Abstract
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 immunoflourescence 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.—Field, F. J., E. Born, S. Murthy, and S. N. Mathur. Caveolin is present in intestinal cells: role in cholesterol trafficking? J. Lipid Res. 1998. 39: 1938–1950.
Highlights
It was postulated that specialized microdomains of the plasma membrane, consistent with caveolae, might play a role in cholesterol trafficking in intestinal cells
To address whether caveolin mRNA was present in human intestine, RNA extracted from small intestinal biopsies and brushings were amplified by Reverse transcription-polymerase chain reaction (RT-PCR)
The results of this study clearly demonstrate the presence of caveolin in the small intestine and CaCo-2 cells
Summary
It was postulated that specialized microdomains of the plasma membrane, consistent with caveolae, might play a role in cholesterol trafficking in intestinal cells. During the influx of oleic acid and/or cholesterol, the amount of cholesterol transported from the plasma membrane to the endoplasmic reticulum was increased and this cholesterol was found as part of the secreted lipoprotein particle [2]. To maintain cholesterol homeostasis during the influx of fatty acid, more newly synthesized cholesterol was transported to the plasma membrane to replenish the cholesterol that was being utilized for lipoprotein assembly. While performing these studies, we found that the absorption of cholesterol from a micellar solution at the apical membrane caused plasma membrane cholesterol to move to the endoplasmic reticulum where it was esterified
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