Tyagi S, Joshi V, Alrefai W A, Gill R K, Ramaswamy K, Dudeja P K
Department of Medicine, University of Illinois at Chicago and Chicago VA Medical Center, 60612, USA.
Dig Dis Sci. 2000 Dec;45(12):2282-9. doi: 10.1023/a:1005670404456.
The mechanism(s) of electrolyte transport across the human colonic contraluminal domain is not well understood. Current studies were undertaken to develop a technique for the isolation and purification of the human colonic basolateral membrane vesicles (BLMV) and to examine the presence of a Na+-H+ exchange process in these membranes. BLMV were purified from mucosal scrapings of organ donor proximal colons utilizing a Percoll density gradient centrifugation technique, and Na+ transport was examined utilizing a rapid filtration, technique. Our data demonstrate that purified basolateral membranes were enriched 10- to 11-fold in Na+, K+-ATPase activity compared to crude homogenate. Results consistent with the Na+-H+ exchange in BLMV are as follows: (1) an outwardly directed H+ gradient stimulated 22Na uptake; (2) 22Na uptake was markedly inhibited by EIPA and amiloride; (3) H+-gradient-stimulated 22Na uptake was not inhibited by bumetanide, SITS, DIDS, acetazolamide, phenamil and benzamil; (4) 22Na uptake was voltage insensitive; (5) 22Na uptake demonstrated saturation kinetics; (6) 22 Na uptake was markedly inhibited by Na+ and Li+ but was unaffected by N-methyl glucamine+, choline+, and NH4+. Immunoblotting studies demonstrated this Na+-H+ exchanger isoform to be represented by NHE1. In conclusion, a technique has been established for the purification of functional human proximal colonic BLMV, and an electroneutral Na+-H+ exchange process has been demonstrated in these membranes.
电解质跨人结肠腔外区域转运的机制尚未完全明确。目前开展了多项研究，旨在开发一种分离和纯化人结肠基底外侧膜囊泡（BLMV）的技术，并检测这些膜中是否存在Na⁺-H⁺交换过程。利用Percoll密度梯度离心技术从器官捐献者近端结肠的黏膜刮片中纯化BLMV，并采用快速过滤技术检测Na⁺转运。我们的数据表明，与粗匀浆相比，纯化后的基底外侧膜中Na⁺,K⁺-ATP酶活性提高了10至11倍。与BLMV中Na⁺-H⁺交换相符的结果如下：（1）外向的H⁺梯度刺激²²Na摄取；（2）²²Na摄取受到EIPA和阿米洛利的显著抑制；（3）H⁺梯度刺激的²²Na摄取不受布美他尼、SITS、DIDS、乙酰唑胺、非那明和苄胺的抑制；（4）²²Na摄取对电压不敏感；（5）²²Na摄取呈现饱和动力学；（6）²²Na摄取受到Na⁺和Li⁺的显著抑制，但不受N-甲基葡糖胺⁺、胆碱⁺和NH₄⁺影响。免疫印迹研究表明，这种Na⁺-H⁺交换异构体由NHE1代表。总之，已建立了一种纯化功能性人近端结肠BLMV的技术，并在这些膜中证实了一种电中性的Na⁺-H⁺交换过程。
