did not change, the guanosine triphosphate (GTP) levels increased and increased GTP levels have previously been associated with insulin release in INS-1 cells. These results provide evidence that ACBPS can have significant biological effects on cells. In the case of INS-1 cells, ACBPS promotes insulin release without causing cytotoxicity.     

Secretion of intracellular IL-1 receptor antagonist (type 1) is dependent on P2X7 receptor activation

Inflammatory mechanisms are critical in the arterial response to injury. Both IL-1 and the naturally occurring inhibitor of IL-1, IL-1R antagonist (IL-1ra), are expressed in the arterial wall, and in particular in the endothelium. Previous studies suggest that endothelial cells only make the intracellular type I isoform of IL-1ra (icIL-1ra1), an isoform known to lack a secretory signal peptide. It is unclear how icIL-1ra is released from the endothelial cell to act as an antagonist on cell surface IL-1 type I receptors. IL-1beta, which also lacks a secretory signal peptide, may be released by ATP stimulation of the P2X(7)R. Therefore, we examined whether icIL-1ra1 release occurs in an analogous manner, using both the mouse macrophage cell line RAW264.7 and HUVECs. P2X(7)R activation caused icIL-1ra1 release from LPS-primed RAW264.7 macrophages and from HUVECs. This release was inhibited in the absence of extracellular calcium, and attenuated by preincubation with oxidized ATP, KN62, and apyrase. Endogenous ATP release, which also facilitated release of icIL-1ra1, was detected during LPS treatment of both RAW264.7 macrophages and HUVECs. Annexin V assays showed that ATP stimulation resulted in a rapid phosphatidylserine (PS) exposure on the cell surface of RAW264.7 macrophages, and that PS-exposed microvesicles contained icIL-1ra1. However, PS flip and microvesicle shedding was not apparent in ATP-treated HUVECs. These data support a general role for the P2X(7)R in the release of leaderless cytokines into the extracellular medium, and indicate how icIL-1ra1 may act upon its extracellular target, the IL-1R.     

Prostacyclin receptor-mediated ATP release from erythrocytes requires the voltage-dependent anion channel

Erythrocytes have been implicated as controllers of vascular caliber by virtue of their ability to release the vasodilator ATP in response to local physiological and pharmacological stimuli. The regulated release of ATP from erythrocytes requires activation of a signaling pathway involving G proteins (G(i) or G(s)), adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator as well as a final conduit through which this highly charged anion exits the cell. Although pannexin 1 has been shown to be the final conduit for ATP release from human erythrocytes in response to reduced oxygen tension, it does not participate in transport of ATP following stimulation of the prostacyclin (IP) receptor in these cells, which suggests that an additional protein must be involved. Using antibodies directed against voltage-dependent anion channel (VDAC)1, we confirm that this protein is present in human erythrocyte membranes. To address the role of VDAC in ATP release, two structurally dissimilar VDAC inhibitors, Bcl-x(L) BH4(4-23) and TRO19622, were used. In response to the IP receptor agonists, iloprost and UT-15C, ATP release was inhibited by both VDAC inhibitors although neither iloprost-induced cAMP accumulation nor total intracellular ATP concentration were altered. Together, these findings support the hypothesis that VDAC is the ATP conduit in the IP receptor-mediated signaling pathway in human erythrocytes. In addition, neither the pannexin inhibitor carbenoxolone nor Bcl-x(L) BH4(4-23) attenuated ATP release in response to incubation of erythrocytes with the β-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes.     

Botulinum toxin A inhibits ATP release from bladder urothelium after chronic spinal cord injury

The effects of mechanoreceptor stimulation and subsequent ATP release in spinal cord injured and normal bladders was examined to demonstrate if spinal cord injury (SCI) modulates the basal or evoked release of ATP from bladder urothelium and whether intravesical botulinum toxin A (BTX-A) administration inhibits urothelial ATP release, a measure of sensory nerve activation. A Ussing chamber was used to isolate and separately measure resting and mechanoreceptor evoked (e.g. hypoosmotic stimulation) ATP release from urothelial and serosal sides of the bladder. Following spinal cord injury, resting urothelial release of ATP was ninefold higher than that of normal rats. Botulinum toxin A instillation did not significantly affect the resting release of ATP after spinal cord injury. Evoked ATP release following hypoosmotic stimulation was significantly higher in chronic spinal cord injured compared to normal rat bladders. However, botulinum toxin A treatment markedly reduced ATP release in spinal cord injured bladders by 53% suggesting that ATP release by mechanoreceptor stimulation, as opposed to basal release, occurs by exocytotic mechanisms. In contrast, there was no significant difference in basal or evoked ATP release from bladder serosa following spinal cord injury. Moreover, intravesical instillation of botulinum toxin A did not affect ATP release from the serosal side after spinal cord injury, suggesting that its effects were confined to the urothelial side of the bladder preparation. In summary: (1) increased release of ATP from the urothelium of spinal cord injured bladders may contribute to the development of bladder hyperactivity and, (2) mechanoreceptor stimulated vesicular ATP release, as opposed to basal non-vesicular release of ATP, is significantly inhibited in spinal cord injured bladders by intravesical instillation of botulinum toxin A. These results may have important relevance in our understanding of the mechanisms underlying plasticity of bladder afferent pathways following SCI.     

Exocytotic release of ATP from cultured astrocytes

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca(2+)](i) also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca(2+)](i) attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca(2+)-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X(3) receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca(2+). Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared.     

Neuroglial ATP release through innexin channels controls microglial cell movement to a nerve injury

Microglia, the immune cells of the central nervous system, are attracted to sites of injury. The injury releases adenosine triphosphate (ATP) into the extracellular space, activating the microglia, but the full mechanism of release is not known. In glial cells, a family of physiologically regulated unpaired gap junction channels called innexons (invertebrates) or pannexons (vertebrates) located in the cell membrane is permeable to ATP. Innexons, but not pannexons, also pair to make gap junctions. Glial calcium waves, triggered by injury or mechanical stimulation, open pannexon/innexon channels and cause the release of ATP. It has been hypothesized that a glial calcium wave that triggers the release of ATP causes rapid microglial migration to distant lesions. In the present study in the leech, in which a single giant glial cell ensheathes each connective, hydrolysis of ATP with 10 U/ml apyrase or block of innexons with 10 µM carbenoxolone (CBX), which decreased injury-induced ATP release, reduced both movement of microglia and their accumulation at lesions. Directed movement and accumulation were restored in CBX by adding ATP, consistent with separate actions of ATP and nitric oxide, which is required for directed movement but does not activate glia. Injection of glia with innexin2 (Hminx2) RNAi inhibited release of carboxyfluorescein dye and microglial migration, whereas injection of innexin1 (Hminx1) RNAi did not when measured 2 days after injection, indicating that glial cells’ ATP release through innexons was required for microglial migration after nerve injury. Focal stimulation either mechanically or with ATP generated a calcium wave in the glial cell; injury caused a large, persistent intracellular calcium response. Neither the calcium wave nor the persistent response required ATP or its release. Thus, in the leech, innexin membrane channels releasing ATP from glia are required for migration and accumulation of microglia after nerve injury.     

mTOR and differential activation of mitochondria orchestrate neutrophil chemotaxis

Neutrophils use chemotaxis to locate invading bacteria. Adenosine triphosphate (ATP) release and autocrine purinergic signaling via P2Y2 receptors at the front and A2a receptors at the back of cells regulate chemotaxis. Here, we examined the intracellular mechanisms that control these opposing signaling mechanisms. We found that mitochondria deliver ATP that stimulates P2Y2 receptors in response to chemotactic cues, and that P2Y2 receptors promote mTOR signaling, which augments mitochondrial activity near the front of cells. Blocking mTOR signaling with rapamycin or PP242 or mitochondrial ATP production (e.g., with CCCP) reduced mitochondrial Ca²⁺ uptake and membrane potential, and impaired cellular ATP release and neutrophil chemotaxis. Autocrine stimulation of A2a receptors causes cyclic adenosine monophosphate accumulation at the back of cells, which inhibits mTOR signaling and mitochondrial activity, resulting in uropod retraction. We conclude that mitochondrial, purinergic, and mTOR signaling regulates neutrophil chemotaxis and may be a pharmacological target in inflammatory diseases.     

Mechanisms of ATP release by human trabecular meshwork cells, the enabling step in purinergic regulation of aqueous humor outflow

Our guiding hypothesis is that ecto-enzymatic conversion of extracellular ATP to adenosine activates A(1) adenosine receptors, reducing resistance to aqueous humor outflow and intraocular pressure. The initial step in this purinergic regulation is ATP release from outflow-pathway cells by mechanisms unknown. We measured similar ATP release from human explant-derived primary trabecular meshwork (TM) cells (HTM) and a human TM cell line (TM5). Responses to 21 inhibitors indicated that pannexin-1 (PX1) and connexin (Cx) hemichannels and P2X(7) receptors (P2RX(7) ) were comparably important in modulating ATP release induced by hypotonic swelling, whereas vesicular release was insignificant. Consistent with prior studies of PX1 activity in certain other cells, ATP release was lowered by the reducing agent dithiothreitol. Overexpressing PX1 in HEK293T cells promoted, while partial knockdown (KD) in both HEK293T and TM5 cells inhibited hypotonicity-activated ATP release. Additionally, KD reduced the pharmacologically defined contribution of PX1 and enhanced those of Cx and P2RX(7) . ATP release was also triggered by raising intracellular Ca(2+) activity with ionomycin after a prolonged lag time and was unaffected by the PX1 blocker probenecid, but nearly abolished by P2RX(7) antagonists. We conclude that swelling-stimulated ATP release from human TM cells is physiologically mediated by PX1 and Cx hemichannels and P2X(7) receptors, but not by vesicular release. PX1 appears not to be stimulated by intracellular Ca(2+) in TM cells, but can be modulated by oxidation-reduction state. The P2RX(7) -dependent component of swelling-activated release may be mediated by PX1 hemichannels or reflect apoptotic magnification of ATP release, either through itself and/or hemichannels.