Effect of lipopolysaccharide on expression and characterization of cholecystokinin receptors in rat pulmonary interstitial macrophages

AIM: To investigate the effect of lipopolysaccharide (LPS) on the expression and the binding characteristics of cholecystokinin receptors (CCK-R) in rat pulmonary interstitial macrophages (PIMs). METHODS: The PIMs isolated from rat lung tissues were purified by the collagenase digestion method combined with alveolar lavage and pulmonary vessel perfusion. The expression of CCK-R mRNA was detected by RT-PCR and Southern blot analysis and the binding experiments were performed by radioligand binding assay (RBA). RESULTS: CCK-A receptor (CCK-AR) and CCK-B receptor (CCK-BR) mRNA were detected in rat PIMs and their RT-PCR amplified products had a size of approximately 1.37 kb and 480 bp, respectively. The relative expression of CCK-BR mRNA was higher than that of CCK-AR mRNA after incubation with LPS for 0.5, 2, and 6 h. The expression of CCK-R mRNA could be upregulated obviously by LPS. Southern blot analysis of RT-PCR amplified CCK-AR and CCK-BR mRNA products using gamma-32P]ATP 5'-end-labelled probe showed specific hybridization bands. The specific binding of 3H]CCK-8S to rat PIM membranes was detected in the rats administered with LPS for 48 h, but not in normal rats. Scatchard analysis of the saturation curves suggested the presence of CCK-R with a high affinity (Kd = 0.68 +/- 0.28 nmol/L) and a low binding capacity (Bmax = 32.5 +/- 2.7 fmol/g protein) in rat PIMs. The specific binding of 3H]CCK-8S to rat PIM membranes was inhibited by unlabelled CCK-8S (IC50 = 2.3 +/- 0.8 nmol/L), CCK-AR specific antagonist CR1409 (IC50 = 0.19 +/- 0.06 micromol/L) and CCK-BR specific antagonist CR2945 (IC50 = 3.2 +/- 0.1 nmol/L). CONCLUSION: Two types of functional CCK-AR and CCK-BR existed in rat PIMs and their expression could be upregulated by LPS.