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T Bourcier, N Rondeau, S Paquet, P Forgez, A Lombet, F Pouzaud, W Rostène, V Borderie, L Laroche; Expression of Neurotensin Receptors in Human Corneal Keratocytes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3209.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:The aim of the study was to investigate whether cultured human keratocytes express the neurotensin receptors (NTR1, NTR2, and NTR3), to determine the presence of neurotensin (NT) in keratocytes, and to assess the influence of NT on these cells. Methods:Human keratocytes were cultured in culture medium treated with various concentrations (10-7 to 10-9 M) of JMV449 (a weakly degradable NT agonist). Cell proliferation and viability were analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay. Apoptosis was studied by nucleus labeling using a fluorescent dye (Hoescht 33342) and cold light fluorometry. NT, NTR1, NTR2, NTR3 mRNA were detected in human keratocytes by means of reverse transcriptase polymerase chain reaction (RT-PCR). NTR1 protein was detected by Western-blot analysis. Functionality of NTR1 was assessed by intracellular calcium measurement using a dynamic imaging microscopy system. Results:RT-PCR and Western-blot showed the expression of the NTR1 (mRNA and protein), and NTR3 mRNA in human corneal keratocytes. NT and NTR2 mRNA were undetectable. JMV449 induced a rapid and transient intracellular calcium increase in human corneal keratocytes that was blocked by the specific antagonist SR48692. JMV449 significantly increased cell proliferation and viability after 72, 96 and 120 hours of culture with a maximum effect at 10-7 M (p < 0.005). Finally, JMV449 decreased keratocyte apoptosis whatever the concentration used (p < 0.005). Conclusion:These results indicate that cultured human keratocytes express NTR1 and NTR3, and that NT may exert physiological effects on cornea such as regulation of keratocyte proliferation and apoptosis.
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