The bidirectional flux of cholesterol between cells and lipoproteins. Effects of phospholipid depletion of high density lipoprotein

The bidrectional surface transfer of free cholesterol (FC) between Fu5AH rat hepatoma cells and human high density lipoprotein (HDL) was studied. Cells and HDL were prelabeled with [4-¹⁴C]FC and [7-³H]FC, respectively. Influx and efflux of FC were measured simultaneously from the appearance of ³H counts in cells and ¹⁴C counts in medium. Results were analyzed by a computerized procedure which fitted sets of kinetic data to a model assuming that cell and HDL FC populations each formed a single homogeneous pool and that together the pools formed a closed system. This analysis yielded values for the first-order rate constants of FC influx and efflux (k(i) and k(e)), from which influx and efflux of FC mass (F(i) and F(e)) could be calculated. With normal HDL, the uptake and release of FC tracers conformed well to the above-described model; F(i) and F(e) were approximately equal, suggesting an exchange of FC between cells and HDL. HDL was depleted of phospholipid (PL) by treatment withe either phospholipase A₂ or heparin-releasable rat hepatic lipase, followed by incubation with bovine serum albumin. PL depletion of HDL had little or no effect on k(i), but reduced k(e), indicating that PL-deficient HDL is a relatively poor acceptor of cell choelsterol. The reduction in k(e) resulted in initial F(i) > F(e) and, thus, in net uptake of FC by the cells. This result explained previous results demonstrating net uptake of FC from PL-depleted HDL. In the presence of an inhibitor of acyl coenzyme A:cholesterol acyltransferase, the steady state distribution of FC mass between cells and HDL was accurately predicted by the ratio of rate constants for FC flux. This result provided additional validation for describing FC flux in terms of first-order rate constants and homogeneous cell and HLD FC pools.