Abstract
The transport of newly synthesized cholesterol from its site of synthesis, the endoplasmic reticulum, to the plasma membrane was studied in CaCo-2 cells. The appearance of newly synthesized cholesterol on the cell surface was rapid. By 30 min, 50% of the total labeled cholesterol was observed in the plasma membrane. The arrival of cholesterol at the plasma membrane was independent of new protein synthesis, a functional Golgi apparatus, or microtubular function. Progesterone, verapamil, and trifluoperazine, inhibitors of p-glycoprotein which are known to inhibit cholesterol transport from the plasma membrane to the endoplasmic reticulum, reduced the amount of newly synthesized cholesterol reaching the plasma membrane. The p-glycoprotein inhibitors, however, caused the accumulation of sterol intermediates in the plasma membrane, suggesting that sterol trafficking to the plasma membrane remained intact, but that trafficking from the plasma membrane to the endoplasmic reticulum was disrupted. In contrast, nigericin, another potent inhibitor of cholesterol movement from the plasma membrane to the endoplasmic reticulum, did not alter the transport of newly synthesized cholesterol to the plasma membrane. Moreover, promoting cholesterol transport from the plasma membrane to the endoplasmic reticulum by sphingomyelin hydrolysis or by micellar cholesterol influx did not alter the percent of newly synthesized cholesterol transported to the plasma membrane. Likewise, preventing plasma membrane cholesterol from reaching the endoplasmic reticulum by incubating cells with lysophosphatidylcholine, filipin, or digitonin did not alter the arrival of newly synthesized cholesterol to the plasma membrane. The results suggest that the amount of cholesterol moving to the plasma membrane from the endoplasmic reticulum is constitutive and regulated at the level of cholesterol synthesis and not at the level of the transport process. The pathways of cholesterol transport to and from the plasma membrane are distinct.—Field, F. J., E. Born, S. Murthy, and S. N. Mathur. Transport of cholesterol from the endoplasmic reticulum to the plasma membrane is constitutive in CaCo-2 cells and differs from the transport of plasma membrane cholesterol to the endoplasmic reticulum.
Highlights
The transport of newly synthesized cholesterol from its site of synthesis, the endoplasmic reticulum, to the plasma membrane was studied in CaCo-2 cells
There is no information in intestinal cells as to whether this flux of newly synthesized cholesterol from the endoplasmic reticulum to the plasma membrane is a regulated process, whether it differs from the vesicular pathway of plasma membrane cholesterol influx, or whether p-glycoprotein is involved
To address the rate of movement of newly synthesized cholesterol from the endoplasmic reticulum to the plasma membrane in CaCo-2 cells, cells were incubated for 5 h with labeled acetate
Summary
[14C]acetate was purchased from New England Nuclear (Boston, MA). Cholesterol, cholesterol oxidase, cholestenone, acetate, sodium taurocholate, verapamil, and progesterone were from Sigma Chemical Co. After washing two more times with 10 mm sodium phosphate buffer (pH 7.4), the cells were incubated for 10 min at 4ЊC with water. The cells were washed twice with 10 mm sodium phosphate buffer (pH 7.4) containing 310 mm sucrose that had been warmed to 37ЊC. They were incubated in this buffer for 15 min at 37ЊC. The radioactivity in the cholesterol and cholestenone bands was added to determine the amount of total newly synthesized cholesterol. CaCo-2 cells grown on micropore membranes were incubated for 5 h with 60 m [14C]acetate (130 dpm/ pmol) with or without treatments. The data were analyzed by Dunnett's method to compare treatment groups with the control group at alpha value of 0.05 using SIGMASTAT software from Jandel Scientific (San Rafael, CA)
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