Abstract
The activity of substance P (SP) in the rat thymus seems to be tightly controlled by its bioavailability. In this study, we provide evidence for the expression of the SP-degrading enzyme, neutral endopeptidase (NEP)/CD10, by rat thymocyte subsets, and we illustrate its involvement in the in vivo SP/neurokinin-1 receptor (NK1R)-mediated regulation of thymocyte survival and proliferation. NEP/CD10 was expressed at both mRNA and protein levels on a substantial portion (45.5%) of CD5+ thymocytes, namely on the CD4+CD8+ (double positive; DP) and CD4+ subsets. Continuous administration of thiorphan, a specific NEP/CD10 inhibitor, by means of miniosmotic pumps, enhanced rat thymocyte preprotachykinin-A (PPT-A) and NK1R mRNA expression as well as SP and NK1R protein levels in an NK1R-dependent manner. Thiorphan increased CD10+CD4+ and CD10+DP thymocyte numbers, and an NK1R antagonist, (S)1-{2-[3(3-4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)-piperidine-3-yl]ethyl}-4-pheny-1-azoniabicyclo[2.2.2]octane, chloride (SR140333), abrogated these stimulatory effects. In addition, the NEP/CD10 inhibitor stimulated interleukin (IL)-2 production, IL-2 receptor α chain expression, and concanavalin A-induced proliferation of CD5+ thymocytes, and it inhibited spontaneous and NK1R-dependent thymocyte apoptosis. The thiorphan-protective antiapoptotic and proliferative effects involved the activation of Akt serine-threonine kinase, subsequent up-regulation of survivin mRNA, down-regulation of procaspase-3 mRNA levels, and suppression of caspase-3 activity, which were inhibited by SR140333 and mimicked by exogenous SP administration. Overall, our findings suggest that by controlling SP availability, NEP/CD10 negatively regulates thymocyte homeostasis and development.
Footnotes
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This work was supported in part by the University of Camerino.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.138719.
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ABBREVIATIONS: NEP, neutral endopeptidase; ET-1, endothelin-1; TMPO, thymopoietin; SP, substance P; CGRP, calcitonin gene-related peptide; NK1R, neurokinin-1 receptor; PPT-A, preprotachykinin A; DP, double positive; SR140333, (S)1-{2-[3(3-4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidine-3-yl]ethyl}-4-pheny-1-azoniabicyclo[2.2.2]octane, chloride; PI3-K, phosphatidylinositol-3 kinase; IAP, inhibitor of apoptosis; FACS, fluorescent-activated cell sorting; mAbs, monoclonal antibodies; PE, phycoerythrin; FITC, fluorescein isothiocyanate; RAG, rabbit anti-goat; GAR, goat anti-rabbit; HRP, horseradish peroxidase; Ab, antibody; PAGE, polyacrylamide gel electrophoresis; ConA, concanavalin A; [3H]TdR, tritiated thymidine; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; AnnV, annexin V; PI, propidium iodine; PCR, polymerase chain reaction; ANOVA, analysis of variance; RT, real time; IL-2R, IL-2 receptor.
- Received March 7, 2008.
- Accepted July 9, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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