CCK attenuates LPS-induced iNOS expression interacting predominantly through CCK-1R 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 analysis of CCK-Rs expression by western blot. (a) Representative immunoblots with densitometry values were shown for CCK-1R/β-actin ratio. The β-actin levels were used as an internal control. The relevance of CCK-1R subtype was pharmacologically tested, incubating macrophage culture with selective antagonist [devazepide (CCK-1R antagonist; 10−9–10−6 M)] for 30 min before the addition of CCK at 10−10 or 10−6 M. Thirty minutes later, cells were stimulated by LPS. The culture supernatant was collected for nitrite quantification by Griess method (b and c) and the iNOS synthesis was determined in cell lysates by ELISA (d). 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.
