[Ca
            2+
            ]
            i
            oscillations in renal epithelia caused by α‐hemolysin from
            Escherichia coli
            require ATP release and P2 receptor mediated signaling

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1–1 µM). Exposure to MST7 and MEL enhanced ATP release by 3–7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6–7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

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P2 receptors in the regulation of renal transport mechanisms

AJP Renal Physiology ◽

10.1152/ajprenal.00432.2007 ◽

2008 ◽

Vol 294 (1) ◽

pp. F10-F27 ◽

Cited By ~ 85

Author(s):

Volker Vallon

Keyword(s):

Collecting Duct ◽

P2 Receptors ◽

Extracellular Nucleotides ◽

Receptor Subtype ◽

Receptor Subtypes ◽

Inhibitory Influence ◽

P2 Receptor ◽

Transport Mechanisms ◽

Renal Epithelia ◽

Regulation Of Transport

Extracellular nucleotides (e.g., ATP) regulate physiological and pathophysiological processes through activation of nucleotide P2 receptors in the plasma membrane. Examples include such diverse processes as communication from taste buds to gustatory nerves, platelet aggregation, nociception, or neutrophil chemotaxis. Over approximately the last 15 years, evidence has also accumulated that cells in renal epithelia release nucleotides in response to physiological stimuli and that these nucleotides act in a paracrine and autocrine way to activate P2 receptors and play a significant role in the regulation of transport mechanisms and cell volume regulation. This review discusses potential stimuli and mechanisms involved in nucleotide release in renal epithelia and summarizes the available data on the expression and function of nucleotide P2 receptors along the native mammalian tubular and collecting duct system. Using established agonist profiles for P2 receptor subtypes, significant insights have been gained particularly into a potential role for P2Y2-like receptors in the regulation of transport mechanisms in the collecting duct. Due to the lack of receptor subtype-specific antagonists, however, the in vivo relevance of P2 receptor subtypes is unclear. Studies in gene knockout mice provided first insights including an antihypertensive activity of P2Y2receptors that is linked to an inhibitory influence on renal Na+and water reabsorption. We are only beginning to unravel the important roles of extracellular nucleotides and P2 receptors in the regulation of the diverse transport mechanisms of the kidney.

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Astrocytes in the Retrotrapezoid Nucleus Sense H+ by Inhibition of a Kir4.1–Kir5.1-Like Current and May Contribute to Chemoreception by a Purinergic Mechanism

Journal of Neurophysiology ◽

10.1152/jn.00544.2010 ◽

2010 ◽

Vol 104 (6) ◽

pp. 3042-3052 ◽

Cited By ~ 89

Author(s):

Ian C. Wenker ◽

Orsolya Kréneisz ◽

Akiko Nishiyama ◽

Daniel K. Mulkey

Keyword(s):

Atp Release ◽

Ph Sensitive ◽

Equilibrium Potential ◽

P2 Receptor ◽

Bicarbonate Buffer ◽

Hepes Buffer ◽

Retrotrapezoid Nucleus ◽

Baseline Activity ◽

Patch Clamp Electrophysiology ◽

Brain Slice Preparation

Central chemoreception is the mechanism by which CO2/pH sensors regulate breathing in response to tissue pH changes. There is compelling evidence that pH-sensitive neurons in the retrotrapezoid nucleus (RTN) are important chemoreceptors. Evidence also indicates that CO2/H+-evoked adenosine 5′-triphosphate (ATP) release in the RTN, from pH-sensitive astrocytes, contributes to chemoreception. However, mechanism(s) by which RTN astrocytes sense pH is unknown and their contribution to chemoreception remains controversial. Here, we use the brain slice preparation and a combination of patch-clamp electrophysiology and immunohistochemistry to confirm that RTN astrocytes are pH sensitive and to determine mechanisms by which they sense pH. We show that pH-sensitive RTN glia are immunoreactive for aldehyde dehydrogenase 1L1, a marker of astrocytes. In HEPES buffer the pH-sensitive current expressed by RTN astrocytes reversed near EK+ (the equilibrium potential for K+) and was inhibited by Ba2+ and desipramine (blocker of Kir4.1-containing channels), characteristics most consistent with heteromeric Kir4.1–Kir5.1 channels. In bicarbonate buffer, the sodium/bicarbonate cotransporter also contributed to the CO2/H+-sensitive current in RTN astrocytes. To test the hypothesis that RTN astrocytes contribute to chemoreception by a purinergic mechanism, we used fluorocitrate to selectively depolarize astrocytes while measuring neuronal activity. We found that fluorocitrate increased baseline activity and pH sensitivity of RTN neurons by a P2-receptor–dependent mechanism, suggesting that astrocytes may release ATP to activate RTN chemoreceptors. We also found in bicarbonate but not HEPES buffer that P2-receptor antagonists decreased CO2 sensitivity of RTN neurons. We conclude that RTN astrocytes sense CO2/H+ in part by inhibition of a Kir4.1–Kir5.1-like current and may provide an excitatory purinergic drive to pH-sensitive neurons.

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Inhibition of P2X Receptors Protects Human Monocytes against Damage by Leukotoxin from Aggregatibacter actinomycetemcomitans and α-Hemolysin from Escherichia coli

Infection and Immunity ◽

10.1128/iai.00674-16 ◽

2016 ◽

Vol 84 (11) ◽

pp. 3114-3130 ◽

Cited By ~ 12

Author(s):

Steen K. Fagerberg ◽

Martin R. Jakobsen ◽

Marianne Skals ◽

Helle A. Praetorius

Keyword(s):

Escherichia Coli ◽

Plasma Membranes ◽

Cell Damage ◽

Aggregatibacter Actinomycetemcomitans ◽

P2x Receptors ◽

Receptor Expression ◽

Human Monocytes ◽

P2 Receptor ◽

Content Type ◽

Tract Infections

α-Hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans are important virulence factors in ascending urinary tract infections and aggressive periodontitis, respectively. The extracellular signaling molecule ATP is released immediately after insertion of the toxins into plasma membranes and, via P2X receptors, is essential for the erythrocyte damage inflicted by these toxins. Moreover, ATP signaling is required for the ensuing recognition and phagocytosis of damaged erythrocytes by the monocytic cell line THP-1. Here, we investigate how these toxins affect THP-1 monocyte function. We demonstrate that both toxins trigger early ATP release and a following increase in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) in THP-1 monocytes. The HlyA- and LtxA-induced [Ca 2+ ] i response is diminished by the P2 receptor antagonist in a pattern that fits the functional P2 receptor expression in these cells. Both toxins are capable of lysing THP-1 cells, with LtxA being more aggressive. Either desensitization or blockage of P2X 1 , P2X 4 , or P2X 7 receptors markedly reduces toxin-induced cytolysis. This pattern is paralleled in freshly isolated human monocytes from healthy volunteers. Interestingly, only a minor fraction of the toxin-damaged THP-1 monocytes eventually lyse. P2X 7 receptor inhibition generally prevents cell damage, except from a distinct cell shrinkage that prevails in response to the toxins. Moreover, we find that preexposure to HlyA preserves the capacity of THP-1 monocytes to phagocytose damaged erythrocytes and may induce readiness to discriminate between damaged and healthy erythrocytes. These findings suggest a new pharmacological target for protecting monocytes during exposure to pore-forming cytolysins during infection or injury.

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P2Y Receptor Modulation of ATP Release in the Urothelium

BioMed Research International ◽

10.1155/2014/830374 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 10

Author(s):

Kylie J. Mansfield ◽

Jessica R. Hughes

Keyword(s):

Atp Release ◽

Receptor Activation ◽

P2y Receptor ◽

P2 Receptor ◽

Urothelial Cells ◽

Receptor Agonists ◽

Receptor Signalling ◽

Receptor Modulation ◽

High Concentrations ◽

Stimulation Of

The release of ATP from the urothelium in response to stretch during filling demonstrates the importance of the purinergic system for the physiological functioning of the bladder. This study examined the effect of P2 receptor agonists on ATP release from two urothelial cell lines (RT4 and UROtsa cells). Hypotonic Krebs was used as a stretch stimulus. Incubation of urothelial cells with high concentrations of the P2Y agonist ADP induced ATP release to a level that was 40-fold greater than hypotonic-stimulated ATP release (P< 0.0011, ADP EC50 1.8 µM). Similarly, an increase in ATP release was also observed with the P2Y agonist, UTP, up to a maximum of 70% of the hypotonic response (EC50 0.62 µM). Selective P2 receptor agonists,αβ-methylene-ATP, ATP-γ-S, and 2-methylthio-ADP had minimal effects on ATP release. ADP-stimulated ATP release was significantly inhibited by suramin (100 µM,P= 0.002). RT4 urothelial cells break down nucleotides (100 µM) including ATP, ADP, and UTP to liberate phosphate. Phosphate liberation was also demonstrated from endogenous nucleotides with approximately 10% of the released ATP broken down during the incubation. These studies demonstrate a role for P2Y receptor activation in stimulation of ATP release and emphasize the complexity of urothelial P2 receptor signalling.

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Two pathways for ATP release from host cells in enteropathogenicEscherichia coliinfection

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00137.2005 ◽

2005 ◽

Vol 289 (3) ◽

pp. G407-G417 ◽

Cited By ~ 19

Author(s):

John K. Crane ◽

Tonniele M. Naeher ◽

Shilpa S. Choudhari ◽

Elisa M. Giroux

Keyword(s):

Escherichia Coli ◽

Cell Death ◽

Host Cell ◽

Atp Release ◽

Host Cells ◽

Specific Cell ◽

Hypotonic Medium ◽

Enteropathogenic Escherichia Coli ◽

Extracellular Adenosine ◽

Transfected Cells

We previously reported that enteropathogenic Escherichia coli (EPEC) infection triggered a large release of ATP from the host cell that was correlated with and dependent on EPEC-induced killing of the host cell. We noted, however, that under some circumstances, EPEC-induced ATP release exceeded that which could be accounted for on the basis of host cell killing. For example, EPEC-induced ATP release was potentiated by noncytotoxic agents that elevate host cell cAMP, such as forskolin and cholera toxin, and by exposure to hypotonic medium. These findings and the performance of the EPEC espF mutant led us to hypothesize that the CFTR plays a role in EPEC-induced ATP release that is independent of cell death. We report the results of experiments using specific, cell-permeable CFTR activators and inhibitors, as well as transfection of the CFTR into non-CFTR-expressing cell lines, which incriminate the CFTR as a second pathway for ATP release from host cells. Increased ATP release via CFTR is not accompanied by an increase in EPEC adherence to transfected cells. The CFTR-dependent ATP release pathway becomes activated endogenously later in EPEC infection, and this activation is mediated, at least in part, by generation of extracellular adenosine from the breakdown of released ATP.

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Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes.

Journal of Experimental Medicine ◽

10.1084/jem.169.3.737 ◽

1989 ◽

Vol 169 (3) ◽

pp. 737-754 ◽

Cited By ~ 78

Author(s):

S Bhakdi ◽

S Greulich ◽

M Muhly ◽

B Eberspächer ◽

H Becker ◽

...

Keyword(s):

Escherichia Coli ◽

Low Density Lipoprotein ◽

Atp Release ◽

Density Lipoprotein ◽

Kinetic Studies ◽

Membrane Permeabilization ◽

Human Organism ◽

High Doses ◽

Escherichia Coli Hemolysin ◽

Protein Rich

The contribution of Escherichia coli hemolysin (ECH) to bacterial virulence has been considered mainly in context with its hemolytic properties. We here report that this prevalent bacterial cytolysin is the most potent leukocidin known to date. Very low concentrations (approximately 1 ng/ml) of ECH evoke membrane permeability defects in PMN (2-10 x 10(6) cells/ml) leading to an efflux of cellular ATP and influx of propidium iodide. The attacked cells do not appear to repair the membrane lesions. Human serum albumin, high density and low density lipoprotein, and IgG together protect erythrocytes and platelets against attack by even high doses (5-25 micrograms/ml) of ECH. In contrast, PMN are still permeabilized by ECH at low doses (50-250 ng/ml) in the presence of these plasma inactivators. Thus, PMN become preferred targets for attack by ECH in human blood and protein-rich body fluids. Kinetic studies demonstrate that membrane permeabilization is a rapid process, ATP-release commencing within seconds after application of toxin to leukocytes. It is estimated that membrane permeabilization ensues upon binding of approximately 300 molecules ECH/PMN. This process is paralleled by granule exocytosis, and by loss of phagocytic killing capacity of the cells. The recognition that ECH directly counteracts a major immune defence mechanism of the human organism through its attack on granulocytes under physiological conditions sheds new light on its possible role and potential importance as a virulence factor of E. coli.

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Chemical activation of the Piezo1 channel drives mesenchymal stem cell migration via inducing ATP release and activation of P2 receptor purinergic signaling

Stem Cells ◽

10.1002/stem.3114 ◽

2020 ◽

Vol 38 (3) ◽

pp. 410-421 ◽

Cited By ~ 4

Author(s):

Fatema Mousawi ◽

Hongsen Peng ◽

Jing Li ◽

Sreenivasan Ponnambalam ◽

Sébastien Roger ◽

...

Keyword(s):

Stem Cell ◽

Cell Migration ◽

Mesenchymal Stem Cell ◽

Purinergic Signaling ◽

Chemical Activation ◽

Atp Release ◽

P2 Receptor ◽

Stem Cell Migration

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Altered urothelial ATP signaling in a major subset of human overactive bladder patients with pyuria

AJP Renal Physiology ◽

10.1152/ajprenal.00339.2015 ◽

2016 ◽

Vol 311 (4) ◽

pp. F805-F816 ◽

Cited By ~ 8

Author(s):

Alberto Contreras-Sanz ◽

Louise Krska ◽

Aswini A. Balachandran ◽

Natasha L. Curtiss ◽

Rajvinder Khasriya ◽

...

Keyword(s):

Overactive Bladder ◽

Bacterial Colonization ◽

Atp Release ◽

Receptor Expression ◽

Intracellular Bacteria ◽

P2 Receptor ◽

Urothelial Cells ◽

Urgency Incontinence ◽

Major Subset ◽

Atp Signaling

Overactive Bladder (OAB) is an idiopathic condition, characterized by urgency, urinary frequency, and urgency incontinence, in the absence of routinely traceable urinary infection. We have described microscopic pyuria (≥10 wbc/μl) in patients suffering from the worst symptoms. It is established that inflammation is associated with increased ATP release from epithelial cells, and extracellular ATP originating from the urothelium following increased hydrostatic pressure is a mediator of bladder sensation. Here, using bladder biopsy samples, we have investigated urothelial ATP signaling in OAB patients with microscopic pyuria. Basal, but not stretch-evoked, release of ATP was significantly greater from the urothelium of OAB patients with pyuria than from non-OAB patients or OAB patients without pyuria (<10 wbc/μl). Basal ATP release from the urothelium of OAB patients with pyuria was inhibited by the P2 receptor antagonist suramin and abolished by the hemichannel blocker carbenoxolone, which differed from stretch-activated ATP release. Altered P2 receptor expression was evident in the urothelium from pyuric OAB patients. Furthermore, intracellular bacteria were visualized in shed urothelial cells from ∼80% of OAB patients with pyuria. These data suggest that increased ATP release from the urothelium, involving bacterial colonization, may play a role in the heightened symptoms associated with pyuric OAB patients.

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