Figure 8.

CRH promotes nitric oxide production from CRH-R1α-expressing cells. A: HMVECs were nucleofected with or without pc-CRH-R1α and seeded into 12-well plates (300,000 cells/well). Cells were left unstimulated or stimulated with increasing concentrations of CRH (10⁻⁸ to 10⁻⁶ mol/L) or a combination of IL-1β (25 ng/ml), TNFα (25 ng/ml), and interferon-γ (1000 U/ml) for 24 hours. Cell culture medium was harvested, and nitric oxide production was determined by measuring nitrite production using the Griess reaction. Values are the means ± SEM from three separate experiments. B: Synoviocytes transfected with pc-CRH-R1α for 24 hours were treated with CRH (10⁻⁸ mol/L) for a further 1 to 3 hours. Expression of NOS III and GAPDH mRNA was analyzed using total RNA. Each gel is representative of three separate experiments.