Zusammenfassung
Hintergrund
Muskarinerge Cholinozeptoren lassen sich im gesamten Auge, nicht zuletzt in der Hornhaut nachweisen. Die jüngste Forschung hat sich überwiegend mit dem m5-Subtyp beschäftigt, obwohl mehrfach auch über das Vorkommen des m2- bzw. m4-Subtypes in kornealen Geweben berichtet wurde. Ziel der vorliegenden Arbeit war der Nachweis einer verminderten cAMP-Bildung und einer reduzierten Proteinkinase-A-Aktivität (PKA) nach Stimulation von m2- bzw. m4-Cholinozeptoren in bovinem Hornhautepithel und -endothel.
Material und Methoden
Der Nachweis muskarinerger Cholinozeptoren erfolgte mittels polyklonaler Antikörper. Die cAMP-Konzentration wurde mit einem handelsüblichen Enzymimmunoassay bestimmt, die PKA-Aktivität anhand des Verbrauchs von ATP ermittelt.
Ergebnisse
Immunhistochemie, Immunfluoreszenz und Western-Blot zeigten das Vorkommen des m4-Cholinozeptors. Der m2-Subtyp ließ sich an bovinen kornealen Epithel- und Endothelzellen nicht nachweisen. Ferner ließ sich eine dosisabhängige Hemmung der cAMP-Bildung sowie der PKA-Aktivität nach Stimulation mit Acetylcholin aufzeigen (p<0,001).
Schlussfolgerung
Die vorliegenden Ergebnisse zeigen eine Hemmung des cAMP-PKA-Signalweges nach Stimulation muskarinerger m4-Cholinozeptoren in bovinen Hornhautepithel und -endothelzellen. Über die physiologische Rolle dieses Signalweges kann allerdings derzeit nur spekuliert werden.
Abstract
Background
Muscarinic cholinoceptors have been found in all types of ocular tissue, e.g. in corneal epithelium and endothelium. Latest research has focused only on the m5 cholinoceptor subtype. However, previous studies have also indicated the presence of m2 or m4 receptor subtypes in corneal tissue. The aim of this study was to show the decrease of intracellular cAMP formation and protein kinase A (PKA) activity after stimulation of m2 or m4 cholinoceptors in bovine corneal epithelial and endothelial cells.
Materials und methods
Muscarinic cholinoceptors were studied using polyclonal antibodies. The cAMP concentration was determined with an enzyme immunoassay and PKA activity was estimated by the consumption of ATP.
Results
Immunocytochemistry, immunofluorescence and immunoblotting revealed the presence of the m4 muscarinic cholinoceptor subtype but not of the m2 receptor subtype in bovine corneal epithelial and endothelial cells. In bovine corneal epithelium and endothelium protein cAMP formation was decreased and PKA activity was inhibited by acetylcholine in a dose-dependent manner (p<0.001).
Conclusion
The findings indicate that stimulation of m4 muscarinic cholinoceptors inhibits the cAMP-PKA pathway in corneal epithelial and endothelial cells resulting in decreased protein kinase A activity. Further work will be needed to clarify the physiological role of this signaling pathway in corneal epithelium and endothelium.
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Grüb, M., Mielke, J. & Rohrbach, J. m4-muskarinerge Rezeptoren der Hornhaut. Ophthalmologe 108, 651–657 (2011). https://doi.org/10.1007/s00347-011-2356-3
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DOI: https://doi.org/10.1007/s00347-011-2356-3