George C. Fuller

CELL-MEDIATED CONTRACTION OF COLLAGEN LATTICES IN SERUM-FREE MEDIUM: EFFECT OF SERUM AND NONSERUM FACTORS

SummaryThis study was conducted to identify a defined, serum-free culture medium that supports cell dependent contraction of a collagen lattice. Collagen lattices were found to contract in cultures containing human foreskin fibroblasts (HFF) or rabbit aortic smooth muscle (RASM) cells incubated in serum-free medium. HFF and RASM cells required different supplements to contract the collagen gels. HFF cultured in Dulbecco’s modified Eagle’s (DME) medium supplemented with bovine serum albumin (BSA) and either endothelial cell growth supplement (EnGS), insulin (In), or platelet derived growth factor (PDGF) supported collagen lattice contraction. Replacement of BSA with casein without the addition of other supplements improved contraction. In contrast, RASM cells supplemented with BSA, EnGS, In, and PDGF were able to contract collagen gels only minimally. Similar to HFF, RASM cells cultured in DME medium supplemented with casein, but without the addition of other supplements, contracted collagen lattices. HFF-mediated collagen contraction was inhibited by prostaglandins E1 or E2, fibronectin, or ascorbic acid. The reported serum-free model provides a useful in vitro method to investigate the role of serum and nonserum factors regulating cell mediated-contraction of insoluble collagen fibrils.

Enzyme markers of collagen synthesis in carbon tetrachloride-induced fibrosis and during colchicine modification of CCl4-induced liver injury

Serum galactosylhydroxylysyl glucosyltransferase (S-Glu-Gal-Hyl-Tase), liver galactosylhydroxylysyl glucosyltransferase (L-Glu-Gal-Hyl-Tase), liver hydroxylysyl galactosyltransferase (L-Gal-Hyl-Tase), and liver prolyl hydroxylase (L-PH) activities were measured in rats during the development of CCl4-induced cirrhosis (0.2 ml of 33% CCl4 in light mineral oil two times weekly for 10 weeks followed by 6 weeks of no treatment). Serum and liver markers of collagen synthesis increased in a time-dependent manner reaching maximum activity at 6 weeks (S-Glu-Gal-Hyl-Tase, two times; L-PH, two times). These enzyme levels returned to normal during the 4-week recovery period. In a separate 4-week experiment, colchicine (10 micrograms/rat/day) was administered with CCl4. Colchicine prevented the increase in S-Glu-Gal-Hyl-Tase, L-Glu-Gal-Hyl-Tase, and L-Gal-Hyl-Tase induced by CCl4 and resulted in a smaller increase in L-PH. These results demonstrate that S-Glu-Gal-Hyl-Tase elevation occurs following CCl4 because of increased liver collagen synthetic activity and the hepatocellular injury produced by CCl4.

Effect of the stable endoperoxide analog U-46619 on prostacyclin production and cyclic AMP levels in bovine endothelial cells

The effects of the stable endoperoxide analog U46619 (U) on the regulation of prostacyclin (PGI2) formation and cyclic adenosine monophosphate (cAMP) were investigated in cultured bovine aortic endothelial (BAE) cells. Incubation of U (0.3, 3.0 and 30 microM) with BAE cells for 5 min results in a dose-dependent increase in PGI2. Cyclic AMP levels were not changed at 0.3 and 3.0 microM but were stimulated at 30 microM U. When cells were exposed to U for a second and third 5 min period, PGI2 formation at 0.3 and 3.0 microM U remained stimulated while at 30 microM, PGI2 was not increased. Five min incubation of BAE cells with the cyclooxygenase inhibitor indomethacin blocked the stimulation of PGI2 at all concentrations of U and also prevented the increase of cAMP levels at 30 microM. In cells prelabeled with 3H-arachidonate, U stimulated release of labeled products at 0.3 and 3.0 microM but not at 30 microM U. In cells treated with bradykinin in the presence of U, PGI2 production was stimulated at 0.3 and 3.0 microM but not 30 microM U. When cells were exposed to U and stimulated with PGI2 (with and without phosphodiesterase inhibition), U caused significant increases in cAMP. We conclude that incubation of BAE cells with U results in an initial dose-dependent increase in PGI2 formation. Cyclic AMP levels are increased at high concentrations of U. This increase in cAMP is mediated by the initial stimulated PGI2 and results in decreased PGI2 on further exposure to U. Data suggest that U stimulates phospholipase activity and, at high concentrations, inhibits phosphodiesterase.

Dibutyryl cyclic guanosine monophosphate elevates bovine endothelial cell thromboxane production without affecting prostacyclin metabolism.

Cloned bovine aortic endothelial cells (BAEC) were grown to confluence then treated for 24 hours with dibutyryl cyclic AMP (dbcAMP) or dibutyryl cyclic GMP (dbcGMP) in serum free medium. Submillimolar concentrations of dbcGMP caused a significant enhancement of thromboxane (TXB2) synthesis in washed cells exposed to arachidonate. DbcAMP had no effect on the production of either metabolite. TXB2 synthesis was inhibited by 3 micrograms/ml cycloheximide, whether or not the cells were pretreated with dbcGMP. Prostacyclin production was inhibited to a much lesser extent by cycloheximide. We conclude that dbcGMP elevates TXB2 production by increasing the amount of thromboxane synthetase available, and that this effect is inhibited by cycloheximide. Data are described which suggest that dbcGMP increases the degradation rate of TXB2 by BAEC, so that the observed increase in TXB2 may be an underestimate of the true effects of dbcGMP on TXB2 production.

Serum and liver enzymes of collagen synthesis in hepatic murine schistosomiasis mansoni

Serum galactosylhydroxylysyl glucosyltransferase (S-GGT), liver galactosylhydroxylysyl glucosyltransferase (L-GGT), liver hydroxylysyl galactosyltransferase (L-GGal-T) and liver prolyl hydroxylase (L-PH) activities were measured in CF1 female mice each week for 10 weeks after infection with Schistosoma mansoni (50 cercariae per mouse). None of the enzyme activities measured were significantly increased in the first six weeks following the infection. However, the activities increased thereafter reaching a maximum of seven-fold for S-GGT, five-fold for L-GGT, three-fold for L-GGal-T and ten-fold for L-PH compared with control animals. The increase in S-GGT activity was positively correlated with L-GGT (r = 0.788), L-GGal-T (r = 0.774) and L-PH (r = 0.800). The present data suggest that during the first six weeks of the development of murine hepatic schistosomiasis mansoni infection, few eggs bombard the liver. The biochemical changes during this period are mild. During weeks 7 to 10, which is the active fibrotic stage, the liver biochemical markers of collagen synthesis increase rapidly and lead to fibrotic morphological features that are the chronic irreversible hepatic changes characteristic of this infection. The data reported here indicate that the aggressive fibrotic changes in liver during weeks 7 to 10 of murine schistosomiasis infection are predicted by S-GGT activity.

Enzymes of collagen synthesis in lung tissues of bleomycin-induced pulmonary fibrosis

The activities of prolyl hydroxylase (Pro-OHase), galactosylhydroxylysyl glucosyltransferase (Glu-Gal-Hyl-Tase), and hydroxylysyl galactosyltransferase (Gal-Hyl-Tase) were assayed in lung tissues of hamsters with bleomycin-induced experimental pulmonary fibrosis. Serum Glu-Gal-Hyl-Tase and aspartate transaminase (Asp-NH2-Tase) were measured in the same animals. Lung fibrosis was induced by intratracheal bleomycin instillation, and the enzyme activities were assayed 2, 3, and 4 weeks after bleomycin administration. The activities of the three lung enzymes increased significantly after bleomycin instillation. However, no difference in the values of serum Glu-Gal-Hyl-Tase or Asp-NH2-Tase were observed. Histologic examination of lung sections indicated progressive fibrotic foci. These results thus indicate that the activities of collagen processing enzymes are elevated in the fibrotic lung tissues as a reflection of the increased rate of collagen synthesis during the period of active fibrogenesis, but unlike liver fibrosis, the elevation of tissue Glu-Gal-Hyl-Tase is not predicted by a corresponding increase in serum levels of this enzyme.

The susceptibility of various cultured cells to induction of clear cytoplasmic vacuoles by disobutamide

Cultured cells were found to be highly useful for investigating intracellular storage of amphiphilic compounds using disobutamide as a model agent. To select types of cultured cells most suitable for investigations, cells of dog coronary artery muscle, rabbit aorta muscle, rat urinary bladder carcinoma, rat basophilic leukaemia, human skin fibroblasts, bovine aorta endothelium, Chinese hamster ovary tumour and mouse fibroblasts were incubated with 0, 1, 2, 4, 6, 8 and 10 x 10(-4)m-disobutamide for 24 hr. Cultures were examined in situ by phase light microscopy for the presence of clear cytoplasmic vacuoles, cell death (cell detachment), and for drug effect on confluency/cell count. Disobutamide induced vacuoles in all cell types except rat leukaemia. The drug induced cell death and reduction in confluency or cell count in cultures of all cell types except rat carcinoma and rabbit aorta muscle. Release of lactic dehydrogenase from cells confirmed the relative resistance of the rat carcinoma and rabbit cells, and susceptibility of rat leukaemia, to drug-induced cell death. By means of electron microscopy of rat carcinoma and rabbit cells, it was established that vacuoles were membrane-bound and their content was predominantly electron-lucent.