Properties of heart fibroblasts of adult rats in culture.

In the heart of the adult rat, fibroblasts are mainly responsible for the synthesis and deposition of the collagenous matrix. Because these cells in vitro may serve as an important model system for studies of collagen metabolism in heart tissue, we have cultured and characterized rat-heart fibroblasts from young adult and old animals. Conditions included use of media of different compositions with and without addition of ascorbate. Cells used were either cultured directly from fresh tissues or thawed previously frozen cells. Cultured cells were studied with respect to growth properties, morphology and ultrastructure and patterns of collagen. Heart fibroblasts generally resembled fibroblasts cultured from other tissues, but were more like skeletal muscle fibroblasts in that they deposited, in addition to type I collagen, type IV collagen and laminin. The fibroblasts showed a typical appearance in phase-contrast microscopy and electron microscopy. In the case of cells grown with added ascorbate, aligned collagen fibrils in the extracellular matrix showed a periodicity typical of type I collagen. The deposition of type I collagen occurred only in medium supplemented with ascorbate, and in that circumstance increased as a function of time past confluence; this was independent of the age of the animal from which the cells were obtained or of other changes of medium composition studied. Immunofluorescence studies with specific antibodies revealed that the cells deposited types I and IV collagens, laminin and fibronectin. In contrast to the case of type I collagen, the deposition of type IV collagen occurred in cells grown either with or without ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)

Collagen chain mRNAs in isolated heart cells from young and adult rats.

Collagen is the predominant component of the extracellular matrix of the heart, where it is organized in a hierarchy of structures. To establish the cellular origin of the various collagen types, type I-procollagen alpha 2 chain and types III and IV collagen mRNAs were examined in preparations of myocytes and non-myocyte heart cells freshly isolated from rats 1 to 6 months old. The cardiomyocytes appeared morphologically intact and functionally competent. Fibroblast-like cells predominated in the non-myocyte cell fractions but endothelial and smooth muscle cells were also present. RNA from whole ventricular tissue served as a control. Northern and dot blot analyses were used to establish the presence or absence of mRNAs. In RNA prepared from whole ventricular tissue, the mRNAs for alpha-, beta-, and gamma-actin isotypes were detected whereas mRNA for alpha-actin was found in myocytes and those for beta- and gamma-actins were found in non-myocyte cells, confirming further the nature of the cell populations. Procollagen types I and III mRNAs were not detected in the total RNA of cardiomyocytes but mRNA for type IV collagen was present. The mRNAs for all three collagen types were present in the non-myocyte cells. These results suggest that in the rat heart the non-myocyte cells, probably fibroblasts, are responsible for interstitial collagen production. Both cell populations may engage in the formation of basement membrane collagen type IV.

Collagenase and elastase production by mouse mammary adenocarcinoma primary cultures and cloned cells.

We have shown previously that an increase in tumor invasion and metastases occurred concurrently with a decrease in collagen content of the extracellular matrix surrounding the C3H mouse mammary adenocarcinoma borne by C3H/HeJ mice. In this paper we report the production of collagenase and elastase activities by the primary tumor cultures and three types of cloned C3H mouse mammary adenocarcinoma cell cultures. The primary tumor cell cultures and tumor-associated stromal cultures produced large amounts of collagenase and elastase activities. On the other hand, the primary tumor capsule cultures produced little or no collagenase and elastase activities even though they produced type I collagen. The production of proteases by the primary tumor cultures decreased along with time and with an alteration in the morphology of cell populations and/or passage of the cultures. The three clones of tumor cell cultures produced variable amounts of collagenase in response to induction by phorbol myristate acetate, an agent that stimulates maximal collagenase production. In contrast, all three cloned cultures elaborated significant amounts of elastase that degraded insoluble ligamental elastin, and most of the elastase production was increased further in response to induction by phorbol myristate acetate. Each cloned cell population exhibited differences in their production of collagenase and elastase in parallel with their difference in growth kinetics, yet these cells still possess the distinctive properties of the tumor. However, a unit amount of collagenase produced by each of the cloned cultures, with or without induction by phorbol myristate acetate, was less than that of the primary tumor cultures. Results suggest that some cell types or combination of cell types in the heterogeneous cell population of the tumor and/or their products appear to be responsible for the increased production of collagenase and elastase activities and for the invasiveness of a malignant tumor.

Ammonia production by IMR-90 fibroblast cultures: effects of ammonia on glutathione, gamma-glutamyl transpeptidase, lysosomal enzymes, and cell division.

gamma-Glutamyl transpeptidase has multi-catalytic activities. It degrades glutathione and can produce ammonia from glutamine. The present study was designed to examine whether the decreased cell proliferation, cellular glutathione content and concurrent increase in ammonia production in senescent cells in culture are the result of increased gamma-glutamyl transpeptidase activity. We used IMR-90 fibroblast and 3T3 LI preadipocyte cultures. The cellular glutathione content depended upon cell proliferation and cell density. The glutathione content was higher in cells at logarithmic growth, and lower at stationary growth or post confluency; dead cells had no detectable glutathione by the method currently used. The glutathione content was minimal in "old" IMR-90 cells, regardless of cell density. On the other hand, an increase occurred in the unit number of molecules of bound 5-iodoacetoamidofluorescein, an active-site directed stoichiometric inhibitor of transpeptidase. That result corresponded favorably with the increased enzyme activity, suggesting that the number of enzyme molecules per cell was increased. The inhibition of ammonia production of the cultures by inhibition of gamma-glutamyl transpeptidase by 5-iodoacetoamidofluorescein and reversible inhibition of ammonia production by a serine-borate mixture were consistent with our postulate. Addition of NH4Cl (0.1 mM) to IMR-90 cultures caused increased activities of transpeptidase and some of the lysosomal enzymes; concurrently, the amount of cellular glutathione and the number of cell divisions decreased. This suggests that the increased ammonia production presumably resulting from glutaminase activity of the observed increase of transpeptidase may profoundly affect certain cellular functions.