Mechanisms of Kinase-dependent Regulation of the V-ATPase in Kidney

肾脏中 V-ATP 酶的激酶依赖性调节机制

• 批准号: 8539782
• 负责人: NURIA M. PASTOR-SOLER
• 金额: $ 28.66万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8539782
• 关键词: 5' -AMP-activated protein kinase; 6-carboxyfluorescein; AMP-activated protein kinase kinase; ATP phosphohydrolase; Abbreviations; Acids; Address; Adenylate Cyclase; Apical; Bicarbonates; Biological Assay; Cell Culture Techniques; Cell Line; Cell membrane; Cells; Chronic; Clear Cell; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Disease; Distal renal tubular acidosis Type 1; Duct (organ) structure; Embryo; Environment; Enzymes; Epithelial; Epithelial Cells; Esters; Excretory function; Functional disorder; Generations; Genitourinary system; Horseradish Peroxidase; Human; In Vitro; Individual; Intercalated Cell; Kidney; Lead; Maintenance; Male Infertility; Maps; Mass Spectrum Analysis; Mediating; Metabolic; Metabolic Activation; Metabolic acidosis; Metabolism; Organ; Oryctolagus cuniculus; Patients; Peanut Agglutinin; Phospholipase C; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Physiology; Protein Kinase Inhibitors; Protons; Pump; Recovery; Regulation; Role; Site-Directed Mutagenesis; Sperm Maturation; Stimulus; Tight Junctions; Vas deferens structure; Whole Organism; abstracting; apical membrane; base; bone; carbonate dehydratase; collecting tubule structure; complement C2b; epithelial Na+ channel; extracellular; in vivo; inhibitor/antagonist; insight; interest; male; pH Homeostasis; prevent; protein complex; protein kinase inhibitor; reproductive; sensor; sperm cell; vacuolar H+-ATPase; water channel

Abstract. V-ATPase function in kidney is important for excretion of non-volatile acids generated by metabolism. Individuals with defective V-ATPase activity in kidney intercalated cells (ICs) may suffer from severe chronic metabolic acidosis with potential severe sequelae in bone, kidney, and other organs. V-ATPase activity is also critical to the function of epithelial proton-secreting epididymal clear cells, which contribute to the acidic environment where spermatozoa mature. Therefore, understanding V-ATPase regulation may provide insights involving the physiology and pathophysiology of proton secretion in the urogenital tract and lead to better treatments for severe metabolic acidosis and male infertility. V-ATPase activity is likely orchestrated by a number of regulatory mechanisms; direct V-ATPase phosphorylation in mammalian proton- secreting epithelial cells, however, has been addressed by only a few studies. We and others have shown that in clear cells, V-ATPase translocation to the apical membrane responds to alkaline luminal pH, increased luminal HCO3- concentration, soluble adenylyl cyclase (sAC), and protein kinase A (PKA) activity. Our preliminary studies demonstrate that PKA is also required for apical V-ATPase in ICs and that sAC regulates V-ATPase-mediated intracellular pH (pHi) recovery in isolated perfused outer medullary collecting ducts (OMCDs). In addition, activation of the metabolic sensor AMP-activated protein kinase (AMPK) prevents V- ATPase translocation in clear cells and ICs, and that both PKA and AMPK phosphorylate V-ATPase A and C2b subunits in vitro and in vivo. Based on these findings, we hypothesize that subcellular localization and V-ATPase activity in ICs depend on the direct phosphorylation of its subunits by PKA and AMPK, and that V-ATPase activity may be coupled to the sensing of extracellular acid-base status via PKA and metabolic status via AMPK. The Aims of this proposal are to: 1) determine the mechanisms involved in the modulation of V-ATPase subcellular localization and activity by PKA in ICs; and 2) determine the mechanisms involved in the modulation of V-ATPase subcellular localization and activity by AMPK in ICs. Initially, we will focus our efforts toward mapping PKA and AMPK candidate phosphorylation sites in the V1 sector A subunit. To identify relevant phosphorylation sites, we will use site-directed mutagenesis followed by in vitro and in vivo phosphorylation assays, as well as mass spectrometry. Additionally, we will examine the roles of relevant PKA and AMPK phosphorylation sites in the A subunit on V-ATPase subcellular localization and activity using ICs in cell culture. We will determine the effects of PKA or AMPK activation on V-ATPase-dependent pHi changes in OMCDs. Finally, we will examine the effects of AMPK activation on PKA-mediated phosphorylation of the V- ATPase A subunit in vitro and in vivo. The successful completion of the Aims of this application will contribute to new strategies to treat disorders of acidification in the kidney and in the urogenital tract.

抽象的。 肾脏中的V-ATPase功能对于排泄代谢产生的非易失性酸很重要。 肾脏插入细胞（IC）中V-ATPase活性有缺陷的个体可能患有严重的慢性 代谢性酸中毒具有潜在的骨，肾脏和其他器官的潜在后遗症。 V-ATPase活性也是 对上皮质子分泌的附睾透明细胞的功能至关重要，这有助于酸性 精子成熟的环境。因此，了解V-ATPase调节可能会提供 涉及泌尿生殖道质子分泌的生理学和病理生理学的见解 为严重的代谢性酸中毒和男性不育症提供更好的治疗方法。 V-ATPase活动可能是 由多种监管机制精心策划；哺乳动物质子的直接V-ATPase磷酸化 然而，仅少数研究已经解决了分泌上皮细胞。我们和其他人表明 在透明细胞中，V-ATPase转移到顶膜对碱性腔pH的反应，增加 腔HCO3-浓度，可溶性腺苷酸环化酶（SAC）和蛋白激酶A（PKA）活性。我们的 初步研究表明，PKA在IC中的顶端V-ATPase也需要PKA，并且SAC调节了PKA。 V-ATPase介导的细胞内pH（PHI）回收 （OMCD）。另外，代谢传感器AMP激活蛋白激酶（AMPK）的激活可防止V- 透明细胞和ICS中的ATPase易位，并且PKA和AMPK磷酸化V-ATPase A和 C2B体外和体内亚基。基于这些发现，我们假设该亚细胞定位和 IC中的V-ATPase活性取决于PKA和AMPK的亚基的直接磷酸化，以及 V-ATPase活性可以与PKA和 代谢状态通过AMPK。该提议的目的是：1）确定 PKA在IC中调节V-ATPase亚细胞定位和活性； 2）确定机制 参与IC中AMPK对V-ATPase亚细胞定位和活性的调节。最初，我们会的 将我们的努力集中在V1部门的一个亚基中绘制PKA和AMPK候选磷酸化位点。 为了识别相关的磷酸化位点，我们将使用位置定向的诱变，然后体外和体内 磷酸化测定以及质谱法。此外，我们将研究相关PKA的作用 A和AMPK磷酸化位点在V-ATPase亚细胞亚基上使用ICS中的V-ATPase亚基定位和活性 细胞培养。我们将确定PKA或AMPK激活对V-ATPase依赖性PHI变化的影响 OMCD。最后，我们将研究AMPK激活对V- PKA介导的磷酸化的影响 ATPase在体外和体内亚基。该申请的目标成功完成将有助于 采取新的策略来治疗肾脏和泌尿生殖道中的酸化疾病。