ION TRANSPORT IN AIRWAY SMOOTH MUSCLE

气道平滑肌中的离子传输

• 批准号: 2332535
• 负责人: KERRY J RHODEN
• 金额: $ 9.66万
• 依托单位: JOHN B. PIERCE LABORATORY, INC.
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2332535
• 关键词: adenosinetriphosphatase; bicarbonates; calcium flux; calcium metabolism; carbon dioxide; guinea pigs; human tissue; hydrogen potassium exchanging ATPase; hydrogen transport; ion transport; membrane transport proteins; muscle contraction; muscle relaxation; muscle tension; muscle tone; phosphatase inhibitor; potassium; respiratory muscles; smooth muscle; sodium potassium exchanging ATPase; tissue /cell culture

Airway smooth muscle (ASM) is subjected to changes in intracellular pH (pHi) due to physiological and pathological perturbations in acid/base balance, and such changes can exert a profound influence on ASM function. The overall purpose of this proposal is to determine the pathways of pHi regulation in ASM, the effect of alterations in pHi on ASM contractile function, and the cellular mechanisms by which pHi influences excitation- contraction coupling. Specific Aim 1 is to define and characterize ion transport pathways involved in maintaining resting pHi in ASM and in mediating pHi recovery following an acid- and alkaline-load. The contribution of H+-transporting pathways (the Na-H exchanger, the H-K ATPase and the v-type H ATPase) and HCO3--transporting pathways (Cl-HCO3 transporters) will be assessed. This will be accomplished through spectrofluorimetric measurements of pHi in cultured ASM cells with BCECF. One ion transport pathway of particular interest is the H-K ATPase, since preliminary data suggests that selective inhibitors of this pathway cause relaxation of ASM. Specific Aim 2 is identify a putative H-K ATPase in ASM by immunofluorescent staining with H-K ATPase antibodies, and by the measurement of H+ and K+ transport across the ASM cell membrane. It is hypothesized that ASM relaxation induced by H-K ATPase inhibitors is mediated by a change in pHi. Specific Aim 3, therefore, is to determine the influence of pHi on ASM contractile function. This will be accomplished by examining the effect of physiological and pharmacological manipulations of pHi on tone and contractility of freshly isolated airways in vitro. It is hypothesized that an intracellular acidifications promotes ASM relaxation and alkalinization promotes contraction, and that these effects are mediated through an alterations in intracellular Ca2+ metabolism. Specific Aim 4 is to investigate the relationship between pHi and intracellular Ca2+ concentration in ASM. This will be accomplished by the spectrofluorimetric measurement of Ca2+ in cultured ASM cells with Fura 2. Finally, it is hypothesized that ASM pHi, in addition to exerting an influence on muscle tone, may itself be influenced by receptor agonists which contract or relax ASM. Specific Aim 5 is to determine the effect of receptor agonists on ASM pHi and to investigate the role that agonist- induced changes in pHi may play in excitation-contraction coupling.

气道平滑肌（ASM）受细胞内pH的变化 （PHI）由于酸/碱的生理和病理扰动 平衡，这种变化会对ASM功能产生深远的影响。 该提案的总体目的是确定PHI的途径 ASM的调节，PHI改变对ASM收缩的影响 功能以及PHI影响激发的细胞机制 收缩耦合。特定目标1是定义和表征离子 涉及维持ASM和ASM中静止PHI的运输途径 酸和碱负载后介导PHI恢复。这 H+运输途径的贡献（Na-H交换器，H-K ATPase和V型H ATPase）和HCO3-运输途径（CL-HCO3 将评估转运蛋白）。这将通过 用BCECF培养的ASM细胞中PHI的光谱测量值。 H-K ATPase是一种特别感兴趣的离子运输途径，因为 初步数据表明，这种途径的选择性抑制剂 放松ASM。特定目标2是识别ASM中推定的H-K ATPase 通过使用H-K ATPase抗体进行免疫荧光染色，并通过 跨ASM细胞膜的H+和K+转运的测量。这是 假设H-K ATPase抑制剂诱导的ASM松弛是 由PHI的变化介导。因此，特定目标3是确定 PHI对ASM收缩功能的影响。这将是 通过检查生理和药理的影响来完成 对PHI的操纵对新鲜隔离气道的音调和收缩力 体外。假设细胞内酸化会促进 ASM放松和碱化会促进收缩，而这些 效应是通过细胞内Ca2+的改变介导的 代谢。特定目的4是调查PHI之间的关系 ASM中的细胞内Ca2+浓度。这将通过 培养的ASM细胞中Ca2+的光谱测量 Fura 2。最后，假设ASM Phi除了施加 对肌肉张力的影响本身可能受到受体激动剂的影响 哪个收缩或放松ASM。具体目标5是确定 ASM PHI上的受体激动剂，并研究激动剂的作用 诱导的PHI变化可能会在激发反应耦合中发挥作用。