【摘要】 目的探讨八肽胆囊收缩素(CCK-8)对大鼠肺间质巨噬细胞(PIMs)cAMP-PKA信号通路的激活作用。方法分离纯化大鼠PIMs,采用放射免疫分析法测定细胞内cAMP含量,放射激酶法测定PKA活性,受体拮抗剂的IC50值由对数-几率单位法求得。结果正常对照组大鼠静息状态下PIMscAMP含量和PKA活性分别为(2.04±0.13)nmol·g-1和(118.3±11.2)nmol·min-1·g-1。低浓度CCK-8[(10-12～10-10)mol·L-1]对细胞内cAMP含量和PKA活性没有影响(与正常对照组比较:P>0.05);高浓度CCK-8[(10-9～10-5)mol.L-1]可明显提高细胞内cAMP含量和PKA活性(与正常对照组比较:P<0.05)。10mg·L-1脂多糖(LPS)刺激大鼠PIMs,可明提高细胞内cAMP含量和PKA活性,分别为(5.15±0.12)nmol·g-1和(188.6±13.5)nmol·min-1·g-1。不同浓度的CCK-8与LPS共同孵育PIMs,细胞内cAMP含量和PKA活性的变化趋势与CCK-8作用于静息状态下大鼠PIMs的变化趋势完全相同。CCK受体拮抗剂丙谷胺、CR-1409、CR-2945可呈剂量依赖性地抑制CCK导致的cAMP含量的升高,它们的IC50值分别为(0.5×10-6、4.1×10-6、7.2×10-4)mol·L-1。丙谷胺、CR-1409、CR-2945也可明显减弱CCK-8所导致的PKA活性的升高;其中,丙谷胺的抑制作用最强,CR-1409次之,CR-2945的抑制作用最小。结论CCK-8可剂量依赖性地激活静息状态和LPS诱导的大鼠PIMscAMP-PKA信号转导途径,这可能是CCK抗炎作用的分子机制之一。CCK激活cAMP-PKA通路是通过CCK受体来实现的,且CCK-AR的作用比CCK-BR的作用更为明显。更多还原

【Abstract】 Aim To investigate the activation of CCK-8 on cAMP-PKA signaling pathway in rat pulmonary interstitial macrophages (PIMs). Methods PIMs were isolated and purified, and radioimmunoassay was used to detect the cAMP content and radioenzymatic assay to detect the protein kinase A (PKA) activity. Half-maximal inhibition (IC50) of receptor antagonist was calculated by log-probit method. Results Under quiescent condition, the cAMP content and PKA activity in PIMs of normal control group were(2.04±0.13)nmol·g-1 and(118.3±11.2)nmol·min-1·g-1 respectively. CCK-8 did not affect cAMP content and PKA activity significantly at low concentration [(10-12~10-10) mol·L-1](compared with normal control group: P>0.05); but significantly increased cAMP content and PKA activity at high concentration [(10-9~10-5) mol·L-1] (compared with normal control group:P<0.05). Stimulating PIMs with 10 mg·L-1 lipopolysaccharide (LPS) resulted in significant increase of cAMP content and PKA activity in the concentration of(5.15±0.12)nmol·g-1 and(188.6±13.5)nmol·min-1·g-1. When PIMs were incubated with different dosages of CCK-8 and LPS, the changes of cAMP content and PKA activity were similar to PIMs in quiescent condition under action of CCK-8. The inhibitory effects of CCK receptor antagonist proglumide, CR-1409 and CR-2945 on the CCK-8-resulted increasing of cAMP content were dose-dependent and the IC50 was(0.5×10-6,4.1×10-6,and 7.2×10-4)mol·L-1 respectively. The effects of CCK-8 on PKA activity were significantly attenuated by proglumide, CR-1409 and CR-2945, their inhibitory effects decreased gradually from proglumide to CR-1409 and CR-2945. Conclusion CCK-8 activates cAMP-PKA signaling pathway in a dose-dependent manner in rat PIMs under quiescent condition or induced by LPS through its receptors, this may be one of the mechanisms of CCK anti-inflammatory effects. Both CCK-AR and CCK-BR may be involved in this pathway, but CCK-AR may play a main role in this process.更多还原