大鼠肺间质巨噬细胞CCK受体的结合特性

目的:观察大鼠肺间质巨噬细胞(PIMs)胆囊收缩素(CCK)受体的表达亚型和结合特性。方法:用酶消化法结合肺泡耗竭灌洗和肺循环灌洗技术分离纯化大鼠PIMs,超速离心法提取细胞膜,与标记的硫酸化CCK-8(-CCK-8S)进行放射配基结合实验,用非标记的CCK-8S、CCK-A受体(CCK-AR)特异性拮抗剂CR1409及CCK-B受体(CCK-BR)特异性拮抗剂CR2945进行竞争抑制实验,观察配体受体结合的特异性及CCK受体表达亚型,观察孵育时间和温度对特异性结合的影响。结果:正常大鼠PIMs未能检出特异性结合,静脉注射脂多糖(LPS)48h出现特异性结合,且对孵育时间与温度有依赖性。经Scatchard分析,平衡解离常数(Kd)值为:(0.68±0.28)nmol·L^-1,最大结合容量(Bmax)值为(32.50±2.70)pmol·g^-1蛋白。通过竞争抑制实验,-CCK-8S与膜的结合可被CCK-8S、CR1409、CR2945所抑制,其IC₅₀值分别为:(3.20±1.13)nmol·L^-1,(0.19±0.06)μmol·L^-1和(2.30±0.80)nmol·L^-1。结论:大鼠PIMs存在CCK-A和CCK-B两种受体亚型,为CCK对生理及病理条件下巨噬细胞发挥效应提供了直接的结构依据。

Binding characterization of cholecystokinin receptors in rat pulmonary interstitial macrophages

AIM: To investigate the binding characteristics of cholecystokinin receptors in rat pulmonary interstitial macrophages (PIMs). METHODS: The PIMs were isolated from rat lung tissues, purified using the collagenase digestion method combined with alveolar lavage and pulmonary vessel perfusion. The PIM membrane was obtained by supercentrifuge. Receptors for CCK in PIMs were examined using labeled CCK-8S as ligand. The specificity of -CCK-8S binding to PIMs membrane and the subtypes of CCK receptors were determined by competitive inhibition experiments with CCK-8S, CCK-A and CCK-B receptors selective antagonists (CR1409 and CR2945). The effects of time and incubation temperature on the specific binding were also observed. RESULTS: The specific binding of -CCK-8S was not detected in normal rat PIMs, but was detected in the rat administrated with LPS for 48 h. The capacity of ligand-receptor binding was dependent on the incubation temperature and time. Scatchard analysis of the saturation curves suggested that the presence of CCK receptors with high affinity and low binding capacity in PIMs. By means of competitive inhibition studies, the specific binding of -CCK-8S to rat PIMs was inhibited by unlabelled CCK-8S ,CCK-AR specific antagonist CR 1 409 and CCK-BR specific antagonist CR 2945 . CONCLUSION: These results suggest the presence of two subtypes of CCK-AR and CCK-BR and provide a structural basis for CCK to play a pivotal role in PIMs.