Coux G, Trumper L, Elías M M
Farmacología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, República Argentina.
Nephron. 2001 Sep;89(1):82-9. doi: 10.1159/000046048.
The aim of our work was to study the changes in activity, abundance and distribution of sodium, potassium-adenosine triphosphatase (Na+,K+-ATPase) in membranes of cortical tubular cells in an in vivo model of ischemic injury without reperfusion. Na+,K+-ATPase, alkaline phosphatase (AP) activities and their distribution in membranes isolated from renal cortex using a Percoll gradient were studied after different ischemic periods. Na+,K+-ATPase alpha-subunit protein abundance was analysed by Western-blot. Plasma urea and cortical adenosine 5' triphosphate (ATP) were also measured. In cortical homogenates 5 min of ischemia promoted a diminution in ATP content. Na+,K+-ATPase activity diminished after 40 min and AP after 100 min of ischemia. Na+,K+-ATPase activity in the Percoll gradient fractions after 5 min peaked at a higher density and was significantly decreased after 40 min. AP activity was decreased in typically enriched apical membranes after both times of ischemia. At each time studied Na+,K+-ATPase abundance was increased in cortical homogenates and membranes. Our results showed opposite effects of ischemia on Na+,K+-ATPase activity and abundance. Increased levels of Na+,K+-ATPase protein were observed. The enzyme would be rapidly delivered to membrane domains and become inactivated as ischemia persists.
我们研究的目的是在无再灌注的缺血性损伤体内模型中，研究皮质肾小管细胞膜中钠钾 - 三磷酸腺苷酶（Na +，K + -ATPase）的活性、丰度和分布变化。在不同缺血时间后，研究了使用Percoll梯度从肾皮质分离的膜中Na +，K + -ATPase、碱性磷酸酶（AP）的活性及其分布。通过蛋白质印迹分析Na +，K + -ATPaseα亚基蛋白的丰度。还测量了血浆尿素和皮质三磷酸腺苷（ATP）。在皮质匀浆中，5分钟的缺血导致ATP含量降低。缺血40分钟后Na +，K + -ATPase活性降低，缺血100分钟后AP活性降低。5分钟后Percoll梯度组分中的Na +，K + -ATPase活性在较高密度处达到峰值，40分钟后显著降低。两次缺血后，典型富集的顶端膜中的AP活性均降低。在研究的每个时间点，皮质匀浆和膜中Na +，K + -ATPase的丰度均增加。我们的结果显示缺血对Na +，K + -ATPase活性和丰度有相反的影响。观察到Na +，K + -ATPase蛋白水平升高。随着缺血持续，该酶会迅速转运到膜结构域并失活。
