Zhao Y, Levin S S, Wein A J, Levin R M
Division of Urology, University of Pennsylvania, Philadelphia, USA.
Urology. 1997 Feb;49(2):293-300. doi: 10.1016/S0090-4295(96)00452-9.
In the rabbit, both experimental ischemia and partial outlet obstruction of the urinary bladder induce similar dysfunctions with regard to the contractile responses to both field (neuronal) stimulation and postsynaptic receptor stimulation. Circumstantial evidence indicates that the pathologic response to both conditions is related to two connected processes-tissue ischemia and reperfusion injury-that result in a marked increase in intracellular calcium ([Ca2+]i), followed by the activation of the Ca(2+)-dependent neutral protease calpain. Calpain activation results in the proteolysis of specific membrane proteins, including those of neuronal membranes (resulting in progressive denervation of the detrusor) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), resulting in the previously reported decrease in SERCA. The current study is designed to generate direct support for the theory that both ischemia and partial outlet obstruction result in the activation of calpain.
Separate sets of rabbits were subjected to 1 or 2 hours of ischemia, followed by reperfusion for different lengths of time, or partial outlet obstruction for different lengths of time. We determined the state of calpain activation by quantitating tissue proteolysis of alpha-spectrin by Western blot analysis. Correlative organ bath studies were conducted to observe the contractile responses of bladder strips to field stimulation and bethanechol administration.
(1) Sixty minutes of ischemia followed by 30 minutes of reperfusion resulted in (a) a reduction in the contractile responses to field stimulation and bethanechol (89% and 57%, respectively), and (b) a 72% decrease in native alpha-spectrin, with a concomitant 300% increase in its breakdown products (BDPs). Neither alpha-spectrin nor its BDPs had returned to control levels after 72 hours of reperfusion. (2) Twenty-four hours after the creation of a partial obstruction, alpha-spectrin BDP levels were increased 330%, then gradually fell to 130% of control levels by 14 days after obstruction. Concomitantly, the native alpha-spectrin level was decreased 74% 24 hours after obstruction and remained low through 7 days after obstruction. At 14 days after obstruction, the alpha-spectrin levels had recovered to 75% of control levels.
These findings suggest that Ca(2+)-dependent proteolysis of the preferred calpain substrate alpha-spectrin in urinary bladder tissues is increased significantly by both ischemia/reperfusion and partial outlet obstruction. Temporally, proteolysis precedes the reduced muscle function resulting from these pathologic conditions.
在兔体内，实验性缺血和膀胱部分出口梗阻在对场（神经元）刺激和突触后受体刺激的收缩反应方面会引发相似的功能障碍。间接证据表明，对这两种情况的病理反应与两个相关过程有关——组织缺血和再灌注损伤，这会导致细胞内钙（[Ca2+]i）显著增加，随后激活钙（2+）依赖性中性蛋白酶钙蛋白酶。钙蛋白酶激活会导致特定膜蛋白的蛋白水解，包括神经元膜蛋白（导致逼尿肌进行性去神经支配）和肌浆网钙（2+）-ATP酶（SERCA），从而导致先前报道的SERCA减少。本研究旨在为缺血和部分出口梗阻均导致钙蛋白酶激活这一理论提供直接支持。
将不同组的兔分别进行1或2小时的缺血，随后再灌注不同时长，或进行不同时长的部分出口梗阻。我们通过蛋白质印迹分析定量α-血影蛋白的组织蛋白水解来确定钙蛋白酶的激活状态。进行相关的器官浴研究以观察膀胱条对场刺激和给予氨甲酰甲胆碱的收缩反应。
（1）缺血60分钟后再灌注30分钟导致：（a）对场刺激和氨甲酰甲胆碱的收缩反应降低（分别降低89%和57%），以及（b）天然α-血影蛋白减少72%，同时其降解产物（BDPs）增加300%。再灌注72小时后，α-血影蛋白及其BDPs均未恢复到对照水平。（2）部分梗阻形成24小时后，α-血影蛋白BDP水平增加330%，然后在梗阻后14天逐渐降至对照水平的130%。同时，梗阻后24小时天然α-血影蛋白水平降低74%，并在梗阻后7天内一直保持较低水平。在梗阻后14天，α-血影蛋白水平恢复到对照水平的75%。
这些发现表明，缺血/再灌注和部分出口梗阻均会显著增加膀胱组织中钙蛋白酶的首选底物α-血影蛋白的钙（2+）依赖性蛋白水解。在时间上，蛋白水解先于这些病理状况导致的肌肉功能降低出现。
