Faculty Bibliography

Insulin-like growth factor-I (IGF-I) in concentration as low as 10 ng/ml significantly increased basal testosterone formation and 100 ng/ml of IGF-I increased testosterone production more than two fold in primary cultures of purified mature Leydig cells. IGF-I also markedly potentiated hCG-induced testosterone formation in a dose-dependent manner. Furthermore, IGF-I enhanced 8-bromo cyclic AMP induced steroidogenesis and hCG-stimulated cyclic AMP formation. The binding of 125I-IGF-I to purified Leydig cells was linear with a binding affinity of 0.56 +/- 0.07 X 10(9) M-1 and a capacity of 167 +/- 10.2 fmol/mg protein. Insulin and multiplication-stimulating activity were less potent than IGF-I in competing the binding of 125I-IGF-I to purified Leydig cells. This suggests that Leydig cells contain specific type I IGF receptor and IGF-I could modulate Leydig cell steroidogenesis.

Rat serum albumin has been labeled with dilactitol-125I-tyramine, (125I-DLT) a radioactive tracer which remains entrapped within lysosomes following cellular uptake and degradation of the carrier protein. Similar kinetics of clearance from the rat circulation were observed for albumin labeled conventionally with 125I or 125I-DLT-albumin, both proteins having circulating half-lives of approximately 2.2 days. In contrast, the recovery of whole body radioactivity had half-lives of approximately 2.2 and 5.1 days, respectively, for the two protein preparations, indicating substantial retention of degradation products derived from catabolism of 125I-DLT-albumin. Measurement of total and acid-soluble radioactivity in tissues 2 or 4 days after injection of 125I-DLT-albumin revealed that skin and muscle accounted for the largest fraction (50-60%) of degradation products in the body. Fibroblasts were identified by autoradiography as the major cell type containing radioactive degradation products in skin and muscle. Fibroblasts were isolated from skin by collagenase digestion, followed by density gradient centrifugation. The amount of acid-soluble radioactivity recovered in these cells was in excellent agreement with that predicted based on acid precipitation of solubilized whole skin preparations. These studies demonstrate for the first time that fibroblasts are a major cell type involved in the degradation of albumin in vivo.

Primary cultures of fibroblasts and epithelial cells were established from rat ventral prostate (RVP), canine (CP), baboon (BP), and human (HP) prostates, and were used in an assay system to evaluate cadmium chloride (CdCl2) cytotoxicity in vitro. Fibroblasts were always more susceptible to CdCl2 cytotoxicity than the epithelial cells of the same species. There was a distinct species variability to CdCl2 cytotoxicity, with RVP cells being greater than 200 times more susceptible than HP. Primary cultures treated with CdCl2 were subcultivated to establish cell lines. Only RVP fibroblast and epithelial cells resulted in permanent cell lines. Two fibroblast and two epithelial cell lines were derived from CdCl2-treated RVP cell cultures. The epithelial cell lines possessed tonofilaments, desmosomes and keratin. All four cell lines were resistant to CdCl2, had different karyotypes and an excess of chromosome 13. These results demonstrate the transforming potential of cadmium on prostate cells. The role of metallothionein and the significance of extra chromosomes 13 are discussed as possible factors of cadmium resistance.

Isoenzymes of rat ventral prostate (RVP) acid phosphatase were isolated and partially purified by ultracentrifugation, Sephadex G-100 column chromatography, and isoelectric focusing. Antisera were raised to the isoenzymes of prostatic acid phosphatase by immunization of New Zealand white rabbits. Rabbit antisera reacting specifically to homologous but not heterologous isoenzymes of acid phosphatase were then reacted with a variety of tissues using indirect immunofluorescence. The tissues included prostate, spleen, bone marrow, liver, kidney, salivary gland complex, small intestine, and adrenal glands. An antiserum against a RVP acid phosphatase isoenzyme with a pI of 4.5 (A-PAP) localized acid phosphatase only in the supranuclear region of rat ventral prostate epithelial cells, and did not react with acid phosphatase in any of the other organs tested. A-PAP did not localize acid phosphatase in the ventral prostate from rats 14 days after castration. A-PAP did localize acid phosphatase in the ventral prostate from castrated animals that were treated with testosterone. These results indicate the A-PAP localized an androgen-dependent isoenzyme of acid phosphatase in RVP epithelial cells that may be secretory in nature. This antiserum should prove to be an ideal marker for studies involving hormonal regulation of prostatic epithelial function in vivo and in vitro.

Calcium-tolerant myocytes were isolated from adult rat hearts by collagenase perfusion and plated on various substrates in serum-free medium and their adhesion to various extracellular matrix (ECM) components was determined. The myocytes attached readily to dishes coated with collagen type IV (C-IV), laminin (LN), and to fetal bovine serum (FBS) in a manner dependent on the concentration of the components. Substantially fewer myocytes adhered to dishes coated with fibronectin (FN) or to uncoated plastic dishes. Cells adhered equally well to dishes coated with C-IV, LN and FBS within 1-4 h. However, when examined after 2 weeks in culture it was found that only C-IV and LN could support survival of the attached myocytes, and when cultured on C-IV or LN the myocytes were spread and had formed a dense monolayer. The actin filaments had at this time reorganized linearly along the long axis of the cell and the myocytes contracted spontaneously. Rabbit antibodies were raised against myocyte membranes and their ability to inhibit attachment to ECM components was studied. Purified IgG inhibited attachment to C-IV, while having only a minor effect on attachment to LN. These data are compatible with the presence of a specific cell surface component(s) that interacts with ECM substrates and influences cell shape and possibly thereby influences cellular functions.

The interaction of ECM components with adult cardiac myocytes is not well understood, but is of physiological importance. Most physiological studies are conducted on myocytes in suspension yet in vivo the cells are attached to each other and to the ECM. In this paper, we further define the interaction of isolated adult myocytes with the ECM substrates. Of interest is not only the short-term attachment of cells to ECM substrates but also the ability of ECM components to support the long-term maintenance of cardiac myocytes in cultures.

Lamellar body ultrastructure was examined in cultured type II alveolar epithelial cells processed by a method of rapid freezing and freeze drying in the absence of both chemical fixation and solvent dehydration. This method of specimen preparation was chosen to optimize the retention of soluble substances within the type II cell. The use of cultured cell aggregates in which type II cells line the free surface facilitated the effectiveness of rapid freezing for the preservation of lamellar body fine structure. Lamellar bodies of frozen/frozen dried type II cells showed none of the often profound lipid extraction artifact produced by conventional processing. Instead they exhibited a substructure with noteworthy characteristics in common with lamellar bodies processed by resin dehydration lipid retention methods (Stratton, 1976). Importantly, the lamellae of frozen/frozen dried lamellar bodies were contiguous, with no interlamellar space, as is commonly observed in solvent-processed (extracted) specimens. The dimensions of lamellar components in frozen/frozen dried lamellar bodies were, however, different from published values for resin-dehydrated lipid-retained specimens. Lamellar width and the widths of component phospholipid head and fatty acid tail regions in frozen/frozen dried lamellar bodies were approximately 35% smaller than values reported for resin-dehydrated lamellar bodies. This difference was attributed to shrinkage of lamellar components as water was removed from the unfixed tissue during the freeze-drying process. Lamellar bodies preserved by rapid freezing/freeze drying to optimize the in situ retention of intracellular components possess closely adherent concentric membranous lamellae. This supports the contention that the widely appreciated lamellar pattern of the pulmonary lamellar body represents the in vivo molecular organization of intracellular surfactant phospholipids.

Sequential changes in epithelial cells of collagenase-dissociated rat ventral prostate were studied by thin-section and freeze-fracture electron microscopy. Epithelial cells did not attach to the substrate for 48 h. Pelleted cells obtained 1, 24, and 48 h after dissociation were assigned to three categories depending on morphology and cellular associations. (a) Solitary epithelial cells degenerated as determined by extensive vacuolization in the cytoplasm and aggregation of intramembranous particles (IMP). (b) Epithelial clusters consisted of a homogeneous population of well-maintained, closely packed cells. Aggregation of IMP was minimal. Tight junctions that formed between cells at the periphery of the clusters appeared normal and provided an effective permeability barrier demonstrated by the exclusion of ruthenium red tracer. (c) Tissue fragments were comprised of varying combinations of epithelial, endothelial, and smooth muscle cells as well as fibroblasts and erythrocytes. Maintenance of tissue fragments was variable. Plasma membranes often displayed aggregated IMP and proliferated tight junctional strands. An effective permeability barrier was absent. After the 48 h "latent period," epithelial cells in the clusters lost interdependence, disassociated from one another, and attached to the substrate. These isolated cells, which did not display aggregated IMP, retained the ability to form an effective permeability barrier upon reaching confluency. During the first 48 h, epithelial cells did not tolerate solitary existence, yet as participants in clusters they were well maintained. After this interval, they no longer required interactions with neighbors in order to survive. These results indicate that under our experimental conditions, an adaptation period is required by prostatic epithelial cells. The enhanced quality of maintenance associated with epithelial clusters suggests that control over the internal microenvironment, provided by a tight junctional barrier, may be important during the initial period of adaptation in vitro.