Li D, Cheng S X, Fisone G, Caplan M J, Ohtomo Y, Aperia A
Departments of Woman and Child Health, Karolinska Institute, S-171 76 Stockholm, Sweden.
Am J Physiol. 1998 Dec;275(6):F863-9. doi: 10.1152/ajprenal.1998.275.6.F863.
Several indirect lines of evidence suggest that protein kinases and phosphatases modulate the activity of renal Na+-K+-ATPase. The aim of this study was to examine whether such regulation may occur via modulation of the state of phosphorylation of Na+-K+-ATPase. Slices from rat renal cortex were prelabeled with [32P]orthophosphate and incubated with the inhibitors of protein phosphatase (PP)-1 and PP-2A, okadaic acid (OA) and calyculin A (CL-A), respectively, the protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), or the PP-2B inhibitor, FK-506. Phosphorylation of Na+-K+-ATPase alpha-subunit was evaluated by measuring the amount of [32P]phosphate incorporation into the immunoprecipitated protein. Incubation with either OA, CL-A, or PDBu caused four- to fivefold increases in the amount of [32P]phosphate incorporation into immunoprecipitated Na+-K+-ATPase alpha-subunit. OA and PDBu had a synergistic effect on the state of phosphorylation of Na+-K+-ATPase alpha-subunit. FK-506 did not affect Na+-K+-ATPase phosphorylation, neither alone nor in the presence of PDBu. Each of the drugs, OA, CL-A, and PDBu, inhibited the activity of Na+-K+-ATPase in microdissected proximal tubules. PDBu potentiated OA-induced inhibition of Na+-K+-ATPase activity. Inhibition of Na+-K+-ATPase required a lower dose of CL-A than of OA. On the basis of the inhibitory constant values of CL-A and OA for PP-1 and PP-2A, it is concluded that the tubular effect is mainly due to inhibition of PP-1. The PP-1 activity in rat renal cortex was approximately 1.5 nmol Pi. mg protein-1. min-1. Using a monoclonal anti-alpha antibody that fails to recognize the subunit when Ser23 is phosphorylated by PKC, we demonstrated that the dose response of PDBu inhibition of Na+-K+-ATPase correlated with the dose response of phosphorylation of the enzyme. The results suggest that the state of phosphorylation and activity of proximal tubular Na+-K+-ATPase are determined by the balance between the activities of protein kinases and phosphatases.
几条间接证据表明，蛋白激酶和磷酸酶可调节肾钠钾ATP酶的活性。本研究的目的是检验这种调节是否可能通过调节钠钾ATP酶的磷酸化状态来实现。用[32P]正磷酸盐对大鼠肾皮质切片进行预标记，然后分别与蛋白磷酸酶（PP）-1和PP-2A的抑制剂冈田酸（OA）和花萼海绵诱癌素A（CL-A）、蛋白激酶C（PKC）激活剂佛波醇12,13-二丁酸酯（PDBu）或PP-2B抑制剂FK-506一起孵育。通过测量免疫沉淀蛋白中[32P]磷酸盐的掺入量来评估钠钾ATP酶α亚基的磷酸化情况。用OA、CL-A或PDBu孵育会使免疫沉淀的钠钾ATP酶α亚基中[32P]磷酸盐的掺入量增加4至5倍。OA和PDBu对钠钾ATP酶α亚基的磷酸化状态有协同作用。FK-506单独或与PDBu一起存在时均不影响钠钾ATP酶的磷酸化。每种药物，OA、CL-A和PDBu，均抑制显微解剖的近端小管中钠钾ATP酶的活性。PDBu增强了OA诱导的钠钾ATP酶活性抑制作用。抑制钠钾ATP酶所需的CL-A剂量低于OA。根据CL-A和OA对PP-1和PP-2A的抑制常数，得出肾小管效应主要是由于PP-1的抑制。大鼠肾皮质中的PP-1活性约为1.5 nmol Pi·mg蛋白-1·min-1。使用一种单克隆抗α抗体，当Ser23被PKC磷酸化时该抗体无法识别该亚基，我们证明PDBu抑制钠钾ATP酶的剂量反应与该酶磷酸化的剂量反应相关。结果表明，近端小管钠钾ATP酶的磷酸化状态和活性由蛋白激酶和磷酸酶活性之间的平衡决定。
