scholarly journals P2X1 receptor activation in HL60 cells

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2659-2664 ◽  
Author(s):  
G Buell ◽  
AD Michel ◽  
C Lewis ◽  
G Collo ◽  
PP Humphrey ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Radioligand Binding ◽  
Atp Release ◽  
Receptor Activation ◽  
P2x Receptor ◽  
Luciferase Assay ◽  
Receptor Protein ◽  
Hl60 Cells ◽  
P2x1 Receptor ◽  
Alpha Beta

Recent cloning of the human P2X1 receptor revealed high levels of its messenger RNA in differentiated promyelocytes (HL60 cells). We found expression of P2X1 receptor protein in HL60 cells by radioligand binding, by immunohistochemistry, using a receptor specific antibody, and by electrophysiology. The currents elicited by adenosine triphosphate (ATP) had the expected properties of P2X1 receptors (rapid desensitization, mimicked by alpha,beta-methylene-ATP). However, these currents were only observed in cells that were pretreated with apyrase, which destroys extracellular ATP, or with suramin, a P2X receptor antagonist. This implies that HL60 cells release ATP, which chronically desensitizes the receptor. ATP release was detected by direct measurement, using the luciferin-luciferase assay. It is concluded that functional P2X1 receptors are present in the membrane of differentiated HL60 cells.

scholarly journals Shiga toxin signals via ATP and its effect is blocked by purinergic receptor antagonism

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Karl E. Johansson ◽  
Anne-Lie Ståhl ◽  
Ida Arvidsson ◽  
Sebastian Loos ◽  
Ashmita Tontanahal ◽  
...  
Keyword(s):  
Hela Cells ◽  
Shiga Toxin ◽  
Calcium Influx ◽  
Purinergic Receptor ◽  
Atp Release ◽  
Retrograde Transport ◽  
P2x Receptor ◽  
P2x1 Receptor ◽  
Uremic Syndrome ◽  
Induced Apoptosis

Abstract Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli (EHEC), that cause gastrointestinal infection leading to hemolytic uremic syndrome. The aim of this study was to investigate if Stx signals via ATP and if blockade of purinergic receptors could be protective. Stx induced ATP release from HeLa cells and in a mouse model. Toxin induced rapid calcium influx into HeLa cells, as well as platelets, and a P2X1 receptor antagonist, NF449, abolished this effect. Likewise, the P2X antagonist suramin blocked calcium influx in Hela cells. NF449 did not affect toxin intracellular retrograde transport, however, cells pre-treated with NF449 exhibited significantly higher viability after exposure to Stx for 24 hours, compared to untreated cells. NF449 protected HeLa cells from protein synthesis inhibition and from Stx-induced apoptosis, assayed by caspase 3/7 activity. The latter effect was confirmed by P2X1 receptor silencing. Stx induced the release of toxin-positive HeLa cell- and platelet-derived microvesicles, detected by flow cytometry, an effect significantly reduced by NF449 or suramin. Suramin decreased microvesicle levels in mice injected with Stx or inoculated with Stx-producing EHEC. Taken together, we describe a novel mechanism of Stx-mediated cellular injury associated with ATP signaling and inhibited by P2X receptor blockade.

scholarly journals Extracellular ATP and P2Y Receptor Activation Induce a Proinflammatory Host Response in the Human Urinary Tract

2010 ◽  
Vol 78 (8) ◽  
pp. 3609-3615 ◽  
Author(s):  
Susanne Säve ◽  
Katarina Persson
Keyword(s):  
Urinary Tract ◽  
Cell Line ◽  
Atp Release ◽  
Receptor Activation ◽  
Extracellular Atp ◽  
Luciferase Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Epithelial Cell Line ◽  
P2y Receptor ◽  
Uroepithelial Cells

ABSTRACT Extracellular ATP can be released by many cell types under conditions of cellular stress and signals through activation of purinergic receptors. Bladder uroepithelial cells grown in vitro have previously been shown to release ATP in response to stretch. In the present study, we investigated ATP release from uroepithelial cells infected with bacteria and the effect of ATP on the host cell proinflammatory interleukin 8 (IL-8) response. The human kidney epithelial cell line A498 and the human uroepithelial cell line UROtsa were grown in culture and stimulated by the uropathogenic Escherichia coli (UPEC) IA2 strain or the stable ATP analogue ATP-γ-S. ATP and IL-8 levels were measured in cell culture medium with a luciferin-luciferase assay and enzyme-linked immunosorbent assay (ELISA), respectively. The results showed that UPEC infection of uroepithelial cells for 1 h significantly increased (P < 0.01) the extracellular ATP levels. ATP-γ-S (10 and 100 μM) stimulated release of IL-8 from UROtsa and A498 cells after 6 and 24 h. Experiments with different purinoceptor agonists suggested that P2Y receptors, and not P2X receptors, were responsible for the ATP-γ-S-induced IL-8 release. The potency profile further suggested involvement of P2Y1, P2Y2, and/or P2Y11 receptors, and reverse transcription-PCR (RT-PCR) studies confirmed that the cells expressed these receptors. The amount of IL-8 released increased 12-fold in UPEC-infected cells, and apyrase, an enzyme that degrades ATP, reduced this increase by approximately 50%. The present study suggests that enhanced ATP release and P2Y receptor activation during urinary tract infection may represent a novel, non-TLR4-mediated mechanism for production of proinflammatory IL-8 in human urinary tract epithelial cells.

scholarly journals Ca2+ Signaling Triggered by Shear-Autocrine P2X Receptor Pathway in Rat Atrial Myocytes

2018 ◽  
Vol 50 (6) ◽  
pp. 2296-2313 ◽  
Author(s):  
Joon-Chul Kim ◽  
Min-Jeong Son ◽  
Sun-Hee Woo
Keyword(s):  
Shear Stress ◽  
Membrane Potential ◽  
Atp Release ◽  
P2x Receptor ◽  
Luciferase Assay ◽  
Atrial Myocytes ◽  
Patch Clamp Technique ◽  
T Wave ◽  
Connexin Hemichannels ◽  
T Waves

Background/Aims: The atrium is exposed to high shear stress during heart failure and valvular diseases. We aimed to understand atrial shear-induced Ca2+ signaling and its underlying mechanisms. Methods: Pressurized micro-flow was applied to single rat atrial myocytes, and Ca2+ signal, membrane potential, and ATP release were assessed using confocal imaging, patch clamp technique, and luciferin-luciferase assay, respectively. Results: Shear stress (∼16 dyn/cm2) induced global Ca2+ waves (∼0.1 events/s) from the periphery to the center of cells in a transverse direction (“T-wave”; ∼145 μm/s). Pharmacological interventions and simultaneous recording of membrane potential and Ca2+ demonstrated that shear-induced T-waves resulted from action potential (AP)-triggered Ca2+ release from the sarcoplasmic reticulum. T-waves were not sensitive to inhibitors of known shear signaling mechanisms except connexin hemichannels and ATP release. Shear stress caused ATP release from these myocytes (∼1.1x10-17 moles/unit membrane, µm2); ATP release was increased by enhancement of connexin hemichannels and suppressed by inhibition of the hemichannels, but not affected by inhibitors of other ATP release pathways. Blockade of P2X receptor, but not pannexin or the Na+-Ca2+ exchanger, eliminated shear-induced T-wave initiation. Conclusion: Our data suggest that shear stress triggers APs and concomitant Ca2+ signaling via activation of P2X receptors by connexin hemichannel-mediated ATP release in atrial myocytes.

Blocking ATP releasing channels prevents high extracellular ATP levels and airway hyperreactivity in an asthmatic mouse model

2021 ◽  
Author(s):  
Llilian Arzola Martínez ◽  
Rebeca Benavente ◽  
Génesis Vega ◽  
Mariana Ríos ◽  
Wendy Fonseca ◽  
...  
Keyword(s):  
Mouse Model ◽  
Airway Inflammation ◽  
Airway Epithelium ◽  
Atp Release ◽  
Specific Inhibitor ◽  
Extracellular Atp ◽  
Airway Hyperreactivity ◽  
Luciferase Assay ◽  
Asthmatic Patients ◽  
Allergic Mouse

Allergic asthma is a chronic airway inflammatory response to different triggers like inhaled allergens. Excessive ATP in fluids from asthmatic patients is considered an inflammatory signal and an important autocrine/paracrine modulator of airway physiology. Here we investigated the deleterious effect of increased extracellular ATP (eATP) concentration on the mucociliary clearance (MCC) effectiveness and determined the role of ATP releasing channels during airway inflammation in an ovalbumin (OVA)-sensitized mouse model. Our allergic mouse model exhibited high levels of eATP measured in the tracheal fluid with a luciferin-luciferase assay and reduced MCC velocity determined by microspheres tracking in the trachea ex vivo. Addition of ATP had a dual effect on MCC, where lower ATP concentration (µM) increased microspheres velocity, while higher concentration (mM) transiently stopped microspheres movement. Also, an augmented ethidium bromide uptake by the allergic tracheal airway epithelium suggests an increase in ATP release channel functionality during inflammatory conditions. The use of carbenoxolone, a non-specific inhibitor of connexin and pannexin1channels reduced the eATP concentration in the allergic mouse tracheal fluid and dye uptake by the airway epithelium, providing evidence that these ATP release channels are facilitating the net flux of ATP to the lumen during airway inflammation. However, only the specific inhibition of pannexin1 with 10Panx peptide significantly reduced eATP in bronchoalveolar lavage and decreased airway hyperresponsiveness in OVA-allergic mouse model. These data provide evidence that blocking eATP may be a pharmacological alternative to be explored in rescue therapy during episodes of airflow restriction in asthmatic patients.

Amiloride-sensitive Na+channels in pelvic uroepithelium involved in renal sensory receptor activation

1998 ◽  
Vol 275 (6) ◽  
pp. R1780-R1792 ◽  
Author(s):  
Ulla C. Kopp ◽  
Kazumichi Matsushita ◽  
Rita D. Sigmund ◽  
Lori A. Smith ◽  
Shigeru Watanabe ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Collecting Duct ◽  
Receptor Activation ◽  
Sensory Receptor ◽  
Renal Nerve ◽  
Pelvic Wall ◽  
Renal Nerve Activity ◽  
Collecting Duct Cells ◽  
Pelvic Perfusion ◽  
Pelvic Pressure

Stretching the renal pelvic wall increases ipsilateral afferent renal nerve activity (ARNA). This response is enhanced by inhibiting Na+-K+-ATPase with ouabain, suggesting a modulatory role for intracellular Na+ in the activation of mechanosensitive neurons. The messenger RNA for α-, β-, and γ-subunits of epithelial Na+channels (ENaC) is found in collecting duct cells. Because ENaC subunits show homology with genes involved in mechanosensation, we examined whether ENaC mRNA could be found in the pelvic wall and whether the ARNA response to increased renal pelvic pressure was modulated by blockers of the Na+channel. α-, β-, and γ-subunits are present in the pelvis. The messenger RNA for the β- and γ-subunits is readily detected by in situ hybridization throughout the uroepithelium. The ARNA response to increased renal pelvic pressure was reduced by 53 ± 10% and 40 ± 10% ( P < 0.01) by renal pelvic perfusion with the inhibitors amiloride and benzamil, respectively. Amiloride inhibited the ouabain-induced enhancement of the ARNA response to increased renal pelvic pressure. The magnitude of this inhibition was inversely correlated with the magnitude of the amiloride-mediated blockade of the ARNA response to increased renal pelvic pressure ( P < 0.001). Amiloride also reduced the ARNA response to renal pelvic administration of substance P, a mediator of the ARNA response to increased renal pelvic pressure. We conclude that the ENaC complex in the pelvic uroepithelium participates in the activation of renal pelvic mechanosensitive neurons.

Temporal decrease in renal sensory responses in rats after chronic ligation of the bile duct

2002 ◽  
Vol 283 (1) ◽  
pp. F164-F172 ◽  
Author(s):  
Ming-Chieh Ma ◽  
Ho-Shiang Huang ◽  
Chiang-Ting Chien ◽  
Ming-Shiou Wu ◽  
Chau-Fong Chen
Keyword(s):  
Bile Duct ◽  
Receptor Activation ◽  
Receptor Protein ◽  
Sensory Receptor ◽  
Mrna Levels ◽  
Sensory Receptors ◽  
Common Bile Duct Ligation ◽  
Renal Nerve ◽  
Duct Ligation ◽  
Sensory Responses

Renal responses to renal sensory receptor activation were examined in rats after 1 and 4 wk of common bile duct ligation (CBDL). Compared with sham-operated rats (Sham), urine and sodium excretion after acute saline loading was significantly reduced at both times after CBDL. The blunted excretory responses in CBDL rats, accompanied by less activation of afferent renal nerve activity (ARNA), were already apparent at 1 wk and became severe at 4 wk. The defect in ARNA activation in CBDL rats was further studied using specific stimuli to activate renal sensory receptors. Graded increases in intrapelvic pressure or renal pelvic perfusion of substance P (SP) elicited an increase in ARNA in Sham rats, these responses being temporally attenuated in CBDL rats. Despite no significant change in renal pelvic SP release, no renorenal reflex was demonstrable in 4-wk CBDL rats. Immunoblotting showed that expression of renal pelvic neurokinin 1 (NK-1) receptors was 32 and 47% lower in 1- and 4-wk CBDL rats, respectively, than in Sham rats, this decrease correlating well with plasma SP levels. The quantitative real-time RT-PCR showed similar levels of NK-1 receptor mRNA in the renal pelvis in the Sham and 4-wk CBDL groups. We conclude that impairment of renal excretory and sensory responses increases with the duration of cirrhosis. An impaired renorenal reflex in cirrhotic rats is involved in the defective activation of the renal sensory receptors could be due, in part, to the low expression of NK-1 receptors, which is dependent on the duration of CBDL. The decrease in NK-1 receptor protein levels is not due to a decrease in mRNA levels.

scholarly journals Heterogeneous immunophenotype of granular lymphocyte expansions: differential expression of the CD8 alpha and CD8 beta chains [see comments]

Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1765-1773 ◽  
Author(s):  
D de Totero ◽  
PL Tazzari ◽  
JP DiSanto ◽  
PF di Celle ◽  
D Raspadori ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Interleukin 2 ◽  
Rare Condition ◽  
Cell Receptor ◽  
Beta Chain ◽  
Phenotypic Analysis ◽  
Gamma Chain ◽  
Membrane Expression ◽  
Variable Regions ◽  
Alpha Beta

Abstract In this study, we have evaluated 14 large granular lymphocyte (LGL) expansions, 11 of which were CD8+. Analysis of the membrane expression of the alpha and beta chains of the CD8 antigen, using specific monoclonal antibodies (MoAbs), has shown that LGL expansions with the CD3+, CD4+, CD8+, CD57+ T-cell receptor (TcR) alpha beta phenotype bear the CD8 alpha/alpha isoform, while the CD3+, CD4-, CD8+, CD57+ TcR alpha beta samples were positive for both the CD8 alpha and CD8 beta chains. These data were confirmed also by messenger RNA analysis. One additional case, with a peculiar phenotype (CD3-, CD2-, CD4-, CD8+, CD57-) and a germline configuration of the TcR beta and gamma chain genes, expressed only the CD8 alpha chain. After additional phenotypic analysis with a wider panel of MoAbs, it was found that the beta chain of the interleukin-2 receptor was constitutively expressed on the majority of the samples tested, and that most of the monoclonal samples coexpressed CD45RA/R0 antigens. Using MoAbs directed against the variable regions of the TcR beta chain, we could show a preferential V beta region restriction in the CD8+ monoclonal cases. This more extensive characterization of CD8+ LGL expansions has further documented the marked heterogeneity within this rare condition and allowed a better phenotypic dissection between the monoclonal and polyclonal cases.

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