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Involvement of CD73 (ecto-5'-nucleotidase) in Adenosine Generation by Human Gingival Fibroblasts

¹ Department of Periodontology, Division of Oral Biology and Disease Control, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan; and
² Oklahoma Medical Research Foundation, Immunobiology and Cancer Program, Oklahoma City, OK 73104, USA;

View larger version (36K): [in this window] [in a new window]   Figure 1. Surface expression of CD73 on HGF. (A) Analysis of surface expression of CD73 on HGF by FCM. HGF were stained with anti-human CD73 Ab (solid line) plus FITC-goat anti-mouse IgG. Staining with the isotype-matched control is shown in the shaded peak. The data shown are representative of three separate experiments. (B) Cytochemical localization of CD73 on HGF. HGF cultured in the glass-bottomed dish were stained with anti-human CD73 mAb, fixed in 4% paraformaldehyde, incubated with Alexa Fluor 594 goat anti-mouse IgG, and counterstained with DAPI. The data shown are representative of three separate experiments. Scale bars = 100 µm.

View larger version (21K): [in this window] [in a new window]   Figure 2. Adenosine levels converted from 5'-AMP by ecto-5'-nucleotidase (CD73) on HGF. (A) Kinetics of extracellular adenosine levels. HGF were treated with 300 nM 5'-AMP for different time intervals. The data shown are representative of three separate experiments. (B) Effects of AOPCP on extracellular adenosine levels. HGF were treated with 20 µM 5'-AMP in the presence or absence of 0.2 µM, 2 µM, 20 µM, and 200 µM AOPCP for 10 min. The data shown are the mean ± SD of 4 adenosine concentration determinations and are representative of three separate experiments. *p < 0.05 compared with group of AMP.

View larger version (26K): [in this window] [in a new window]   Figure 3. cAMP responses to adenosine converted from 5'-AMP by CD73. (A) Cells were pre-incubated with 10 µM 4-(3-butoxy-4-methoxy-benzyl)imidazolidin-2-one for 10 min at 37°C and stimulated with 20 µM adenosine (open bar) or 5'-AMP (closed bar) for 5 min at 37°C in the presence or absence of 10 µM XAC. The data shown are the mean ± SD of 4 cAMP determinations and are representative of three separate experiments. *p < 0.05 compared with the adenosine group; **p < 0.05 compared with the AMP group. (B) Effects of AOPCP on cAMP responses. HGF were treated with 20 µM 5'-AMP in the presence or absence of 0.2 µM, 2 µM, 20 µM, and 200 µM AOPCP for 10 min. The data shown are the mean ± SD of 4 cAMP determinations and are representative of three separate experiments. *p < 0.05 compared with the AMP group.

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