Selective down-regulation by protein kinase C inhibitors of apolipoprotein-mediated cellular cholesterol efflux in macrophages.
Extracellular apolipoprotein A-I removed cholesterol and phospholipid from cholesterol-loaded mouse peritoneal macrophage and thereby generated a prebeta high-density lipoprotein (HDL) particle having a weight ratio of cholesterol to phosphatidylcholine of approximately 1:1. Treatment of the cells with phorbol myristate slightly increased cholesterol efflux by this mechanism without influencing the nonspecific cholesterol efflux to the lipid microemulsion. When the cells were treated by protein kinase C (PKC) inhibitors, H7 and staurosporine, apolipoprotein-mediated cellular cholesterol efflux was substantially reduced without a significant change in phosphatidylcholine efflux, resulting in generation of cholesterol-poor prebeta-HDL particles having a weight ratio of cholesterol to phosphatidylcholine as low as 1:10. In spite of this change, specific binding of apoA-I to the cellular surface was unaffected. Cellular cholesterol available for acylCoA:cholesterol acyltransferase (ACAT) was rapidly depleted by adding apoA-I to the medium, and the PKC inhibitor treatment reversed this effect. In contrast, nonspecific cellular cholesterol efflux to the lipid microemulsion did not influence the ACAT-available cellular cholesterol pool, and it was not influenced by the PKC inhibitors. Thus, we concluded that apolipoprotein-mediated cellular cholesterol efflux is linked to mobilization of cholesterol from an intracellular pool used by ACAT to a specific pool for apolipoprotein-mediated prebeta-HDL generation, in response to apolipoprotein-cell interaction and subsequent intracellular signaling. Binding of apolipoprotein to the cell surface is required for assembly of the prebeta-HDL particle with cellular phospholipid, and the intracellular cholesterol mobilization is needed for enrichment with cholesterol of the prebeta-HDL. These reactions are largely independent of diffusion-mediated nonspecific cell cholesterol efflux.
Li
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Biochemistry I, Nagoya City University Medical School, Japan.
Tsujita
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M
Yokoyama
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S
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism; D001053:Apolipoproteins / Q000378:metabolism / Q000502:physiology*; D001692:Biological Transport / Q000187:drug effects; D002784:Cholesterol / Q000378:metabolism*; D002788:Cholesterol Esters / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects*; D053442:High-Density Lipoproteins, Pre-beta; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D008075:Lipoproteins, HDL / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*
D016632:Apolipoprotein A-I; D001053:Apolipoproteins; D002788:Cholesterol Esters; D053442:High-Density Lipoproteins, Pre-beta; D008075:Lipoproteins, HDL; D010743:Phospholipids; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002784:Cholesterol; D011493:Protein Kinase C