组胺刺激的气道平滑肌细胞钙离子浓度和长度变化及机制

The cytosolic calcium comcentration and length changes of airway smooth muscle cells induced by histamine and its mechanism

目的 观察组胺刺激后大鼠气道平滑肌细胞 (ASMC)内钙离子浓度 ( [Ca2 +]i)和长度的变化 ,并初步探讨其机制。方法 以Fluo 3/AM作为细胞内游离Ca2 +示踪剂 ,通过激光共聚焦显微镜观察不同浓度组胺对大鼠ASMC [Ca2 +]i和长度的影响 ,观察胞外Ca2 +、G蛋白、蛋白激酶C(PKC)、丝裂素活化蛋白 (MAPK)在组胺上述生理效应中的作用。结果 组胺刺激后 ,[Ca2 +]i变化呈现双相反应。组胺刺激后 [Ca2 +]i快速上升至峰值 ,随后稍下降 ,进入一个维持期。 [Ca2 +]i增加的峰值 ( 13.39%± 3.35 %、36 .35 %± 13.35 %、6 4 .6 8%± 11.4 2 % )与组胺浓度 ( 1× 10 -6、1× 10 -5、1× 10 -4mol/L)显著相关 (P <0 .0 5 )。无钙培养基不影响组胺刺激的 [Ca2 +]i上升 ,而在有钙培养基中加入乙二醇二乙醚二胺四乙酸 (EGTA)也不影响组胺刺激诱导的 [Ca2 +]i上升。随组胺浓度 ( 1× 10 -6、1× 10 -5、1× 10 -4mol/L)的上升 ,ASMC最大缩短程度 ( 2 3.14 %、37.6 2 %、4 1.75 % )仅有增加的趋势 (P >0 .0 5 ) ,但在同一组胺浓度下 ,其 [Ca2 +]i变化与细胞缩短程度显著相关 (P <0 .0 1)。百日咳毒素对组胺刺激ASMC [Ca2 +]i和长度变化均有明显的抑制作用。Staurosporine和PD0 980 5 9对组胺刺激的 [Ca2

Objective To observe the cytosolic calcium comcentration ([Ca 2+ ]i) and length changes of airway smooth muscle cells(ASMC) induced by histamine and to explore its mechanism. Methods With Fluo 3/AM as the indicator of intracellular free Ca 2+ , the [Ca 2+ ]i and changes in length of ASMC induced by histamine at different concentrations were investigated by laser scan confocal microscopy and the role of extracellular Ca 2+ , G protein, PKC, MAPK in the histamine physiological effect were examined. Results [Ca 2+ ]i changes induced by histamine presented a biphasic response. [Ca 2+ ]i rose rapidly to the peak, then maintained at a lower level. The maximal [Ca 2+ ]i changes (13.39%, 36.35%, 64.68%) correlated significantly with histamine concentrations (1×10 -6 , 1×10 -5 , 1×10 -4 mol/L)(P<0.05). Ca 2+ free medium had no influence on changes of [Ca 2+ ]i and EGTA also did not inhibit the [Ca 2+ ]i changes induced by histamine. The maximal shortening of ASMC had an increasing tendency with the increment of histamine concentrations ( 37.62% , 23.14% , 41.75% )( P > 0.05) . But the degree of shortening at different concentrations was correlated with [Ca 2+ ]i ( P < 0.01 ). PTX significantly inhibited both [Ca 2+ ]i and length changes. Staurosporine and PD098059 had no effect on [Ca 2+ ]i changes, but inhibited the changes in length of the muscle cells. Conclusion In rat ASMC, histamine induces a typical biphasic [Ca 2+ ]i response, [Ca 2+ ]i rises rapidly to the peak, then maintains at a lower level。[Ca 2+ ]i changes induced by histamine mainly depends on the release of intracellular store of [Ca 2+ ]i, [Ca 2+ ]i plays a vital roles in ASMC contraction induced by histamine. G protein, PKC, MAPK also play important roles in the physiological effects of histamine.