CCK increases cAMP formation which attenuates LPS-induced iNOS expression by peritoneal macrophages. The macrophage culture was pretreated with CCK (10−10 or 10−6 M) for 30 min before the addition of LPS (1 μg/mL) to the culture medium (time zero). At the indicated time-points, whole cell lysates were obtained for quantification of cAMP content by using ELISA kit (a), and samples were normalized with total protein concentration by Bradford method. To associate changes on intracellular cAMP with LPS-induced iNOS expression, the macrophage culture was incubated with an adenylyl cyclase inhibitor (SQ-22,536) (10−8 –10−5 M) (b, c, and d) for 30 min before the addition of CCK at 10−10 or 10−6 M. Thirty minutes later, cells were stimulated by LPS. Additionally, the peritoneal macrophage culture was pretreated with an adenylyl cyclase activator (forskolin) (10 and 25 μM) 30 min prior to LPS (e and f). The culture supernatant was collected for nitrite quantification by Griess method and the iNOS synthesis was determined in cell lysates by ELISA. Data is a representative of three independent experiments with similar results. Values are expressed as means ± S.E.M. 4–12 samples per group. * versus saline, # versus LPS, and a versus respective CCK group.
