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1
Shirai, Y., K. Kashiwagi, N. Sakai, and N. Saito. "Phospholipase A(2) and its products are involved in the purinergic receptor-mediated translocation of protein kinase C in CHO-K1 cells." Journal of Cell Science 113, no. 8 (2000): 1335–43. http://dx.doi.org/10.1242/jcs.113.8.1335.
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The signal transduction involved in the purinergic stimuli-induced activation of protein kinase C (PKC) in CHO-K1 cells was investigated. Purinergic stimuli such as adenosine triphosphate and uridine triphosphate induced a transient translocation of PKC epsilon, gamma, and delta from the cytoplasm to the plasma membrane. These translocations were blocked by an inhibitor of phosphatidylinositol-specific phospholipase C (PLC), but not by an inhibitor of phosphatidylcholine-specific PLC. A diacylglycerol (DAG) analogue also induced reversible translocations of PKC gamma, epsilon, and delta from the cytoplasm to the plasma membrane, while the calcium ionophore A23187 caused a similar translocation of only the gamma subtype. These results confirm that the hydrolysis of phosphatidylinositol-2-phosphate by PLC and the subsequent generation of DAG and increase in Ca(2+)are involved in the purinergic stimuli-induced translocation of PKC. A DAG antagonist, 1-o-hexadecyl-2-o-acetyl-glycerol, blocked the DAG analogue-induced translocations of all PKC subtypes tested but failed to inhibit the purinergic stimuli-induced translocations of PKC epsilon and gamma. The DAG antagonist could not block the ATP- and UTP-induced translocation of PKC epsilon even in the absence of extracellular Ca(2+). Co-application of the DAG antagonist and a phospholipase A(2) (PLA(2)) inhibitor such as aristolochic acid, arachidonyltrifluoromethyl ketone, or bromoenol lactone inhibited the purinergic receptor-mediated translocation of PKC epsilon although each PLA(2) inhibitor alone did not block the translocation. In contrast to the epsilon subtype, ATP-induced translocation of PKC gamma was observed in the presence of both the PLA(2) inhibitor and the DAG antagonist. However, it is noteworthy that re-translocation of PKC gamma was hastened by the PLA(2) inhibitor. Furthermore products of PLA(2), such as lysophospholipids and fatty acids, induced the translocation of PKC gamma and epsilon in a dose dependent manner, but not delta. These results indicate that, in addition to PLC and DAG, PLA(2) and its products are involved in the purinergic stimuli-induced translocation of PKC epsilon and gamma in CHO-K1 cells. Each subtype of PKC in CHO-K1 cell is individually activated in response to a purinergic stimulation.
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Spek, Annabel, Bingsheng Li, Beata Rutz, et al. "Purinergic smooth muscle contractions in the human prostate: estimation of relevance and characterization of different agonists." Naunyn-Schmiedeberg's Archives of Pharmacology 394, no. 6 (2021): 1113–31. http://dx.doi.org/10.1007/s00210-020-02044-4.
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AbstractNon-adrenergic prostate smooth muscle contractions may account for the limited effectiveness of α1-adrenoceptor antagonists, which are the first-line option for medical treatment of voiding symptoms suggestive of benign prostatic hyperplasia. In non-human prostates, purinergic agonists induce contractions reaching similar magnitudes as α1-adrenergic contractions. However, evidence for the human prostate is highly limited, and pointed to much weaker purinergic contractions. Here, we examined contractions of different purinergic agonists in human prostate tissues. Tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath, and expression of purinergic receptors was studied by RT-PCR. Electric field stimulation (EFS)–induced contractions amounted to 104% of KCl-induced contractions (95% CI: 84–124%). From all tested agonists, only ATP induced concentration-dependent contractions, reaching an average maximum of 18% (12–24%) of KCl. Maximum tensions following application of other agonists averaged to 7.1% of KCl for α,β-methylene-ATP (1.8–12.4%), 3.9% for β,γ-methylene-ATP (2.0–5.4%), 3.1% for 2-methylthio-ATP (− 0.1–6.3%), and 5.1% for ATPγS (1.0–9.2%). Responses were not affected by the P2X antagonist NF023 or the P2Y antagonist PPADS. mRNA expression of P2X1-4 correlated with expression of a marker for catecholaminergic nerves, although neither ATP, NF023, nor PPADS changed EFS-induced contractions. Correlation between expression of receptors and the smooth muscle marker calponin was not observed. Our findings point to a low relevance of purinergic contractions in the human prostate, compared to other contractile stimuli in the human prostate and compared to purinergic contractions in non-human prostates. Purinergic contractions in the human prostate are not sensitive to NF023 or PPADS.
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Klausen, Polina, Daria Semenova, Daria Kostina, Vladimir Uspenskiy, and Anna Malashicheva. "Purinergic Signaling in Pathologic Osteogenic Differentiation of Aortic Valve Interstitial Cells from Patients with Aortic Valve Calcification." Biomedicines 11, no. 2 (2023): 307. http://dx.doi.org/10.3390/biomedicines11020307.
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Purinergic signaling is associated with a vast spectrum of physiological processes, including cardiovascular system function and, in particular, its pathological calcifications, such as aortic valve stenosis. Aortic valve stenosis (AS) is a degenerative disease for which there is no cure other than surgical replacement of the affected valve. Purinergic signaling is known to be involved in the pathologic osteogenic differentiation of valve interstitial cells (VIC) into osteoblast-like cells, which underlies the pathogenesis of AS. ATP, its metabolites and related nucleotides also act as signaling molecules in normal osteogenic differentiation, which is observed in pro-osteoblasts and leads to bone tissue development. We show that stenotic and non-stenotic valve interstitial cells significantly differ from each other, especially under osteogenic stimuli. In osteogenic conditions, the expression of the ecto-nucleotidases ENTPD1 and ENPP1, as well as ADORA2b, is increased in AS VICs compared to normal VICs. In addition, AS VICs after osteogenic stimulation look more similar to osteoblasts than non-stenotic VICs in terms of purinergic signaling, which suggests the stronger osteogenic differentiation potential of AS VICs. Thus, purinergic signaling is impaired in stenotic aortic valves and might be used as a potential target in the search for an anti-calcification therapy.
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Zhang, Yong, and William G. Paterson. "Characterization of the peristaltic reflex in murine distal colon." Canadian Journal of Physiology and Pharmacology 94, no. 2 (2016): 190–98. http://dx.doi.org/10.1139/cjpp-2015-0086.
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Ascending and descending neuromuscular reflexes play an important role in gastrointestinal motility. However, the underlying mechanisms in colon are incompletely understood. Nerve stimulation (NS)- and balloon distention (BD)-mediated reflexes in distal colonic circular smooth muscle (CSM) and longitudinal smooth muscle (LSM) of mice were investigated using conventional intracellular recordings. In the CSM, NS evoked ascending purinergic inhibitory junction potentials (IJPs), whereas BD induced atropine-sensitive ascending depolarization with superimposed action potentials (APs). The ascending depolarization reached a peak ∼4–7 s after the onset of distention and gradually returned to baseline after termination of the distention. In the LSM, NS produced an ascending biphasic IJP followed by a train of atropine-sensitive APs. Both stimuli produced similar descending IJPs in CSM and LSM, which were blocked by MRS-2500 and MRS-2179, putative purinergic receptor blockers. These data indicate that in the murine distal colon, descending purinergic inhibition in both CSM and LSM occurs. Ascending responses are more complex, with NS producing both inhibition and excitation to CSM and LSM, and BD evoking only cholinergic excitation.
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Guzman-Aranguez, Ana, Xavier Gasull, Yolanda Diebold, and Jesús Pintor. "Purinergic Receptors in Ocular Inflammation." Mediators of Inflammation 2014 (2014): 1–11. http://dx.doi.org/10.1155/2014/320906.
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Inflammation is a complex process that implies the interaction between cells and molecular mediators, which, when not properly “tuned,” can lead to disease. When inflammation affects the eye, it can produce severe disorders affecting the superficial and internal parts of the visual organ. The nucleoside adenosine and nucleotides including adenine mononucleotides like ADP and ATP and dinucleotides such as P1,P4-diadenosine tetraphosphate (Ap4A), and P1,P5-diadenosine pentaphosphate (Ap5A) are present in different ocular locations and therefore they may contribute/modulate inflammatory processes. Adenosine receptors, in particularA2Aadenosine receptors, present anti-inflammatory action in acute and chronic retinal inflammation. Regarding the A3receptor, selective agonists like N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine (CF101) have been used for the treatment of inflammatory ophthalmic diseases such as dry eye and uveoretinitis. Sideways, diverse stimuli (sensory stimulation, large intraocular pressure increases) can produce a release of ATP from ocular sensory innervation or after injury to ocular tissues. Then, ATP will activate purinergic P2 receptors present in sensory nerve endings, the iris, the ciliary body, or other tissues surrounding the anterior chamber of the eye to produce uveitis/endophthalmitis. In summary, adenosine and nucleotides can activate receptors in ocular structures susceptible to suffer from inflammatory processes. This involvement suggests the possible use of purinergic agonists and antagonists as therapeutic targets for ocular inflammation.
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Chen, Zhiyong, Chi Zhang, Xiaodan Song, et al. "BzATP Activates Satellite Glial Cells and Increases the Excitability of Dorsal Root Ganglia Neurons In Vivo." Cells 11, no. 15 (2022): 2280. http://dx.doi.org/10.3390/cells11152280.
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The purinergic system plays an important role in pain transmission. Recent studies have suggested that activation of P2-purinergic receptors (P2Rs) may be involved in neuron-satellite glial cell (SGC) interactions in the dorsal root ganglia (DRG), but the details remain unclear. In DRG, P2X7R is selectively expressed in SGCs, which closely surround neurons, and is highly sensitive to 3’-O-(4-Benzoyl) benzoyl-ATP (BzATP). Using calcium imaging in intact mice to survey a large number of DRG neurons and SGCs, we examined how intra-ganglionic purinergic signaling initiated by BzATP affects neuronal activities in vivo. We developed GFAP-GCaMP6s and Pirt-GCaMP6s mice to express the genetically encoded calcium indicator GGCaM6s in SGCs and DRG neurons, respectively. The application of BzATP to the ganglion induced concentration-dependent activation of SGCs in GFAP-GCaMP6s mice. In Pirt-GCaMP6s mice, BzATP initially activated more large-size neurons than small-size ones. Both glial and neuronal responses to BzATP were blocked by A438079, a P2X7R-selective antagonist. Moreover, blockers to pannexin1 channels (probenecid) and P2X3R (A317491) also reduced the actions of BzATP, suggesting that P2X7R stimulation may induce the opening of pannexin1 channels, leading to paracrine ATP release, which could further excite neurons by acting on P2X3Rs. Importantly, BzATP increased the responses of small-size DRG neurons and wide-dynamic range spinal neurons to subsequent peripheral stimuli. Our findings suggest that intra-ganglionic purinergic signaling initiated by P2X7R activation could trigger SGC-neuron interaction in vivo and increase DRG neuron excitability.
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Muñoz, Manuel F., Theanne N. Griffith, and Jorge E. Contreras. "Mechanisms of ATP release in pain: role of pannexin and connexin channels." Purinergic Signalling 17, no. 4 (2021): 549–61. http://dx.doi.org/10.1007/s11302-021-09822-6.
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AbstractPain is a physiological response to bodily damage and serves as a warning of potential threat. Pain can also transform from an acute response to noxious stimuli to a chronic condition with notable emotional and psychological components that requires treatment. Indeed, the management of chronic pain is currently an important unmet societal need. Several reports have implicated the release of the neurotransmitter adenosine triphosphate (ATP) and subsequent activation of purinergic receptors in distinct pain etiologies. Purinergic receptors are broadly expressed in peripheral neurons and the spinal cord; thus, purinergic signaling in sensory neurons or in spinal circuits may be critical for pain processing. Nevertheless, an outstanding question remains: what are the mechanisms of ATP release that initiate nociceptive signaling? Connexin and pannexin channels are established conduits of ATP release and have been suggested to play important roles in a variety of pathologies, including several models of pain. As such, these large-pore channels represent a new and exciting putative pharmacological target for pain treatment. Herein, we will review the current evidence for a role of connexin and pannexin channels in ATP release during nociceptive signaling, such as neuropathic and inflammatory pain. Collectively, these studies provide compelling evidence for an important role of connexins and pannexins in pain processing.
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Scarpellino, Giorgia, Tullio Genova, Daniele Avanzato, et al. "Purinergic Calcium Signals in Tumor-Derived Endothelium." Cancers 11, no. 6 (2019): 766. http://dx.doi.org/10.3390/cancers11060766.
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Tumor microenvironment is particularly enriched with extracellular ATP (eATP), but conflicting evidence has been provided on its functional effects on tumor growth and vascular remodeling. We have previously shown that high eATP concentrations exert a strong anti-migratory, antiangiogenic and normalizing activity on human tumor-derived endothelial cells (TECs). Since both metabotropic and ionotropic purinergic receptors trigger cytosolic calcium increase ([Ca2+]c), the present work investigated the properties of [Ca2+]c events elicited by high eATP in TECs and their role in anti-migratory activity. In particular, the quantitative and kinetic properties of purinergic-induced Ca2+ release from intracellular stores and Ca2+ entry from extracellular medium were investigated. The main conclusions are: (1) stimulation of TECs with high eATP triggers [Ca2+]c signals which include Ca2+ mobilization from intracellular stores (mainly ER) and Ca2+ entry through the plasma membrane; (2) the long-lasting Ca2+ influx phase requires both store-operated Ca2+ entry (SOCE) and non-SOCE components; (3) SOCE is not significantly involved in the antimigratory effect of high ATP stimulation; (4) ER is the main source for intracellular Ca2+ release by eATP: it is required for the constitutive migratory potential of TECs but is not the only determinant for the inhibitory effect of high eATP; (5) a complex interplay occurs among ER, mitochondria and lysosomes upon purinergic stimulation; (6) high eUTP is unable to inhibit TEC migration and evokes [Ca2+]c signals very similar to those described for eATP. The potential role played by store-independent Ca2+ entry and Ca2+-independent events in the regulation of TEC migration by high purinergic stimula deserves future investigation.
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Scarpellino, Giorgia, Tullio Genova, Elisa Quarta, et al. "P2X Purinergic Receptors Are Multisensory Detectors for Micro-Environmental Stimuli That Control Migration of Tumoral Endothelium." Cancers 14, no. 11 (2022): 2743. http://dx.doi.org/10.3390/cancers14112743.
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The tumoral microenvironment often displays peculiar features, including accumulation of extracellular ATP, hypoxia, low pH-acidosis, as well as an imbalance in zinc (Zn2+) and calcium (Ca2+). We previously reported the ability of some purinergic agonists to exert an anti-migratory activity on tumor-derived human endothelial cells (TEC) only when applied at a high concentration. They also trigger calcium signals associated with release from intracellular stores and calcium entry from the external medium. Here, we provide evidence that high concentrations of BzATP (100 µM), a potent agonist of P2X receptors, decrease migration in TEC from different tumors, but not in normal microvascular ECs (HMEC). The same agonist evokes a calcium increase in TEC from the breast and kidney, as well as in HMEC, but not in TEC from the prostate, suggesting that the intracellular pathways responsible for the P2X-induced impairment of TEC migration could vary among different tumors. The calcium signal is mainly due to a long-lasting calcium entry from outside and is strictly dependent on the presence of the receptor occupancy. Low pH, as well as high extracellular Zn2+ and Ca2+, interfere with the response, a distinctive feature typically found in some P2X purinergic receptors. This study reveals that a BzATP-sensitive pathway impairs the migration of endothelial cells from different tumors through mechanisms finely tuned by environmental factors.
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Dahl, Gerhard. "ATP release through pannexon channels." Philosophical Transactions of the Royal Society B: Biological Sciences 370, no. 1672 (2015): 20140191. http://dx.doi.org/10.1098/rstb.2014.0191.
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Extracellular adenosine triphosphate (ATP) serves as a signal for diverse physiological functions, including spread of calcium waves between astrocytes, control of vascular oxygen supply and control of ciliary beat in the airways. ATP can be released from cells by various mechanisms. This review focuses on channel-mediated ATP release and its main enabler, Pannexin1 (Panx1). Six subunits of Panx1 form a plasma membrane channel termed ‘pannexon’. Depending on the mode of stimulation, the pannexon has large conductance (500 pS) and unselective permeability to molecules less than 1.5 kD or is a small (50 pS), chloride-selective channel. Most physiological and pathological stimuli induce the large channel conformation, whereas the small conformation so far has only been observed with exclusive voltage activation of the channel. The interaction between pannexons and ATP is intimate. The pannexon is not only the conduit for ATP, permitting ATP efflux from cells down its concentration gradient, but the pannexon is also modulated by ATP. The channel can be activated by ATP through both ionotropic P2X as well as metabotropic P2Y purinergic receptors. In the absence of a control mechanism, this positive feedback loop would lead to cell death owing to the linkage of purinergic receptors with apoptotic processes. A control mechanism preventing excessive activation of the purinergic receptors is provided by ATP binding (with low affinity) to the Panx1 protein and gating the channel shut.
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Boncoeur, Émilie, Guillaume F. Bouvet, Francis Migneault та ін. "Induction of nitric oxide synthase expression by lipopolysaccharide is mediated by calcium-dependent PKCα-β1 in alveolar epithelial cells". American Journal of Physiology-Lung Cellular and Molecular Physiology 305, № 2 (2013): L175—L184. http://dx.doi.org/10.1152/ajplung.00295.2012.
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Nitric oxide (NO) plays an important role in innate host defense and inflammation. In response to infection, NO is generated by inducible nitric oxide synthase (iNOS), a gene product whose expression is highly modulated by different stimuli, including lipopolysaccharide (LPS) from gram-negative bacteria. We reported recently that LPS from Pseudomonas aeruginosa altered Na+ transport in alveolar epithelial cells via a suramin-dependent process, indicating that LPS activated a purinergic response in these cells. To further study this question, in the present work, we tested whether iNOS mRNA and protein expression were modulated in response to LPS in alveolar epithelial cells. We found that LPS induced a 12-fold increase in iNOS mRNA expression via a transcription-dependent process in these cells. iNOS protein, NO, and nitrotyrosine were also significantly elevated in LPS-treated cells. Ca2+ chelation and protein kinase C (PKCα-β1) inhibition suppressed iNOS mRNA induction by LPS, implicating Ca2+-dependent PKC signaling in this process. LPS evoked a significant increase of extracellular ATP. Because PKC activation is one of the signaling pathways known to mediate purinergic signaling, we evaluated the hypothesis that iNOS induction was ATP dependent. Although high suramin concentration inhibited iNOS mRNA induction, the process was not ATP dependent, since specific purinergic receptor antagonists could not inhibit the process. Altogether, these findings demonstrate that iNOS expression is highly modulated in alveolar epithelial cells by LPS via a Ca2+/PKCα-β1 pathway independent of ATP signaling.
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Buchanan, Kelly L., Laura E. Rupprecht, M. Maya Kaelberer, et al. "The preference for sugar over sweetener depends on a gut sensor cell." Nature Neuroscience 25, no. 2 (2022): 191–200. http://dx.doi.org/10.1038/s41593-021-00982-7.
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AbstractGuided by gut sensory cues, humans and animals prefer nutritive sugars over non-caloric sweeteners, but how the gut steers such preferences remains unknown. In the intestine, neuropod cells synapse with vagal neurons to convey sugar stimuli to the brain within seconds. Here, we found that cholecystokinin (CCK)-labeled duodenal neuropod cells differentiate and transduce luminal stimuli from sweeteners and sugars to the vagus nerve using sweet taste receptors and sodium glucose transporters. The two stimulus types elicited distinct neural pathways: while sweetener stimulated purinergic neurotransmission, sugar stimulated glutamatergic neurotransmission. To probe the contribution of these cells to behavior, we developed optogenetics for the gut lumen by engineering a flexible fiberoptic. We showed that preference for sugar over sweetener in mice depends on neuropod cell glutamatergic signaling. By swiftly discerning the precise identity of nutrient stimuli, gut neuropod cells serve as the entry point to guide nutritive choices.
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Brayden, Joseph E., Yao Li, and Matthew J. Tavares. "Purinergic Receptors Regulate Myogenic Tone in Cerebral Parenchymal Arterioles." Journal of Cerebral Blood Flow & Metabolism 33, no. 2 (2012): 293–99. http://dx.doi.org/10.1038/jcbfm.2012.169.
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Myogenic tone is a fundamental aspect of vascular behavior in resistance arteries. This contractile response to changes in intravascular pressure is critically involved in blood flow autoregulation in tissues such as the brain, kidneys, and heart. Myogenic tone also helps regulate precapillary pressure and provides a level of background tone upon which vasodilator stimuli act to increase tissue perfusion when appropriate. Despite the importance of these processes in the brain, little is known about the mechanisms involved in control of myogenic tone in the cerebral microcirculation. Here, we report that pharmacological inhibition of P2Y4 and P2Y6 pyrimidine receptors nearly abolished myogenic tone in cerebral parenchymal arterioles (PAs). Molecular suppression of either P2Y4 or P2Y6 receptors using antisense oligodeoxynucleotides reduced myogenic tone by 44% ± 8% and 45% ± 7%, respectively. These results indicate that both receptor isoforms are activated by increased intravascular pressure, which enhances the activity of voltage-dependent calcium channels and increases myogenic tone in PAs. Enhancement or inhibition of ectonucleotidase activity had no effect on parenchymal arteriolar myogenic tone, indicating that this response is not mediated by local release of nucleotides, but rather may involve direct mechanical activation of P2Y receptors in the smooth muscle cells.
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Yang, Lijing, Mengjia Hu, Yukai Lu, Songling Han, and Junping Wang. "Inflammasomes and the Maintenance of Hematopoietic Homeostasis: New Perspectives and Opportunities." Molecules 26, no. 2 (2021): 309. http://dx.doi.org/10.3390/molecules26020309.
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Hematopoietic stem cells (HSCs) regularly produce various blood cells throughout life via their self-renewal, proliferation, and differentiation abilities. Most HSCs remain quiescent in the bone marrow (BM) and respond in a timely manner to either physiological or pathological cues, but the underlying mechanisms remain to be further elucidated. In the past few years, accumulating evidence has highlighted an intermediate role of inflammasome activation in hematopoietic maintenance, post-hematopoietic transplantation complications, and senescence. As a cytosolic protein complex, the inflammasome participates in immune responses by generating a caspase cascade and inducing cytokine secretion. This process is generally triggered by signals from purinergic receptors that integrate extracellular stimuli such as the metabolic factor ATP via P2 receptors. Furthermore, targeted modulation/inhibition of specific inflammasomes may help to maintain/restore adequate hematopoietic homeostasis. In this review, we will first summarize the possible relationships between inflammasome activation and homeostasis based on certain interesting phenomena. The cellular and molecular mechanism by which purinergic receptors integrate extracellular cues to activate inflammasomes inside HSCs will then be described. We will also discuss the therapeutic potential of targeting inflammasomes and their components in some diseases through pharmacological or genetic strategies.
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Yang, Lijing, Mengjia Hu, Yukai Lu, Songling Han, and Junping Wang. "Inflammasomes and the Maintenance of Hematopoietic Homeostasis: New Perspectives and Opportunities." Molecules 26, no. 2 (2021): 309. http://dx.doi.org/10.3390/molecules26020309.
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Hematopoietic stem cells (HSCs) regularly produce various blood cells throughout life via their self-renewal, proliferation, and differentiation abilities. Most HSCs remain quiescent in the bone marrow (BM) and respond in a timely manner to either physiological or pathological cues, but the underlying mechanisms remain to be further elucidated. In the past few years, accumulating evidence has highlighted an intermediate role of inflammasome activation in hematopoietic maintenance, post-hematopoietic transplantation complications, and senescence. As a cytosolic protein complex, the inflammasome participates in immune responses by generating a caspase cascade and inducing cytokine secretion. This process is generally triggered by signals from purinergic receptors that integrate extracellular stimuli such as the metabolic factor ATP via P2 receptors. Furthermore, targeted modulation/inhibition of specific inflammasomes may help to maintain/restore adequate hematopoietic homeostasis. In this review, we will first summarize the possible relationships between inflammasome activation and homeostasis based on certain interesting phenomena. The cellular and molecular mechanism by which purinergic receptors integrate extracellular cues to activate inflammasomes inside HSCs will then be described. We will also discuss the therapeutic potential of targeting inflammasomes and their components in some diseases through pharmacological or genetic strategies.
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He, Jin-Rong, Shu-Guang Yu, Yong Tang, and Peter Illes. "Purinergic signaling as a basis of acupuncture-induced analgesia." Purinergic Signalling 16, no. 3 (2020): 297–304. http://dx.doi.org/10.1007/s11302-020-09708-z.
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Abstract This review summarizes experimental evidence indicating that purinergic mechanisms are causally involved in acupuncture (AP)-induced analgesia. Electroacupuncture (EAP) and manual AP release at pain-relevant acupoints ATP which may activate purinergic P2X receptors (Rs) especially of the P2X3 type situated at local sensory nerve endings (peripheral terminals of dorsal root ganglion [DRG] neurons); the central processes of these neurons are thought to inhibit via collaterals of ascending dorsal horn spinal cord neurons, pain-relevant pathways projecting to higher centers of the brain. In addition, during AP/EAP non-neuronal P2X4 and/or P2X7Rs localized at microglial cells of the CNS become activated at the spinal or supraspinal levels. In consequence, these microglia secrete bioactive compounds such as growth factors, cytokines, chemokines, reactive oxygen, and nitrogen species, which modulate the ascending neuronal pathways conducting painful stimuli. Alternatively, ATP released at acupoints by AP/EAP may be enzymatically degraded to adenosine, stimulating in loco presynaptic A1Rs exerting an inhibitory influence on the primary afferent fibers (the above mentioned pain-sensing peripheral terminals of DRG neurons) which thereby fail to conduct action potentials to the spinal cord dorsal horn. The net effect of the stimulation of P2X3, P2X4, P2X7, and A1Rs by the AP/EAP-induced release of ATP/adenosine at certain acupoints will be analgesia.
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Sangsiri, Sutheera, Hui Xu, Roxanne Fernandes, et al. "Spinal cord injury alters purinergic neurotransmission to mesenteric arteries in rats." American Journal of Physiology-Heart and Circulatory Physiology 318, no. 2 (2020): H223—H237. http://dx.doi.org/10.1152/ajpheart.00525.2019.
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Complications associated with spinal cord injury (SCI) result from unregulated reflexes below the lesion level. Understanding neurotransmission distal to the SCI could improve quality of life by mitigating complications. The long-term impact of SCI on neurovascular transmission is poorly understood, but reduced sympathetic activity below the site of SCI enhances arterial neurotransmission (1). We studied sympathetic neurovascular transmission using a rat model of long-term paraplegia (T2–3) and tetraplegia (C6–7). Sixteen weeks after SCI, T2–3 and C6–7 rats had lower blood pressure (BP) than sham rats (103 ± 2 and 97 ± 4 vs. 117 ± 6 mmHg, P < 0.05). T2–3 rats had tachycardia (410 ± 6 beats/min), and C6–7 rats had bradycardia (299 ± 10 beats/min) compared with intact rats (321 ± 4 beats/min, P < 0.05). Purinergic excitatory junction potentials (EJPs) were measured in mesenteric arteries (MA) using microlectrodes, and norepinephrine (NE) release was measured using amperometry. NE release was similar in all groups, while EJP frequency-response curves from T2–3 and C6–7 rats were left-shifted vs. sham rats. EJPs in T2–3 and C6–7 rats showed facilitation followed by run-down during stimulation trains (10 Hz, 50 stimuli). MA reactivity to exogenous NE and ATP was similar in all rats. In T2–3 and C6–7 rats, NE content was increased in left cardiac ventricles compared with intact rats, but was not changed in MA, kidney, or spleen. Our data indicate that peripheral purinergic, but not adrenergic, neurotransmission increases following SCI via enhanced ATP release from periarterial nerves. Sympathetic BP support is reduced after SCI, but improving neurotransmitter release might maintain cardiovascular stability in individuals living with SCI. NEW & NOTEWORTHY This study revealed increased purinergic, but not noradrenergic, neurotransmission to mesenteric arteries in rats with spinal cord injury (SCI). An increased releasable pool of ATP in periarterial sympathetic nerves may contribute to autonomic dysreflexia following SCI, suggesting that purinergic neurotransmission may be a therapeutic target for maintaining stable blood pressure in individuals living with SCI. The selective increase in ATP release suggests that ATP and norepinephrine may be stored in separate synaptic vesicles in periarterial sympathetic varicosities.
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Iwanaga, Koichi, Takahisa Murata, Masatoshi Hori, and Hiroshi Ozaki. "Purinergic P2Y1 receptor signaling mediates wound stimuli-induced cyclooxygenase-2 expression in intestinal subepithelial myofibroblasts." European Journal of Pharmacology 702, no. 1-3 (2013): 158–64. http://dx.doi.org/10.1016/j.ejphar.2013.01.025.
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LIGHT, ALAN R., YING WU, RONALD W. HUGHEN, and PETER B. GUTHRIE. "Purinergic receptors activating rapid intracellular Ca2+ increases in microglia." Neuron Glia Biology 2, no. 2 (2005): 125–38. http://dx.doi.org/10.1017/s1740925x05000323.
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We provide both molecular and pharmacological evidence that the metabotropic, purinergic, P2Y6, P2Y12 and P2Y13 receptors and the ionotropic P2X4 receptor contribute strongly to the rapid calcium response caused by ATP and its analogues in mouse microglia. Real-time PCR demonstrates that the most prevalent P2 receptor in microglia is P2Y6 followed, in order, by P2X4, P2Y12, and P2X7 = P2Y13. Only very small quantities of mRNA for P2Y1, P2Y2, P2Y4, P2Y14, P2X3 and P2X5 were found. Dose-response curves of the rapid calcium response gave a potency order of: 2MeSADP>ADP=UDP=IDP=UTP>ATP>BzATP, whereas A2P4 had little effect. Pertussis toxin partially blocked responses to 2MeSADP, ADP and UDP. The P2X4 antagonist suramin, but not PPADS, significantly blocked responses to ATP. These data indicate that P2Y6, P2Y12, P2Y13 and P2X receptors mediate much of the rapid calcium responses and shape changes in microglia to low concentrations of ATP, presumably at least partly because ATP is rapidly hydrolyzed to ADP. Expression of P2Y6, P2Y12 and P2Y13 receptors appears to be largely glial in the brain, so that peripheral immune cells and CNS microglia share these receptors. Thus, purinergic, metabotropic, P2Y6, P2Y12, P2Y13 and P2X4 receptors might share a role in the activation and recruitment of microglia in the brain and spinal cord by widely varying stimuli that cause the release of ATP, including infection, injury and degeneration in the CNS, and peripheral tissue injury and inflammation which is signaled via nerve signaling to the spinal cord.
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Bradley, Eamonn, Andrea Law, David Bell, and Christopher D. Johnson. "Effects of varying impulse number on cotransmitter contributions to sympathetic vasoconstriction in rat tail artery." American Journal of Physiology-Heart and Circulatory Physiology 284, no. 6 (2003): H2007—H2014. http://dx.doi.org/10.1152/ajpheart.01061.2002.
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We examined the contributions of the cotransmitters norepinephrine (NE), ATP, and neuropeptide Y (NPY) to sympathetically evoked vasoconstriction in the rat tail artery in isolated vascular rings by using 1–100 stimulation impulses at 20 Hz. Phentolamine (2 μM), the α-adrenoceptor antagonist, markedly reduced responses to all stimuli, although responses to lower impulse numbers were reduced less than responses to longer trains. The purinergic receptor antagonist suramin (100 μM) reduced all responses, but to a much greater extent with few impulse trains. Responses were further reduced or abolished by addition of the second antagonist. Any remaining responses were abolished by the NPY-Y1 receptor antagonist BIBP-3226 (75 nM). NPY had a direct agonist action and potentiated sympathetically mediated responses. NPY (75 nM) potentiated responses and BIBP-3226 decreased responses to 2- and 20-impulse trains. Both affected responses from 2 impulses to >20 impulses, but there was no preferential effect on purinergic contributions to responses because neurally released NPY potentiated both “pure” NE and ATP responses equally. We conclude that all three cotransmitters contribute significantly to vascular responses and their contribution varies markedly with impulse numbers. There is considerable synergy between cotransmitters, especially with lower impulse numbers where NPY contributions are greater than expected.
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Hanna, Ramy L., and Marc P. Kaufman. "Role played by purinergic receptors on muscle afferents in evoking the exercise pressor reflex." Journal of Applied Physiology 94, no. 4 (2003): 1437–45. http://dx.doi.org/10.1152/japplphysiol.01011.2002.
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The exercise pressor reflex is believed to be evoked, in part, by multiple metabolic stimuli that are generated when blood supply to exercising muscles is inadequate to meet metabolic demand. Recently, ATP, which is a P2 receptor agonist, has been suggested to be one of the metabolic stimuli evoking this reflex. We therefore tested the hypothesis that blockade of P2 receptors within contracting skeletal muscle attenuated the exercise pressor reflex in decerebrate cats. We found that popliteal arterial injection of pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS; 10 mg/kg), a P2 receptor antagonist, attenuated the pressor response to static contraction of the triceps surae muscles. Specifically, the pressor response to contraction before PPADS averaged 36 ± 3 mmHg, whereas afterward it averaged 14 ± 3 mmHg ( P < 0.001; n = 19). In addition, PPADS attenuated the pressor response to postcontraction circulatory occlusion ( P < 0.01; n = 11). In contrast, popliteal arterial injection of CGS-15943 (250 μg/kg), a P1 receptor antagonist, had no effect on the pressor response to static contraction of the triceps surae muscles. In addition, popliteal arterial injection of PPADS but not CGS-15943 attenuated the pressor response to stretch of the calcaneal (Achilles) tendon. We conclude that P2 receptors on the endings of thin fiber muscle afferents play a role in evoking both the metabolic and mechanoreceptor components of the exercise pressor reflex.
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Henriques, Tiago, Emilio Agostinelli, Andres Hernandez-Clavijo, et al. "TMEM16A calcium-activated chloride currents in supporting cells of the mouse olfactory epithelium." Journal of General Physiology 151, no. 7 (2019): 954–66. http://dx.doi.org/10.1085/jgp.201812310.
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Glial-like supporting (or sustentacular) cells are important constituents of the olfactory epithelium that are involved in several physiological processes such as production of endocannabinoids, insulin, and ATP and regulation of the ionic composition of the mucus layer that covers the apical surface of the olfactory epithelium. Supporting cells express metabotropic P2Y purinergic receptors that generate ATP-induced Ca2+ signaling through the activation of a PLC-mediated cascade. Recently, we reported that a subpopulation of supporting cells expresses also the Ca2+-activated Cl− channel TMEM16A. Here, we sought to extend our understanding of a possible physiological role of this channel in the olfactory system by asking whether Ca2+ can activate Cl− currents mediated by TMEM16A. We use whole-cell patch-clamp analysis in slices of the olfactory epithelium to measure dose–response relations in the presence of various intracellular Ca2+ concentrations, ion selectivity, and blockage. We find that knockout of TMEM16A abolishes Ca2+-activated Cl− currents, demonstrating that TMEM16A is essential for these currents in supporting cells. Also, by using extracellular ATP as physiological stimuli, we found that the stimulation of purinergic receptors activates a large TMEM16A-dependent Cl− current, indicating a possible role of TMEM16A in ATP-mediated signaling. Altogether, our results establish that TMEM16A-mediated currents are functional in olfactory supporting cells and provide a foundation for future work investigating the precise physiological role of TMEM16A in the olfactory system.
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Giannantoni, Antonella, Silvia Proietti, Silvia Giovannozzi, and Massimo Porena. "Vesical Urothelium and New Concepts." Urologia Journal 79, no. 1 (2012): 14–18. http://dx.doi.org/10.5301/ru.2012.9021.
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Vesical urothelium was long considered to simply be a protection barrier, which passively separates the urinary content from the underlying smooth muscle and the blood stream. Recent observations, though, have pointed out that vesical urothelium cells have clear active and sensory functions, in response to various physical and chemical stimuli. Among these characteristics are the expression of several neurotransmitters and receptors: Acetylcholine, Nitric Oxide, VIP, CGRP, NKA, SP and cholinergic, vanilloid, purinergic, and tachykinin receptors. Urothelium-produced neurotransmitters are likely supposed to act through a receptor stimulation of the afferent nerve fibers within the sub-urothelial spaces. Sub-urothelial myofibroblasts are considered to play a mediation role between urothelium-produced neurotransmitters and the underlying receptors. According to these observations, a pharmacologic modulation, directly affecting the urothelium, can be hypothesized.
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Moschetta, Donato, Enrico Di Maria, Vincenza Valerio, et al. "Purinergic Receptor P2Y2 Stimulation Averts Aortic Valve Interstitial Cell Calcification and Myofibroblastic Activation." Biomedicines 10, no. 2 (2022): 457. http://dx.doi.org/10.3390/biomedicines10020457.
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Rationale—Calcific aortic valve stenosis (CAVS) is a pathological condition of the aortic valve with a prevalence of 3% in the general population. It is characterized by massive rearrangement of the extracellular matrix, mostly due to the accumulation of fibro-calcific deposits driven by valve interstitial cells (VIC), and no pharmacological treatment is currently available. The aim of this study was to evaluate the effects of P2Y2 receptor (P2RY2) activation on fibro-calcific remodeling of CAVS. Methods—We employed human primary VICs isolated from CAVS leaflets treated with 2-thiouridine-5′-triphosphate (2ThioUTP, 10 µM), an agonist of P2RY2. The calcification was induced by inorganic phosphate (2 mM) and ascorbic acid (50 µg/mL) for 7 or 14 days, while the 2ThioUTP was administered starting from the seventh day. 2ThioUTP was chronically administered for 5 days to evaluate myofibroblastic activation. Results—P2RY2 activation, under continuous or interrupted pro-calcific stimuli, led to a significant inhibition of VIC calcification potential (p < 0.01). Moreover, 2ThioUTP treatment was able to significantly reduce pro-fibrotic gene expression (p < 0.05), as well as that of protein α-smooth muscle actin (p = 0.004). Conclusions—Our data suggest that P2RY2 activation should be further investigated as a pharmacological target for the prevention of CAVS progression, acting on both calcification and myofibroblastic activation.
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Oyasu, Miho, Mineko Fujimiya, Kaori Kashiwagi, Shiho Ohmori, Hirotsugu Imaeda та Naoaki Saito. "Immunogold Electron Microscopic Demonstration of Distinct Submembranous Localization of the Activated γPKC Depending on the Stimulation". Journal of Histochemistry & Cytochemistry 56, № 3 (2007): 253–65. http://dx.doi.org/10.1369/jhc.7a7291.2007.
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We examined the precise intracellular translocation of γ subtype of protein kinase C (γPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12- O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged γPKC (γPKC–GFP) on the plasma membrane. In contrast, treatment with Ca2+ ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of γPKC–GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that γPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of γPKC–GFP on the plasma membrane, Ca2+ ionophore and NMDA rapidly translocated γPKC–GFP to the plasma membrane and then restricted γPKC–GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca2+ influx alone induced the association of γPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of γPKC in response to various stimuli suggests a novel mechanism for PKC activation.
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Khan, Amna, Salman Khan, and Yeong Shik Kim. "Insight into Pain Modulation: Nociceptors Sensitization and Therapeutic Targets." Current Drug Targets 20, no. 7 (2019): 775–88. http://dx.doi.org/10.2174/1389450120666190131114244.
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Pain is a complex multidimensional concept that facilitates the initiation of the signaling cascade in response to any noxious stimuli. Action potential generation in the peripheral nociceptor terminal and its transmission through various types of nociceptors corresponding to mechanical, chemical or thermal stimuli lead to the activation of receptors and further neuronal processing produces the sensation of pain. Numerous types of receptors are activated in pain sensation which vary in their signaling pathway. These signaling pathways can be regarded as a site for modulation of pain by targeting the pain transduction molecules to produce analgesia. On the basis of their anatomic location, transient receptor potential ion channels (TRPV1, TRPV2 and TRPM8), Piezo 2, acid-sensing ion channels (ASICs), purinergic (P2X and P2Y), bradykinin (B1 and B2), &#945;-amino-3-hydroxy-5- methylisoxazole-4-propionate (AMPA), N-methyl-D-aspartate (NMDA), metabotropic glutamate (mGlu), neurokinin 1 (NK1) and calcitonin gene-related peptide (CGRP) receptors are activated during pain sensitization. Various inhibitors of TRPV1, TRPV2, TRPM8, Piezo 2, ASICs, P2X, P2Y, B1, B2, AMPA, NMDA, mGlu, NK1 and CGRP receptors have shown high therapeutic value in experimental models of pain. Similarly, local inhibitory regulation by the activation of opioid, adrenergic, serotonergic and cannabinoid receptors has shown analgesic properties by modulating the central and peripheral perception of painful stimuli. This review mainly focused on various classes of nociceptors involved in pain transduction, transmission and modulation, site of action of the nociceptors in modulating pain transmission pathways and the drugs (both clinical and preclinical data, relevant to targets) alleviating the painful stimuli by exploiting nociceptor-specific channels and receptors.
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Murakami, Makoto, Yasuyuki Furukawa, and Shigetoshi Chiba. "Effects of Glibenclamide on Negative Cardiac Responses to Cholinergic and Purinergic Stimuli and Cromakalim in the Isolated Dog Heart." Japanese Journal of Pharmacology 65, no. 3 (1994): 215–22. http://dx.doi.org/10.1016/s0021-5198(19)35752-x.
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Murakami, Makoto, Yasuyuki Furukawa, and Shigetoshi Chiba. "Effects of Glibenclamide on Negative Cardiac Responses to Cholinerg and Purinergic Stimuli and Cromakalim in the Isolated Dog Heart." Japanese Journal of Pharmacology 65, no. 3 (1994): 215–22. http://dx.doi.org/10.1254/jjp.65.215.
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Panicucci, Chiara, Lizzia Raffaghello, Santina Bruzzone, et al. "eATP/P2X7R Axis: An Orchestrated Pathway Triggering Inflammasome Activation in Muscle Diseases." International Journal of Molecular Sciences 21, no. 17 (2020): 5963. http://dx.doi.org/10.3390/ijms21175963.
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In muscle ATP is primarily known for its function as an energy source and as a mediator of the “excitation-transcription” process, which guarantees muscle plasticity in response to environmental stimuli. When quickly released in massive concentrations in the extracellular space as in presence of muscle membrane damage, ATP acts as a damage-associated molecular pattern molecule (DAMP). In experimental murine models of muscular dystrophies characterized by membrane instability, blockade of eATP/P2X7 receptor (R) purinergic signaling delayed the progression of the dystrophic phenotype dampening the local inflammatory response and inducing Foxp3+ T Regulatory lymphocytes. These discoveries highlighted the relevance of ATP as a harbinger of immune-tissue damage in muscular genetic diseases. Given the interactions between the immune system and muscle regeneration, the comprehension of ATP/purinerigic pathway articulated organization in muscle cells has become of extreme interest. This review explores ATP release, metabolism, feedback control and cross-talk with members of muscle inflammasome in the context of muscular dystrophies.
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Sikora, Andrew, Judy Liu, Celia Brosnan, Gary Buell, Iain Chessel, and Barry R. Bloom. "Cutting Edge: Purinergic Signaling Regulates Radical-Mediated Bacterial Killing Mechanisms in Macrophages Through a P2X7-Independent Mechanism." Journal of Immunology 163, no. 2 (1999): 558–61. http://dx.doi.org/10.4049/jimmunol.163.2.558.
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Abstract Signaling by extracellular nucleotides through P2 purinergic receptors affects diverse macrophage functions; however, its role in regulating antimicrobial radicals during bacterial infection has not been investigated. Mycobacterium tuberculosis-infected macrophages released ATP in a dose-dependent manner, which correlated with nitrite accumulation. P2 receptor inhibitors, including oxidized ATP, blocked NO synthase (NOSII) up-regulation and NO production induced by infection with M. tuberculosis or bacille Calmette-Guérin, or treatment with LPS or TNF-α. Oxidized ATP also inhibited oxygen radical production and activation of NF-κB and AP-1 in response to infection and inhibited NO-dependent killing of bacille Calmette-Guérin by macrophages. Experiments using macrophages derived from P2X7 gene-disrupted mice ruled out an essential role for P2X7 in NOSII regulation. These data demonstrate that P2 receptors regulate macrophage activation in response to bacteria and proinflammatory stimuli, and suggest that extracellular nucleotides released from infected macrophages may enhance production of oxygen radicals and NO at sites of infection.
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Rogers, Richard C., David H. McDougal, Sue Ritter, Emily Qualls-Creekmore, and Gerlinda E. Hermann. "Response of catecholaminergic neurons in the mouse hindbrain to glucoprivic stimuli is astrocyte dependent." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 315, no. 1 (2018): R153—R164. http://dx.doi.org/10.1152/ajpregu.00368.2017.
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Hindbrain catecholaminergic (CA) neurons are required for critical autonomic, endocrine, and behavioral counterregulatory responses (CRRs) to hypoglycemia. Recent studies suggest that CRR initiation depends on hindbrain astrocyte glucose sensors (McDougal DH, Hermann GE, Rogers RC. Front Neurosci 7: 249, 2013; Rogers RC, Ritter S, Hermann GE. Am J Physiol Regul Integr Comp Physiol 310: R1102–R1108, 2016). To test the proposition that hindbrain CA responses to glucoprivation are astrocyte dependent, we utilized transgenic mice in which the calcium reporter construct (GCaMP5) was expressed selectively in tyrosine hydroxylase neurons (TH-GCaMP5). We conducted live cell calcium-imaging studies on tissue slices containing the nucleus of the solitary tract (NST) or the ventrolateral medulla, critical CRR initiation sites. Results show that TH-GCaMP5 neurons are robustly activated by a glucoprivic challenge and that this response is dependent on functional astrocytes. Pretreatment of hindbrain slices with fluorocitrate (an astrocytic metabolic suppressor) abolished TH-GCaMP5 neuronal responses to glucoprivation, but not to glutamate. Pharmacologic results suggest that the astrocytic connection with hindbrain CA neurons is purinergic via P2 receptors. Parallel imaging studies on hindbrain slices of NST from wild-type C57BL/6J mice, in which astrocytes and neurons were prelabeled with a calcium reporter dye and an astrocytic vital dye, show that both cell types are activated by glucoprivation but astrocytes responded significantly sooner than neurons. Pretreatment of these hindbrain slices with P2 antagonists abolished neuronal responses to glucoprivation without interruption of astrocyte responses; pretreatment with fluorocitrate eliminated both astrocytic and neuronal responses. These results support earlier work suggesting that the primary detection of glucoprivic signals by the hindbrain is mediated by astrocytes.
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Johnson, Christopher D., Andrew M. Coney, and Janice M. Marshall. "Roles of norepinephrine and ATP in sympathetically evoked vasoconstriction in rat tail and hindlimb in vivo." American Journal of Physiology-Heart and Circulatory Physiology 281, no. 6 (2001): H2432—H2440. http://dx.doi.org/10.1152/ajpheart.2001.281.6.h2432.
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In anesthetized rats, we characterized the contributions of norepinephrine (NE) and ATP to changes in tail and hindlimb (femoral) vascular resistances (TVR and FVR, respectively) evoked by three patterns of sympathetic stimulation: 1) couplets (2 impulses at 20 Hz), 2) short trains (20 impulses at 20 Hz), and 3) a natural irregular pattern previously recorded from a sympathetic fiber innervating the rat tail artery. All stimuli evoked greater changes in TVR than FVR. Judging from the effects of the α-adrenoceptor antagonist phentolamine, the purinergic receptor antagonist suramin, or α,β-methylene ATP (which desensitizes P2X receptors), we propose that NE has a major role in the constriction evoked by the couplet, as well as by the short train and by the low- and high-frequency components of the natural pattern, but that considerable synergy occurred between the actions of ATP and NE. This contrasts with previous in vitro studies that indicated that ATP dominates vascular responses evoked by sympathetic stimulation with a few impulses at low frequency and that NE dominates responses to longer trains or at high frequencies.
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Leiva-Salcedo, Elias, Claudio Coddou, Felipe E. Rodríguez, et al. "Lipopolysaccharide Inhibits the Channel Activity of the P2X7 Receptor." Mediators of Inflammation 2011 (2011): 1–12. http://dx.doi.org/10.1155/2011/152625.
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The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance.
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Birder, Lori, and Karl-Erik Andersson. "Urothelial Signaling." Physiological Reviews 93, no. 2 (2013): 653–80. http://dx.doi.org/10.1152/physrev.00030.2012.
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The urothelium, which lines the inner surface of the renal pelvis, the ureters, and the urinary bladder, not only forms a high-resistance barrier to ion, solute and water flux, and pathogens, but also functions as an integral part of a sensory web which receives, amplifies, and transmits information about its external milieu. Urothelial cells have the ability to sense changes in their extracellular environment, and respond to chemical, mechanical and thermal stimuli by releasing various factors such as ATP, nitric oxide, and acetylcholine. They express a variety of receptors and ion channels, including P2X3 purinergic receptors, nicotinic and muscarinic receptors, and TRP channels, which all have been implicated in urothelial-neuronal interactions, and involved in signals that via components in the underlying lamina propria, such as interstitial cells, can be amplified and conveyed to nerves, detrusor muscle cells, and ultimately the central nervous system. The specialized anatomy of the urothelium and underlying structures, and the possible communication mechanisms from urothelial cells to various cell types within the bladder wall are described. Changes in the urothelium/lamina propria (“mucosa”) produced by different bladder disorders are discussed, as well as the mucosa as a target for therapeutic interventions.
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Lapel, Martin, Philip Weston, Derek Strassheim, et al. "Glycolysis and oxidative phosphorylation are essential for purinergic receptor-mediated angiogenic responses in vasa vasorum endothelial cells." American Journal of Physiology-Cell Physiology 312, no. 1 (2017): C56—C70. http://dx.doi.org/10.1152/ajpcell.00250.2016.
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Angiogenesis is an energy-demanding process; however, the role of cellular energy pathways and their regulation by extracellular stimuli, especially extracellular nucleotides, remain largely unexplored. Using metabolic inhibitors of glycolysis (2-deoxyglucose) and oxidative phosphorylation (OXPHOS) (oligomycin, rotenone, and FCCP), we demonstrate that glycolysis and OXPHOS are both essential for angiogenic responses of vasa vasorum endothelial cell (VVEC). Treatment with P2R agonists, ATP, and 2-methylthioadenosine diphosphate trisodium salt (MeSADP), but not P1 receptor agonist, adenosine, increased glycolytic activity in VVEC (measured by extracellular acidification rate and lactate production). Stimulation of glycolysis was accompanied by increased levels of phospho-phosphofructokinase B3, hexokinase (HK), and GLUT-1, but not lactate dehydrogenase. Moreover, extracellular ATP and MeSADP, and to a lesser extent adenosine, increased basal and maximal oxygen consumption rates in VVEC. These effects were potentiated when the cells were cultured in 20 mM galactose and 5 mM glucose compared with 25 mM glucose. Treatment with P2R agonists decreased phosphorylation of pyruvate dehydrogenase (PDH)-E1α and increased succinate dehydrogenase (SDH), cytochrome oxidase IV, and β-subunit of F1F0 ATP synthase expression. In addition, P2R stimulation transiently elevated mitochondrial Ca2+ concentration, implying involvement of mitochondria in VVEC angiogenic activation. We also demonstrated a critical role of phosphatidylinositol 3-kinase and Akt pathways in lactate production, PDH-E1α phosphorylation, and the expression of HK, SDH, and GLUT-1 in ATP-stimulated VVEC. Together, our findings suggest that purinergic and metabolic regulation of VVEC energy pathways is essential for VV angiogenesis and may contribute to pathologic vascular remodeling in pulmonary hypertension.
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Blasetti Fantauzzi, Claudia, Stefano Menini, Carla Iacobini, et al. "Deficiency of the Purinergic Receptor 2X7 Attenuates Nonalcoholic Steatohepatitis Induced by High-Fat Diet: Possible Role of the NLRP3 Inflammasome." Oxidative Medicine and Cellular Longevity 2017 (2017): 1–14. http://dx.doi.org/10.1155/2017/8962458.
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Molecular mechanisms driving transition from simple steatosis to nonalcoholic steatohepatitis (NASH), a critical step in the progression of nonalcoholic fatty liver disease (NAFLD) to cirrhosis, are poorly defined. This study aimed at investigating the role of the purinergic receptor 2X7 (PR2X7), through the NLRP3 inflammasome, in the development of NASH. To this end, mice knockout for the Pr2x7 gene (Pr2x7−/−) and coeval wild-type (WT) mice were fed a high-fat diet (HFD) or normal-fat diet for 16 weeks. NAFLD grade and stage were lower in Pr2x7−/− than WT mice, and only 1/7 Pr2x7−/− animals showed evidence of NASH, as compared with 4/7 WT mice. Molecular markers of inflammation, oxidative stress, and fibrosis were markedly increased in WT-HFD mice, whereas no or significantly reduced increments were detected in Pr2x7−/− animals, which showed also decreased modulation of genes of lipid metabolism. Deletion of Pr2x7 gene was associated with blunted or abolished activation of NLRP3 inflammasome and expression of its components, which were induced in liver sinusoidal endothelial cells challenged with appropriate stimuli. These data show that Pr2x7 gene deletion protects mice from HFD-induced NASH, possibly through blunted activation of NLRP3 inflammasome, suggesting that PR2X7 and NLRP3 may represent novel therapeutic targets.
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Homolya, László, Thomas H. Steinberg, and Richard C. Boucher. "Cell to Cell Communication in Response to Mechanical Stress via Bilateral Release of Atp and Utp in Polarized Epithelia." Journal of Cell Biology 150, no. 6 (2000): 1349–60. http://dx.doi.org/10.1083/jcb.150.6.1349.
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Airway epithelia are positioned at the interface between the body and the environment, and generate complex signaling responses to inhaled toxins and other stresses. Luminal mechanical stimulation of airway epithelial cells produces a propagating wave of elevated intracellular Ca2+ that coordinates components of the integrated epithelial stress response. In polarized airway epithelia, this response has been attributed to IP3 permeation through gap junctions. Using a combination of approaches, including enzymes that destroy extracellular nucleotides, purinergic receptor desensitization, and airway cells deficient in purinoceptors, we demonstrated that Ca2+ waves induced by luminal mechanical stimulation in polarized airway epithelia were initiated by the release of the 5′ nucleotides, ATP and UTP, across both apical and basolateral membranes. The nucleotides released into the extracellular compartment interacted with purinoceptors at both membranes to trigger Ca2+ mobilization. Physiologically, apical membrane nucleotide-release coordinates airway mucociliary clearance responses (mucin and salt, water secretion, increased ciliary beat frequency), whereas basolateral release constitutes a paracrine mechanism by which mechanical stresses signal adjacent cells not only within the epithelium, but other cell types (nerves, inflammatory cells) in the submucosa. Nucleotide-release ipsilateral and contralateral to the surface stimulated constitutes a unique mechanism by which epithelia coordinate local and distant airway defense responses to mechanical stimuli.
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MIMIKAKIS, L. John, L. David NELSON, and R. Robin PRESTON. "Oscillating response to a purine nucleotide disrupted by mutation in Paramecium tetraurelia." Biochemical Journal 330, no. 1 (1998): 139–47. http://dx.doi.org/10.1042/bj3300139.
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The purine nucleotide GTP, when added extracellularly, induces oscillations in the swimming behaviour of the protist Paramecium tetraurelia. For periods as long as 10 min the cell swims backwards and forwards repetitively. The oscillations in swimming behaviour are driven by changes in membrane potential of the cell, which in turn are caused by periodic activation of inward Mg2+- and Na+-specific currents. We screened for and isolated mutants that are defective in this response, exploiting the fact that the net result of GTP on a population of cells is repulsion. One mutant, GTP-insensitive (gin A), is not repelled by GTP. In addition, GTP fails to induce repetitive backwards swimming in gin A mutants, although they swim backwards normally in response to other stimuli. GTP fails to evoke oscillations in membrane potential or Mg2+ and Na+ currents in the mutant, although the Mg2+ and Na+ conductances are not themselves measurably affected. A small, oscillating Ca2+ current induced by GTP in the wild type, which might be part of the mechanism that generates oscillations, is also missing from gin A cells. To our knowledge, gin A is the first example of a mutant defective in a purinergic response. We discuss the possibility that the gin A lesion affects the oscillator itself.
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Hanidziar, Dusan, and Simon C. Robson. "Synapomorphic features of hepatic and pulmonary vasculatures include comparable purinergic signaling responses in host defense and modulation of inflammation." American Journal of Physiology-Gastrointestinal and Liver Physiology 321, no. 2 (2021): G200—G212. http://dx.doi.org/10.1152/ajpgi.00406.2020.
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Hepatosplanchnic and pulmonary vasculatures constitute synapomorphic, highly comparable networks integrated with the external environment. Given functionality related to obligatory requirements of “feeding and breathing,” these organs are subject to constant environmental challenges entailing infectious risk, antigenic and xenobiotic exposures. Host responses to these stimuli need to be both protective and tightly regulated. These functions are facilitated by dualistic, high-low pressure blood supply of the liver and lungs, as well as tolerogenic characteristics of resident immune cells and signaling pathways. Dysregulation in hepatosplanchnic and pulmonary blood flow, immune responses, and microbiome implicate common pathogenic mechanisms across these vascular networks. Hepatosplanchnic diseases, such as cirrhosis and portal hypertension, often impact lungs and perturb pulmonary circulation and oxygenation. The reverse situation is also noted with lung disease resulting in hepatic dysfunction. Others, and we, have described common features of dysregulated cell signaling during liver and lung inflammation involving extracellular purines (e.g., ATP, ADP), either generated exogenously or endogenously. These metabokines serve as danger signals, when released by bacteria or during cellular stress and cause proinflammatory and prothrombotic signals in the gut/liver-lung vasculature. Dampening of these danger signals and organ protection largely depends upon activities of vascular and immune cell-expressed ectonucleotidases (CD39 and CD73), which convert ATP and ADP into anti-inflammatory adenosine. However, in many inflammatory disorders involving gut, liver, and lung, these protective mechanisms are compromised, causing perpetuation of tissue injury. We propose that interventions that specifically target aberrant purinergic signaling might prevent and/or ameliorate inflammatory disorders of the gut/liver and lung axis.
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Hesse, Julia, Mona K. Rosse, Bodo Steckel, et al. "Mono-ADP-ribosylation sites of human CD73 inhibit its adenosine-generating enzymatic activity." Purinergic Signalling 18, no. 1 (2021): 115–21. http://dx.doi.org/10.1007/s11302-021-09832-4.
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AbstractCD73-derived adenosine plays a major role in damage-induced tissue responses by inhibiting inflammation. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the cellular release of ATP and NAD+ and upregulate the expression of the nucleotide-degrading purinergic ectoenzyme cascade, including adenosine-generating CD73. Extracellular NAD+ also serves as substrate for mono-ADP-ribosylation of cell surface proteins, which in human cells is mediated by ecto-ADP-ribosyltransferase 1 (ARTC1). Here we explored, whether human CD73 enzymatic activity is regulated by mono-ADP-ribosylation, using recombinant human CD73 in the presence of ARTC1 with etheno-labelled NAD+ as substrate. Multi-colour immunoblotting with an anti-etheno-adenosine antibody showed ARTC1-mediated transfer of ADP-ribose together with the etheno label to CD73. HPLC analysis of the enzymatic activity of in vitro-ribosylated CD73 revealed strong inhibition of adenosine generation in comparison to non-ribosylated CD73. Mass spectrometry of in vitro-ribosylated CD73 identified six ribosylation sites. 3D model analysis indicated that three of them (R328, R354, R545) can interfere with CD73 enzymatic activity. Our study identifies human CD73 as target for ARTC1-mediated mono-ADP-ribosylation, which can profoundly modulate its adenosine-generating activity. Thus, in settings with enhanced release of NAD+ as substrate for ARTC1, assessment of CD73 protein expression in human tissues may not be predictive of adenosine formation resulting in anti-inflammatory activity.
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von Muecke-Heim, Iven-Alex, Clemens Ries, Lidia Urbina, and Jan M. Deussing. "P2X7R antagonists in chronic stress-based depression models: a review." European Archives of Psychiatry and Clinical Neuroscience 271, no. 7 (2021): 1343–58. http://dx.doi.org/10.1007/s00406-021-01306-3.
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AbstractDepression affects around 320 million people worldwide. Growing evidence proposes the immune system to be the core interface between psychosocial stress and the neurobiological and behavioural features of depression. Many studies have identified purinergic signalling via the P2X7 receptor (P2X7R) to be of great importance in depression genesis yet only a few have evaluated P2X7R antagonists in chronic stress-based depression models. This review summarizes their findings and analyses their methodology. The four available studies used three to nine weeks of unpredictable, chronic mild stress or unpredictable, chronic stress in male mice or rats. Stress paradigm composition varied moderately, with stimuli being primarily psychophysical rather than psychosocial. Behavioural testing was performed during or after the last week of stress application and resulted in depressive-like behaviours, immune changes (NLRP3 assembly, interleukin-1β level increase, microglia activation) and neuroplasticity impairment. During the second half of each stress paradigm, a P2X7R antagonist (Brilliant Blue G, A-438079, A-804598) was applied. Studies differed with regard to antagonist dosage and application timing. Nonetheless, all treatments attenuated the stress-induced neurobiological changes and depressive-like behaviours. The evidence at hand underpins the importance of P2X7R signalling in chronic stress and depression. However, improvements in study planning and reporting are necessary to minimize experimental bias and increase data purview. To achieve this, we propose adherence to the Research Domain Criteria and the STRANGE framework.
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Jones, Rachel J., David Jourd'heuil, John C. Salerno, Susan M. E. Smith, and Harold A. Singer. "iNOS regulation by calcium/calmodulin-dependent protein kinase II in vascular smooth muscle." American Journal of Physiology-Heart and Circulatory Physiology 292, no. 6 (2007): H2634—H2642. http://dx.doi.org/10.1152/ajpheart.01247.2006.
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Nitric oxide synthase (NOS) expression is regulated transcriptionally in response to cytokine induction and posttranslationally by palmitoylation and trafficking into perinuclear aggresome-like structures. We investigated the effects of multifunctional calcium/calmodulin-dependent protein kinase II protein kinase (CaMKII) on inducible NOS (iNOS) trafficking in cultured rat aortic vascular smooth muscle cells (VSMCs). Immunofluorescence and confocal microscopy demonstrated colocalization of iNOS and CaMKIIδ2 with a perinuclear distribution and concentration in aggresome-like structures identified by colocalization with γ-tubulin. Furthermore, CaMKIIδ2 coimmunoprecipitated with iNOS in a CaMKII activity-dependent manner. Addition of Ca2+-mobilizing stimuli expected to activate CaMKII; a purinergic agonist (UTP) or calcium ionophore (ionomycin) caused a general redistribution of iNOS from cytosolic to membrane and nuclear fractions. Similarly, adenoviral expression of a constitutively active CaMKIIδ2 mutant altered iNOS localization, shifting iNOS from the cytosolic fraction. Suppression of CaMKIIδ2 using an adenovirus expressing a short hairpin, small interfering RNA increased nuclear iNOS localization in resting cells but inhibited ionomycin-induced translocation of iNOS to the nucleus. Following addition of these chronic and acute CaMKII modulators, there were fewer aggresome-like structures containing iNOS. All of the treatments that chronically affected CaMKII activity or expression significantly inhibited iNOS-specific activity following cytokine induction. The results suggest that CaMKIIδ2 may be an important regulator of iNOS trafficking and activity in VSMCs.
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Nunes, Ana R., Raul Chavez-Valdez, Tarrah Ezell, David F. Donnelly, Joel C. Glover, and Estelle B. Gauda. "Effect of development on [Ca2+]i transients to ATP in petrosal ganglion neurons: a pharmacological approach using optical recording." Journal of Applied Physiology 112, no. 8 (2012): 1393–402. http://dx.doi.org/10.1152/japplphysiol.00511.2011.
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ATP, acting through P2X2/P2X3 receptor-channel complexes, plays an important role in carotid body chemoexcitation in response to natural stimuli in the rat. Since the channels are permeable to calcium, P2X activation by ATP should induce changes in intracellular calcium ([Ca2+]i). Here, we describe a novel ex vivo approach using fluorescence [Ca2+]i imaging that allows screening of retrogradely labeled chemoafferent neurons in the petrosal ganglion of the rat. ATP-induced [Ca2+]i responses were characterized at postnatal days (P) 5–8 and P19–25. While all labeled cells showed a brisk increase in [Ca2+]i in response to depolarization by high KCl (60 mM), only a subpopulation exhibited [Ca2+]i responses to ATP. ATP (250–1,000 μM) elicited one of three temporal response patterns: fast (R1), slow (R2), and intermediate (R3). At P5–8, R2 predominated and its magnitude was attenuated 44% by the P2X1 antagonist, NF449 (10 μM), and 95% by the P2X1/P2X3/P2X2/3 antagonist, TNP-ATP (10 μM). At P19–25, R1 and R3 predominated and their magnitudes were attenuated 15% by NF449, 66% by TNP-ATP, and 100% by suramin (100 μM), a nonspecific P2 purinergic receptor antagonist. P2X1 and P2X2 protein levels in the petrosal ganglion decreased with development, while P2X3 protein levels did not change significantly. We conclude that the profile of ATP-induced P2X-mediated [Ca2+]i responses changes in the postnatal period, corresponding with changes in receptor isoform expression. We speculate that these changes may participate in the postnatal maturation of chemosensitivity.
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Adebiyi, Morayo G., Jeanne M. Manalo, and Yang Xia. "Metabolomic and molecular insights into sickle cell disease and innovative therapies." Blood Advances 3, no. 8 (2019): 1347–55. http://dx.doi.org/10.1182/bloodadvances.2018030619.
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Abstract Sickle cell disease (SCD) is an autosomal-recessive hemolytic disorder with high morbidity and mortality. The pathophysiology of SCD is characterized by the polymerization of deoxygenated intracellular sickle hemoglobin, which causes the sickling of erythrocytes. The recent development of metabolomics, the newest member of the “omics” family, has provided a powerful new research strategy to accurately measure functional phenotypes that are the net result of genomic, transcriptomic, and proteomic changes. Metabolomics changes respond faster to external stimuli than any other “ome” and are especially appropriate for surveilling the metabolic profile of erythrocytes. In this review, we summarize recent pioneering research that exploited cutting-edge metabolomics and state-of-the-art isotopically labeled nutrient flux analysis to monitor and trace intracellular metabolism in SCD mice and humans. Genetic, structural, biochemical, and molecular studies in mice and humans demonstrate unrecognized intracellular signaling pathways, including purinergic and sphingolipid signaling networks that promote hypoxic metabolic reprogramming by channeling glucose metabolism to glycolysis via the pentose phosphate pathway. In turn, this hypoxic metabolic reprogramming induces 2,3-bisphosphoglycerate production, deoxygenation of sickle hemoglobin, polymerization, and sickling. Additionally, we review the detrimental role of an impaired Lands’ cycle, which contributes to sickling, inflammation, and disease progression. Thus, metabolomic profiling allows us to identify the pathological role of adenosine signaling and S1P-mediated erythrocyte hypoxic metabolic reprogramming and hypoxia-induced impaired Lands' cycle in SCD. These findings further reveal that the inhibition of adenosine and S1P signaling cascade and the restoration of an imbalanced Lands' cycle have potent preclinical efficacy in counteracting sickling, inflammation, and disease progression.
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Kempson, Stephen A., Jason M. Edwards, Alyssa Osborn, and Michael Sturek. "Acute inhibition of the betaine transporter by ATP and adenosine in renal MDCK cells." American Journal of Physiology-Renal Physiology 295, no. 1 (2008): F108—F117. http://dx.doi.org/10.1152/ajprenal.00108.2008.
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Extracellular ATP interacts with purinergic P2 receptors to regulate a range of physiological responses, including downregulation of transport activity in the nephron. ATP is released from cells by mechanical stimuli such as cell volume changes, and autocrine signaling by extracellular ATP could occur in renal medullary cells during diuresis. This was tested in Madin-Darby canine kidney (MDCK) cells, a model used frequently to study P1 and P2 receptor activity. ATP was released within 1 min after transfer from 500 to 300 mosmol/kgH2O medium. A 30-min incubation with ATP produced dose-dependent inhibition (0.01–0.10 mM) of the renal betaine/GABA transporter (BGT1) with little effect on other osmolyte transporters. Inhibition was reproduced by specific agonists for P2X (α,β-methylene-ATP) and P2Y (UTP) receptors. Adenosine, the final product of ATP hydrolysis, also inhibited BGT1 but not taurine transport. Inhibition by ATP and adenosine was blocked by pertussis toxin and A73122, suggesting involvement of inhibitory G protein and PLC in postreceptor signaling. Both ATP and adenosine (0.1 mM) produced rapid increases in intracellular Ca2+, due to the mobilization of intracellular Ca2+ stores and Ca2+ influx. Blocking these Ca2+ increases with BAPTA-AM also blocked the action of ATP and adenosine on BGT1 transport. Finally, immunohistochemical studies indicated that inhibition of BGT1 transport may be due to endocytic accumulation of BGT1 proteins from the plasma membrane. We conclude that ATP and adenosine, through stimulation of PLC and intracellular Ca2+, may be rapidly acting regulators of BGT1 transport especially in response to a fall in extracellular osmolarity.
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Leonard, Erin M., and Colin A. Nurse. "Expanding Role of Dopaminergic Inhibition in Hypercapnic Responses of Cultured Rat Carotid Body Cells: Involvement of Type II Glial Cells." International Journal of Molecular Sciences 21, no. 15 (2020): 5434. http://dx.doi.org/10.3390/ijms21155434.
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Dopamine (DA) is a well-studied neurochemical in the mammalian carotid body (CB), a chemosensory organ involved in O2 and CO2/H+ homeostasis. DA released from receptor (type I) cells during chemostimulation is predominantly inhibitory, acting via pre- and post-synaptic dopamine D2 receptors (D2R) on type I cells and afferent (petrosal) terminals respectively. By contrast, co-released ATP is excitatory at postsynaptic P2X2/3R, though paracrine P2Y2R activation of neighboring glial-like type II cells may boost further ATP release. Here, we tested the hypothesis that DA may also inhibit type II cell function. When applied alone, DA (10 μM) had negligible effects on basal [Ca2+]i in isolated rat type II cells. However, DA strongly inhibited [Ca2+]i elevations (Δ[Ca2+]i) evoked by the P2Y2R agonist UTP (100 μM), an effect opposed by the D2/3R antagonist, sulpiride (1–10 μM). As expected, acute hypercapnia (10% CO2; pH 7.4), or high K+ (30 mM) caused Δ[Ca2+]i in type I cells. However, these stimuli sometimes triggered a secondary, delayed Δ[Ca2+]i in nearby type II cells, attributable to crosstalk involving ATP-P2Y2R interactions. Interestingly sulpiride, or DA store-depletion using reserpine, potentiated both the frequency and magnitude of the secondary Δ[Ca2+]i in type II cells. In functional CB-petrosal neuron cocultures, sulpiride potentiated hypercapnia-induced Δ[Ca2+]i in type I cells, type II cells, and petrosal neurons. Moreover, stimulation of type II cells with UTP could directly evoke Δ[Ca2+]i in nearby petrosal neurons. Thus, dopaminergic inhibition of purinergic signalling in type II cells may help control the integrated sensory output of the CB during hypercapnia.
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Furuya, Kishio, Ju Jing Tan, Francis Boudreault, Masahiro Sokabe, Yves Berthiaume, and Ryszard Grygorczyk. "Real-time imaging of inflation-induced ATP release in the ex vivo rat lung." American Journal of Physiology-Lung Cellular and Molecular Physiology 311, no. 5 (2016): L956—L969. http://dx.doi.org/10.1152/ajplung.00425.2015.
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Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that regulate diverse processes critical for lung function, including mucociliary clearance, surfactant secretion, and local blood flow. Cellular ATP release is mechanosensitive; however, the impact of physical stimuli on ATP release during breathing has never been tested in intact lungs in real time and remains elusive. In this pilot study, we investigated inflation-induced ATP release in rat lungs ex vivo by real-time luciferin-luciferase (LL) bioluminescence imaging coupled with simultaneous infrared tissue imaging to identify ATP-releasing sites. With LL solution introduced into air spaces, brief inflation of such edematous lung (1 s, ∼20 cmH2O) induced transient (<30 s) ATP release in a limited number of air-inflated alveolar sacs during their recruitment/opening. Released ATP reached concentrations of ∼10−6 M, relevant for autocrine/paracrine signaling, but it remained spatially restricted to single alveolar sacs or their clusters. ATP release was stimulus dependent: prolonged (100 s) inflation evoked long-lasting ATP release that terminated upon alveoli deflation/derecruitment while cyclic inflation/suction produced cyclic ATP release. With LL introduced into blood vessels, inflation induced transient ATP release in many small patchlike areas the size of alveolar sacs. Findings suggest that inflation induces ATP release in both alveoli and the surrounding blood capillary network; the functional units of ATP release presumably consist of alveolar sacs or their clusters. Our study demonstrates the feasibility of real-time ATP release imaging in ex vivo lungs and provides the first direct evidence of inflation-induced ATP release in lung air spaces and in pulmonary blood capillaries, highlighting the importance of purinergic signaling in lung function.
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Brozmanova, M., L. Mazurova, F. Ru, et al. "Mechanisms of the adenosine A2Areceptor-induced sensitization of esophageal C fibers." American Journal of Physiology-Gastrointestinal and Liver Physiology 310, no. 3 (2016): G215—G223. http://dx.doi.org/10.1152/ajpgi.00350.2014.
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Clinical studies indicate that adenosine contributes to esophageal mechanical hypersensitivity in some patients with pain originating in the esophagus. We have previously reported that the esophageal vagal nodose C fibers express the adenosine A2Areceptor. Here we addressed the hypothesis that stimulation of the adenosine A2Areceptor induces mechanical sensitization of esophageal C fibers by a mechanism involving transient receptor potential A1 (TRPA1). Extracellular single fiber recordings of activity originating in C-fiber terminals were made in the ex vivo vagally innervated guinea pig esophagus. The adenosine A2Areceptor-selective agonist CGS21680 induced robust, reversible sensitization of the response to esophageal distention (10–60 mmHg) in a concentration-dependent fashion (1–100 nM). At the half-maximally effective concentration (EC50: ≈3 nM), CGS21680 induced an approximately twofold increase in the mechanical response without causing an overt activation. This sensitization was abolished by the selective A2Aantagonist SCH58261. The adenylyl cyclase activator forskolin mimicked while the nonselective protein kinase inhibitor H89 inhibited mechanical sensitization by CGS21680. CGS21680 did not enhance the response to the purinergic P2X receptor agonist α,β-methylene-ATP, indicating that CGS21680 does not nonspecifically sensitize to all stimuli. Mechanical sensitization by CGS21680 was abolished by pretreatment with two structurally different TRPA1 antagonists AP18 and HC030031 . Single cell RT-PCR and whole cell patch-clamp studies in isolated esophagus-specific nodose neurons revealed the expression of TRPA1 in A2A-positive C-fiber neurons and demonstrated that CGS21682 potentiated TRPA1 currents evoked by allylisothiocyanate. We conclude that stimulation of the adenosine A2Areceptor induces mechanical sensitization of nodose C fibers by a mechanism sensitive to TRPA1 antagonists indicating the involvement of TRPA1.
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Olson, Kim E., Marinus Johan Broekman, Ashley E. Olson, Dianne Pulte, and Aaron J. Marcus. "Nucleotidase Activity of CD39/NTPDase1 Is Dependent on Internal Proteolytic Cleavage Which Contributes to Lipid Raft Localization." Blood 110, no. 11 (2007): 3634. http://dx.doi.org/10.1182/blood.v110.11.3634.3634.
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Abstract Brief trypsin exposure increases apyrase activity in hCD39 expressing cells, as previously reported (Schulte am Esch et al, Biochemistry38:2248, 1999). Since regulated proteolytic cleavage of CD39 would allow for a rapid response to extracellular stimuli, we studied the relationship between observed CD39 cleavage and enzymatic activity. We generated N- and C-terminal VP16-tagged hCD39 to study CD39 expression, processing, and activity in transiently transfected HEK 293 cells. We found that optimal enzymatic activity of CD39 indeed depends on incorporation into cholesterol-rich plasma membrane domains (lipid "rafts"). Membrane fractions from hCD39 -transfected 293 cells readily hydrolyze ATP. Pretreatment of 293 cells with the cholesterol-depleting agent methyl β cyclodextrin (MBCD) results in a dose-dependent decrease in ATPase activity. In addition, treatment of isolated membranes with MBCD also decreases enzymatic activity. We next performed Western blot analyses of membranes prepared from hCD39-transfected 293 cells treated with membrane-impermeant crosslinking agents. These experiments demonstrated a dose-dependent, MBCD-reversible decrease in monomeric CD39. Taken together, these data demonstrate that CD39 enzyme activity resides in raft-localized CD39. Western blots of membrane fractions from cells transfected with N- or C-terminal VP16-tagged hCD39 show partial cleavage of full-length CD39 to yield a 20kDa N-terminal and 50 kDa C-terminal fragments. Biotinylation studies established that both fragments are expressed on the cell surface. As with full-length CD39, crosslinking results in dose-dependent decreases of both monomeric species. Moreover, prior cholesterol depletion with MBCD abolishes crosslinking. Since the cleavage products of full-length CD39 are expressed on the cell surface and localize to lipid rafts, we examined the relation between CD39 cleavage, ATPase activity and lipid raft localization using a panel of cell permeable protease inhibitors. 293 cells transfected with N-terminal VP16-tagged CD39 were treated with AEBSF (serine protease inhibitor), zYVAD.fmk (caspase inhibitor), zLLY.fmk (calpain inhibitor) or the furin inhibitor Furin I. All inhibitors resulted in dose-dependent decreases in formation of the VP16-tagged N-terminal fragment. Concomitantly, ATPase assays of the membrane fractions demonstrated a corresponding dose-dependent decrease in enzymatic activity. Finally, we established that CD39 cleavage promotes raft localization, since protease inhibition decreased the fraction of CD39 susceptible to crosslinking with all inhibitors tested. In summary, we have established that generation of optimally active, raft-localized CD39 requires prior limited proteolysis of the full-length molecule. Activation of caspase-1 by exposure of cells to ATP leads to processing and release of interleukin family members. We propose that purinergic signaling might also enhance CD39 cleavage in vascular cells by an as yet unidentified protease. Our data suggest that subsequent increased cell surface apyrase activity leads to dampening of purinergic signaling and a resulting increase in antithrombotic activity. Of note, we identified an alternately spliced isoform of CD39 which inhibits cleavage of the full-length molecule.
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Gentile, Chiara, Arianna Finizio, Guendalina Froechlich, et al. "Generation of a Retargeted Oncolytic Herpes Virus Encoding Adenosine Deaminase for Tumor Adenosine Clearance." International Journal of Molecular Sciences 22, no. 24 (2021): 13521. http://dx.doi.org/10.3390/ijms222413521.
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Background: Oncolytic viruses are immunotherapeutic agents that can be engineered to encode payloads of interest within the tumor microenvironment to enhance therapeutic efficacy. Their therapeutic potential could be limited by many avenues for immune evasion exerted by the tumor. One such is mediated by adenosine, which induces pleiotropic immunosuppression by inhibiting antitumor immune populations as well as activating tolerogenic stimuli. Adenosine is produced starting from the highly immunostimulatory ATP, which is progressively hydrolyzed to ADP and adenosine by CD39 and CD73. Cancer cells express high levels of CD39 and CD73 ectoenzymes, thus converting immunostimulatory purinergic signal of ATP into an immunosuppressive signal. For this reason, CD39, CD73 and adenosine receptors are currently investigated in clinical trials as targets for metabolic cancer immunotherapy. This is of particular relevance in the context of oncovirotherapy, as immunogenic cell death induced by oncolytic viruses causes the secretion of a high amount of ATP which is available to be quickly converted into adenosine. Methods: Here, we took advantage of adenosine deaminase enzyme that naturally converts adenosine into the corresponding inosine derivative, devoid of immunoregulatory function. We encoded ADA into an oncolytic targeted herpes virus redirected to human HER2. An engineered ADA with an ectopic signal peptide was also generated to improve enzyme secretion (ADA-SP). Results: Insertion of the expression cassette was not detrimental for viral yield and cancer cell cytotoxicity. The THV_ADA and THV_ADA-SP successfully mediated the secretion of functional ADA enzyme. In in vitro model of human monocytes THP1, this ability of THV_ADA and THV_ADA-SP resulted in the retrieval of eADO-exposed monocytes replication rate, suggesting the proficiency of the viruses in rescuing the immune function. Conclusions: Encoding ADA into oncolytic viruses revealed promising properties for preclinical exploitation.