Calmodulin antagonist W-7 inhibits de novo synthesis of cholesterol and suppresses secretion of de novo synthesized and preformed lipids from cultured hepatocytes.
The effects of a calmodulin antagonist W-7 were studied on the synthesis and secretion of lipids in primary rat hepatocytes and McArdle-RH7777 cells. In time course experiments, W-7 (20 microM) inhibited secretion of newly synthesized triacyl[(3)H]glycerol by 35%. When the cells were pre-treated overnight with W-7 (20 microM), followed by incubation with [(3)H]oleate, a significant decrease in the secretion of triacylglycerol (TG) and cholesteryl ester (CE) was observed. De novo synthesis of cholesterol from acetate or mevalonolactone was inhibited by W-7, but not glycerolipid synthesis from glycerol and oleic acid precursors. Concentration-response curves for the effects of overnight pre-incubation with W-7 followed labeling with [(3)H]glycerol and [(14)C]mevalonolactone revealed that: (1). the inhibitory effect of W-7 was concentration-dependent and appeared even at the lowest concentration examined (1 microM). W-7 at a concentration of 20 microM suppressed secretion of TG by 60% (P<or=0.002), phosphatidylcholine (PC) by 31% (P<or=0.05), CE by 59% (P<or=0.002) and cholesterol by 64% (P<or=0.002). (2). The incorporation of [(14)C]mevalonolactone into cellular cholesterol and CE was decreased significantly, while W-7 did not have any significant effect upon incorporation of [(3)H]glycerol into glycerolipids, except at the highest concentration examined (50 microM), where synthesis of both TG and PC was significantly suppressed. (3). While the percentage of secreted de novo synthesized glycerolipids and CE decreased proportionally with increasing concentration of W-7, the percentage of secreted newly made cholesterol remained unaffected at any concentration of W-7. In the absence of W-7, about 19% of newly formed cholesterol became esterified into CE, whereas W-7 increased cholesterol esterification in a concentration-dependent manner. (4) W-7 (20 microM) also suppressed the secretion of preformed cholesterol by 24% and CE by 55% but did not affect the recruitment of preformed cholesterol for esterification. About 6.5% of pre-labeled cholesterol and 20% of CE were directed to secretion, which was suppressed in the presence of W-7 by 17% (P<or=0.09) and 48% (P<or=0.001), respectively. These results suggest that, W-7 in the range of 1-20 microM inhibited de novo synthesis of cholesterol and the secretion of both de novo synthesized and preformed lipids.
Rasouli
Mehdi
M
Departments of Pediatrics and Cell Biology, University of Alberta, 328 Heritage Medical Research Centre, Edmonton, AB, Canada T6G 2S2.
Trischuk
Timothy C
TC
Lehner
Richard
R
D000818:Animals; D001055:Apolipoproteins B / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002784:Cholesterol / Q000096:biosynthesis*; D005989:Glycerides / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism*; D008079:Lipoproteins, VLDL / Q000378:metabolism; D008798:Mevalonic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D019301:Oleic Acid / Q000378:metabolism; D051381:Rats; D013449:Sulfonamides / Q000378:metabolism*; D014316:Tritium
D001055:Apolipoproteins B; D002147:Calmodulin; D002250:Carbon Radioisotopes; D005989:Glycerides; D008079:Lipoproteins, VLDL; D013449:Sulfonamides; D014316:Tritium; D019301:Oleic Acid; C017967:W 7; C015367:mevalonolactone; D002784:Cholesterol; D008798:Mevalonic Acid