Lefler David, Mukhin Yurii V, Pettus Tobiah, Leeb-Lundberg L M Fredrik, Garnovskaya Maria N, Raymond John R
Department of Medicine (Nephrology Division) of the Medical University of South Carolina, Charleston, South Carolina, USA.
Assay Drug Dev Technol. 2003 Apr;1(2):281-9. doi: 10.1089/15406580360545099.
Na(+)/H(+) exchangers are ubiquitous in mammalian cells, carrying out key functions, such as cell volume defense, acid-base homeostasis, and regulation of the cytoskeleton. We used two screening technologies (FLIPR and microphysiometry) to characterize the signal transduction pathway used by the bradykinin B(2) receptor to activate Na(+)/H(+) exchange in two cell lines, KNRK and CHO. In both cell types, B(2) receptor activation resulted in rapid increases in the rate of proton extrusion that were sodium-dependent and could be blocked by the Na(+)/H(+) exchange inhibitors EIPA and MIA or by replacing extracellular sodium with TMA. Activation of Na(+)/H(+) exchange by bradykinin was concentration-dependent and could be blocked by the selective B(2) receptor antagonist HOE140, but not by the B(1) receptor antagonist des-Arg10-HOE140. Inhibitors of Jak2 tyrosine kinase (genistein and AG490) and of CAM (W-7 and calmidazolium) attenuated bradykinin-induced activation of Na(+)/H(+) exchange. Bradykinin induced formation of a complex between CAM and Jak2, supporting a regulatory role for Jak2 and CAM in the activation of Na(+)/H(+) exchange in KNRK and CHO cells. We propose that this pathway (B(2) receptor --> Jak2 --> CAM --> Na(+)/H(+) exchanger) is a fundamental regulator of Na(+)/H(+) exchange activity.
钠/氢交换体在哺乳动物细胞中广泛存在，执行着诸如细胞容积防御、酸碱平衡及细胞骨架调节等关键功能。我们运用两种筛选技术（荧光成像板读数器和微生理测定法）来表征缓激肽B2受体在两种细胞系（KNRK和CHO）中激活钠/氢交换所使用的信号转导途径。在这两种细胞类型中，B2受体激活均导致质子外排速率迅速增加，这种增加依赖于钠，并且可被钠/氢交换抑制剂EIPA和MIA阻断，或者通过用三甲胺替代细胞外钠来阻断。缓激肽对钠/氢交换的激活呈浓度依赖性，并且可被选择性B2受体拮抗剂HOE140阻断，但不能被B1受体拮抗剂去-精氨酸10-HOE140阻断。Jak2酪氨酸激酶抑制剂（金雀异黄素和AG490）以及钙调蛋白抑制剂（W-7和氯米帕明）可减弱缓激肽诱导的钠/氢交换激活。缓激肽诱导钙调蛋白与Jak2之间形成复合物，这支持了Jak2和钙调蛋白在KNRK和CHO细胞中钠/氢交换激活过程中的调节作用。我们提出这条途径（B2受体→Jak2→钙调蛋白→钠/氢交换体）是钠/氢交换活性的一个基本调节因子。
