- Poster presentation
In vivo and in vitro role of cholecystokinin in nitric oxide
Critical Care volume 14, Article number: P10 (2010)
Nitric oxide (NO) plays a key role in innate immune system controlling microbial infection; however, during septic shock its exacerbate formation is associated with several deleterious complications. Cholecystokinin (CCK) was first described as a gastrointestinal hormone, but immune cells express their receptor, suggesting a possible involvement of this hormone in modulation of inflammatory response. Our aim was to evaluate the role of CCK on NO production during endotoxemia in rats as well as lipopolysaccharide (LPS)-stimulated macrophages.
Male Wistar rats received an intravenous injection of CCK (0.4 and 40 μg/kg) 10 minutes before LPS (1.5 mg/kg) administration. The mean arterial pressure was monitored during 6 hours after endotoxin injection. Blood was collected for plasma nitrate level and vasopressin measurement at 2, 4 and 6 hours after LPS. Thioglicollate-elicited macrophages were obtained by peritoneal lavage and cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum and antibiotics. Macrophage culture was treated with CCK (10-14, 10-12, 10-10, 10-8, 10-6 M) 30 minutes before LPS stimulation (1 μg/ml) and supernatant nitrite concentration was determined at 6, 24 and 48 hours. The iNOS expression was evaluated by quantitative real-time PCR and the amount of gene transcription was measured using the delta-delta method. The presence of iNOS was analyzed by indirect immunofluorescence at 12 and 24 hours after LPS incubation.
The LPS-induced hypotension was reverted by the pretreatment with CCK only at the lower dose. Moreover, CCK increased vasopressin levels at 2 and 4 hours after LPS administration and reduced nitrate levels during 2 and 6 hours. LPS-stimulated macrophages increased rapidly nitrite levels in supernatant and also iNOS expression. The pretreatment with CCK at all tested concentrations significantly reduced nitrite levels at 6, 24 and 48 hours after LPS stimulation when compared with the LPS group (P < 0.05). The iNOS/GAPDH expression ratio were also lower in CCK-treated cells at 6 and 24 hours (P < 0.001). The qualitative analysis of iNOS protein was assessed at 12 and 24 hours after LPS stimulus by immunocytochemistry. In CCK-treated macrophages, a reduction of fluorescence emission in comparison with the LPS group was observed. In control groups (without LPS), fluorescence was not observed, suggesting the absence of iNOS protein in non-inflammatory conditions.
These data suggest that CCK restores hypotension and reduces NO formation during endotoxemia in rats. Furthermore, CCK regulates negatively iNOS expression and also NO synthesis in LPS-activated peritoneal macrophage culture.
About this article
Cite this article
Saia, R.S., Cárnio, E. In vivo and in vitro role of cholecystokinin in nitric oxide. Crit Care 14, P10 (2010). https://doi.org/10.1186/cc9113
- Nitric Oxide
- iNOS Expression
- Nitrate Level
- Nitrite Level