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ATP and adenosine in experimental urinary tract infection
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Place, publisher, year, edition, pages
Linnaeus University Press, 2010. , p. 59
Series
Linnaeus University Dissertations ; 4
National Category
Urology and Nephrology
Research subject
Biomedical Sciences, Immunology
Identifiers
URN: urn:nbn:se:lnu:diva-110331Libris ID: 11809099ISBN: 9789186491048 (print)OAI: oai:DiVA.org:lnu-110331DiVA, id: diva2:1637399
Public defence
2010-02-19, N2007, Smålandsgatan 26B, Kalmar, 09:00 (English)
Opponent
Supervisors
Available from: 2022-02-14 Created: 2022-02-14 Last updated: 2022-12-07Bibliographically approved
List of papers
1. Studies of the Extracellular ATP-Adenosine Pathway in Human Urinary Tract Epithelial Cells
Open this publication in new window or tab >>Studies of the Extracellular ATP-Adenosine Pathway in Human Urinary Tract Epithelial Cells
2009 (English)In: Pharmacology, ISSN 0031-7012, E-ISSN 1423-0313, Vol. 84, no 4, p. 196-202Article in journal (Refereed) Published
Abstract [en]

Aims: Extracellular ATP may be metabolized to AMP and adenosine by the ectonucleotidases CD39 and CD73 and, in this study, we characterized the pathways for adenosine formation in human urinary tract epithelial cells. Methods: Bladder (RT4) and kidney (A498) epithelial cells were grown in cell culture and the expression of CD39 and CD73 was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. High-performance liquid chromatography was used to determine adenosine formation in cell medium. Results: RT-PCR and immunohistochemistry revealed a high CD73 and a low CD39 expression in human urinary tract epithelial cells, whereas neutrophils had a higher CD39 than CD73 expression. Adenosine was produced when the cells were exposed to 5'-AMP (substrate for CD73), but not when exposed to 5'-ATP (substrate for CD39). A pronounced inhibition of 5'-AMP-induced adenosine formation by the CD73 inhibitor AMP-CP confirmed the involvement of CD73. Adenosine production from 5'-ATP was slightly increased (p < 0.05) when epithelial cells were cocultured with neutrophils. Conclusions: The data demonstrate that adenosine formation from extracellular ATP is negligible in urinary tract epithelial cells due to low CD39 expression in this cell type. However, the epithelial cells express CD73 and are able to convert extracellular AMP to adenosine.

National Category
Medical and Health Sciences
Research subject
Biomedical Sciences, Pharmacology
Identifiers
urn:nbn:se:lnu:diva-2023 (URN)10.1159/000235908 (DOI)
Available from: 2010-04-06 Created: 2010-04-06 Last updated: 2022-02-14Bibliographically approved
2. Adenosine receptor expression in Escherichia coli-infected and cytokine-stimulated human urinary tract epithelial cells
Open this publication in new window or tab >>Adenosine receptor expression in Escherichia coli-infected and cytokine-stimulated human urinary tract epithelial cells
Show others...
2009 (English)In: BJU International, ISSN 1464-4096, E-ISSN 1464-410X, Vol. 104, no 11, p. 1758-1765Article in journal (Refereed) Published
Abstract [en]

OBJECTIVETo assess the expression and regulation of adenosine receptors in unstimulated, uropathogenic Escherichia coli (UPEC)-infected and cytokine-stimulated human urinary tract epithelial cells, and to examine the regulation of interleukin (IL)-6 secretion in response to A(2A) receptor activation.

MATERIALS AND METHODSHuman urinary tract epithelial cells (A498, T24 and RT4) were grown in cell culture and stimulated with a mixture of pro-inflammatory cytokines (CM) or UPEC. The expression of adenosine receptors was evaluated using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunocytochemistry. IL-6 secretion was measured with an enzyme-linked immunosorbent assay.

RESULTSRT-PCR analysis showed the presence of transcripts for the A(1), A(2A) and A(2B) receptor subtypes but not for the A(3) receptor in A498 kidney epithelial cells. The expression of A(2A) receptor mRNA increased in A498 epithelial cells exposed to CM and UPEC, while A(1) and A(2B) receptor transcripts decreased or remained unchanged. Up-regulation of A(2A) receptors was confirmed at the protein level using Western blot analysis and immunocytochemistry. There was also an increase in A(2A) receptor mRNA in human bladder epithelial cells (T24 and RT4) and in mouse bladder uroepithelium in response to cytokines and UPEC. IL-6 secretion in UPEC-infected A498 cells was decreased by 38% when exposed to the A(2A) receptor agonist CGS 21680.

CONCLUSIONOur data showed a subtype-selective plasticity among adenosine receptors in urinary tract epithelial cells in response to UPEC-infection and cytokines. There was a consistent up-regulation of A(2A) receptors in kidney and bladder epithelial cells. Functionally, A(2A) receptor activation reduced UPEC-induced IL-6 secretion. These findings suggest that adenosine might be a previously unrecognized regulator of the mucosal response in urinary tract infection.

National Category
Medical and Health Sciences
Research subject
Biomedical Sciences, Pharmacology
Identifiers
urn:nbn:se:lnu:diva-2022 (URN)10.1111/j.1464-410X.2009.08638.x (DOI)
Available from: 2010-04-06 Created: 2010-04-06 Last updated: 2022-02-14Bibliographically approved
3. Effects of adenosine A2A and A2B receptor activation on signalling pathways and cytokine production in the human urothelium
Open this publication in new window or tab >>Effects of adenosine A2A and A2B receptor activation on signalling pathways and cytokine production in the human urothelium
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Extracellular adenosine is formed in response to inflammation and the adenosine A2A and A2Breceptor subtypes have both been implicated in modulation of inflammation. In the presentstudy, we examined adenosine A2A and A2B receptor expression and signalling pathways inhuman uroepithelial cells. Modulation of adenosine A2A and A2B receptor activation on theuropathogenic Escherichia coli (UPEC)‐stimulated IL‐8 host response was also evaluated. Thehuman uroepithelial cell line UROtsa was grown in cell culture and stimulated with a mixture ofpro‐inflammatory cytokines (CM) or UPEC. Receptor expression was examined by RT‐PCR andIL‐8, intracellular cAMP and phosphoproteins were measured by ELISA, EIA and multipleximmunoassay (Luminex), respectively. The mRNA expression of the adenosine A2A, but not A2B,receptor was up‐regulated in response to CM and UPEC. The adenosine A2A receptor agonist, CGS21680 did not stimulate cAMP production but CREB phosphorylation was slightly increased. Bycontrast, the adenosine A2 receptor agonist CPCA induced a pronounced cAMP and CREBresponse. Furthermore, adenosine A2A, but not A2B, receptor activation decreased ERK1/2, JNK,p38 and STAT3 phosphorylation. UPEC‐infection stimulated the host IL‐8 production but CPCAor CGS 21680 had no impact on basal or UPEC‐evoked IL‐8 production. In conclusion, our dataidentified marked differences in signalling pathways by activation of the adenosine A2A and A2Breceptors. Activation of the adenosine A2A receptor inhibited STAT3 and MAPK‐signalling, whilethe cAMP‐CREB pathway was induced by adenosine A2B receptor activation. No anti‐ or proinflammatoryeffects were found for uroepithelial adenosine A2A or A2B receptors.

National Category
Medical and Health Sciences
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-11744 (URN)
Available from: 2011-12-16 Created: 2011-05-18 Last updated: 2022-02-14Bibliographically approved
4. Activation of adenosine A2A receptors inhibit neutrophil transuroepithelial migration
Open this publication in new window or tab >>Activation of adenosine A2A receptors inhibit neutrophil transuroepithelial migration
(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-11746 (URN)
Available from: 2011-06-16 Created: 2011-05-18 Last updated: 2022-02-14Bibliographically approved
5. Extracellular ATP and P2Y Receptor Activation Induce a Proinflammatory Host Response in the Human Urinary Tract
Open this publication in new window or tab >>Extracellular ATP and P2Y Receptor Activation Induce a Proinflammatory Host Response in the Human Urinary Tract
2010 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 78, no 8, p. 3609-3615Article in journal (Refereed) Published
Abstract [en]

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-gamma-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-gamma-S (10 and 100 mu M) 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-gamma-S-induced IL-8 release. The potency profile further suggested involvement of P2Y(1), P2Y(2), and/or P2Y(11) 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.

National Category
Medical and Health Sciences
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-16181 (URN)10.1128/IAI.00074-10 (DOI)000279990400029 ()2-s2.0-77955298675 (Scopus ID)
Available from: 2011-12-16 Created: 2011-12-16 Last updated: 2022-02-14Bibliographically approved

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