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Activation of Adenosine Receptor on 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 (10K): [in a new window]   Figure 1. FACS analysis of surface expression of CD26 and ecto-ADA on human gingival fibroblasts. (A) Human gingival fibroblasts (1 x 106 cells) were incubated with (bold line) or without (solid line) Jurkat cell lysate for 30 min at 37°C, washed twice, and stained with anti-ADA plus FITC-donkey anti-goat IgG. Normal goat IgG was utilized as a control (dotted line). (B) Untreated human gingival fibroblasts were stained with PE-anti-human CD26 mAb (solid line) or isotype-matched murine myeloma protein (dotted line). The data shown are representative of 3 separate experiments.

View larger version (29K): [in a new window]   Figure 2. Regulation of adenosine receptor engagement by ecto-ADA. (A) Regulation of cAMP responses by ecto-ADA. Human gingival fibroblasts (5.0 x 105 cells/well) were incubated with or without Jurkat cell lysate for 30 min at 37°C and stimulated with 20 µM adenosine for 5 min at 37°C in the presence or absence of 5 µM dCF. The data shown are the mean ± SD of 4 cAMP determinations and are representative of 3 separate experiments. *p < 0.05 compared with the lysate (–) dCF (–) group. **p < 0.05 compared with the lysate (+) dCF (–) group. (B) Effects of adenosine (Ado) on HAS mRNA expression in human gingival fibroblasts. Human gingival fibroblasts were cultured with or without Ado (20 µM) for 3 hrs, and then RT-PCR was carried out for the detection of HAS1, HAS2, and HAS3 mRNA expression. The results shown are representative of 3 separate experiments. The number of PCR cycles is shown above each lane. Each band was standardized against the amount of HPRT. (C) Effects of ecto-ADA on adenosine (Ado)-induced HAS1 mRNA expression in human gingival fibroblasts. Human gingival fibroblasts were pre-incubated with or without Jurkat cell lysate for 30 min at 37°C, and were treated with Ado (20 µM) with or without dCF (5 µM) for 2.5 hrs. HAS1 mRNA expression was examined by RT-PCR. Results of 1 representative experiment from among 3 identical experiments are shown. The number of PCR cycles is shown above each lane. Each band was standardized against the amount of HPRT.

View larger version (16K): [in a new window]   Figure 3. Regulation of cAMP responses by ecto-ADA and CD73. Human gingival fibroblasts (5.0 x 105 cells/well) were incubated with or without Jurkat lysate for 30 min at 37°C and stimulated with 20 µM AMP for 5 min at 37°C in the presence or absence of 5 µM dCF. The data shown are the mean ± SD of 4 cAMP determinations and are representative of 3 separate experiments. *p < 0.05 compared with the lysate (–) dCF (–) group. **p < 0.05 compared with the lysate (+) dCF (–) group.

View larger version (42K): [in a new window]   Figure 4. Regulation of adenosine receptor engagement by ecto-ADA and CD73. (A) Effect of XAC on adenosine (Ado) or AMP-induced HAS1 mRNA expression in human gingival fibroblasts. Human gingival fibroblasts (5.0 x 105 cells/well) were treated with Ado (20 µM) or AMP (20 µM) for 2.5 hrs in the presence or absence of XAC (5 µM). HAS1 mRNA expression in human gingival fibroblasts was examined by RT-PCR. The results shown are representative of 3 separate experiments. The number of PCR cycles is shown above each lane. Each band was standardized against the amount of HPRT. (B) Effects of ecto-ADA on AMP-induced HAS1 mRNA expression in human gingival fibroblasts. Human gingival fibroblasts were pre-incubated with or without Jurkat lysate for 30 min at 37°C, and treated with AMP (20 µM) in the presence and absence of dCF (5 µM) for 2.5 hrs. HAS1 mRNA expression in human gingival fibroblasts was examined by RT-PCR. The results shown are representative of 3 separate experiments. The number of PCR cycles is shown above each lane. Each band was standardized against the amount of HPRT.