CATION TRANSPORT, ANTIGENS AND RED CELL MATURATION

阳离子运输、抗原和红细胞成熟

基本信息

批准号：
3235925
负责人：
PETER K LAUF
金额：
$ 24.38万
依托单位：
WRIGHT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-09-01 至 1990-11-30
项目状态：
已结题

项目摘要

The cation dimorphism of high K+ (HK) and low K+ (LK) sheep red cells is genetically associated with differences in a) Na+K+ pumps, b) ouabain-sensitive Cl- dependent K+ transport which is activated through reaction of N-ethylmaleimide (NEM) with its sulfhydryl (SH) groups or by cell swelling in hyposmotic media, and c) the presence of the M and L membrane antigens. Both HK and LK red cells possess a ouabain-insensitive Na+/Cl exchange flux whose role is unexplained. The process of maturation of the reticulocy to the prospective LK red cell may involve all of the above transport systems operating consecutively or simultaneously to exchange cellular K+ for Na+ (HK-LK transition). For the study of regulation and development of membrane transport and its pathophysiology and its genetic basis in disease, the HK/LK sheep red cell system offers a unique, genetically defined model. To understand the precise mechanism of the HK-LK transition a further analysis is proposed of the individual transport systems in mature HK and LK red cells as well as an in vitro follow-up study of the maturational changes of all transport activities. In particular, it is proposed: 1. A further characterization of the SH-dependent, volume sensitive K/Cl pathway in LK red cells with respect to a) its biochemical and antigenic nature as ascertained by covalent binding of 3H-NEM or 3H-bumetanide, an inhibitory loop diuretic, and attachment of radiolabeled anti-L1 specifically reducing SH-dependent K/Cl flux; b) the hypothesis of regulation of SH-dependent K/Cl flux by cytoplasmic factors (ATP) or cellular metabolism; and c) the physiologic basis of Cl-requirement and inhibition by loop diuretics. These analyses will shed new light on the mechanism of other coupled ion transporters recently described in renal and intestinal physiology. 2. An analysis of ouabain-insensitive Na+ pathways of HK and LK red cells, in particular of the Na+/Na+ counter transport as a potential candidate for Na+/H+ exchange that together with outward K/Cl flux may be important for the development of the LK cell. 3. An investigation of the specific site of action of cellular K+ to inhibit the Na+K+ pump in LK cells, and an evaluation of the role of protons. 4. A further characterization of the M/L surface antigens with the aid of new electrophoretic techniques (electro blotting). 5. To test experimentally the hypothesis that all of the above transporters studied simultaneously or consecutively down-regulate cellular K+ levels from that in HK reticulocytes to those in mature LK red cells, and to understand the mode of inactivation of SH-dependent K/Cl transport in HK and of K+ pump fluxes in LK erythrocytes.

高 K+ (HK) 和低 K+ (LK) 绵羊红细胞的阳离子二态性为遗传上与 a) Na+K+ 泵、b) 的差异相关哇巴因敏感的 Cl 依赖性 K+ 转运通过 N-乙基马来酰亚胺 (NEM) 与其巯基 (SH) 基团的反应或通过低渗介质中的细胞肿胀，以及 c) M 和 L 的存在膜抗原。 HK 和 LK 红细胞均具有哇巴因不敏感蛋白 Na+/Cl 交换通量，其作用尚不清楚。成熟的过程网状细胞向预期 LK 红细胞的转变可能涉及所有上述运输系统连续或同时运行将细胞 K+ 交换为 Na+（HK-LK 转换）。为了研究膜运输的调控和发展及其病理生理学及其疾病的遗传基础，HK/LK 绵羊红细胞系统提供了独特的、基因定义的模型。为了解其精确机制 HK-LK 过渡建议对个人进行进一步分析成熟 HK 和 LK 红细胞以及体外运输系统对所有运输活动的成熟变化进行跟踪研究。特别是，建议： 1. 进一步表征 LK 红细胞中 SH 依赖性、体积敏感的 K/Cl 通路 a) 通过共价结合确定其生化和抗原性质 3H-NEM 或 3H-布美他尼（一种抑制性袢利尿剂）和附件放射性标记的抗 L1 特异性降低 SH 依赖性 K/Cl 通量； b) 的细胞质因子调节 SH 依赖性 K/Cl 通量的假说 (ATP) 或细胞代谢； c) 生理基础 Cl-需求和袢利尿剂的抑制。这些分析将揭示最近对其他偶联离子转运蛋白机制的新认识肾脏和肠道生理学中描述。 2. 分析 HK 和 LK 红细胞的哇巴因 Na+ 通路不敏感，特别是 Na+/Na+ 逆向转运作为 Na+/H+ 交换的潜在候选者与向外的 K/Cl 通量一起可能对开发很重要 LK 细胞。 3.具体作用部位的调查细胞 K+ 抑制 LK 细胞中的 Na+K+ 泵，并评估质子的作用。 4. M/L 表面抗原的进一步表征借助新的电泳技术（电印迹）。 5. 到通过实验检验上述所有转运蛋白的假设同时或连续研究下调细胞 K+ 水平从 HK 网织红细胞中的细胞到成熟 LK 红细胞中的细胞，以及了解香港 SH 依赖性 K/Cl 运输的失活模式以及 LK 红细胞中的 K+ 泵通量。