The coordination of sterol and phospholipid synthesis in cultured myogenic cells. Effect of cholesterol synthesis inhibition on the synthesis of phosphatidylcholine.

The coordination of biosynthesis of cholesterol and phosphatidylcholine has been investigated in a myoblast cell line L6, grown in lipid-depleted medium. The addition of 25-hydroxycholesterol or compactin to this medium inhibits cholesterol synthesis by over 95%. The rate of [3H]choline incorporation into phosphatidylcholine begins to decline after 6 h and eventually falls to 45% of control. Measurements of choline flux through the CDPcholine pathway and of the pool sizes of choline-containing intermediates indicate that the formation of CDPcholine is the rate-limiting step in phosphatidylcholine synthesis in L6. The rate of CDPcholine synthesis was measured in vivo by pulse-chase experiments. Culturing cells with 25-hydroxycholesterol or compactin results in an inhibition of this step, which parallels the inhibition of incorporation of [3H]choline into phosphatidylcholine. The specific activities of the enzymes of phosphatidylcholine synthesis were assayed under optimal substrate conditions. Growth in the presence of sterol-synthesis inhibitors for 24 h has a significant, but variable, effect on the activity of microsomal and cytosolic cholinephosphate cytidylytransferase. Inhibition is seen in approximately one-half of the preparations and ranges up to 60%. The degree of inhibition of the enzyme in vitro correlates with an elevation of cytosolic triacylglycerol and phospholipid levels, and is not eliminated by the inclusion of excess stimulatory phospholipids in the assay. The pool sizes of the substrates, cholinephosphate and CTP, are unaffected by cholesterol synthesis inhibition. In contrast to the effects on cholinephosphate cytidylytransferase, the microsomal enzymes glycerol-3-phosphate acyltransferase and choline phosphotransferase are stimulated 2-fold or more. Choline kinase specific activity was inhibited 2-fold after 24 h of treatment with 25-hydroxycholesterol; however, no effect on this step was observed in vivo. These results indicate that the coordination of cholesterol and phosphatidylcholine synthesis involves regulation at the cytidylytransferase-catalyzed step.