Koichiro Nakashima

Metabolic changes in LDL receptors and an appearance of scavenger receptors after phorbol ester-induced differentiation of U937 cells

Metabolic changes in lipoprotein receptors after cell differentiation were investigated using U937 cells, a human tumor cell line with monoblastic characteristics. After inducing the differentiation of U937 cells into monocyte-macrophage-like cells using TPA (12-tetradecanoyl-phorbol-13-acetate), the incorpotation of [14C]oleate into cellular cholesteryl [14C]oleate was increased in comparison with U937 cells when incubated with r-beta VLDL, h-VLDL or h-LDL. A marked down-regulation of LDL receptors was observed in U937 cells upon addition of 25-hydroxycholesterol. However, this down-regulation of LDL receptors was poor in monocyte-macrophage-like cells that had been induced to differentiate from U937 cells with TPA. Acyl coenzyme A:cholesterol acyltransferase activity was increased after TPA-induced differentiation of U-937 cells. The incorporation of [14C]oleate into cellular cholesteryl [14C]oleate was also increased when incubated with acetylated h-LDL in monocyte-macrophage-like cells in comparison with U937 cells. These results suggest that a poor down-regulation of LDL receptors, which is attributable to increased acyl coenzyme A:cholesterol acyltransferase activity, and scavenger receptors are induced and that these metabolic changes in lipoprotein receptors and an increased acyl coenzyme A:cholesterol acyltransferase activity contribute to cholesterol ester accumulation in monocyte-macrophage-like cells.

Role of cholesterol-accumulating macrophages on vascular smooth muscle cell proliferation

The proliferation of vascular smooth muscle cells (VSMC) was stimulated by co-incubation with macrophages. Further stimulation was observed when co-incubated macrophages were supplied with LDL or cholesterol. However, the stimulation of VSMC proliferation did not result from co-incubation with macrophages supplemented with acetylated LDL or delipidated LDL. The addition of anti-PDGF antibody partially abolished the stimulation of VSMC proliferation induced by co-incubation with macrophages supplemented with LDL or cholesterol. A high concentration of prostaglandin E2 inhibited the proliferation of VSMC stimulated by PDGF and plasma-derived serum when they were at the G0/G1 stage. However, the inhibitory effect of prostaglandin E2 on proliferation was not observed when cells were incubated with macrophages supplemented with LDL or cholesterol in spite of the promotion under these conditions of prostaglandin E2 production. These results suggest that cholesterol-accumulating macrophages may exert a regulatory effect on the proliferation of VSMC through the synthesis and secretion of platelet-derived growth factor (PDGF) and prostaglandin E2, besides foam-cell formation.

Analysis of neutral amino acid trasnport systems in the small intestine: A study of brush border membrane vesicles

SummaryTransport of L-proline, L-leucine and L-cysteine was studied in brush border membrane vesicles prepared from guinea pig ileum. Concentrative transport of L-proline, L-leucine and L-cysteine was obtained in the presence of an Na+ gradient from, outside to inside of the vesicles, which indicated contribution of either system A (alanine-preferring) or system ASC (alanine-, serine- and cysteine-preferring) to the transport. When Na+ was replaced by Li+, L-leucine and L-cysteine maintained the same concentrative transport. However, the concentrative transport of L-proline was markedly decreased by Li+-for-Na+ substitution. Strong exchange properties of L-leucine transport via system L (leucine-preferring) was observed with brush border membrane vesicles, in which preloaded L-methionine could be exchanged with labeled L-leucine added outside the vesicles. These results suggest that the small intestine of the guinea pig possesses classical neutral amino acid transport systems such as systems A, ASC and L.

Identification of a functional receptor differing from the LDL receptor that catabolizes chylomicron remnant in Hep G2 cells

We investigated types of lipoprotein receptors on Hep G2 cells using a monoclonal antibody against the LDL receptor. IgG-C7 inhibited the binding and internalization of 125I-labeled low density lipoprotein (LDL) in Hep G2 cells with upregulated and downregulated LDL receptors by 90% of control values. Binding and internalization of 125I-labeled chylomicron remnant in Hep G2 cells with upregulated and downregulated LDL receptors was 50% and 85%, respectively, of control values after exposure to IgG-C7. Excess unlabeled chylomicron remnant inhibited binding and internalization of 125I-labeled chylomicron remnant in Hep G2 cells with downregulated LDL receptors completely. Pronase treatment abolished binding and internalization of 125I-labeled LDL and 125I-labeled chylomicron remnant in Hep G2 cells. When solubilized fractions of Hep G2 cells were immunoprecipitated with IgG-C7, the binding activity of 125I-labeled chylomicron remnant to reconstituted vesicles was unchanged. 45Ca blotting analysis showed the presence of 45Ca binding protein (approximately 600 kDa) in Hep G2 cells. The amount of 45Ca binding protein was not affected by cholesterol and was abolished by pronase treatment. These results suggest the existence of a functional receptor other than the LDL receptor that catabolizes chylomicron remnant in Hep G2 cells and that this receptor may correspond to LDL receptor-related protein.