Mechanisms and treatment of impaired erythrocyte ATP release in older adults

老年人红细胞 ATP 释放受损的机制和治疗

• 批准号: 8979328
• 负责人: Matthew Racine
• 金额: $ 3.4万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8979328
• 关键词: Activities of Daily Living; Adenine Nucleotides; Adenosine Triphosphate; Adult; Age; Aging; Ascorbic Acid; Binding; Biology; Blood; Blood Circulation; Blood Gas Analysis; Blood Vessels; Blood flow; Blood gas; Cardiovascular system; Cells; Cilostazol; Clinical Research; Cyclic AMP; Diamide; Disease; Doppler Ultrasonography; Doppler Ultrasound; Elderly; Electrolytes; Endothelium; Erythrocyte Membrane; Erythrocytes; Exercise; Fatigue; Forearm; Goals; Hemoglobin; Human; Hypertension; Hypoxia; Impairment; In Vitro; Intervention; Lead; Link; Luciferases; Measures; Membrane; Methodology; Mission; Muscle; Outcome; Oxidative Stress; Oxygen; Pathway interactions; Pharmaceutical Preparations; Physiological; Plasma; Property; Purinoceptor; Quality of life; Regulation; Research Priority; Saline; Science; Signal Transduction; Skeletal Muscle; Stimulus; Structure; Techniques; Testing; Time; Tissues; Training; Translating; Vasodilation; cardiovascular disorder risk; cardiovascular risk factor; fasudil; improved; in vivo; indexing; insight; luciferin; new therapeutic target; novel; public health relevance; response; stressor; young adult

 DESCRIPTION (provided by applicant): The adenine nucleotide ATP is released from erythrocytes (RBCs) into the circulation in response to hemoglobin (Hb) deoxygenation and cell deformation, and binds directly to purinergic receptors along the endothelium to evoke vasodilation and increase tissue blood flow and oxygen delivery. Isolated RBC ATP release during Hb deoxygenation, and plasma [ATP] and forearm blood flow (FBF) responses during exercise and systemic hypoxia, are impaired in healthy older vs. young adult humans. However, the underlying cause of these age-associated impairments remains unclear. Thus, the overall aim of this proposal is to identify changes within the RBC that contribute to impaired ATP release and vascular control with advancing age. Specific Aim 1 will test the hypothesis that age-associated changes in RBC structure and function - specifically, membrane deformability, oxidative stress, and cyclic AMP signaling - are mechanistically-linked to impaired ATP release during Hb deoxygenation in isolated RBCs from older adults. To test this, isolated RBC ATP release in response to normoxic (pO2 ~110-120mmHg) and hypoxic (pO2 ~15-25mmHg) stimuli will be quantified in RBCs from young and older adults using the luciferin-luciferase technique after incubation with either saline (control) or pharmacological agents to modify membrane deformability (Y-27632/ diamide), oxidative stress (ascorbic acid), or intracellular [cAMP] (cilostazol). Specific Aim 2 will test the hypothesis that in vivo treatment of impaired RBC ATP release with age will improve isolated RBC ATP release during Hb deoxygenation and in vivo vascular and plasma ATP responsiveness to systemic hypoxia and graded-intensity handgrip exercise in older adults, and that this will be associated with improved exercise capacity. To test this, the treatment from Specific Aim 1 that elicits the greatest improvement in Hb deoxygenation-induced ATP release in RBCs from older adults will be identified, and the corresponding pathway will be targeted using in vivo, systemic administration of either fasudil (deformability), ascorbic acid (oxidative stress), or cilostazol (cAMP signaling), in young and older adults. When peak plasma concentration of the drug is achieved, RBCs will be isolated and Hb deoxygenation-induced ATP release will be quantified as in Specific Aim 1; in conjunction, FBF responses to systemic isocapnic hypoxia (80% SpO2) and graded-intensity handgrip exercise (5%, 15%, 25% MVC) will be quantified using Doppler ultrasound and plasma [ATP] will be measured using the luciferin-luciferase technique. Submaximal rhythmic handgrip exercise time-to-fatigue will be assessed as an index of exercise capacity. The expected outcomes will provide novel insight into the impairments in RBC function and subsequent in vivo vascular control that occur with normal human aging; this insight is relevant to NHLBI's mission and research priorities, particularly those of the Vascular Biology and Hypertension Branch in the Division of Cardiovascular Sciences, given that impaired vascular control with advancing age leads to an increased risk of cardiovascular/ischemic disease and a decline in functional capacity and overall quality of life.

 描述（由申请人提供）：腺嘌呤核苷酸ATP响应血红蛋白（Hb）脱氧和细胞变形从红细胞（RBC）释放到循环中，并直接与沿着内皮的嘌呤能受体结合，以引起血管舒张并增加组织血流量和氧输送。Hb脱氧期间的分离RBC ATP释放，以及运动和全身缺氧期间的血浆[ATP]和前臂血流（FBF）反应在健康的老年人与年轻人中受损。然而，这些与年龄相关的损伤的根本原因仍不清楚。因此，本提案的总体目标是确定红细胞内的变化，这些变化有助于随着年龄的增长而受损的ATP释放和血管控制。具体目标1将检验以下假设：RBC结构和功能的年龄相关变化-特别是膜变形性、氧化应激和环AMP信号传导-与老年人分离RBC中Hb脱氧期间ATP释放受损存在机械联系。为了测试这一点，分离的RBC ATP释放响应于常氧（pO 2 ~110- 120 mmHg）和缺氧在与生理盐水（对照）或药理学试剂孵育以改变膜变形性后，将使用荧光素酶技术对来自年轻人和老年人的RBC中的（pO 2 ~15- 25 mmHg）刺激进行定量（Y-27632/二酰胺）、氧化应激（抗坏血酸）或细胞内[cAMP]（西洛他唑）。具体目标2将检验以下假设：随着年龄的增长，RBC ATP释放受损的体内治疗将改善Hb脱氧期间的孤立RBC ATP释放，以及老年人体内血管和血浆ATP对全身缺氧和分级强度握力运动的反应性，并且这将与运动能力的改善相关。测试 因此，将鉴定出特异性目标1的治疗，其最大程度地改善了老年人RBC中Hb脱氧诱导的ATP释放，并且将在年轻人和老年人中使用法舒地尔（可变形性）、抗坏血酸（氧化应激）或西洛他唑（cAMP信号传导）的体内全身给药靶向相应的途径。当达到药物的血浆峰浓度时，将分离RBC，并如特定目的1中所述定量Hb脱氧诱导的ATP释放;同时，将使用多普勒超声定量FBF对全身等二氧化碳缺氧（80% SpO 2）和分级强度握力运动（5%、15%、25% MVC）的反应，并使用荧光素酶技术测量血浆[ATP]。将评估次极量节律性握力运动至疲劳时间作为运动能力指标。预期的结果将提供新的见解，在红细胞功能的损害和随后的体内血管控制，发生与正常的人类衰老;这一认识与NHLBI的使命和研究重点有关，特别是心血管科学部的血管生物学和高血压分支，因为随着年龄的增长，血管控制受损导致心血管/缺血性疾病风险增加，功能能力和总体生活质量下降。