哮喘气道重塑中钙调神经磷酸酶与蛋白激酶活性的相互调节

Cross-talk between calcineurin and protein kinases in airway remodeling in asthma

目的研究钙调神经磷酸酶(CaN)信号途径与蛋白激酶C(PKC)、丝裂素活化蛋白激酶(MAPK)和蛋白激酶A(PKA)的相互调节在哮喘气道重塑发病中的作用。方法建立卵蛋白诱导的哮喘豚鼠模型,实验分为哮喘组、CaN抑制剂环孢霉素A(CsA)组和对照组,采用磷酸化和去磷酸化方法测定肺组织CaN活性、MAPK活性、PKC活性、PKA活性。在离体培养的大鼠气道平滑肌细胞(ASMC)中,以尾加压素Ⅱ(UⅡ)为刺激因素,测定CaN,PKC和MAPK活性以及它们之间的相互调节。结果(1)哮喘组CaN活性、MAPK活性和PKC活性分别较对照组高19%(P<0.01)、28%(P<0.01)和35%(P<0.05),PKA活性较对照组低53%(P<0.01)。CsA组CaN活性、MAPK活性和PKC活性分别较哮喘组低52%(P<0.01)、18%(P<0.05)和52%(P<0.01),PKA活性较哮喘组高2.65倍(P<0.01)。(2)UⅡ10-7mol/L孵育20min引起ASMC PKC和MAPK活性分别较对照组增加44%和24%(P<0.01),并呈时间依赖性引起ASMC中CaN活性增加,24h为对照组的1.67倍(P<0.01)。(3)与单用UⅡ10-7mol/L组比,CaN抑制剂CsA10-6mol/L使CaN活性降低了45%(P<0.01),MAPK抑制剂PD9805950μmol/L对CaN活性无明显影响(P>0.05),PKC抑制剂H750μmol/L使CaN活性下降21%(P<0.05)。(4)CsA10-6mol/L作用使UⅡ10-7mol/L刺激的ASMC PKC活性下降了14%(P<0.05),对MAPK活性无明显影响(P>0.05)。结论在哮喘气道重塑的发生过程中,CaN与MAPK、PKC和PKA之间存在相互调节。

Objective： To determine the role of cross-talk between ca｜cineurin-dependent signal transduction pathway and protein kinase C （PKC）, mitogen-activated protein kinase （MAPK） and protein kinase A （PKA） in airway remodeling in asthma. Methods： Male guinea pigs were sensitized with intraperitoneal injections of ovabumin （OVA）, then treated with cyclosporin A （CsA,5 mg/kg）, an inhibitor of calcineurin, then inhaled OVA for 2 weeks 14 clays later. Activities of calcineurin, PKC, MAPK, and PKA were was analyzed by phosphorylation and dephosphorylation. In primary cultures of rat airway smooth muscle ceils （ASMC）, activities of calcineurin, PKC, MAPK, and cross-talk induced by urotensin Ⅱ （ U Ⅱ ）, a recently identified strong mitogen, were measured. Results： （ 1 ） The activities of calcineurin, MAPK and PKC increased by 19% （P 〈0.01）, 28% （P 〈0.01） and 35% （P 〈0.05）, respectively, in the asthmatic group compared with controls but decreased by 52% （P 〈0.01 ） , 18% （P 〈0.05） and 52% （P 〈0.01 ）, respectively, in the CsA group compared with asthmatic group. PKA activity in the asthma group decreased by 53% （P 〈 0.01 ） compared with controls but increased by 2.65-fold （P〈0.01） in the CsA group compared with the asthma group. （2） U Ⅱ 10^-7 mol/L stimulated ASMC PKC and MAPK activities by 44% and 24% （P 〈0.01 ） , respectively, after incubating for 20 min. It increased CaN activity in a time-dependent manner, being 1.67 times that of the control for 24 h （P 〈0.01 ）. （3） CsA 10 -6 mol/L and H7 50 μmol/L, an inhibitor of PKC, inhibited U Ⅱ -stimulated CaN activity by 45% （P 〈 0.01 ） and 21% （ P 〈 0.05 ）, respectively, while PD98059 50 μmol/L, an inhibitor of MAPK, had no effect on CaN activity （P 〉0.05）. （4） CsA 10-6 mol/L inhibited U ll- stimulated PKC activity by 14% （ P 〈 0.05 ）, while having no effect on MAPK activity （ P 〉 0.05 ）. Conclusion ： The signal transduction pathways between calcineurin and other protein kinases such as PKC, MAPK and PKA have cross-talk in airway remodeling in asthma.