ATP hydrolysis has been regarded as a general requirement for internalization processes in mammalian cells. We found, however, that treatment of ATP-depleted macrophages and fibroblasts with exogenous sphingomyelinase (SMase) rapidly induces formation of numerous vesicles that pinch off from the plasma membrane; the process is complete within 10 min after adding SMase. By electron microscopy, the SMase-induced vesicles are ∼400 nm in diameter and lack discernible coats. 15–30% of plasma membrane is internalized by SMase treatment, and there is no detectable enrichment of either clathrin or caveolin in these vesicles. When ATP is restored to the cells, the SMase-induced vesicles are able to deliver fluid-phase markers to late endosomes/lysosomes and return recycling receptors, such as transferrin receptors, back to the plasma membrane. We speculate that hydrolysis of sphingomyelin on the plasma membrane causes inward curvature and subsequent fusion to form sealed vesicles. Many cell types express a SMase that can be secreted or delivered to endosomes and lysosomes. The hydrolysis of sphingomyelin by these enzymes is activated by several signaling pathways, and this may lead to formation of vesicles by the process described here.

The stimulation of the intracellular cholesterol esterification pathway by atherogenic lipoproteins in macrophages is a key step in the development of atheroma foam cells. The esterification pathway can also be stimulated by hydrolysis of cell-surface sphingomyelin by the enzyme sphingomyelinase (SMase). In both cases, intracellular cholesterol transport to the cholesterol esterifying enzyme, acyl-CoA:cholesterol O-acyltransferase (ACAT), is thought to be critical, although the mechanism of cholesterol transport is not known. In this report, we explore two fundamental properties of the cholesterol esterification pathway, namely its dependence on energy and the effect of other treatments that block membrane vesicle trafficking. After the atherogenic lipoprotein, beta-very low density lipoprotein (beta-VLDL), was internalized by macrophages and hydrolyzed in lysosomes, the cells were depleted of energy by treatment with sodium azide and 2-deoxyglucose or by permeabilization. Under these conditions, which allowed equal beta-VLDL-cholesteryl ester hydrolysis, cholesterol esterification was markedly decreased in the energy-depleted cells. This effect was not due to blockage of lysosomal cholesterol export. In the permeabilized cell system, energy repletion restored beta-VLDL-induced cholesterol esterification. Remarkably, stimulation of cholesterol esterification by SMase was not inhibited by energy depletion. Energy depletion also inhibited beta-VLDL-induced, but not SMase-induced, cholesterol esterification in Chinese hamster ovary cells. Similar experiments were carried out using N-ethylmaleimide, low potassium medium, or inhibitors of phosphatidylinositol 3-kinase, each of which blocks intracellular membrane vesicle trafficking. These treatments also inhibited beta-VLDL-induced, but not SMase-induced, cholesterol esterification. Finally, we show here that SMase treatment of cells leads to an increase in plasma membrane vesiculation that is relatively resistant to energy depletion. In summary, the stimulation of cholesterol esterification by lipoproteins, but not by SMase, is energy-dependent, N-ethylmaleimide-sensitive, and blocked by both low potassium and phosphatidylinositol 3-kinase inhibitors. The affected step or steps are distal to cholesterol export from lysosomes and not due to direct inhibition of the ACAT enzyme. Thus, the mechanisms involved in lipoprotein-induced versus SMase-induced cholesterol esterification are different, perhaps due to the involvement of energy-dependent vesicular cholesterol transport in the lipoprotein pathway and a novel, energy-independent vesicular transport mechanism in the SMase pathway.

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Understanding the interaction of the atherogenic lipoprotein, lipoprotein(a) [Lp(a)], with macrophages may provide important insight into the physiology and pathophysiology of this lipoprotein. We have recently shown that cholesterol loading of macrophages, such as occurs in atheroma foam cells, leads to marked upregulation of a novel receptor activity for native Lp(a) and its plasminogen-like protein component, apoprotein(a) [apo(a)]. We show here that the Lp(a)/apo(a) receptor activity on cholesterol-loaded macrophages is trypsin sensitive, indicating that a cell-surface protein is involved and that the upregulation by cholesterol loading requires new protein synthesis. Ligand studies revealed that the foam cell receptor activity recognizes Lp(a) containing both small and large isoforms of apo(a) as well as rhesus monkey Lp(a), which contains an inactive kringle-4 37 (K4 37 ) lysine-binding domain. Elastase degradation products of plasminogen did not compete for 12 Elevated plasma levels of Lp(a) occur in «=20% of the adult Caucasian population, and there appears to be an association between elevated Lp(a) levels and the occurrence of coronary atherosclerosis.1 -2 Furthermore, cholesterolfed apo(a) transgenic mice develop significantly more atherosclerosis than nontransgenic controls.3 Despite several important hypotheses and in vitro observations regarding possible roles of Lp(a) in atherosclerosis, 47 neither the mechanism of Lp(a) atherogenicity nor the normal physiological roles of the lipoprotein are definitively known. Nonetheless, the interaction of Lp(a) with macrophages is thought to be important, since cholesteryl ester-filled macrophages (foam cells) are a prominent feature of atherosclerotic lesions. 810 In fact, apo(a) has been found to colocalize with foam cells in these lesions. © 1994 American Heart Association, Inc.interaction. Consistent with these data, the degradation of 125 I-r-apo(a) was completely blocked by an anti-Lp(a) polyclonal antibody that does not cross-react with plasminogen. Furthermore, the multiple sialic residues of apo(a) are also not involved in receptor interaction, since desialylated r-apo(a) interacted with foam cells as well as native r-apo(a Previous studies have shown that cultured macrophages internalize and degrade native Lp(a) and apo(a) poorly. 1215 Recent work from our laboratory, however, revealed an Lp(a) receptor activity, different from known lipoprotein receptors, that can be induced in macrophages by cholesterol loading. 16 The interaction of Lp(a) with cholesterol-loaded macrophages is solely dependent on the apo(a) moiety of Lp(a), since lipid-free recombinant apo(a) [r-apo(a)] but not apo(a)-free Lp(a-) is recognized by the foam cell receptor activity. 16In the present study, we investigated whether a cell-surface protein is involved in the foam cell Lp(a)/ apo(a) receptor activity and whether upregulation induced by cholesterol loading requires new protein synthesis. Furthermore, we explored several key properties of ligands that are need...