Initiation of purinergic signaling by exocytosis of ATP-containing vesicles in liver epithelium.
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ABSTRACT:
SUBMITTER: Feranchak AP
PROVIDER: S-EPMC2832965 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ATP in bile is a potent secretogogue, stimulating biliary epithelial cell (BEC) secretion through binding apical purinergic receptors. In response to mechanosensitive stimuli, BECs release ATP into bile, although the cellular basis of ATP release is unknown. The aims of this study in human and mouse BECs were to determine whether ATP release occurs via exocytosis of ATP-enriched vesicles and to elucidate the potential role of the vesicular nucleotide transporter SLC17A9 in purinergic signaling. Dynamic, multiscale, live cell imaging (confocal and total internal reflection fluorescence microscopy and a luminescence detection system with a high sensitivity charge-coupled device camera) was utilized to detect vesicular ATP release from cell populations, single cells, and the submembrane space of a single cell. In response to increases in cell volume, BECs release ATP, which was dependent on intact microtubules and vesicular trafficking pathways. ATP release occurred as stochastic point source bursts of luminescence consistent with exocytic events. Parallel studies identified ATP-enriched vesicles ranging in size from 0.4 to 1 ?m that underwent fusion and release in response to increases in cell volume in a protein kinase C-dependent manner. Present in all models, SLC17A9 contributed to ATP vesicle formation and regulated ATP release. The findings are consistent with the existence of an SLC17A9-dependent ATP-enriched vesicular pool in biliary epithelium that undergoes regulated exocytosis to initiate purinergic signaling.
| S-EPMC3137107 | biostudies-literature
Project description:Extracellular nucleosides and nucleotides activate a group of G protein-coupled receptors (GPCRs) known as purinergic receptors, comprising adenosine and P2Y receptors. Furthermore, purinergic P2X ion channels are activated by ATP. These receptors are expressed in liver resident cells and play a critical role in maintaining liver function. In the normal physiology, these receptors regulate hepatic metabolic processes such as insulin responsiveness, glycogen and lipid metabolism, and bile secretion. In disease states, ATP and other nucleotides serve as danger signals and modulate purinergic responses in the cells. Recent studies have demonstrated that purinergic receptors play a significant role in the development of metabolic syndrome associated non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fibrosis, hepatocellular carcinoma (HCC) and liver inflammation. In this concise review, we dissect the role of purinergic signaling in different liver resident cells involved in maintaining healthy liver function and in the development of the above-mentioned liver pathologies. Moreover, we discuss potential therapeutic strategies for liver diseases by targeting adenosine, P2Y and P2X receptors.
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Project description:Adenosine triphosphate (ATP) is essential for the myriad of metabolic processes upon which life is based and is known widely as the universal energy currency unit of intracellular biologic reactions. ATP, adenosine diphosphate, adenosine, as well as other purines and pyrimidines also serve as ubiquitous extracellular mediators which function through the activation of specific receptors (viz. P2 receptors for nucleotides and purinergic P1 receptors for adenosine). Extracellular nucleotides are rapidly converted to nucleosides, such as adenosine, by highly regulated plasma membrane ectonucleotidases that modulate many of the normal biological and metabolic processes in the liver - such as gluconeogenesis and insulin signaling. Under inflammatory conditions, as with ischemia reperfusion, sepsis or metabolic stress, ATP and other nucleotides can also act as 'damage-associated molecular patterns' causing inflammasome activation in innate immune cells and endothelium resulting in tissue damage. The phosphohydrolysis of ATP by ectonucleotidases, such as those of the CD39/ENTPD family, results in the generation of immune suppressive adenosine, which in turn markedly limits inflammatory processes. Experimental studies by others and our group have implicated purinergic signaling in experimental models of hepatic ischemia reperfusion and inflammation, transplant rejection, hepatic regeneration, steatohepatitis, fibrosis and cancer, amongst others. Expression of ectonucleotidases on sinusoidal endothelial, stellate or immune cells allows for homeostatic integration and linking of the control of vascular inflammatory and immune cell reactions in the liver. CD39 expression also identifies hepatic myeloid dendritic cells and efficiently distinguishes T-regulatory-type cells from other resting or activated T cells. Our evolving data strongly indicate that CD39 serves as a key 'molecular switch' and is an integral component of the suppressive machinery of myeloid, dendritic and T cells. Increased understanding of mechanisms of extracellular ATP scavenging and specifically conversion to nucleosides by ectonucleotidases of the CD39 family have also led to novel insights into the exquisite balance of nucleotide P2-receptor and adenosinergic P1-receptor signaling in inflammatory and hepatic diseases. Further, CD39 and other ectonucleotidases exhibit genetic polymorphisms in humans which alter levels of expression/function and are associated with predisposition to inflammatory and immune diseases, diabetes and vascular calcification, amongst other problems. Development of therapeutic strategies targeting purinergic signaling and ectonucleotidases offers promise for the management of disordered inflammation and aberrant immune reactivity.
| S-EPMC6310169 | biostudies-literature
Project description:Adenosine triphosphate (ATP) modulates mechanosensitive vagal afferent nerves in the gastrointestinal tract. ATP is stored in secretory vesicles via the ATP transporter VNUT. Recently, the bisphosphate clodronate was reported to inhibit VNUT and was suggested to be a safe potent therapeutic option for chronic pain. Transient receptor potential vanilloid 4 (TRPV4) is activated by mechanical stimuli and some epoxyeicosatrienoic acids and becomes sensitized under inflammatory conditions. We have previously reported that TRPV4 and VNUT are expressed in mouse esophageal keratinocytes and that TRPV4 activation induces ATP release in gastric epithelial cells. Here we show the expression of TRPV4 and VNUT in normal human gastrointestinal cell derived cell lines (GES-1 and CCD 841) and in tissues from normal and VNUT-KO mice. TRPV4 agonists (GSK101 or 8,9-EET) induced an increase in cytosolic Ca2+ and/or current responses in mouse primary colonic epithelial cells and CCD 841 cells, but not in cells isolated from TRPV4-KO mice. TRPV4 agonists (GSK101 or 5.6-EET) also induced ATP release in GES-1 and CCD 841 cells, which could be blocked by the VNUT inhibitor, clodronate. Thus, VNUT inhibition with clodronate could represent a novel therapeutic option for visceral pain.
| S-EPMC6203352 | biostudies-literature
Project description:Following central nervous system (CNS) injury, activated astrocytes form glial scars, which inhibit axonal regeneration, leading to long-term functional deficits. Engineered nanoscale scaffolds guide cell growth and enhance regeneration within models of spinal cord injury. However, the effects of micro-/nanosize scaffolds on astrocyte function are not well characterized. In this study, a high throughput (HTP) microscale platform was developed to study astrocyte cell behavior on micropatterned surfaces containing 1 μm spacing grooves with a depth of 250 or 500 nm. Significant changes in cell and nuclear elongation and alignment on patterned surfaces were observed, compared to on flat surfaces. The cytoskeleton components (particularly actin filaments and focal adhesions) and nucleus-centrosome axis were aligned along the grooved direction as well. More interestingly, astrocytes on micropatterned surfaces showed enhanced mitochondrial activity with lysosomes localized at the lamellipodia of the cells, accompanied by enhanced adenosine triphosphate (ATP) release and calcium activities. These data indicate that the lysosome-mediated ATP exocytosis and calcium signaling may play an important role in astrocytic responses to substrate topology. These new findings have furthered our understanding of the biomechanical regulation of astrocyte cell-substrate interactions, and may benefit the optimization of scaffold design for CNS healing.
| S-EPMC4297955 | biostudies-literature
Project description:Deposition of uric acid crystals in joints causes the acute and chronic inflammatory disease known as gout and prolonged airway exposure to silica crystals leads to the development of silicosis, an irreversible fibrotic pulmonary disease. Aluminum salt (Alum) crystals are frequently used as vaccine adjuvant. The mechanisms by which crystals activate innate immunity through the Nlrp3 inflammasome are not well understood. Here, we show that uric acid, silica and Alum crystals trigger the extracellular delivery of endogenous ATP, which just precedes the secretion of mature interleukin-1β (IL-1β) by macrophages, both events depending on purinergic receptors and connexin/pannexin channels. Interestingly, not only ATP but also ADP and UTP are involved in IL-1β production upon these Nlrp3 inflammasome activators through multiple purinergic receptor signaling. These findings support a pivotal role for nucleotides as danger signals and provide a new molecular mechanism to explain how chemically and structurally diverse stimuli can activate the Nlrp3 inflammasome.
| S-EPMC3481132 | biostudies-literature
Project description:Purinergic signaling regulates a diverse and biologically relevant group of processes in the liver. However, progress of research into functions regulated by purinergic signals in the liver has been hampered by the complexity of systems probed. Specifically, there are multiple liver cell subpopulations relevant to hepatic functions, and many of these have been effectively modeled in human cell lines. Furthermore, there are more than 20 genes relevant to purinergic signaling, each of which has distinct functions. Hence, we felt the need to categorize genes relevant to purinergic signaling in the best characterized human cell line models of liver cell subpopulations. Therefore, we investigated the expression of adenosine receptor, P2X receptor, P2Y receptor, and ecto-nucleotidase genes via RT-PCR in the following cell lines: LX-2, hTERT, FH11, HepG2, Huh7, H69, and MzChA-1. We believe that our findings will provide an excellent resource to investigators seeking to define functions of purinergic signals in liver physiology and liver disease pathogenesis.
| S-EPMC4272373 | biostudies-literature
Project description:Conventional chemical synapses in the nervous system involve a presynaptic accumulation of neurotransmitter-containing vesicles, which fuse with the plasma membrane to release neurotransmitters that activate postsynaptic receptors. In taste buds, type II receptor cells do not have conventional synaptic features but nonetheless show regulated release of their afferent neurotransmitter, ATP, through a large-pore, voltage-gated channel, CALHM1. Immunohistochemistry revealed that CALHM1 was localized to points of contact between the receptor cells and sensory nerve fibers. Ultrastructural and super-resolution light microscopy showed that the CALHM1 channels were consistently associated with distinctive, large (1- to 2-?m) mitochondria spaced 20 to 40 nm from the presynaptic membrane. Pharmacological disruption of the mitochondrial respiratory chain limited the ability of taste cells to release ATP, suggesting that the immediate source of released ATP was the mitochondrion rather than a cytoplasmic pool of ATP. These large mitochondria may serve as both a reservoir of releasable ATP and the site of synthesis. The juxtaposition of the large mitochondria to areas of membrane displaying CALHM1 also defines a restricted compartment that limits the influx of Ca2+ upon opening of the nonselective CALHM1 channels. These findings reveal a distinctive organelle signature and functional organization for regulated, focal release of purinergic signals in the absence of synaptic vesicles.
| S-EPMC5966022 | biostudies-literature
Project description:BackgroundAugmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signaling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation.MethodsUnilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for 10 minutes after intravenous injection of 2×108 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signaling pathways were assessed by studying interventions that (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or KATP channels; or (3) inhibited downstream signaling pathways involving endothelial nitric oxide synthase or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease.ResultsTherapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hours in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with sickle cell disease. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced an ≈40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of endothelial nitric oxide synthase abolished the effects of therapeutic ultrasound, indicating downstream signaling through both nitric oxide and prostaglandins.ConclusionsTherapeutic ultrasound using microbubble cavitation to increase muscle perfusion relies on shear-dependent increases in ATP, which can act through a diverse portfolio of purinergic signaling pathways. These events can reverse hindlimb ischemia in mice for >24 hours and increase muscle blood flow in patients with sickle cell disease.Clinical trial registrationURL: http://clinicaltrials.gov. Unique identifier: NCT01566890.
| S-EPMC5373943 | biostudies-literature
Project description:T cells play a central role in host defense. ATP release and autocrine feedback via purinergic receptors has been shown to regulate T cell function. However, the sources of the ATP that drives this process are not known. We found that stimulation of T cells triggers a spike in cellular ATP production that doubles intracellular ATP levels in <30 s and causes prolonged ATP release into the extracellular space. Cell stimulation triggered rapid mitochondrial Ca(2+) uptake, increased oxidative phosphorylation, a drop in mitochondrial membrane potential (??m), and the accumulation of active mitochondria at the immune synapse of stimulated T cells. Inhibition of mitochondria with CCCP, KCN, or rotenone blocked intracellular ATP production, ATP release, intracellular Ca(2+) signaling, induction of the early activation marker CD69, and IL-2 transcription in response to cell stimulation. These findings demonstrate that rapid activation of mitochondrial ATP production fuels the purinergic signaling mechanisms that regulate T cells and define their role in host defense.
| S-EPMC4162192 | biostudies-literature