CCK-8对大鼠肺间质巨噬细胞cAMP-PKA信号通路的激活作用

Activation of cAMP-PKA signal pathway by CCK-8 in rat pulmonary interstitial macrophages

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。

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 （LOS） 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.