Borghini I, Geering K, Gjinovci A, Wollheim C B, Pralong W F
Département de Médecine, Centre Médical Universitaire, CH-1211 Geneva, Switzerland.
Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6211-5. doi: 10.1073/pnas.91.13.6211.
The phosphorylation state of the Na,K-ATPase alpha subunit has been examined in 32P-labeled sciatic nerves of control and streptozotocin-treated diabetic rats. Intact nerves were challenged with protein kinase (PK) modulators and alpha-subunit 32P labeling was analyzed after immunoprecipitation. In control nerves, the PKC activator phorbol 12-myristate 13-acetate (PMA) had little effect on alpha-subunit 32P labeling. In contrast, staurosporine, a PKC inhibitor, and extracellular calcium omission decreased it. In Ca(2+)-free conditions, PMA restored the labeling to basal levels. The cAMP-raising agent forskolin reduced the 32P labeling of the alpha subunit. The results suggest that nerve Na,K-ATPase is tonically phosphorylated by PKC in a Ca(2+)-dependent manner and that PKA modulates the phosphorylation process. In nerves of diabetic rats, PMA increased 32P labeling of the alpha subunit. In contrast to staurosporine or extracellular calcium omission, the decreased state of phosphorylation seen with forskolin was no longer significant in diabetic nerves. No change in the level of alpha-subunit isoforms (alpha 1 or alpha 2) was detected by Western blot analysis in such nerves. In conclusion, the altered effect of PK activators on Na,K-ATPase phosphorylation state is consistent with the view that a defect in PKC activation exists in diabetic nerves.
在对照大鼠和经链脲佐菌素治疗的糖尿病大鼠的32P标记坐骨神经中，研究了钠钾ATP酶α亚基的磷酸化状态。完整神经用蛋白激酶（PK）调节剂进行刺激，免疫沉淀后分析α亚基的32P标记。在对照神经中，蛋白激酶C激活剂佛波醇12 -肉豆蔻酸酯13 -乙酸酯（PMA）对α亚基32P标记影响很小。相比之下，蛋白激酶C抑制剂星形孢菌素和细胞外钙缺失则使其降低。在无钙条件下，PMA将标记恢复到基础水平。提高环磷酸腺苷（cAMP）的试剂福斯高林降低了α亚基的32P标记。结果表明，神经钠钾ATP酶在钙离子依赖的方式下被蛋白激酶C持续磷酸化，且蛋白激酶A调节磷酸化过程。在糖尿病大鼠的神经中，PMA增加了α亚基的32P标记。与星形孢菌素或细胞外钙缺失不同，福斯高林导致的磷酸化降低状态在糖尿病神经中不再显著。通过蛋白质印迹分析未在此类神经中检测到α亚基同工型（α1或α2）水平的变化。总之，蛋白激酶激活剂对钠钾ATP酶磷酸化状态的改变效应与糖尿病神经中存在蛋白激酶C激活缺陷的观点一致。
