MacLeod R J, Hamilton J R
Department of Pediatrics, McGill University-Montreal Children's Hospital, Research Institute, Quebec, Canada.
Am J Physiol. 1990 May;258(5 Pt 1):G665-74. doi: 10.1152/ajpgi.1990.258.5.G665.
We assessed ion transport mechanisms operative during regulatory volume increase (RVI) in villus enterocytes isolated in suspension from guinea pig jejunum and examined with electronic cell sizing and 86Rb influx. After validation of the electronic-sizing technique with direct measurements of cell water, the response of cell volume to hypertonic media was evaluated in detail. When shrunk by exposure to hyperosmotic media (455 mosmol/kg medium) cells demonstrated a RVI that was complete in 20 min. RVI required extracellular Na+, K+, and Cl-; this cell swelling showed the following ion sensitivity; Na+ greater than Li+ greater than choline, K+ = Rb+, and Cl- greater than or equal to Br- greater than NO3- = acetate = gluconate. Bumetanide inhibition of villus cell swelling was concentration dependent from 10(-10) to 10(-5) M (7.0 +/- 4.5% vs. 87.8 +/- 0.3%); furosemide (10(-3)M) inhibited RVI (74.1 +/- 9.5%), but amiloride (10(-4) M) had little effect on cell swelling. Disulfonic stilbenes, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (10(-4) M), generated the same inhibition of RVI in either nominally HCO3(-)-free phosphate-buffered saline (PBS) or HCO3(-)-buffered PBS, suggesting anion exchange was not involved. Ouabain (10(-4) M) stimulated cell swelling. Hypertonic shrinkage increased the initial rate of bumetanide-sensitive 86Rb influx (80 +/- 38 vs. 1,011 +/- 241 pmol.mg protein-1.min-1; P less than 0.005) and required extracellular Na+ and Cl- (11 +/- 16 vs. 28 +/- 61 pmol.mg protein-1.min-1). RVI was prevented in low-K+ media (0.2 mM), but the addition of KCl initiated cell swelling. Our data strongly suggest that RVI in jejunal villus enterocytes occurs because of the hypertonic activation of NaKCl2 cotransport.
我们评估了从豚鼠空肠分离的悬浮绒毛肠上皮细胞在调节性容积增加（RVI）过程中起作用的离子转运机制，并通过电子细胞大小测定和86Rb内流进行检测。在用直接测量细胞内水的方法验证了电子大小测定技术后，详细评估了细胞容积对高渗介质的反应。当细胞暴露于高渗介质（455 mosmol/kg介质）而收缩时，表现出在20分钟内完成的RVI。RVI需要细胞外的Na+、K+和Cl-；这种细胞肿胀表现出以下离子敏感性：Na+大于Li+大于胆碱，K+ = Rb+，Cl-大于或等于Br-大于NO3- = 乙酸根 = 葡萄糖酸根。布美他尼对绒毛细胞肿胀的抑制作用在10(-10)至10(-5) M范围内呈浓度依赖性（7.0 +/- 4.5%对87.8 +/- 0.3%）；呋塞米（10(-3)M）抑制RVI（74.1 +/- 9.5%），但阿米洛利（10(-4) M）对细胞肿胀影响很小。二磺基芪类化合物，4-乙酰氨基-4'-异硫氰酸芪-2,2'-二磺酸和4,4'-二异硫氰酸芪-2,2'-二磺酸（10(-4) M），在名义上无HCO3(-)的磷酸盐缓冲盐水（PBS）或HCO3(-)缓冲的PBS中对RVI产生相同程度的抑制，表明不涉及阴离子交换。哇巴因（10(-4) M）刺激细胞肿胀。高渗性收缩增加了布美他尼敏感的86Rb内流的初始速率（80 +/- 38对1,011 +/- 241 pmol·mg蛋白-1·min-1；P < 0.005），且需要细胞外的Na+和Cl-（11 +/- 16对28 +/- 61 pmol·mg蛋白-1·min-1）。在低钾培养基（0.2 mM）中RVI被阻止，但添加KCl可引发细胞肿胀。我们的数据强烈表明，空肠绒毛肠上皮细胞中的RVI是由于NaKCl2协同转运的高渗激活所致。
