Abstract
Herein, we provide the first evidence on the capsaicin (CPS) receptor vanilloid receptor type-1 (VR1) by rat thymocytes, and its involvement in CPS-induced apoptosis. VR1 mRNA was identified by quantitative RT-PCR in CD5+ thymocytes. By immunofluorescence and flow cytometry, we found that a substantial portion of CD5+ thymocytes, namely CD4+ and double negative (DN) cell subsets, express VR1 that was present on plasma membrane on discrete spots. By Western blot, VR1 protein was identified as a single band of 95 kDa. We also described that CPS could trigger two distinct pathways of thymocyte death, namely apoptosis and necrosis depending on the dose of CPS exposure. CPS-induced apoptosis involved intracellular free calcium (Ca2+) influx, phosphatidylserine exposure, mitochondrial permeability transmembrane pore (PTP) opening and mitochondrial transmembrane potential (ΔΨm) dissipation leading to cytochrome c release, activation of caspase-9 and -3 and oligonucleosomal DNA fragmentation. VR1 was functionally implicated in these events as they were completely abrogated by the VR1 antagonist, capsazepine (CPZ). Finally, we demonstrated that VR1 expression on distinct thymocytes was associated with the selective ability of CPS to trigger DNA fragmentation in VR1+ CD4+ and DN thymocytes. Overall, our results suggest that the expression of VR1 on thymocytes may function as a sensor of harmful stimuli present in the thymic environment.
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Abbreviations
- AEA:
-
anandamide
- AIF:
-
apoptose-inducing factor
- [Ca2+]:
-
intracellular free calcium
- CPS:
-
capsaicin
- CPZ:
-
capsazepine
- CSA:
-
cyclosporin A
- DEPC:
-
diethyl pyrocarbonate
- DMSO:
-
dimethylsulfoxide
- DN:
-
double negative
- DP:
-
double positive
- EDTA:
-
ethilenediaminetetra-acetic acid
- FACS:
-
fluorescent-activated cell sorter
- FCS:
-
fetal calf serum
- FDA:
-
fluorescent diacetate
- FITC:
-
fluorescein isothiocyanate
- GAPDH:
-
glyceraldehyde-3-phosphate dehydrogenase
- HRP:
-
horseradish peroxidase
- JC-1:
-
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanineiodide
- mAb:
-
monoclonal antibody
- MFI:
-
mean fluorescence intensity
- PBMC:
-
peripheral blood mononuclear cells
- PBS:
-
phosphate-buffered saline
- PE:
-
phicoerythrin
- PI:
-
propidium iodide
- PMSF:
-
phenylmethylsulfonylfluoride
- PS:
-
phosphatidylserine
- PTP:
-
permeability transmembrane pore
- RAG:
-
rabbit anti-goat
- VR1:
-
vanilloid receptor type-1
- ΔΨm:
-
mitochondrial transmembrane potential
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Acknowledgements
This study was partially supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MIUR), University of Camerino.
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Amantini, C., Mosca, M., Lucciarini, R. et al. Distinct thymocyte subsets express the vanilloid receptor VR1 that mediates capsaicin-induced apoptotic cell death. Cell Death Differ 11, 1342–1356 (2004). https://doi.org/10.1038/sj.cdd.4401506
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DOI: https://doi.org/10.1038/sj.cdd.4401506
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