Swine Arterial Smooth Muscle Research Articles

Distribution of membrane marker enzymes in cultured arterial endothelial and smooth muscle cells. The subcellular location of oleoyl-CoA:1-acyl-sn-glycero-3-phosphocholine acyltransferase.

The subcellular distribution of oleoyl-CoA:1-acyl-sn-glycero-3-phosphocholine acyltransferase (E.C.2.3.1.23) in cultured swine aorta endothelial cells and smooth muscle cells was investigated. Isolated membrane pellets were centrifuged through linear sucrose gradients, and the distributions of the activities of seven membrane-bound enzymes were measured. The distribution of acyltrasferase activity was similar to that of the endoplasmic reticulum enzymes. Gradient fractions which contained intact mitochondria had very low activities of acyltransferase. Experiments using mixed fractions and measurements made under conditions which inhibit phospholipase A2 showed that no acyltransferase activity from this location was masked by competing activities. When membranes were treated with digitonin, plasma membranes specifically increased in density, facilitating their separation from endoplasmic reticulum membranes. The plasma membranes were free of acyltransferase activity. We conclude that in cultured swine arterial smooth muscle and endothelial cells, acyltransferase is located primarily in the endoplasmic reticulum.

Lipoprotein and cholesterol metabolism in rabbit arterial endothelial cells in culture

Like all other peripheral cells types thus far studied in culture, endothelial cells derived from the rabbit aorta bind, internalize and degrade low density lipoprotein (LDL) at a significant rate. At any given LDL concentration, the metabolism by rabbit endothelial cells was slower than that by fibroblasts or smooth muscle cells. Thus, longer incubations were required to achieve a net increment in cell cholesterol content or to suppress endogenous sterol synthesis; after 18–24 h incubation in the presence of LDL at 100 μg LDL protein/ml inhibition was greater than 80% relative to the rate in cells incubated in the absence of lipoproteins. High density lipoproteins (HDL) were also taken up and degraded but did not inhibit sterol synthesis. Studies of LDL binding to the cell surface suggested the presence of at least two classes of binding sites; the high-affinity binding sites were fully saturated at very low LDL concentrations (about 5 μg LDL protein/ml). However, the degree of inhibition of endogenous sterol synthesis increased progressively with increasing LDL concentrations from 5 to 100 μg LDL/ml, suggesting that uptake from the low affinity sites in this cell line contributes to the suppression of endogenous sterol synthesis. The internalization and degradation of LDL also increased with concentrations as high as 700 μg/ml. Thus, in vivo, where the cells are exposed to LDL concentrations far above that needed to saturate the high affinity sites, most of the LDL degradation would be attributable to LDL taken up from low affinity sites. As noted previously in swine arterial smooth muscle cells and in human skin fibroblasts, unlabeled HDL reduced the binding, internalization and degradation of labeled LDL. Cells incubated for 24 h in the presence of high concentrations of LDL alone showed a net increment in cell cholesterol content; the simultaneous presence of HDL in the medium significantly reduced this LDL-induced increment in cell cholesterol content. The possible relationship between LDL uptake and degradation by these cells in vitro is discussed in relationship to their transport function in vivo.

Uptake and degradation of low density lipoprotein by swine arterial smooth muscle cells with inhibition of cholesterol biosynthesis

We have previously proposed on the basis of studies in hepatectomized animals that low density lipoproteins are degraded at a significant rate by peripheral tissues. To test the capacity of one peripheral cell type to catabolize low density lipoprotein, cultures of swine aortic smooth muscle cells were incubated with homologous 125I-labeled low density lipoprotein and uptake and degradation measured. Degradation of 125I-labeled low density lipoprotein to products soluble in trichloroacetic acid showed an initial lag period of 1–2 h after which the rate increased and remained linear for the following 15 h. Rates of degradation increased sharply with low density lipoprotein concentration over the lower range (from 0–25μg protein/ml) and then more slowly up to the highest concentration tested, 300 μg protein/ml. Even at very low concentrations, 1 μg low density lipoprotein protein/ml (less than 10% of the plasma low density lipoprotein concentration), the in vitro degradation rate (per kg of smooth muscle cells) exceeded the in vivo degradation rate (per kg of total body weight). To the extent that smooth muscle cells are representative of other peripheral cells, the results support the proposal that peripheral degradation of low density lipoprotein apoprotein may be quantitatively important. The rate of incorporation of labeled acetate into sterols was suppressed in cells incubated with whole serum, low density and very low density lipoproteins, or suspensions of free cholesterol. In this respect, the results were similar to those observed in human skin fibroblasts studied concurrently. However, high density lipoprotein inhibited sterol synthesis by about 25% in swine smooth muscle cells while it had no effect in human skin fibroblasts. Cells incubated overnight in lipoprotein-deficient medium showed a net decrease in cholesterol content which could be restored by incubating with low density lipoprotein but not with high density lipoprotein. At high concentrations of low density lipoprotein, when uptake and degradation proceed at very high rates, the amount of cholesterol delivered to the cell is in excess of observed net changes in cell cholesterol content. These findings suggest that there may be a very active “reverse cholesterol transport” in vivo carrying cholesterol from peripheral cells (in which low density lipoprotein is being degraded) back to the liver for ultimate metabolism and excretion.