Early changes in phosphatidylinositol and arachidonic acid metabolism in quiescent swiss 3T3 cells stimulated to divide by platelet-derived growth factor.

We added platelet-derived growth factor to cultures of quiescent Swiss 3T3 cells to investigate early changes in lipid metabolism related to initiation of cell cycle traverse. In a series of experiments that focused on lipid degradation we added the growth factor to cells that had been prelabeled with myoinositol, glycerol, or arachidonic acid. We observed the following mitogen-dependent effects: a decline of radioactivity in cell phosphatidylinositol within 2 to 5 min that progressed to 25 to 50% during the 1st h, a transient rise of radioactivity in cell diacylglycerol that peaked at 10 min, a gradual increase of radioactivity in monoacylglycerol in the medium, and a concomitant increase of radioactivity in medium-free fatty acid. In experiments that focused on lipid biosynthesis, we added the growth factor to cells and pulse-labeled them with radioactive precursors. We observed increased incorporation within 60 min of myoinositol into phosphatidylinositol, arachidonic acid into phosphatidylinositol, diacylglycerol, and phosphatidylethanolamine, and choline into phosphatidylcholine. These results support the possibility that action of platelet-derived growth factor on Swiss 3T3 cells leads to release of diacylglycerol from phosphatidylinositol, that some of the released diacylglycerol is hydrolyzed to monoacylglycerol and arachidonic acid, and that these lipid products are in part reconverted to phosphatidylinositol and other lipids.

Reversible ultrastructural changes in human fibroblasts grown in hepes buffered MCDB-104 supplemented with human serum.

Prolonged maintenance of human dermal fibroblasts in MCDB-104 medium supplemented with pooled human serum or platelet factor deficient preparations of human serum led to appearance of a large number of membrane bound inclusions, resembling lysosomes, and proliferation of small, Golgi associated vesicles. These inclusions did not appear if the cells were grown in Dulbecco-Vogt's modification of Eagle's minimal essential medium or in minimal essential medium supplemented with the same human serum fractions. Cells that acquired inclusions during a 10 d incubation in MCDB-104 subsequently lost these inclusions when transferred to Dulbecco'Vogt's medium for 4 d. Similar reversal of effects of MCDB-104 was also produced by MCDB-104 buffered with bicarbonate instead of HEPES.

Physiological quiescence in plasma-derived serum: influence of platelet-derived growth factor on cell growth in culture.

A platelet-derived growth factor can be shown to be the principal stimulant of DNA synthesis in whole blood serum for those cells that require serum for maintenance and growth in culture. Cell free plasma-derived serum lacks such platelet-derived material. 3T3 cells and primate arterial smooth muscle cells can be maintained in a quiescent state in culture for as long as six weeks in plasma-derived serum. Such cells can grow logarithmically after exposure to 5% whole blood serum or as little as 100 ng/ml of partially purified platelet factor. The cell cycle of smooth muscle cells has been studied in the quiescent (5% plasma-derived serum) and growing state (5% whole blood serum or 5% plasma-derived serum plus platelet factor). The generation time of smooth muscle cells is 16 to 18 hours as shown by autoradiographic frequency of labelled mitoses. The generation time is the same for cells in the growth fraction in either 5% whole blood serum or 5% plasma-derived serum. Thus, platelet factor acts by recruiting cells into the growth fraction rather than effecting a change in the duration of the cell cycle. Flow microfluorimetry studies on cells growing logarithmically in 5% whole blood serum give the following phase durations: G1 = 5.6 hours; S = 7.6 hours; and G2 + M = 3.8 hours. Based on these studies the argument is presented that cells cultured in 5% plasma-derived serum provide a more physiological base for the study of quiescence than do cells in low concentrations of whole blood serum or confluent, density inhibited cells at high (5% or greater) concentrations of whole blood serum. Furthermore, 5% plasma-derived serum represents an appropriate state to examine the perturbation of quiescent cells.

Location of procollagen in chick corneal and tendon fibroblasts with ferritin-conjugated antibodies.

Three distinct antiprocollagen preparations were characterized and used in immunocytochemical staining of chick embryo corneal and tendon cells. The several ferritin-conjugated antibody preparations permitted similar location of procollagen in the cisternae of the rough endoplasmic reticulum and in Golgi elements in both cell types. The ability to demonstrate and interpret specific ferritin staining was dependent on the extent of membrane breakage in each of those organelles, coupled with adequate retention of cell morphology. Corneal fibroblasts appeared to suffer more extensive intracellular membrane damage under controlled conditions of homogenization than tendon fibroblasts, facilitating the identification of procollagen in Golgi vacuoles of these cells. None of the labeled material appeared to by cytoplasmic in origin since ferritin was observed in the cytoplasm only in the vicinity of Golgi elements that were extensively broken. This study extends previous immunological evidence for the presence of procollagen in the Golgi complex and calls attention to the problems to be encountered in locating the antigen in small Golgi vesicles and lamellae.