Field F J, Born E, Murthy S, Mathur S N
Department of Internal Medicine and the Veterans Administration, University of Iowa, Iowa City 52242, USA.
J Lipid Res. 1998 Oct;39(10):1938-50.
It was postulated that specialized microdomains of the plasma membrane, consistent with caveolae, might play a role in cholesterol trafficking in intestinal cells. The existence, therefore, of caveolin and the role of detergent-resistant microdomains of the plasma membrane in cholesterol trafficking were investigated in human small intestine and CaCo-2 cells. Caveolin mRNA was detected by RT-PCR in small intestinal brushings and biopsies and in CaCo-2 cells. Northern hybridization of caveolin mRNA detected 3 kb and 0.8 kb transcripts in CaCo-2 cells. From brushings of distal duodenum and in CaCo-2 cells, Western analysis for detection of caveolin protein demonstrated a 21 kDa-sized protein and a 600 kDa homooligomer. In CaCo-2 cells, caveolin was demonstrated by immunofluorescence in apical membranes as well as within cells. Using sucrose-density gradients, caveolin was localized to detergent-resistant microdomains of the plasma membrane. As determined by cholesterol oxidase-accessible cholesterol, 3-5% of plasma membrane cholesterol in CaCo-2 cells was estimated to be in these detergent-resistant microdomains. After the absorption of cholesterol from bile-salt micelles, more plasma membrane cholesterol moved to these specialized microdomains within the plasma membrane and was esterified. In CaCo-2 cells, filipin, N-ethyl maleimide, and cholesterol depletion, treatments that disrupt caveolar function, interfered with the transport of plasma membrane cholesterol to the endoplasmic reticulum, whereas okadaic acid, sphingomyelinase, and cholesterol oxidase did not. Changes in cholesterol flux at the apical membrane of the cell did not alter mRNA levels or mass of caveolin. The results suggest that caveolin is present in intestinal and CaCo-2 cells and is associated with detergent-resistant microdomains of cellular membranes. With the influx of micellar cholesterol from the lumen, plasma membrane cholesterol moves or "clusters" to these microdomains and is transported to the endoplasmic reticulum for esterification and eventual transport. Caveolin/caveolae may play a role in cholesterol trafficking in intestinal cells.
据推测，质膜上与小窝一致的特殊微区可能在肠道细胞的胆固醇转运中发挥作用。因此，在人小肠和CaCo-2细胞中研究了小窝蛋白的存在以及质膜耐去污剂微区在胆固醇转运中的作用。通过RT-PCR在小肠刷取物、活检组织和CaCo-2细胞中检测到小窝蛋白mRNA。小窝蛋白mRNA的Northern杂交在CaCo-2细胞中检测到3 kb和0.8 kb的转录本。在十二指肠远端刷取物和CaCo-2细胞中，用于检测小窝蛋白的Western分析显示出一种21 kDa大小的蛋白和一种600 kDa的同型寡聚体。在CaCo-2细胞中，通过免疫荧光在顶端膜以及细胞内证实了小窝蛋白的存在。使用蔗糖密度梯度，小窝蛋白定位于质膜的耐去污剂微区。通过胆固醇氧化酶可及胆固醇测定，估计CaCo-2细胞质膜胆固醇的3-5%存在于这些耐去污剂微区中。从胆盐微团吸收胆固醇后，更多的质膜胆固醇转移到质膜内的这些特殊微区并被酯化。在CaCo-2细胞中，破坏小窝功能的处理，如制霉菌素、N-乙基马来酰胺和胆固醇耗竭，干扰了质膜胆固醇向内质网的转运，而冈田酸、鞘磷脂酶和胆固醇氧化酶则没有。细胞顶端膜胆固醇通量的变化并未改变小窝蛋白的mRNA水平或质量。结果表明，小窝蛋白存在于肠道和CaCo-2细胞中，并与细胞膜的耐去污剂微区相关。随着来自肠腔的微团胆固醇的流入，质膜胆固醇移动或“聚集”到这些微区，并被转运到内质网进行酯化和最终转运。小窝蛋白/小窝可能在肠道细胞的胆固醇转运中发挥作用。
