Isolated principal and intercalated cells: hormone responsiveness and Na+-K+-ATPase activity.
Isolated principal and intercalated cells: hormone responsiveness and Na+-K+-ATPase activity.
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分离的主细胞和闰细胞：激素反应性和 Na -K -ATP 酶活性。
DOI：
10.1152/ajprenal.1989.256.4.f742
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Naray-Fejes-Toth,A
中科院分区：
文献类型：
--
作者：
Fejes-Toth,G;Naray-Fejes-Toth,A
为了探讨主细胞（PC）和闰间细胞（ICC）在生化和功能上的差异，我们建立了一种从兔肾分离主细胞和闰间细胞的方法。皮质集合管的碎片分离免疫解剖，并从这些集群获得的单细胞与荧光染料共轭，细胞特异性标记物染色。然后通过荧光激活细胞分选分离PC和ICC。通过用其他细胞特异性单克隆抗体（MCAB）或花生凝集素染色来确认分选的细胞的身份。ICC的纯度大于99%，PC的纯度大于96%。精氨酸加压素（AVP）增加腺苷3 '，5'-环磷酸（cAMP）的积累在两种细胞类型，但最大刺激与PC（约20倍）比ICC（2.7倍）显着更大。两种细胞类型在约2 x 10（-10）M AVP时均观察到半最大刺激。异丙肾上腺素仅增加ICC的cAMP水平（从1.23 +/- 0.16至12.06 +/- 1.25 fmol/cell; P <0.001）。分选后的PC中哇巴因结合位点的数量和Na+-K+-ATP酶的活性显著高于ICC（分别为2.2 × 10（6）和9.6 × 10（5）结合位点; PC和ICC中ATP水解分别为19.2和9.6 fmol·min-1·cell-1）。这些结果证明了分离PC和ICC的同质群体的可行性，这对于进一步研究其生化和功能特性是有用的。
To explore biochemical and functional differences between principal (PC) and intercalated cells (ICC), we have developed a method for separating them from rabbit kidney. Fragments of cortical collecting ducts were isolated by immunodissection, and single cells obtained from these clusters were stained with fluorochrome-conjugated, cell-specific markers. PC and ICC were then separated by fluorescence-activated cell sorting. Identity of the sorted cells was confirmed by staining with other cell-specific monoclonal antibodies (MCABs) or peanut lectin. Purity was greater than 99% for ICC and greater than 96% for PC. Arginine vasopressin (AVP) increased adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in both cell types, but maximal stimulation was significantly greater with PC (approximately 20-fold) than with ICC (2.7-fold). Half-maximal stimulation was seen at approximately 2 x 10(-10) M AVP with both cell types. Isoproterenol increased cAMP levels only with ICC (from 1.23 +/- 0.16 to 12.06 +/- 1.25 fmol/cell; P less than 0.001). The number of ouabain binding sites and the activity of Na+-K+-ATPase was significantly higher in sorted PC than ICC (2.2 X 10(6) vs. 9.6 X 10(5) binding sites; 19.2 vs. 9.6 fmol.min-1.cell-1 ATP hydrolyzed in PC vs. ICC, respectively). These results demonstrate the feasibility of isolating homogeneous populations of PC and ICC, which is useful for further studies of their biochemical and functional characterization.