Chamberlin M E, LeFurgey A, Mandel L J
Am J Physiol. 1984 Dec;247(6 Pt 2):F955-64. doi: 10.1152/ajprenal.1984.247.6.F955.
A procedure for isolating a suspension of tubules derived from the rabbit medullary thick ascending limb is described. The purity of the preparation was assessed by microscopy and enzyme assays and the viability of the preparation was assessed by measuring oxygen consumption. Microscopy revealed that the suspension contains 95% thick ascending limbs and that the isolation procedure preserves the structure of the epithelium except for the loss of the basement membrane. The preparation had a high activity of calcitonin-sensitive adenylate cyclase, a marker enzyme for the medullary thick ascending limb. Control oxygen consumption was considerably higher than that reported for proximal tubules in the literature, and nystatin or carbonyl cyanide p-trifluoromethoxyphenylhydrazone addition produced a more than 100% increase in oxygen consumption. Furosemide inhibited the oxygen consumption by 43% and ouabain inhibited it by 42%. Furosemide inhibited sodium chloride entry without directly affecting the Na-K-ATPase or cellular metabolism. Chloride removal depressed oxygen consumption to the same extent as furosemide, but some of this action was through direct inhibition of cellular metabolism.
本文描述了一种从兔髓袢升支粗段分离肾小管悬液的方法。通过显微镜检查和酶分析评估制备物的纯度，通过测量耗氧量评估制备物的活力。显微镜检查显示，悬液中含有95%的髓袢升支粗段，且除基底膜缺失外，分离过程保留了上皮结构。该制备物具有高活性的降钙素敏感腺苷酸环化酶，这是髓袢升支粗段的一种标记酶。对照耗氧量显著高于文献报道的近端小管，添加制霉菌素或羰基氰对三氟甲氧基苯腙后耗氧量增加超过100%。呋塞米抑制耗氧量43%，哇巴因抑制42%。呋塞米抑制氯化钠进入，而不直接影响钠钾ATP酶或细胞代谢。去除氯离子使耗氧量降低的程度与呋塞米相同，但部分作用是通过直接抑制细胞代谢实现的。
