P2Y11 impairs cell proliferation by induction of cell cycle arrest and sensitizes endothelial cells to cisplatin-induced cell death

Extracellular ATP mediates a wide range of physiological effects, including cell proliferation, differentiation, maturation, and migration. However, the effect of ATP on cell proliferation has been contradictory, and the mechanism is not fully understood. In the current study, we found that extracellular ATP significantly inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). Treatment with ATP did not induce cell apoptosis but instead induced cell cycle arrest in S phase. ATP induced the phosphorylation of ERK1/2, but the ERK inhibitors, U0126 and PD9809, did not regulate the inhibition of cell proliferation induced by ATP. However, ATP-induced inhibition of cell proliferation was blocked by suramin, a nonspecific antagonist of the P2Y receptors, and endothelial cells expressed P2Y11, a P2Y receptor that specifically binds ATP. Moreover, the down-regulation of P2Y11 by RNA interference not only reversed the inhibition of cell proliferation but also ameliorated cell cycle arrest in S phase. In addition, P2Y11 sensitized endothelial cells to cisplatin-induced cell death by down-regulation of the expression of Bcl-2. Taken together, these results suggest that extracellular ATP impairs cell proliferation by triggering signaling to induce cell cycle arrest and sensitizes cell to death via P2Y11 in endothelial cells.     

H(2)O(2) modulates purinergic-dependent calcium signalling in osteoblast-like cells

Reactive oxygen species (ROS) have long been considered as toxic by-products of aerobic metabolism and appear involved in the pathogenesis of degenerative diseases. The physiological role of ROS as second messengers in cell signal transduction is, on the other hand, increasingly recognized. Here we investigated the effects of H(2)O(2) and extracellular nucleotides on calcium signalling in four osteoblastic cell lines. In the highly differentiated HOBIT cells, sensitive to nanomolar concentrations of ADP and UTP, millimolar H(2)O(2) induced oscillatory increases of the cytosolic calcium concentration followed by a steady and sustained calcium increase. Long lasting rhythmic calcium activity was induced by micromolar H(2)O(2) doses. The H(2)O(2)-induced calcium signals, due to both release from intracellular stores and influx from the extracellular milieu, were totally prevented by incubating the cells with the P2 receptor antagonist suramin or with the ATP/ADP hydrolyzing enzyme apyrase. In the osteosarcoma SaOS-2 cells micromolar H(2)O(2) failed to evoke calcium signals and millimolar H(2)O(2) induced a slowly developing calcium influx which was unaffected by suramin and apyrase. These cells responded to micromolar concentrations of ATP and ADP, but were largely insensitive to UTP. ROS 17/2.8 osteosarcoma cells were totally insensitive to ATP, ADP and UTP in keeping with the evidence that these cells lack functional purinergic receptors. In these cells, H(2)O(2) up to 1mM did not increase the cytosolic calcium concentration. In ROS/P2Y(2) cells, stably expressing the P2Y(2) receptor, spontaneous calcium oscillations were observed in 38% of the population and nanomolar concentration of extracellular ATP or UTP activated oscillations in quiescent cells. Spontaneous calcium signals were inhibited by suramin and apyrase. In these cells H(2)O(2) induced oscillatory calcium activity that was blocked by suramin and apyrase. The sensitivity of ROS/P2Y(2) cells to UTP decreased significantly in the presence of DTT, which was effective also in inhibiting spontaneous calcium oscillations. On the other hand, the membrane-impermeant thiol oxidant DTNB induced calcium oscillations that were inhibited by incubating the cells with suramin or apyrase. Since peroxide did not increase extracellular ATP in these cell lines, we propose that, in osteoblasts, mild oxidative conditions could activate purinergic signalling through the sensitization of P2Y(2) receptor.     

Adenine nucleotides inhibit proliferation of the human lung adenocarcinoma cell line LXF-289 by activation of nuclear factor kappaB1 and mitogen-activated protein kinase pathways

Extracellular nucleotides have a profound role in the regulation of the proliferation of diseased tissue. We studied how extracellular nucleotides regulate the proliferation of LXF-289 cells, the adenocarcinoma-derived cell line from human lung bronchial tumor. ATP and ADP strongly inhibited LXF-289 cell proliferation. The nucleotide potency profile was ATP = ADP = ATPgammaS > > UTP, UDP, whereas alpha,beta-methylene-ATP, beta,gamma-methylene-ATP, 2',3'-O-(4-benzoylbenzoyl)-ATP, AMP and UMP were inactive. The nucleotide potency profile and the total blockade of the ATP-mediated inhibitory effect by the phospholipase C inhibitor U-73122 clearly show that P2Y receptors, but not P2X receptors, control LXF-289 cell proliferation. Treatment of proliferating LXF-289 cells with 100 microm ATP or ADP induced significant reduction of cell number and massive accumulation of cells in the S phase. Arrest in S phase is also indicated by the enhancement of the antiproliferative effect of ATP by coapplication of the cytostatic drugs cisplatin, paclitaxel and etoposide. Inhibition of LXF-289 cell proliferation by ATP was completely reversed by inhibitors of extracellular signal related kinase-activating kinase/extracellular signal related kinase 1/2 (PD98059, U0126), p38 mitogen-activated protein kinase (SB203508), phosphatidylinositol-3-kinase (wortmannin), and nuclear factor kappaB1 (SN50). Western blot analysis revealed transient activation of p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, and nuclear factor kappaB1 and possibly new formation of p50 from its precursor p105. ATP-induced attenuation of LXF-289 cell proliferation was accompanied by transient translocation of p50 nuclear factor kappaB1 and extracellular signal-related kinase 1/2 to the nucleus in a similar time period. In summary, inhibition of LXF-289 cell proliferation is mediated via P2Y receptors by activation of multiple mitogen-activated protein kinase pathways and nuclear factor kappaB1, arresting the cells in the S phase.     

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.     

Effects of ATP on intracellular calcium dynamics of neurons and satellite cells in rat superior cervical ganglia

Adenosine 5'-triphosphate (ATP) which is released from neuronal and non-neuronal tissues interacts with cell surface receptors to produce a broad range of physiological responses. The present study addressed the issue of whether the cells of the superior cervical ganglia (SCG) respond to ATP. To this end, the dynamics of the intracellular calcium ion concentration ([Ca2+]i) of neurons and satellite cells in intact SCG was analyzed by laser scanning confocal microscopy. ATP produced an increase of [Ca2+]i in both neurons and satellite cells; initially, ATP elicited [Ca2+]i increase in satellite cells and, subsequently, a [Ca2+]i change in neurons was observed. P1 purinoceptor agonists had no effect on this process, but P2 purinoceptor agonists induced [Ca2+]i increase and suramin totally inhibited ATP-induced [Ca2+]i dynamics in both neurons and satellite cells. In satellite cells, Ca2+ channel blockers and the removal of extracellular Ca2+, but not thapsigargin pretreatment, abolished ATP-induced [Ca2+]i dynamics. In contrast, thapsigargin pretreatment abolished ATP-induced [Ca2+]i dynamics in neurons. Reactive blue-2 inhibited the ATP-induced reaction on neurons alone. Uridine 5'-triphosphate caused a [Ca2+]i increase in neurons and alpha,beta-methylene ATP caused a [Ca2+]i increase in satellite cells. We concluded that neurons respond to extracellular ATP mainly via P2Y purinoceptors and that satellite cells respond via P2X purinoceptors.     

ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y₁ receptor agonist, induced a large signal while UTPyS (P2Y₂ agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y₁. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.     

Endotoxin activation of macrophages does not induce ATP release and autocrine stimulation of P2 nucleotide receptors

Receptors for extracellular nucleotides (P2, or purinergic receptors) have previously been implicated in the transduction of endotoxin signaling in macrophages. The most compelling evidence has been the observation that inhibitors of ionotropic nucleotide (P2X) receptors, including periodate-oxidized ATP (oATP), attenuate a subset of endotoxin-induced effects such as activation of NF-kappaB and up-regulation of inducible NO synthase. We investigated whether endotoxin induces ATP release from a murine macrophage cell line (BAC1.2F5) using sensitive on-line assays for extracellular ATP. These cells constitutively released ATP, producing steady-state extracellular concentrations of approximately 1 nM when assayed as monolayers of 10(6) adherent cells bathed in 1 ml of medium. However, the macrophages did not release additional ATP during either acute or prolonged endotoxin stimulation. In addition, cellular ecto-ATPase activities were measured following prolonged endotoxin activation and were found not to be significantly altered. Although oATP treatment significantly attenuated the endotoxin-induced production of NO, this inhibitory effect was not reproduced when the cells were coincubated with apyrase, a highly effective ATP scavenger. These results indicate that activation of macrophages by endotoxin does not induce autocrine stimulation of P2 nucleotide receptors by endogenous ATP released to extracellular compartments. Moreover, the data suggest that the ability of oATP to interfere with endotoxin signaling is due to its interaction with molecular species other than ATP-binding P2 receptors.     

Comparison of the effect of ATP on intracellular calcium ion dynamics between rat testicular and cerebral arteriole smooth muscle cells

Adenosine 5'-triphosphate (ATP), when released from neuronal and non-neuronal tissues, interacts with cell surface receptors produces a broad range of physiological responses. The goal of the present study was to examine the issue of whether vascular smooth muscle cells respond to ATP. To this end, the dynamics of the intracellular concentration of calcium ions ([Ca(2+)](i)) in smooth muscle cells in testicular and cerebral arterioles was examined by laser scanning confocal microscopy. ATP produced an increase in [Ca(2+)](i) in arteriole smooth muscle cells. While P1 purinoceptor agonists had no effect on this process, P2 purinoceptor agonists induced a [Ca(2+)](i) increase and a P2 purinoceptor antagonist, suramin, completely inhibited ATP-induced [Ca(2+)](i) dynamics in both arteriole smooth muscle cells.In testicular arterioles, Ca(2+) channel blockers and the removal of extracellular Ca(2+), but not thapsigargin pretreatment, abolished the ATP-induced [Ca(2+)](i) dynamics. In contrast, Ca(2+) channel blockers and the removal of extracellular Ca(2+) did not completely inhibit ATP-induced [Ca(2+)](i) dynamics in cerebral arterioles. Uridine 5'-triphosphate caused an increase in [Ca(2+)](i) only in cerebral arterioles and alpha,beta-methylene ATP caused an increase in [Ca(2+)](i) in both testicular and cerebral arterioles.We conclude that testicular arteriole smooth muscle cells respond to extracellular ATP via P2X purinoceptors and that cerebral arteriole smooth muscle cells respond via P2X and P2Y purinoceptors.     

Extracellular ATP induces fast and transient non-selective cationic currents and cytosolic Ca2+ changes in human umbilical artery smooth muscle cells

Ionotropic purinergic receptors (P2X) are expressed in endothelial and smooth muscle cells of blood vessels. ATP acting on smooth muscle P2X receptors is able to induce vasoconstriction in different kind of vessels. However, to our knowledge, there are no reports that directly show the activity of these purinergic receptors in native human vascular smooth muscle cells. In this work, we describe for the first time an ATP-induced current in freshly isolated human umbilical artery (HUA) smooth muscle cells. The current was measured by patch-clamp technique in whole-cell condition on cells clamped at -50 mV. At 100 μM of ATP the current showed a rapid activation and desensitization, and was carried by both Na(+) and Ca(2+). The current was completely blocked by suramin (300 μM) and partially blocked by 100 μM of Zn(2+) without affecting the kinetic of desensitization. All these properties suggest that the ATP-induced ionic currents are mediated through P2X(1)-like receptors. Moreover, we show that ATP transiently increased cytosolic Ca(2+) in "in situ" smooth muscle cells of intact HUA segments and that this response is dependent of extracellular and intracellular Ca(2+). These data expand the knowledge of purinergic receptors properties in vascular smooth muscle cells and the probable role of ATP as a paracrine modulator of contractile tone in a human artery which is fundamental for feto-placental blood flow.     

P2X7 receptor-dependent cell death is modulated during murine T cell maturation and mediated by dual signaling pathways

Extracellular ATP causes apoptosis and/or necrosis of the hemopoietic lineage through the activation of P2X7 receptors. In this study, we investigated P2X7 receptor-mediated cell death during murine T cell maturation. The expression level and activity of P2X7 receptors, as measured by induction of cell death and pore formation, were higher in splenocytes than thymocytes. Flow cytometric analysis revealed that cell shrinkage was induced by activation of the P2X7 receptor in murine lymphocytes and the responding cells were T cells. Splenic T cells were more responsive than their thymic counterpart. These observations indicate that the system of P2X7 receptor-mediated cell death in T cells could be modulated during T cell maturation. Furthermore, decreased extracellular Cl- suppressed ATP-induced cell shrinkage in splenocytes without inhibiting ERK1/2 phosphorylation, which is reported to mediate necrotic cell death. Treatment with U0126 (a MEK inhibitor) suppressed ATP-induced ERK1/2 phosphorylation without inhibiting cell shrinkage. Moreover, decreased extracellular Cl- and treatment with U0126 suppressed ATP-induced cell death. These observations indicate that the activation of P2X7 receptor leads to T cell death by two independent pathways, one of which is cell shrinkage dependent and the other of which involves the phosphorylation of ERK1/2. In conclusion, we demonstrate increasing P2X7 receptor activity during T cell maturation and the existence of two essential pathways in P2X7 receptor-mediated T cell death. Our findings suggest that ATP-induced cell death of peripheral T lymphocytes is important in P2X7 receptor-regulated immune responses.     

ATP-stimulated interleukin-6 synthesis through P2Y receptors on human osteoblasts

We investigated the effect of extracellular adenosine triphosphate (ATP) on the production of interleukin (IL)-6, whose molecules are capable of stimulating the development of osteoclasts from their hematopoietic precursors as well as are involved in signal transduction systems in human osteoblastic SaM-1 cells. These human osteoblasts constitutively expressed P2X4, P2X5, P2X6, P2Y2, P2Y5, and P2Y6 purinergic receptors. ATP increased gene- and protein-expression of IL-6 in SaM-1 cells. The expression of the IL-6 mRNA was maximal at 1h, and the increase in IL-6 synthesis in response to ATP (10-100 microM) occurred in a concentration-dependent manner. Over the same concentration range of the nucleotide that was effective for IL-6 synthesis, ATP caused an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), which increase was inhibited by pretreatment with suramin, a P2Y receptor antagonist, or 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate receptor blocker, but not by the extracellular Ca(2+)-chelating agent EGTA. The pretreatment of SaM-1 cells with suramin or 2-APB also inhibited the increase in IL-6 synthesis in response to ATP. These findings suggest that extracellular ATP-induced IL-6 synthesis occurs through P2Y receptors and mobilization of Ca(2+) from internal stores in human osteoblastic cells.     

Purinergic-independent calcium signaling mediates recovery from hepatocellular swelling: implications for volume regulation.

Swelling of hepatocytes and other epithelia activates volume-sensitive ion channels that facilitate fluid and electrolyte efflux to restore cell volume, but the responsible signaling pathways are incompletely defined. Previous work in model HTC rat hepatoma cells has indicated that swelling elicits ATP release, which stimulates P2 receptors and activates Cl(-) channels, and that this mechanism is essential for hepatocellular volume recovery. Since P2 receptors are generally coupled to Ca(2+) signaling pathways, we determined whether hepatocellular swelling affected cytosolic [Ca(2+)], and if this involved a purinergic mechanism. Exposure of HTC cells to hypotonic media evoked an increase in cytosolic [Ca(2+)], which was followed by activation of K(+) and Cl(-) currents. Maneuvers that interfered with swelling-induced increases in cytosolic [Ca(2+)], including extracellular Ca(2+) removal and intracellular Ca(2+) store depletion with thapsigargin, inhibited activation of membrane currents and volume recovery. However, the swelling-induced increases in cytosolic [Ca(2+)] were unaffected by either extracellular ATP depletion with apyrase or blockade of P2 receptors with suramin. These findings indicate that swelling elicits an increase in hepatocellular Ca(2+), which is essential for ion channel activation and volume recovery, but that this increase does not stem from activation of volume-sensitive P2 receptors. Collectively, these observations imply that regulatory responses to hepatocellular swelling involve a dual requirement for a purinergic-independent Ca(2+) signaling cascade and a Ca(2+)-independent purinergic signaling pathway.