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CATION TRANSPORT, ANTIGENS AND SHEEP RED CELL MATURATION

阳离子运输、抗原和绵羊红细胞成熟

基本信息

批准号：
3154615
负责人：
PETER K LAUF
金额：
$ 12.13万
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 1989-03-31
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型性是与a）Na+K+泵，B）哇巴因敏感性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，或细胞在低渗培养基中肿胀，和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性， Na+/Cl交换通量，其作用尚不清楚。成熟的过程网织红细胞与预期LK红细胞的比例可能涉及所有上述运输系统连续或同时运行，将细胞中的K+交换为Na+（HK-LK转换）。为研究膜转运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一种独特的基因定义模型为了理解 HK-LK过渡的个人提出了进一步的分析成熟HK和LK红细胞中的转运系统以及体外跟踪研究所有运输活动的成熟变化。具体而言，建议：1.进一步表征了 LK红细胞中的SH依赖性、体积敏感性K/Cl途径， a）通过共价结合确定其生化和抗原性质 3 H-NEM或3 H-布美他尼，一种抑制性袢利尿剂，放射性标记的抗L1特异性降低SH依赖性K/Cl通量; B）细胞质因子调节SH依赖性K/Cl通量假说 (ATP)或细胞代谢;和c）的生理基础氯需求和髓袢利尿剂的抑制。这些分析将使近年来对其他偶联离子转运蛋白的作用机制有了新的认识在肾脏和肠道生理学中有描述。 2.分析 HK和LK红细胞的哇巴因不敏感Na+途径，特别是 Na ~+/Na ~+反向转运作为Na ~+/H ~+交换潜在候选者与外向K/Cl通量一起，可能对发展很重要的LK细胞。 3.研究其作用部位，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于新的电泳技术（电印迹）。 5.到通过实验检验上述所有转运蛋白研究同时或连续下调细胞K+水平从HK网织红细胞到成熟LK红细胞，了解HK中SH依赖性K/Cl转运失活的模式和LK红细胞中K+泵通量。