Involvement of connexin 43 in ATP release from endothelial cells during reoxygenation : Role of PKA signaling pathway
Lade...
Datum
Autor:innen
Betreuer/Gutachter
Weitere Beteiligte
Beteiligte Institutionen
Herausgeber
Zeitschriftentitel
ISSN der Zeitschrift
Bandtitel
Verlag
Lizenz
Zitierlink
Zusammenfassung
Reperfusion-injury impairs endothelial barrier function and may lead to edema formation, impeding recovery of the reperfused heart. Recently is has been shown that ATP, spontaneously released from endothelial cells (EC) during reperfusion protects the endothelial barrier against reperfusion-injury. The aim of the present study was to identify the mechanism of this spontanous ATP release during reoxygenation in endothelia cells. The central hypothesis is that EC release ATP via a connexin-mediated route. It is shown that macrovascular endothelial cells from porcine (PAEC) release ATP via Cx43 hemichannels at the onset of reperfusion. Determination of LDH activity in the culture medium rouled out that the increase in ATP is due to release from lysed cells. Pharmacological inhibitors of connexin function, i.e., mefloquine at concentrations above 10 µM, 200 µM La3+, 50 µM 18-a glycyrrhetinic acid or 50 µM flufenamic acid reduced reperfusion-induced ATP release. In contrast, maneuvers, which inhibit pannexins, like probenecid at concentrations between 10 µM to 1 mM or mefloquine at concentrations below 10 µM, did not affect ATP under the same conditions, indicating that ATP release is mediated via connexins rather than pannexins. These results were confirmed by using a specific Cx43-siRNA transfection strategy. The addition of the connexin hemichannel openers i.e anti-arrythmic peptide 10 (AAP10, 10 µM) or alendronate (10 µM) significantly increased ATP-release compared to the reoxygenated control. Reoxygenation-induced ATP release was also enhanced when a specific PKA-activator like 8-BrcAMP (1 µM), and Sp8CPT (10 µM) was added with the onset of reperfusion, while H-89 (20 µM), KT-5720 (100 nM) as well as two specific protein kinase inhibitor peptides 6-22-amide (100 nM) or 14-22-amide (5 µM) abrogated the effect. In line with that, Cx43 phosphorylation was reduced by PKA inhibitors and increased by activators, indicating that PKA mediated signaling mechanisms are involved in Cx43-mediated ATP release under these conditions. Conclusion: The present study demonstrates that endothelial cells release ATP in a Cx43-dependent process during reoxygenation. Reoxygenation stimulated the cAMP/PKA signaling pathway which targets Cx43 at Ser 368 triggering the ATP release via Cx43. This reoxygenation-induced ATP release is enhanced by two chemically not related connexin channel openers also targeting the cAMP/PKA pathway. Strengthening of this endogenous mechanism by direct activation of cAMP/PKA pathway and temporary opening of Cx43 hemichannels could represent a new therapeutic strategy to prevent acute endothelial barrier failure during reperfusion-injury.Verknüpfung zu Publikationen oder weiteren Datensätzen
Beschreibung
Anmerkungen
Erstpublikation in
Giessen : VVB Laufersweiler