Activation of ERK during ischemia-reperfusion in lung transplantation.

1.大鼠肺移植模型实验：人肺移植缺血再灌注期蛋白酪氨酸磷酸化有明显改变。本研究的目的是确定在大鼠单肺移植模型中是否存在这些变化。冷缺血保存6h后行肺移植。在同种和同种异体移植物中，移植物的肺功能都保存得很好。再灌注2小时后，蛋白酪氨酸磷酸化、蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶活性显著降低。再灌流2小时后，SRC蛋白水平和p38的磷酸化水平均降低。本实验建立的肺移植模型可用于研究肺移植缺血再灌注损伤过程中MAPK的激活情况。这些移植手术是在加拿大多伦多总医院进行的。对15例同种异体肺移植患者：冷缺血保存后、热缺血(植入)后、再灌流1h或2 h后进行肺组织活检。Western blotting检测ERK、p38-MAPK、INK和MEK的磷酸化状态。墨水的磷酸化水平在较低水平时也显著增加。而p38的磷酸化水平则无明显变化。ERK磷酸化的增加与ERK的上游激酶MEK的激活无关。由于MKP-3的蛋白水平与其活性相对应，我们检测了这些样本中MKP-3的蛋白表达。令人惊讶的是，再灌注后MKP-3蛋白水平显著升高。因此，ERK磷酸化的显著增加并不是由于MKP-3的表达受到抑制。

1.Experiment in a rat lung transplant model.Dramatic alterations of protein tyrosine phosphorylation have been found during the ischemia-reperfusion period of human lung transplantation. The object of the present study was to determine whether these changes exist in a rat single-lung transplant model. Lung transplantations were performed after 6 hours of cold ischemic preservation. In both iso-and allografts, the lung function of transplants was very well preserved. Protein tyrosine phosphorylation, protein tyrosine kinase and protein tyrosine phosphatase activities were decreased significantly after 2 hours of reperfusion. Src protein level and phosphorylation of p38 were reduced after 2 hours of reperfusion. This lung transplantation model is useful for studying ischemia-reperfusion injury.MAPK activation in human lung transplantThe phosphorylation status of MAPK during ischemia-reperfusion in human lung transplant was examined. These transplantations were performed in Toronto General Hospital in Canada. Lung tissue biopsy was performed on 15 patients undergoing transplantation: after the cold ischemic preservation, after the warm ischemia (implantation) and after 1 h or 2 h reperfusion. The phosphorylation status of ERK, p38-MAPK, INK and MEK was examined with Western blotting. Phosphorylation of INK also significantly increased at lower levels. In contrast, phosphorylation of p38 had no significant changes. The increase in ERK phosphotylation was not associated with the activation of MEK, the up-stream kinase for ERK. Since MKP-3 protein levels correspond to its activity, we examined the protein expression of MKP-3 in these samples. Surprisingly, the MKP-3 protein level significantly increased after reperfusion. Therefore, the dramatic increase in ERK phosphorylation is not due to the inhibited expression of MKP-3.