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.

Return to article