Brion L P, Schwartz J H, Lachman H M, Zavilowitz B J, Schwartz G J
Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York 10461.
Am J Physiol. 1989 Sep;257(3 Pt 2):F486-501. doi: 10.1152/ajprenal.1989.257.3.F486.
The purpose of these studies was to characterize the development of H+ secretion by cultured inner medullary collecting duct (IMCD) cells. Differentiated IMCD cells derived from rat papillae develop in culture N-ethylmaleimide (NEM)- and dicyclohexylcarbodiimide (DCCD)-sensitive Na+-independent H+ pump, but showed substantial heterogeneity. Cells with pHi greater than or equal to 7.1, measured by 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF), showed more H+ pumping than did those with pHi less than 7.1. Quiescent cells in domes and in the center of growing nests often showed active H+ pumping activity, but rarely exhibited Na+-H+ exchange, whereas dividing cells at the periphery of the nests showed only Na+-H+ exchange. Differentiated IMCD cells also showed Na+-independent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive Cl(-)-HCO3- exchange that functioned best when pHi was greater than or equal to 7.1, and homogenates of IMCD cells expressed carbonic anhydrase (CA) activity and CA II mRNA. A subgroup of IMCD cells avidly bound fluorescent peanut agglutinin (PNA); this binding became more extensive with time in culture. The PNA-labeled cells showed increased BCECF uptake, higher pHi, and more H+ pumping activity than did cells not binding PNA. They could be isolated by gel-immobilized PNA and in culture showed more CA activity and H+ pumping activity than unselected monolayers. The percentage of cells binding PNA correlated well with CA activity, indicating that this subgroup is better specialized to secrete H+. The appearance of PNA binding sites and the development of high CA activity may be characteristic of IMCD cell differentiation in culture.
这些研究的目的是描述培养的髓质内层集合管（IMCD）细胞氢离子分泌的发育过程。从大鼠乳头分离的分化型IMCD细胞在培养过程中会发育出对N-乙基马来酰亚胺（NEM）和二环己基碳二亚胺（DCCD）敏感的非钠依赖性氢离子泵，但表现出很大的异质性。用2',7'-双（羧乙基）-5（6）-羧基荧光素（BCECF）测量，细胞内pH（pHi）大于或等于7.1的细胞比pHi小于7.1的细胞表现出更多的氢离子泵出。穹顶处和生长巢中心的静止细胞通常表现出活跃的氢离子泵出活性，但很少表现出钠-氢离子交换，而巢周边的分裂细胞仅表现出钠-氢离子交换。分化型IMCD细胞还表现出对4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS）敏感的非钠依赖性氯离子-碳酸氢根离子交换，当pHi大于或等于7.1时功能最佳，并且IMCD细胞匀浆表达碳酸酐酶（CA）活性和CA II mRNA。IMCD细胞的一个亚群能强烈结合荧光花生凝集素（PNA）；随着培养时间的延长，这种结合变得更加广泛。与未结合PNA的细胞相比，PNA标记的细胞表现出增加的BCECF摄取、更高的pHi和更多的氢离子泵出活性。它们可以通过凝胶固定的PNA分离，并且在培养中比未选择的单层细胞表现出更多的CA活性和氢离子泵出活性。结合PNA的细胞百分比与CA活性密切相关，表明该亚群更擅长分泌氢离子。PNA结合位点的出现和高CA活性的发展可能是培养中IMCD细胞分化的特征。
