Antiapoptotic Effect of Acetylcholine in Fas-Induced Apoptosis in Human Keratocytes.
OBJECTIVE
To investigate the possible antiapoptotic effect of acetylcholine (ACh) in Fas-mediated apoptosis of primary human keratocytes in vitro, and to explore the underlying mechanism.


METHODS
Primary human keratocytes were isolated from healthy corneas. Fas ligand (FasL) was used to induce apoptosis in keratocytes. Cell death was assessed by ELISA. Activity of caspase-3, -7, -8, and -9 was measured with luminescent caspase activity assays. Expression of nuclear factor-κB (NF-κB) gene was assessed with RT-quantitative (q)PCR. Cytochrome c release apoptosis assay kit was used to extract mitochondria and cytosol. Cytochrome c release, cleavage of Bid, and expression of B-cell lymphoma 2 (Bcl-2) were determined by Western blot.


RESULTS
Cell death ELISA revealed that ACh is able to reduce Fas-induced apoptosis in keratocytes. Analysis of the activity of effector caspases-3 and -7 showed that ACh, when added to Fas-treated cells, decreases the activation of both these enzymes. The activity of initiator caspases -8 and -9 also decreased when ACh was added to Fas-treated cells. This antiapoptotic effect of ACh was dependent on ACh concentration and activation of muscarinic ACh receptors. Analysis of the antiapoptotic mechanisms triggered by ACh showed that ACh downregulates expression of FasL-induced NF-κB RNA expression, upregulates expression of antiapoptotic protein Bcl-2, downregulates expression of proapoptotic protein Bad, reduces cytochrome c release, and prevents proapoptotic Bid protein cleavage.


CONCLUSIONS
Acetylcholine has an antiapoptotic effect in a Fas-apoptosis model of human primary keratocytes in vitro. It is therefore possible that ACh may play a role in corneal wound healing, by modulating its initiation phase.
Department of Integrative Medical Biology, Umeå University, Umeå, Sweden 2Department of Clinical Sciences, Ophthalmology, Umeå University, Umeå, Sweden.
Marta
M
Sloniecka
Department of Integrative Medical Biology, Umeå University, Umeå, Sweden.
Ludvig J
LJ
Backman
Department of Integrative Medical Biology, Umeå University, Umeå, Sweden 2Department of Clinical Sciences, Ophthalmology, Umeå University, Umeå, Sweden.
Patrik
P
Danielson
D000109:Acetylcholine / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D060527:Corneal Keratocytes / Q000187:drug effects*; D045304:Cytochromes c / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D053222:Fas Ligand Protein / Q000494:pharmacology; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D014945:Wound Healing / Q000502:physiology
C000595707:BCL2 protein, human; D053222:Fas Ligand Protein; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D045304:Cytochromes c; D020169:Caspases; D000109:Acetylcholine