证据表明，环鸟苷酸与 NKA（钠-钾 ATP 酶）细胞外结构域的结合介导了利钠作用。
Evidence That Binding of Cyclic GMP to the Extracellular Domain of NKA (Sodium-Potassium ATPase) Mediates Natriuresis.
机构信息
Department of Medicine, Division of Endocrinology and Metabolism (B.A.K., N.L.H., S.R.K., R.M.C.), University of Virginia, Charlottesville.
Department of Pathology (J.J.G.), University of Virginia, Charlottesville.
出版信息
Circ Res. 2023 Apr 28;132(9):1127-1140. doi: 10.1161/CIRCRESAHA.122.321693. Epub 2023 Mar 15.
BACKGROUND
Extracellular renal interstitial guanosine cyclic 3',5'-monophosphate (cGMP) inhibits renal proximal tubule (RPT) sodium (Na) reabsorption via Src (Src family kinase) activation. Through which target extracellular cGMP acts to induce natriuresis is unknown. We hypothesized that cGMP binds to the extracellular α1-subunit of NKA (sodium-potassium ATPase) on RPT basolateral membranes to inhibit Na transport similar to ouabain-a cardiotonic steroid.
METHODS
Urine Na excretion was measured in uninephrectomized 12-week-old female Sprague-Dawley rats that received renal interstitial infusions of vehicle (5% dextrose in water), cGMP (18, 36, and 72 μg/kg per minute; 30 minutes each), or cGMP+rostafuroxin (12 ng/kg per minute) or were subjected to pressure-natriuresis±rostafuroxin infusion. Rostafuroxin is a digitoxigenin derivative that displaces ouabain from NKA.
RESULTS
Renal interstitial cGMP and raised renal perfusion pressure induced natriuresis and increased phosphorylated Src and Erk 1/2 (extracellular signal-regulated protein kinase 1/2); these responses were abolished with rostafuroxin coinfusion. To assess cGMP binding to NKA, we performed competitive binding studies with isolated rat RPTs using bodipy-ouabain (2 μM)+cGMP (10 µM) or rostafuroxin (10 µM) and 8-biotin-11-cGMP (2 μM)+ouabain (10 μM) or rostafuroxin (10 µM). cGMP or rostafuroxin reduced bodipy-ouabain fluorescence intensity, and ouabain or rostafuroxin reduced 8-biotin-11-cGMP staining. We cross-linked isolated rat RPTs with 4-N-PET-8-biotin-11-cGMP (2 μM); 8-N-6-biotin-10-cAMP served as negative control. Precipitation with streptavidin beads followed by immunoblot analysis showed that RPTs after cross-linking with 4-N-PET-8-biotin-11-cGMP exhibited a significantly stronger signal for NKA than non-cross-linked samples and cross-linked or non-cross-linked 8-N-6-biotin-10-cAMP RPTs. Ouabain (10 μM) reduced NKA in cross-linked 4-N-PET-8-biotin-11-cGMP RPTs confirming fluorescence staining. 4-N-PET-8-biotin-11-cGMP cross-linked samples were separated by SDS gel electrophoresis and slices corresponding to NKA molecular weight excised and processed for mass spectrometry. NKA was the second most abundant protein with 50 unique NKA peptides covering 47% of amino acids in NKA. Molecular modeling demonstrated a potential cGMP docking site in the ouabain-binding pocket of NKA.
CONCLUSIONS
cGMP can bind to NKA and thereby mediate natriuresis.
背景
细胞外肾间质鸟苷酸环 3'，5'-单磷酸（cGMP）通过 Src（Src 家族激酶）激活抑制肾近端小管（RPT）钠（Na）重吸收。通过哪种靶细胞外 cGMP 作用于诱导排钠是未知的。我们假设 cGMP 与 RPT 基底外侧膜上 NKA（钠-钾 ATP 酶）的细胞外α1 亚基结合，以抑制 Na 转运，类似于哇巴因 - 一种强心甾体。
方法
在接受肾间质输注载体（5%葡萄糖水）、cGMP（18、36 和 72μg/kg/分钟；每次 30 分钟）或 cGMP+rostafuroxin（12ng/kg/分钟）的 12 周龄雌性 Sprague-Dawley 大鼠中测量单侧肾切除大鼠的尿钠排泄。Rostafuroxin 是一种洋地黄毒苷衍生物，可将哇巴因从 NKA 中置换出来。
结果
肾间质 cGMP 和升高的肾灌注压诱导排钠和增加磷酸化 Src 和 Erk 1/2（细胞外信号调节蛋白激酶 1/2）；这些反应在 rostafuroxin 共输注时被消除。为了评估 cGMP 与 NKA 的结合，我们使用分离的大鼠 RPT 进行了竞争性结合研究，使用 bodipy-ouabain（2μM）+cGMP（10μM）或 rostafuroxin（10μM）和 8-生物素-11-cGMP（2μM）+哇巴因（10μM）或 rostafuroxin（10μM）。cGMP 或 rostafuroxin 降低了 bodipy-ouabain 的荧光强度，哇巴因或 rostafuroxin 降低了 8-生物素-11-cGMP 的染色。我们用 4-N-PET-8-生物素-11-cGMP（2μM）交联分离的大鼠 RPT；8-N-6-生物素-10-cAMP 用作阴性对照。用链霉亲和素珠沉淀后进行免疫印迹分析表明，与 4-N-PET-8-生物素-11-cGMP 交联的 RPT 比非交联样品和交联或非交联的 8-N-6-生物素-10-cAMP RPT 表现出更强的 NKA 信号。哇巴因（10μM）降低了交联的 4-N-PET-8-生物素-11-cGMP RPT 中的 NKA，证实了荧光染色。用 SDS 凝胶电泳分离 4-N-PET-8-生物素-11-cGMP 交联的样品，并切下对应于 NKA 分子量的切片，进行质谱处理。NKA 是第二丰富的蛋白质，具有 50 个独特的 NKA 肽，覆盖 NKA 氨基酸的 47%。分子建模表明 NKA 的哇巴因结合口袋中存在潜在的 cGMP 对接位点。
结论
cGMP 可以与 NKA 结合，从而介导排钠。
Zotero 插件