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Activation of Adenosine-receptor-enhanced iNOS mRNA Expression by Gingival Epithelial Cells

Department of Periodontology, Division of Oral Biology and Disease Control, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan;

View larger version (31K): [in a new window]   Figure 1. Demonstration of adenosine receptor subtypes. (A) Expression of adenosine receptor subtype mRNA in HGEC. We performed RT-PCR analysis to examine adenosine receptor mRNA of the A1, A2a, A2b, and A3 subtypes in HGEC, mononuclear leukocytes, and granulocytes. Representative results of 1 of 3 identical experiments are shown. The number of PCR cycles is shown above each lane. The mean ratios ± SD, which were determined as described in MATERIALS & METHODS, of A1, A2a, A2b, and A3 subtype expression were 2.0 ± 0.4, 13.2 ± 3.0, 8.9 ± 1.4, and 0.0 ± 0.0, respectively. (B) Examination of adenosine receptor A3 subtype mRNA expression in HGEC by nested PCR. Nested amplification primers for the A3 receptor subtype were designed within the primers for the A3 receptor. (C) Detection of adenosine receptor A3 subtype mRNA expression by nested PCR. To detect the adenosine receptor A3 subtype mRNA in HGEC, mononuclear leukocytes, and granulocytes, we performed RT-PCR for 40 cycles and then carried out re-amplification using nested primers. Results of 1 representative experiment from among 3 identical experiments are shown. The number of PCR cycles is shown above each lane.

View larger version (23K): [in a new window]   Figure 2. Regulation of iNOS expression. (A) Ligation of adenosine receptor with 2CADO resulted in an increase of iNOS mRNA expression in HGEC. HGEC were cultured (1 x 106/well in a 60-mm culture dish) with or without 2CADO (100 µM) for 6 hrs at 37°C. HGEC were also cultured (1 x 106/well in a 60-mm culture dish) in the presence of IL-1ß (25 U/mL) plus TNF (10 ng/mL) as a positive control. RT-PCR was then carried out for detection of iNOS and GAPDH mRNA expression in HGEC as described in MATERIALS & METHODS. Results of 1 representative experiment from among 5 identical experiments are shown. The number of PCR cycles is shown above each lane. The mean ratios ± SD, which were determined as described in MATERIALS & METHODS, of iNOS mRNA expression in untreated, IL-1ß plus TNF-treated, and 2CADO-treated HGEC were 0.11 ± 0.16, 0.85 ± 0.25, and 0.64 ± 0.34, respectively. (B) Adenosine receptor antagonist abrogated 2CADO-induced iNOS expression in SV-40-transformed HGEC. SV-40-transformed HGEC (epi 4) were cultured (1 x 105/well in 24-well plates) in the presence or absence of 2CADO (100 µM) with or without XAC (10 µM), an adenosine receptor antagonist, for 2.5 hrs at 37°C, and then RT-PCR was carried out for the detection of iNOS and GAPDH mRNA expression in epi 4. Results of 1 representative experiment from among 3 identical experiments are shown. The number of PCR cycles is shown above each lane. The mean ratios ± SD, which were determined as described in MATERIALS & METHODS, of iNOS mRNA expression in untreated, 2CADO-treated, 2CADO plus XAC-treated, and XAC-treated epi4 were 0.02 ± 0.04, 0.98 ± 0.36, 0.07 ± 0.01, and 0.01 ± 0.02, respectively.

View larger version (20K): [in a new window]   Figure 3. Effects of CPA and CGS21680 on iNOS mRNA expression by HGEC. SV-40-transformed HGEC were cultured (1 x 105/well in 24-well plates) with or without 2CADO (100 µM), CPA (50 µM), or CGS21680 (10 µM), and then RT-PCR was carried out for detection of iNOS and GAPDH mRNA expression. Results of 1 representative experiment from among 3 identical experiments are shown. The number of PCR cycles is shown above each lane. The mean ratios ± SD, which were determined as described in MATERIALS & METHODS, of iNOS mRNA expression in untreated, 2CADO-treated, CPA-treated, and CGS21680-treated epi 4 were 0.09 ± 0.15, 1.42 ± 0.35, 0.81 ± 0.26, and 0.79 ± 0.42, respectively.

View larger version (14K): [in a new window]   Figure 4. Effects of 2CADO, CPA, and CGS21680 on NO production in HGEC. SV-40-transformed HGEC (1 x 105/well in 24-well plates) were cultured with or without 2CADO (100 µM), CPA (50 µM), or CGS21680 (10 µM), and then NO2-/NO3- in the culture supernatants was measured as described in MATERIALS & METHODS. Results of 1 representative experiment from among 3 identical experiments are shown. Statistical analysis was performed by a one-way analysis of variance (ANOVA) with Scheffé's multiple-comparison test to a significance level of p < 0.05. Asterisks (*) indicate significant (p < 0.05) difference as compared with untreated cells.