Role Of Extracellular Nucleotides Research Articles

Crosstalk between P2Y receptors and cyclooxygenase activity in inflammation and tissue repair.

The role of extracellular nucleotides as modulators of inflammation and cell stress is well established. One of the main actions of these molecules is mediated by the activation of purinergic receptors (P2) of the plasma membrane. P2 receptors can be classified according to two different structural families: P2X ionotropic ion channel receptors, and P2Y metabotropic G protein-coupled receptors. During inflammation, damaged cells release nucleotides and purinergic signaling occurs along the temporal pattern of the synthesis of pro-inflammatory and pro-resolving mediators by myeloid and lymphoid cells. In macrophages under pro-inflammatory conditions, the expression and activity of cyclooxygenase 2 significantly increases and enhances the circulating levels of prostaglandin E2 (PGE2), which exerts its effects both through specific plasma membrane receptors (EP1-EP4) and by activation of intracellular targets. Here we review the mechanisms involved in the crosstalk between PGE2 and P2Y receptors on macrophages, which is dependent on several isoforms of protein kinase C and protein kinase D1. Due to this crosstalk, a P2Y-dependent increase in calcium is blunted by PGE2 whereas, under these conditions, macrophages exhibit reduced migratory capacity along with enhanced phagocytosis, which contributes to the modulation of the inflammatory response and tissue repair.

Age-dependent decline in neurogenesis of the hippocampus and extracellular nucleotides

New neurons are continuously generated in the adult brain. This generation primarily occurs in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus. In the SGZ, neural stem cells (NSCs) give rise to glutamatergic granule cells that integrate into the hippocampal circuitry. Reduction of neurogenesis in the hippocampus impairs learning and memory, which suggests that this process is important for adult hippocampal function. Indeed, the neurogenesis is reduced in the progression of aging, which is thought to contribute to age-related cognitive impairment. Although the mechanism of age-dependent decline in neurogenesis remains largely obscure, astrocytes are thought to play a vital role in regulating NSC proliferation and differentiation. Both astrocytes and NSCs secrete nucleotides to the extracellular space and extracellular nucleotides bind to their receptors on the surface of target cells. In this review, the recent knowledge on adult neurogenesis in the hippocampus is summarized briefly, and possible role of extracellular nucleotides in the age-dependent changes of the adult neurogenesis is discussed.

Novel Evidence That Extracellular Nucleotides and Nucleosides Regulate the Expression of Heme Oxygenase 1 (HO-1) in Opposite Ways in Hematopoietic Stem/Progenitor Cells (HSPCs), Which Explains Why ATP Enhances Mobilization of HSPCs, While Its Metabolite Adenosine Inhibits This Process

Background. One of the problems with the mobilization of hematopoietic stem/progenitor cells (HSPCs) in the clinic is that a significant number of patients are poor mobilizers. In order to develop more efficient mobilization strategies, we have to better understand the mobilization process at the molecular and cellular levels. We have reported that HSPCs express heme oxygenase 1 (HO-1), which is a negative regulator of the complement cascade (ComC), which in turn is required for stem cell mobilization (Stem Cell Rev. 2015, 11:110-8, Leukemia. 2017, 31:446-458). Moreover, very recently we became interested in the novel role of extracellular nucleotides (EXNs) and nucleosides in this process (Leukemia 2018, in press, doi: 10.1038/s41375-018-0122-0). The most important EXN is adenosine triphosphate (ATP), which is involved in intracellular energy transfer but if released from activated cells into the extracellular space becomes an important signaling molecule involved in purinergic signaling. Secretion of ATP from activated cells occurs in a pannexin channel-dependent manner and is additionally augmented by a positive regulatory loop after ATP binding to the P2X7 purinergic receptor. We recently found that the ATP concentration during the mobilization process increases to micromolar concentrations in the BM microenvironment. Moreover, we found that in the extracellular BM space ATP is processed by the CD39 and CD73 ectonucleotidases to ADP, AMP, and finally to adenosine, which is also an important mediator of purinergic signaling. Hypothesis. We hypothesized that EXNs and purinergic signaling via extracellular ATP and its metabolite adenosine are involved in triggering the mobilization of HSPCs and that this effect is mediated by the expression of HO-1 in HSPCs. Materials and Methods. To address this question, 2-month-old P2X7-/-, CD39-/-, and CD73-/-mice as well as their normal wild type (WT) littermates were mobilized with G-CSF or AMD3100. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. The secretion of ATP from BM cells was inhibited by employing the pannexin 1 blocking drug probenecid or a synthetic pannexin 1 blocking peptide. In parallel, we evaluated i) the expression of HO-1 in HSPCs by employing RQ-PCR and western blot analysis and ii) the activation of the ComC by C5a ELISA. Results. Here we provide evidence that ATP, as an EXN secreted in a pannexin-1-dependent manner from BM cells, triggers activation of the ComC and initiates mobilization of HSPCs. Inhibition of the pannexin 1 channel by probenecid or a pannexin 1 blocking peptide inhibited this process. Furthermore, mobilization of HSPCs was augmented in a P2X7 receptor-dependent manner, evidenced by the finding that P2X7-/- mice were poor mobilizers. Furthermore, ATP is processed to adenosine in the extracellular space by CD39 and CD73 ectonucleotidases, and to our surprise we observed that CD73-deficient mice mobilize more HSPCs than their control wild type (WT) littermates, which indicates a novel negative role for adenosine in the mobilization process. This finding was confirmed by injecting mice with adenosine during AMD3100 administration. Finally, we found that ATP and adenosine modulate the mobilization process by regulating in opposite ways HSPC expression of HO-1, which is a negative regulator of mobilization. Therefore, while ATP downregulates expression of HO-1 in HSPCs and enhances mobilization, adenosine upregulates its expression and negatively affects the egress of HSPCs from BM into PB. Conclusions. We demonstrate for the first time that purinergic signaling involving ATP and its metabolite adenosine regulate the mobilization of HSPCs in an HO-1-dependent manner. While ATP triggers and promotes this process, adenosine has an inhibitory effect. The opposite effects of ATP and adenosine on the mobilization process can be explained by their different effects on the regulation of HO-1 expression in HSPCs. Finally, administration of ATP as a signaling molecule (together with G-CSF or AMD3100), inhibition of CD73 or HO-1 by small-molecule antagonists may provide the basis for more efficient mobilization strategies. DisclosuresNo relevant conflicts of interest to declare.

New Insights in Plant Biology Gained from Research in Space

ABSTRACT Recent spaceflight experiments have provided many new insights into the role of gravity in plant growth and development. Scientists have been taking seeds and plants into space for decades in an effort to understand how the stressful environment of space affects them. The resultant data have yielded significant advances in the development of advanced life-support systems for long-duration spaceflight and a better understanding of the fundamental role of gravity in directing the growth and development of plants. Experiments have improved as new spaceflight hardware and technology paved the way for progressively more insightful and rigorous plant research in space. The International Space Station (ISS) has provided an opportunity for scientists to both monitor and control their experiments in real-time. Experiments on the ISS have provided valuable insights into endogenous growth responses, light responses, and transcriptomic and proteomic changes that occur in the microgravity environment. In recent years most studies of plants in space have used Arabidopsis thaliana, but the single-celled, Ceratopteris richardii spore is also a valuable model system that has been used to understand plant gravity response. Experiments using these fern spores have revealed a dynamic and gravity-responsive trans-cell Ca2+ current that directs polarization of these spores and a possible role of extracellular nucleotides in establishing or contributing to this current. As technology continues to improve, spaceflight experiments will provide many new insights into the role and effects of gravity on plant growth and development.

Abstract 4144: Extracellular nucleotides and purinergic signaling as novel, underappreciated, pro-metastatic factors for human lung cancer cells

Abstract Background: One of the challenging problems of current radio-chemotherapy is recurrence and metastasis of cancer cells that survive initial treatment. We have proposed that one of the unwanted effects of radio-chemotherapy is release of phospho-sphingolipids in response to treatment and thus induction of pro-metastatic microenvironment in several organs (Mol Cancer Res 2013; 11:793). However, at the same time also intracellular nucleotides such as ATP and UTP are released from damaged (“leaky”) cells exerting chemotactic functions. Hypothesis: Based on these observations that, in addition to phospho-sphingolipids, also nucleotides (e.g., ATP and UTP) are released from damaged cells we hypothesized that these molecules if released into extracellular microenvironment may also direct chemotaxis and metastasis of cancer cells - alone or in synergy with phospho-sphingolipids. Materials and Methods: Several complementary in vitro and in vivo approaches were employed to demonstrate a novel role of extracellular nucleotides (EXN) in a model of metastasis of lung cancer cells. In our studies we employed four large cell and two small cell lung cancer cell lines and a set of nucleotides including ATP, ADP, AMP, UTP, TTP, CTP, and GTP. Real time RT-PCR analysis of the expression of purinergic P1 and P2 receptors as well as chemotaxis, adhesion, proliferation, intracellular calcium flux and cell signaling studies in response to EXN stimulation were performed. Concentrations of ATP and UTP in several organs before and after radio-chemotherapy were measured by colorimetric kits. Purinergic receptor agonists and antagonists, inhibiting all or selected subtypes, were assayed in vitro and in vivo in pro-metastatic assays. Results: We observed that EXN accumulate in in several organs in response to radio-chemotherapy. RT-PCR analysis indicated that most of the P2X, P2Y and adenosine receptor subtypes are expressed in tested lung cancer cell lines. By employing in vitro migration assays, we found that, out of all the nucleotides tested, ATP, AMP and UTP have the strongest chemotactic activity for most of the human lung cancer cell lines correlating with phosphorylation of MAPKp42/44 and AKT. We also observed increased adhesion to fibronectin after stimulation with tested EXN, whereas proliferation was not affected by of EXN. More important, metastasis of lung cancer cells could be inhibited in immune-deficient mice in a presence of specific small molecule inhibitors of nucleotide receptors. Conclusions: Both systemic and local radio-chemotherapy leads to upregulation of EXN release in damaged tissues, and the side effect of such treatment is induction of an unwanted pro-metastatic microenvironment in different organs. Based on this data, EXN are novel pro-metastatic factors and inhibition of their pro-metastatic effects could become an important part of anti-metastatic treatment. Citation Format: Gabriela Schneider, Talita Glaser, Ahmed Abdelbaset Ismail, Henning Ulrich, Mariusz Z. Ratajczak. Extracellular nucleotides and purinergic signaling as novel, underappreciated, pro-metastatic factors for human lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4144. doi:10.1158/1538-7445.AM2015-4144

Extracellular nucleotides from dying cells act as molecular signals to promote wound repair in renal tubular injury.

Acute kidney injury (AKI) often correlates with poor prognosis and is followed by various severe unfavorable systemic outcomes. It is important to understand the pathophysiology of AKI for the development of novel therapeutic approaches toward promoting renal regeneration after injury. Recent studies have indicated that AKI-induced tubular cell death plays an active role in the onset of tissue regeneration; however, the mechanisms underlying renal tubular repair after injury have yet to be understood. In the present study, we explored molecules that might serve as "danger" signals in mediating tubular regeneration. Kidneys of rats systemically administered the nephrotoxicant cisplatin (to induce AKI) exhibited massive cell proliferation. The proportion of proliferating cells in the total cell distribution was highest in the outer stripe of the outer medulla coincided with where the tubular damage was the most severe in this study. This finding suggests that soluble factors may have been released from damaged cells to stimulate the proliferation of neighboring tubular epithelial cells. In elucidating the mechanism of dying cell-to-surviving cell communication using normal rat kidney NRK-52E epithelial cells, we found a significant increase in ATP levels in supernatants of these cells after the induction of cell death using ultraviolet irradiation. Furthermore, treatment of conditioned supernatants with apyrase or suramin, which inhibits purinergic signaling, resulted in significant decreases in cell proliferation and migration activities. These results demonstrate a novel role for extracellular nucleotides, probably as danger signals in aggravating tubular regeneration after AKI.

Abstract P2-09-20: 2’(3’)-O-(4-benzoylbenzoyl)adenosine 5’-triphosphate (BzATP) is a potential drug for triple negative breast cancer: Autophagy and apoptosis inducer

Abstract Triple-negative breast cancer (TNBC) is among the most aggressive and therapy resistant type of breast cancer. New therapeutic strategies and new treatments are intensively sought for TNBC. Here, we report the potential of 2’(3’)-O-(4-Benzoylbenzoyl)adenosine 5’-triphosphate (BzATP), which is ATP analog, as a potential drug for TNBC. Role of extracellular nucleotides as an extracellular signaling molecule and the antitumor effects of ATP have been reported for a few decades, however, the signaling pathways that link from the purinergic receptors to the reported effects including reduced tumor xenografts volume in mice and tumor growth inhibition in vitro is not yet well established. We chose two pairs of TNBC cell lines with matching molecular subtype; one is basal A subtype and another one is basal B subtype. In each pair, one was derived from Caucasian patient and another one was derived from African American patient. This strategy allows us to cover a wide range of cell lines and also to see whether there is any disparity of BzATP treatment between ethnicity. Expression of P2Y receptors, which are one of three families of extracellular receptors for purine and pyrimidine nucleotides, in not only two pairs of TNBC in this study, but also other six breast cancer cell lines, showed five common P2Y receptors including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11. Growth inhibition assay was performed by treating TNBC with agonists including ATP, BzATP, UTP, and NAD from 1 μM to the highest dose at 1000 μM, which is still lower than reported dose in in vivo studies in mice. ATP, BzATP, and NAD, but not UTP, at 100 μM and higher doses inhibited growth of TNBC regardless of ethnicity. Notably, BzATP inhibited growth of all four TNBC cell lines in a greater degree than ATP did. Among five common receptors, P2Y2, P2Y4, and P2Y6, which has UTP or UDP as an agonist, are improbable to be a candidate receptor since UTP failed to inhibit TNBC growth. P2Y11, which has ATP, BzATP, and NAD as an agonist is a first candidate receptor since P2Y1 prefers ADP to ATP. Interestingly, growth inhibition on TNBC was observed along with a morphology change including reduced cell volume and an increase in process number after 24 hours of BzATP treatment at concentration that causes 50% growth reduction (GI50), which is 320 μM, 200 μM, 250 μM, and 200 μM for HCC1143, HCC70, HCC38, and HCC1500 cell lines respectively. Although BzATP also reduced a growth rate of non-malignant human epithelium cell line (MCF10A), no aforementioned morphology change was observed on MCF10A. In addition, BzATP at GI50 induced autophagy through expression of microtubule-associated protein 1 light chain 3 (LC3) and elevated a number of autophagic vesicles. Surprisingly, autophagy-related (Atg) proteins including Atg5, Atg7, and Atg3 did not show an upregulated level. In conjunction with autophagy, BzATP at GI50 also arrested cell cycle of TNBC at G2/M phase. Further, BzATP at GI50 elevated apoptotic marker, cleaved caspase-3 and cleaved PARP, and ER stress marker, CHOP, in TNBC but not in MCF10A. BzATP specifically induced apoptosis without causing necrosis as no elevated released lactate dehydrogenase (LDH) level was detected. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-20.

Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

BackgroundExtracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation.ResultsHere we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells.ConclusionOur results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

Regulatory volume decrease and P receptor signaling in fish cells: mechanisms, physiology, and modeling approaches

For animal cell plasma membranes, the permeability of water is much higher than that of ions and other solutes, and exposure to hyposmotic conditions almost invariably causes rapid water influx and cell swelling. In this situation, cells deploy regulatory mechanisms to preserve membrane integrity and avoid lysis. The phenomenon of regulatory volume decrease, the partial or full restoration of cell volume following cell swelling, is well-studied in mammals, with uncountable investigations yielding details on the signaling network and the effector mechanisms involved in the process. In comparison, cells from other vertebrates and from invertebrates received little attention, despite of the fact that e.g. fish cells could present rewarding model systems given the diversity in ecology and lifestyle of this animal group that may be reflected by an equal diversity of physiological adaptive mechanisms, including those related to cell volume regulation. In this review, we therefore present an overview on the most relevant aspects known on hypotonic volume regulation presently known in fish, summarizing transporters and signaling pathways described so far, and then focus on an aspect we have particularly studied over the past years using fish cell models, i.e. the role of extracellular nucleotides in mediating cell volume recovery of swollen cells. We, furthermore, present diverse modeling approaches developed on the basis of data derived from studies with fish and other models and discuss their potential use for gaining insight into the theoretical framework of volume regulation.

Possible Role of Extracellular Nucleotides in Ectopic Ossification of Human Spinal Ligaments

To reveal the involvement of extracellular nucleotides in the ossification process in ossification of the posterior longitudinal ligament of the spine (OPLL), the mRNA expression profiles of P2 purinoceptors, mechanical stress-induced ATP release, and ATP-stimulated expression of osteogenic genes were analyzed in ligament cells derived from the spinal ligament of OPLL patients (OPLL cells) and non-OPLL cells derived from the spinal ligaments of cervical spondylotic myelopathy patients as a control. The extracellular ATP concentrations of OPLL cells in static culture were significantly higher than those of non-OPLL cells, and this difference was diminished in the presence of ARL67156, an ecto-nuclease inhibitor. Cyclic stretch markedly increased the extracellular ATP concentrations of both cell types to almost the same level. P2Y1 purinoceptor subtypes were intensively expressed in OPLL cells, but only weakly expressed in non-OPLL cells. Not only ATP addition but also cyclic stretch raised the mRNA levels of alkaline phosphatase and osteopontin in OPLL cells, which were blocked by MRS2179, a selective P2Y1 antagonist. These increases in the expression of osteogenic genes were not observed in non-OPLL cells. These results suggest an important role of P2Y1 and extracellular ATP in the progression of OPLL stimulated by mechanical stress.

Disordered Pancreatic Inflammatory Responses and Inhibition of Fibrosis in CD39-Null Mice

Extracellular nucleotides are released from injured cells and bind purinergic-type 2 receptors (P2-Rs) that modulate inflammatory responses. Ectonucleotidases, such as CD39/nucleoside triphosphate diphosphohydrolase-1, hydrolyze extracellular nucleotides to integrate purinergic signaling responses. Because the role of extracellular nucleotides and CD39 in mediating inflammation and fibrosis are understood poorly, we studied the impact of CD39 gene deletion in a model of pancreatic disease. Pancreatitis was induced by cyclosporine pretreatment, followed by cerulein injections (50 mug/kg, 6 intraperitoneal injections/day, 3 times/wk); mice were killed at day 2, week 3, and week 6. Experimental parameters were correlated with cytokine levels in blood, RNA, and protein expression of purinergic and fibrosis markers in tissues. Immunohistochemistry and pancreatic morphometry of fibrosis were performed in wild-type and CD39-null mice. Effects of CD39 deletion on proliferation of primary pancreatic stellate cells (PSCs) were investigated in vitro. Wild-type mice developed morphologic features of pancreatitis with the anticipated development of parenchymal atrophy and fibrosis. CD39 and P2-R became overexpressed in vascular and adventitious wild-type tissues. In contrast, CD39-null mice had inflammatory reactions but developed only minor pancreatic atrophy and limited fibrosis. Interferon-gamma became significantly increased in tissues and plasma of CD39-null mice. Wild-type PSCs expressed high levels of CD39 and P2-R. CD39-null PSCs showed decreased rates of proliferation and the expression of procollagen-alpha1 was inhibited significantly in vitro (P < .03). CD39 deletion decreases fibrogenesis in experimental pancreatitis. Our data implicate extracellular nucleotides as modulators of PSC proliferation and collagen production in pancreatitis.

Intraluminal autocrine purinergic signaling within cysts: implications for the progression of diseases that involve encapsulated cyst formation

purinergic ligands are local mediators, autacoids, or paracrine factors ([5][1], [7][2]–[10][3], [12][4], [16][5], [47][6]–[49][7], [51][8], [63][9], [70][10]). The role of extracellular nucleotides and nucleosides in physiology and pathophysiology has been studied for decades ([5][1], [7][2

A Novel Role of Extracellular Nucleotides in Valve Calcification

Calcific aortic valve disease is a common condition and is associated with inflammatory changes and expression of osteoblast-like cell phenotypes, but the cellular mechanisms are unclear. Recent studies identified extracellular ATP and P2Y receptor cascade as important regulators of bone remodeling, whereas its breakdown product, adenosine, is known to have anti-inflammatory properties. We hypothesize that extracellular ATP and adenosine have important roles in regulating osteoblast differentiation in human valve interstitial cells, and that this can be a potential target for therapy. Method and Results- Primary cultures of human valve interstitial cells (ICs) treated for 21 days with osteogenic media, ATP, and ATP-gamma-S (a stable agonist of the P2Y receptor) revealed a significant increase in alkaline phosphatase (ALP) (an osteoblast marker) activity and expression as measured using spectrophotometric assay and immunocytochemistry staining. Valve ICs treated with adenosine alone did not cause an increase in ALP activity; however, adenosine treatment decreased the ALP activity and expression induced by osteogenic media after 21 days of incubation. In addition, atorvastatin inhibited the activity of ALP induced by ATP in human valve ICs, and enzyme studies revealed that atorvastatin upregulated the breakdown of extracellular ATP into adenosine in human valve ICs after 24-hour treatment. These findings identify a novel role for extracellular nucleotides in inducing osteoblast differentiation in human valve ICs in vitro and provide a potential therapeutic target for preventing the disease progression.

Thrombospondin-1 and indoleamine 2,3-dioxygenase are major targets of extracellular ATP in human dendritic cells

Extracellular adenosine triphosphate affects the maturation of human monocyte-derived dendritic cells (DCs), mainly by inhibiting T-helper 1 (Th1) cytokines, promoting Th2 cytokines, and modulating the expression of costimulatory molecules. In this study, we report that adenosine triphosphate (ATP) can induce immunosuppression through its action on DCs, defining a new role for extracellular nucleotides. Microarray analysis of ATP-stimulated human DCs revealed inter alia a drastic up-regulation of 2 genes encoding mediators involved in immunosuppression: thrombospondin-1 (TSP-1) and indoleamine 2,3-dioxygenase (IDO). The release of TSP-1 by DCs in response to ATP was confirmed at the protein level by enzyme-linked immunosorbent assay (ELISA), immunodetection, and mass spectrometry analysis, and has an antiproliferative effect on T CD4+ lymphocytes through TSP-1/CD47 interaction. Our pharmacologic data support the involvement of purinergic receptor P2Y11 in this ATP-mediated TSP-1 secretion. We demonstrate also that ATP significantly potentiates the up-regulation of IDO--a negative regulator of T lymphocyte proliferation--and kynurenine production initiated by interferon-gamma (IFN-gamma) in human DCs. Thus, extracellular ATP released from damaged cells and previously considered as a danger signal is also a potent regulator of mediators playing key roles in immune tolerance. Consequently, nucleotides' derivatives may be considered as useful tools for DC-based immunotherapies.

Adenosine triphosphate release and purinergic regulation of cholangiocyte transport.

The discovery of purinergic receptors on almost every cell type studied to date suggests that purinergic signaling is a fundamental process regulating cell and organ level functions. Purinergic receptors have been found on all principal liver cell types, including liver parenchymal cells, or hepatocytes, and biliary epithelial cells, or cholangiocytes. Both hepatocytes and cholangiocytes are capable of the regulated release of adenosine triphosphate (ATP), and both cell types express a range of purinergic receptors to mediate cellular processes. The role of extracellular nucleotides in liver function is presently being elucidated. Extracellular ATP, in addition to autocrine regulation of liver cell volume, has recently been shown to play an important role in paracrine signaling to coordinate specific hepatocyte and cholangiocyte cellular responses. The findings that (1) cholangiocytes are capable of the regulated release of ATP into bile, (2) ATP is present in bile in concentrations capable of stimulating purinergic receptors, and (3) P2 receptor stimulation results in brisk Cl(-) channel activation and fluid secretion suggest an important role of extracellular ATP in the regulation of bile formation. This article highlights important developments in our understanding of the role of purinergic signaling in cholangiocyte transport and bile formation.

Metabolism and transport of purinergic receptor agonists in rabbit conjunctival epithelial cells.

The role of extracellular nucleotides as signaling molecules is well established in a number of biological systems. Their function in the regulation of ion1, 2 and fluid3 transport has been investigated in the pigmented rabbit conjunctiva where active Cl− secretion has been implicated as the driving force for fluid secretion.3 These agonists interact with two classes of receptors located on the cell membrane: P2X ligand-gated ion channels and P2Y G-protein-coupled receptors.4 Receptor mapping techniques have revealed the presence of P2Y type receptors in ocular epithelial cells.5 KeywordsContact LensProtein ProfileProtein DepositionContact Lens WearLens MaterialThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

METABOLISM AND TRANSPORT OF PURINERGIC RECEPTOR AGONISTS IN RABBIT CONJUNCTIVAL EPITHELIAL CELLS.

The role of extracellular nucleotides as signaling molecules is well established in a number of biological systems. Their function in the regulation of ion1, 2 and fluid3 transport has been investigated in the pigmented rabbit conjunctiva where active Cl− secretion has been implicated as the driving force for fluid secretion.3 These agonists interact with two classes of receptors located on the cell membrane: P2X ligand-gated ion channels and P2Y G-protein-coupled receptors.4 Receptor mapping techniques have revealed the presence of P2Y type receptors in ocular epithelial cells.5

2.W10.2 Potential roles of extracellular nucleotides in atherogenesis

2.W10.2 Potential roles of extracellular nucleotides in atherogenesis

Purine and pyrimidine nucleotides activate distinct signalling pathways in PC12 cells.

The role of extracellular nucleotides in intracellular signalling and neurosecretion was assessed in PC12 cells. Activation of phospholipase C and increased [Ca2+]i were mediated by purinoceptors with an agonist potency profile, ATP approximately UTP > 2-methylthioadenosine triphosphate (2-MeSATP), typical of P2U. ATP also evoked a rapid acidification followed by a more gradual alkalinization (measured with 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF)), while UTP induced only a gradual alkalinization. The amiloride analogue 5-(N-ethyl-N-isopropyl)amiloride (EIPA) attenuated the alkalinization phase suggesting activation of the Na+/H+ exchanger by ATP and UTP. Using bisoxonol and [3H]tetraphenylphosphonium ([3H]TPP+) as potential-sensitive probes, we showed that while ATP rapidly depolarized PC12 cells in an Na(+)-dependent manner, UTP evoked a much reduced and delayed response. The potency profile (ATP approximately 2-MeSATP approximately adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) >> UTP, alpha, beta-methyleneATP) suggested involvement of a receptor subtype distinct from P2U. Secretion of endogenous dopamine was also assessed. Those nucleotides that induced depolarization (ATP, 2-MeSATP, ATP gamma S) were also the most potent secretagogues. UTP was ineffective. Our results suggest that ATP stimulates distinct purinoceptor subtypes and induces neurosecretion through the activation of multiple signalling pathways.

Evidence for a role of pituitary ATP receptors in the regulation of pituitary function.

Despite a rapidly increasing acceptance for a role of ATP as an extracellular mediator in several biological systems, the present report shows that ATP may mediate physiological responses in pituitary cells. We have now been able to demonstrate a specific action of ATP receptors to mediate the release of luteinizing hormone from gonadotropes and have coupled them with further studies that clearly show that ATP can be exocytotically released from cultured rat pituitary cells. Both ATP and UTP (100 microM) caused a > 14-fold increase in the rate of luteinizing hormone release from superfused cells. Adenosine 5'-[alpha, beta-methylene]triphosphate and 5'-[beta,gamma-methylene triphosphate were ineffective, and 2-methylthio-ATP had only a modest stimulatory effect. Homologous and heterologous desensitization occurred with UTP and ATP, and these did not have additive effects. Thus, nucleotides can be effective stimulators of luteinizing hormone release through a single class of ATP receptor (P2U subtype). The calcium ionophore A23187 provoked release of a substantial amount of ATP from pituitary cells in a concentration- and Ca(2+)-dependent manner, which was desensitized by pretreatment with A23187. This implies a possible paracrine and/or autocrine mechanism by which nucleotides may exert their effects on pituitary cells. In conclusion, we have provided strong evidence for a novel role of extracellular nucleotides as mediators in pituitary--in particular, in gonadotrope--function.