title: [m4 muscarinic receptors of the cornea : muscarinic cholinoceptor-stimulated inhibition of the cAMP-PKA pathway in corneal epithelial and endothelial cells].
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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department für Augenheilkunde, Universität Tübingen, Tübingen, Deutschland. praxis@grueb.eu
authors.forename: M
authors.initials: M
authors.lastname: Grüb
authors.forename: J
authors.initials: J
authors.lastname: Mielke
authors.forename: J M
authors.initials: JM
authors.lastname: Rohrbach
mesh_terms: D000109:Acetylcholine / Q000502:physiology*; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004728:Endothelium, Corneal / Q000473:pathology / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000473:pathology / Q000502:physiology*; D007124:Immunoenzyme Techniques; D008856:Microscopy, Fluorescence; D043585:Receptor, Muscarinic M2 / Q000502:physiology*; D043588:Receptor, Muscarinic M4 / Q000502:physiology*; D015290:Second Messenger Systems / Q000502:physiology; D015398:Signal Transduction
chemical_list: D043585:Receptor, Muscarinic M2; D043588:Receptor, Muscarinic M4; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000109:Acetylcholine