Robert Kruse1, Susanne Säve, Katarina Persson. 1. School of Health and Medical Sciences, Örebro University, Örebro and School of Natural Sciences, Kalmar, Sweden. Robert.Kruse@oru.se
Abstract
PURPOSE: We characterized and identified the uroepithelial P2 receptor responsible for adenosine triphosphate mediated release of the cytokines interleukin-8 and 6. MATERIALS AND METHODS: The human renal epithelial cell line A498 (ATCC™) was cultured and stimulated with different purinergic agonists with or without prior inhibition with different antagonists or signaling pathway inhibitors. Supernatant was analyzed for interleukin-8 and 6 by enzyme-linked immunosorbent assay. P2 receptor mRNA expression was assessed by real-time reverse transcriptase-polymerase chain reaction. The candidate receptor was knocked down with siRNA technology. Interleukin-8 and 6 responses were measured after purinergic stimulation of knocked down cells. RESULTS: ATP and ATP-γ-S (Roche Diagnostics, Mannheim, Germany) were equipotent as inducers of interleukin-8 and 6 release. Agonist profile experiments using different P2 receptor agonists indicated that P2Y(2) was the main contributor to this release, although P2Y(11) and P2X(7) activation could not be excluded. Signaling pathway experiments showed that interleukin-8 release involved phospholipase C and inositol trisphosphate mediated signaling, indicating a P2Y receptor subtype. Antagonist experiments indicated P2Y(2) as the responsible receptor. Gene expression analysis of P2 receptors showed that strong expression of P2Y(2) receptor and subsequent knockdown of P2Y(2) receptor mRNA for 72 and 96 hours abrogated interleukin-8 and 6 release after purinergic stimulation with adenosine triphosphate-γ-S. CONCLUSIONS: Interleukin-8 and 6 release after purinergic stimulation in uroepithelial A498 cells is mediated through P2Y(2) receptor activation.
PURPOSE: We characterized and identified the uroepithelial P2 receptor responsible for adenosine triphosphate mediated release of the cytokines interleukin-8 and 6. MATERIALS AND METHODS: The human renal epithelial cell line A498 (ATCC™) was cultured and stimulated with different purinergic agonists with or without prior inhibition with different antagonists or signaling pathway inhibitors. Supernatant was analyzed for interleukin-8 and 6 by enzyme-linked immunosorbent assay. P2 receptor mRNA expression was assessed by real-time reverse transcriptase-polymerase chain reaction. The candidate receptor was knocked down with siRNA technology. Interleukin-8 and 6 responses were measured after purinergic stimulation of knocked down cells. RESULTS:ATP and ATP-γ-S (Roche Diagnostics, Mannheim, Germany) were equipotent as inducers of interleukin-8 and 6 release. Agonist profile experiments using different P2 receptor agonists indicated that P2Y(2) was the main contributor to this release, although P2Y(11) and P2X(7) activation could not be excluded. Signaling pathway experiments showed that interleukin-8 release involved phospholipase C and inositol trisphosphate mediated signaling, indicating a P2Y receptor subtype. Antagonist experiments indicated P2Y(2) as the responsible receptor. Gene expression analysis of P2 receptors showed that strong expression of P2Y(2) receptor and subsequent knockdown of P2Y(2) receptor mRNA for 72 and 96 hours abrogated interleukin-8 and 6 release after purinergic stimulation with adenosine triphosphate-γ-S. CONCLUSIONS:Interleukin-8 and 6 release after purinergic stimulation in uroepithelial A498 cells is mediated through P2Y(2) receptor activation.