PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells
<p<Abstract</p< <p<Background</p< <p<Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G<sub<q</sub<-and G<su...
Ausführliche Beschreibung
Autor*in: |
Halayko Andrew J [verfasserIn] Gosens Reinoud [verfasserIn] Elzinga Carolina RS [verfasserIn] Menzen Mark H [verfasserIn] Kistemaker Loes EM [verfasserIn] Roscioni Sara S [verfasserIn] Meurs Herman [verfasserIn] Schmidt Martina [verfasserIn] |
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Format: |
E-Artikel |
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Sprache: |
Englisch |
Erschienen: |
2009 |
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Übergeordnetes Werk: |
In: Respiratory Research - BMC, 2003, 10(2009), 1, p 88 |
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Übergeordnetes Werk: |
volume:10 ; year:2009 ; number:1, p 88 |
Links: |
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DOI / URN: |
10.1186/1465-9921-10-88 |
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Katalog-ID: |
DOAJ066084261 |
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520 | |a <p<Abstract</p< <p<Background</p< <p<Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G<sub<q</sub<-and G<sub<s</sub<-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.</p< <p<Methods</p< <p<IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors <it<Clostridium difficile </it<toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.</p< <p<Results</p< <p<The β<sub<2</sub<-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-<it<O</it<-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-<it<O</it<-Me-cAMPS mimicked the effects of 8-pCPT-2'-<it<O</it<-Me-cAMP, whereas the negative control 8-pCPT-2'-<it<O</it<-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-<it<O</it<-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.</p< <p<Conclusion</p< <p<Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.</p< | ||
653 | 0 | |a Diseases of the respiratory system | |
700 | 0 | |a Gosens Reinoud |e verfasserin |4 aut | |
700 | 0 | |a Elzinga Carolina RS |e verfasserin |4 aut | |
700 | 0 | |a Menzen Mark H |e verfasserin |4 aut | |
700 | 0 | |a Kistemaker Loes EM |e verfasserin |4 aut | |
700 | 0 | |a Roscioni Sara S |e verfasserin |4 aut | |
700 | 0 | |a Meurs Herman |e verfasserin |4 aut | |
700 | 0 | |a Schmidt Martina |e verfasserin |4 aut | |
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10.1186/1465-9921-10-88 doi (DE-627)DOAJ066084261 (DE-599)DOAJ1eac4599a13a41f5ae2bffd7434a3893 DE-627 ger DE-627 rakwb eng RC705-779 Halayko Andrew J verfasserin aut PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells 2009 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier <p<Abstract</p< <p<Background</p< <p<Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G<sub<q</sub<-and G<sub<s</sub<-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.</p< <p<Methods</p< <p<IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors <it<Clostridium difficile </it<toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.</p< <p<Results</p< <p<The β<sub<2</sub<-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-<it<O</it<-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-<it<O</it<-Me-cAMPS mimicked the effects of 8-pCPT-2'-<it<O</it<-Me-cAMP, whereas the negative control 8-pCPT-2'-<it<O</it<-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-<it<O</it<-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.</p< <p<Conclusion</p< <p<Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.</p< Diseases of the respiratory system Gosens Reinoud verfasserin aut Elzinga Carolina RS verfasserin aut Menzen Mark H verfasserin aut Kistemaker Loes EM verfasserin aut Roscioni Sara S verfasserin aut Meurs Herman verfasserin aut Schmidt Martina verfasserin aut In Respiratory Research BMC, 2003 10(2009), 1, p 88 (DE-627)326646485 (DE-600)2041675-1 1465993X nnns volume:10 year:2009 number:1, p 88 https://doi.org/10.1186/1465-9921-10-88 kostenfrei https://doaj.org/article/1eac4599a13a41f5ae2bffd7434a3893 kostenfrei http://respiratory-research.com/content/10/1/88 kostenfrei https://doaj.org/toc/1465-9921 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2153 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2009 1, p 88 |
spelling |
10.1186/1465-9921-10-88 doi (DE-627)DOAJ066084261 (DE-599)DOAJ1eac4599a13a41f5ae2bffd7434a3893 DE-627 ger DE-627 rakwb eng RC705-779 Halayko Andrew J verfasserin aut PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells 2009 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier <p<Abstract</p< <p<Background</p< <p<Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G<sub<q</sub<-and G<sub<s</sub<-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.</p< <p<Methods</p< <p<IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors <it<Clostridium difficile </it<toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.</p< <p<Results</p< <p<The β<sub<2</sub<-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-<it<O</it<-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-<it<O</it<-Me-cAMPS mimicked the effects of 8-pCPT-2'-<it<O</it<-Me-cAMP, whereas the negative control 8-pCPT-2'-<it<O</it<-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-<it<O</it<-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.</p< <p<Conclusion</p< <p<Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.</p< Diseases of the respiratory system Gosens Reinoud verfasserin aut Elzinga Carolina RS verfasserin aut Menzen Mark H verfasserin aut Kistemaker Loes EM verfasserin aut Roscioni Sara S verfasserin aut Meurs Herman verfasserin aut Schmidt Martina verfasserin aut In Respiratory Research BMC, 2003 10(2009), 1, p 88 (DE-627)326646485 (DE-600)2041675-1 1465993X nnns volume:10 year:2009 number:1, p 88 https://doi.org/10.1186/1465-9921-10-88 kostenfrei https://doaj.org/article/1eac4599a13a41f5ae2bffd7434a3893 kostenfrei http://respiratory-research.com/content/10/1/88 kostenfrei https://doaj.org/toc/1465-9921 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2153 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2009 1, p 88 |
allfields_unstemmed |
10.1186/1465-9921-10-88 doi (DE-627)DOAJ066084261 (DE-599)DOAJ1eac4599a13a41f5ae2bffd7434a3893 DE-627 ger DE-627 rakwb eng RC705-779 Halayko Andrew J verfasserin aut PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells 2009 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier <p<Abstract</p< <p<Background</p< <p<Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G<sub<q</sub<-and G<sub<s</sub<-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.</p< <p<Methods</p< <p<IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors <it<Clostridium difficile </it<toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.</p< <p<Results</p< <p<The β<sub<2</sub<-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-<it<O</it<-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-<it<O</it<-Me-cAMPS mimicked the effects of 8-pCPT-2'-<it<O</it<-Me-cAMP, whereas the negative control 8-pCPT-2'-<it<O</it<-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-<it<O</it<-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.</p< <p<Conclusion</p< <p<Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.</p< Diseases of the respiratory system Gosens Reinoud verfasserin aut Elzinga Carolina RS verfasserin aut Menzen Mark H verfasserin aut Kistemaker Loes EM verfasserin aut Roscioni Sara S verfasserin aut Meurs Herman verfasserin aut Schmidt Martina verfasserin aut In Respiratory Research BMC, 2003 10(2009), 1, p 88 (DE-627)326646485 (DE-600)2041675-1 1465993X nnns volume:10 year:2009 number:1, p 88 https://doi.org/10.1186/1465-9921-10-88 kostenfrei https://doaj.org/article/1eac4599a13a41f5ae2bffd7434a3893 kostenfrei http://respiratory-research.com/content/10/1/88 kostenfrei https://doaj.org/toc/1465-9921 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2153 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2009 1, p 88 |
allfieldsGer |
10.1186/1465-9921-10-88 doi (DE-627)DOAJ066084261 (DE-599)DOAJ1eac4599a13a41f5ae2bffd7434a3893 DE-627 ger DE-627 rakwb eng RC705-779 Halayko Andrew J verfasserin aut PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells 2009 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier <p<Abstract</p< <p<Background</p< <p<Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G<sub<q</sub<-and G<sub<s</sub<-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.</p< <p<Methods</p< <p<IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors <it<Clostridium difficile </it<toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.</p< <p<Results</p< <p<The β<sub<2</sub<-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-<it<O</it<-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-<it<O</it<-Me-cAMPS mimicked the effects of 8-pCPT-2'-<it<O</it<-Me-cAMP, whereas the negative control 8-pCPT-2'-<it<O</it<-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-<it<O</it<-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.</p< <p<Conclusion</p< <p<Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.</p< Diseases of the respiratory system Gosens Reinoud verfasserin aut Elzinga Carolina RS verfasserin aut Menzen Mark H verfasserin aut Kistemaker Loes EM verfasserin aut Roscioni Sara S verfasserin aut Meurs Herman verfasserin aut Schmidt Martina verfasserin aut In Respiratory Research BMC, 2003 10(2009), 1, p 88 (DE-627)326646485 (DE-600)2041675-1 1465993X nnns volume:10 year:2009 number:1, p 88 https://doi.org/10.1186/1465-9921-10-88 kostenfrei https://doaj.org/article/1eac4599a13a41f5ae2bffd7434a3893 kostenfrei http://respiratory-research.com/content/10/1/88 kostenfrei https://doaj.org/toc/1465-9921 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2153 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2009 1, p 88 |
allfieldsSound |
10.1186/1465-9921-10-88 doi (DE-627)DOAJ066084261 (DE-599)DOAJ1eac4599a13a41f5ae2bffd7434a3893 DE-627 ger DE-627 rakwb eng RC705-779 Halayko Andrew J verfasserin aut PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells 2009 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier <p<Abstract</p< <p<Background</p< <p<Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G<sub<q</sub<-and G<sub<s</sub<-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.</p< <p<Methods</p< <p<IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors <it<Clostridium difficile </it<toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.</p< <p<Results</p< <p<The β<sub<2</sub<-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-<it<O</it<-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-<it<O</it<-Me-cAMPS mimicked the effects of 8-pCPT-2'-<it<O</it<-Me-cAMP, whereas the negative control 8-pCPT-2'-<it<O</it<-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-<it<O</it<-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.</p< <p<Conclusion</p< <p<Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.</p< Diseases of the respiratory system Gosens Reinoud verfasserin aut Elzinga Carolina RS verfasserin aut Menzen Mark H verfasserin aut Kistemaker Loes EM verfasserin aut Roscioni Sara S verfasserin aut Meurs Herman verfasserin aut Schmidt Martina verfasserin aut In Respiratory Research BMC, 2003 10(2009), 1, p 88 (DE-627)326646485 (DE-600)2041675-1 1465993X nnns volume:10 year:2009 number:1, p 88 https://doi.org/10.1186/1465-9921-10-88 kostenfrei https://doaj.org/article/1eac4599a13a41f5ae2bffd7434a3893 kostenfrei http://respiratory-research.com/content/10/1/88 kostenfrei https://doaj.org/toc/1465-9921 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2153 GBV_ILN_2190 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2009 1, p 88 |
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Halayko Andrew J @@aut@@ Gosens Reinoud @@aut@@ Elzinga Carolina RS @@aut@@ Menzen Mark H @@aut@@ Kistemaker Loes EM @@aut@@ Roscioni Sara S @@aut@@ Meurs Herman @@aut@@ Schmidt Martina @@aut@@ |
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<?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>01000caa a22002652 4500</leader><controlfield tag="001">DOAJ066084261</controlfield><controlfield tag="003">DE-627</controlfield><controlfield tag="005">20230309054846.0</controlfield><controlfield tag="007">cr uuu---uuuuu</controlfield><controlfield tag="008">230228s2009 xx |||||o 00| ||eng c</controlfield><datafield tag="024" ind1="7" ind2=" "><subfield code="a">10.1186/1465-9921-10-88</subfield><subfield code="2">doi</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-627)DOAJ066084261</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-599)DOAJ1eac4599a13a41f5ae2bffd7434a3893</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-627</subfield><subfield code="b">ger</subfield><subfield code="c">DE-627</subfield><subfield code="e">rakwb</subfield></datafield><datafield tag="041" ind1=" " ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="050" ind1=" " ind2="0"><subfield code="a">RC705-779</subfield></datafield><datafield tag="100" ind1="0" ind2=" "><subfield code="a">Halayko Andrew J</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">2009</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="a">Text</subfield><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="a">Computermedien</subfield><subfield code="b">c</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="a">Online-Ressource</subfield><subfield code="b">cr</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a"><p<Abstract</p< <p<Background</p< <p<Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G<sub<q</sub<-and G<sub<s</sub<-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.</p< <p<Methods</p< <p<IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors <it<Clostridium difficile </it<toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.</p< <p<Results</p< <p<The β<sub<2</sub<-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-<it<O</it<-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-<it<O</it<-Me-cAMPS mimicked the effects of 8-pCPT-2'-<it<O</it<-Me-cAMP, whereas the negative control 8-pCPT-2'-<it<O</it<-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-<it<O</it<-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.</p< <p<Conclusion</p< <p<Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.</p<</subfield></datafield><datafield tag="653" ind1=" " ind2="0"><subfield code="a">Diseases of the respiratory system</subfield></datafield><datafield tag="700" ind1="0" ind2=" "><subfield code="a">Gosens Reinoud</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="0" ind2=" "><subfield code="a">Elzinga Carolina RS</subfield><subfield code="e">verfasserin</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="0" ind2=" "><subfield code="a">Menzen Mark 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Halayko Andrew J |
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RC705-779 PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells |
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PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells |
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Halayko Andrew J Gosens Reinoud Elzinga Carolina RS Menzen Mark H Kistemaker Loes EM Roscioni Sara S Meurs Herman Schmidt Martina |
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pka and epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells |
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RC705-779 |
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PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells |
abstract |
<p<Abstract</p< <p<Background</p< <p<Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G<sub<q</sub<-and G<sub<s</sub<-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.</p< <p<Methods</p< <p<IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors <it<Clostridium difficile </it<toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.</p< <p<Results</p< <p<The β<sub<2</sub<-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-<it<O</it<-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-<it<O</it<-Me-cAMPS mimicked the effects of 8-pCPT-2'-<it<O</it<-Me-cAMP, whereas the negative control 8-pCPT-2'-<it<O</it<-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-<it<O</it<-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.</p< <p<Conclusion</p< <p<Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.</p< |
abstractGer |
<p<Abstract</p< <p<Background</p< <p<Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G<sub<q</sub<-and G<sub<s</sub<-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.</p< <p<Methods</p< <p<IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors <it<Clostridium difficile </it<toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.</p< <p<Results</p< <p<The β<sub<2</sub<-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-<it<O</it<-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-<it<O</it<-Me-cAMPS mimicked the effects of 8-pCPT-2'-<it<O</it<-Me-cAMP, whereas the negative control 8-pCPT-2'-<it<O</it<-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-<it<O</it<-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.</p< <p<Conclusion</p< <p<Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.</p< |
abstract_unstemmed |
<p<Abstract</p< <p<Background</p< <p<Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G<sub<q</sub<-and G<sub<s</sub<-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.</p< <p<Methods</p< <p<IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors <it<Clostridium difficile </it<toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.</p< <p<Results</p< <p<The β<sub<2</sub<-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-<it<O</it<-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-<it<O</it<-Me-cAMPS mimicked the effects of 8-pCPT-2'-<it<O</it<-Me-cAMP, whereas the negative control 8-pCPT-2'-<it<O</it<-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-<it<O</it<-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.</p< <p<Conclusion</p< <p<Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.</p< |
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PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells |
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https://doi.org/10.1186/1465-9921-10-88 https://doaj.org/article/1eac4599a13a41f5ae2bffd7434a3893 http://respiratory-research.com/content/10/1/88 https://doaj.org/toc/1465-9921 |
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Gosens Reinoud Elzinga Carolina RS Menzen Mark H Kistemaker Loes EM Roscioni Sara S Meurs Herman Schmidt Martina |
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Gosens Reinoud Elzinga Carolina RS Menzen Mark H Kistemaker Loes EM Roscioni Sara S Meurs Herman Schmidt Martina |
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