Maintenance of glucokinase activity in primary hepatocyte cultures

When primary cultures of hepatocytes are maintained for 2 weeks from the time of perfusion, the activity of the enzyme glucokinase decreases rapidly, so that the activity can no longer be detected after the fourth day in culture. Concomitantly, there occurs an increase in the activity of hexokinases, the low-K_M isozymes, which predominate in fetal liver. We have made several modifications of the culture medium in an attempt to prevent the decrease in glucokinase activity. When the medium was supplemented with a mixture of insulin, thyroxine, glucagon, dexamethasone, testosterone, and estradiol, the activity of the enzyme in the hepatocytes was present at approximately 15% of in vivo levels after 2 weeks in culture. When this hormone mixture was present during the first 4 hrs of culture and when the hepatocytes were allowed to attach to the collagen support and were maintained thereafter in medium supplemented with fetal bovine serum, insulin, and dexamethasone, the activity of glucokinase increased after an initial decrease for 3 days and was maintained thereafter at levels comparable to those observed in vivo. This effect of the hormone mixture was found to be the result of the presence of glucagon in the mixture, since the presence of glucagon with no other hormones added, except insulin, during the attachment period produced the same pattern of increased glucokinase activity. Immunoprecipitation of glucokinase from the hepatocytes, using monospecific antibody, indicated that the increase in enzyme activity was the result of increased glucokinase enzyme protein and not an increased synthesis of the other hexokinase isozymes. These studies demonstrate the specific hormonal requirements for the maintenance of glucokinase levels in primary hepatocyte culture at those seen in vivo and lends support to the hypothesis that fetal gene expression in primary hepatocyte cultures is selectively regulated rather than being a general effect with a common regulatory mechanism.     

Rat hepatocyte proliferation is stimulated by insulin-like peptides in defined medium

Amiloride inhibits the growth of mouse mastocytoma cells, the activity of type I and type II protein kinases prepared from the cells and the increase in ⁴⁵Ca²⁺ uptake that occurs after isolated cell membranes are preincubated with ATP and protein kinase. It is suggested that amiloride may inhibit cell growth by inhibiting protein phosphorylation that controls cation availability.     

Rat hepatocyte primary cell cultures. III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium

The conditions for obtaining representative, adult rat hepatocyte primary cell cultures were improved such that viable yields of 50% of the liver were produced which gave rise to cultures representing 30% of the liver. The survival of these cultures in various media was compared revealing that in complex media, particularly containing galactose, survival was improved.     

Tetranectin binds hepatocyte growth factor and tissue-type plasminogen activator.

In the search for new ligands for the plasminogen kringle 4 binding-protein tetranectin, it has been found by ligand blot analysis and ELISA that tetranectin specifically bound to the plasminogen-like hepatocyte growth factor and tissue-type plasminogen activator. The dissociation constants of these complexes were found to be within the same order of magnitude as the one for the plasminogen-tetranectin complex. The study also revealed that tetranectin did not interact with the kindred proteins: macrophage-stimulating protein, urokinase-type plasminogen activator and prothrombin. In order to examine the function of tetranectin, a kinetic analysis of the tPA-catalysed plasminogen activation was performed. The kinetic parameters of the tetranectin-stimulated enhancement of tPA were comparable to fibrinogen fragments, which are so far the best inducer of tPA-catalysed plasminogen activation. The enhanced activation was suggested to be caused by tetranectin's ability to bind and accumulate tPA in an active conformation.     

Growth of functional primary cultures of kidney epithelial cells in defined medium

Primary cultures of baby mouse kidney epithelial cells can grow without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium K-1) designed for an established kidney epithelial cell line, MDCK. The five supplements in Medium K-1 are insulin, transferrin, PGE1, T3, and hydrocortisone. Medium K-1 also supports the growth of kidney epithelial cell cultures from a number of animals, including man, without fibroblast overgrowth. Outgrowth of kidney epithelial cells from kidney explants was also observed with Medium K-1. Thus, the medium appears to be selective for epithelial cell growth. The physiological properties of primary cultures of baby mouse kidney epithelial cells were studied in detail. Baby mouse kidney epithelial cells grew at equal rates (0.5 doublings/day) in Medium K-1 and serum-supplemented medium. Medium K-1 also supported the formation of baby mouse kidney epithelial colonies at low cell densities. The dependence of baby mouse kidney epithelial colony formation upon the five factors in Medium K-1 was examined. These studies indicated that the formation of baby mouse kidney epithelial colonies in defined medium depended upon all the five supplements in Medium K-1, in a manner similar, although not identical, to MDCK colonies. Primary cultures of baby mouse kidney epithelial cells grown in Medium K-1 retained kidney cell-associated properties, including the ability to form multicellular domes, a phenomenon associated with transepithelial salt transport. Amiloride-sensitive Na+ uptake and the mucosal surface enzyme leucine aminopeptidase were also observed in baby mouse kidney cultures. Similar functions were observed in MDCK monolayers.     

Production of proteases type plasminogen activator and their inhibitor in cornea

Corneal epithelial cells secrete tissue plasminogen activator (t-PA), urokinase type plasminogen activator (u-PA) and their inhibitor (PAI), whereas these cell types in other tissues are known to secrete only u-PA hitherto. Endothelial cells in the cornea produce mostly u-PA and only small amounts of t-PA and PAI which remain confined in the cellular compartment contrary to the situation in the vascular endothelial cells where they are liberated into the circulation in the order PAI greater than t-PA greater than U-PA. These unique features of activator/inhibitor secretion and production may play an important role in the remodeling of the corneal matrix.     

Clonal growth of primary cultures of human hyaline chondrocytes in a defined medium

Rapid clonal growth of primary cultures of human costal chondrocytes in a defined medium has been achieved. The basal nutrient medium used for such growth is MCDB 104. It is prepared without linoleic acid and supplemented with 1 microgram/ml insulin, 100 ng/ml fibroblast growth factor, 1.0 x 10(-7) M dexamethasone, and 5 micrograms/ml mixed lipids, presented to the cells in the form of liposomes. The lipid supplement contains soybean lecithin, cholesterol, sphingomyelin, vitamin E, and vitamin E acetate. No expression of cartilage-like differentiation occurs in the defined medium. However, colonies grown for several days in the defined medium and then grown for an additional period of time in medium F12 supplemented with fetal bovine serum and chicken embryo extract synthesize large amounts of refractile matrix that is stained intensely by acidified alcian green, thus demonstrating that the cells growing in the defined medium are capable of expressing cartilage matrix in a permissive environment. Good clonal growth and expression of differentiation can also be obtained by inoculating primary cultures of human chondrocytes directly into the F12-serum-embryo extract medium.     

Modulation of synovial fibroblast plasminogen activator and plasminogen activator inhibitor production by protein kinase C.

Phorbol myristate acetate (PMA) added to human synovial fibroblast cultures caused a dose-dependent increase in the production of plasminogen activator inhibitor-type 1 (PAI-1). In addition, PMA inhibited endogenous and interleukin-1 (IL-1) induced plasminogen activator (PA) activity, while increasing mRNA PAI-1 levels. Other protein kinase C (PKC) activators, mezerein and teleocidin B4, caused similar effects. The simultaneous addition of the PKC antagonists, H-7 or staurosporine, prevented the inhibition of PA activity by PMA. This study shows that activation of PKC inhibits PA and stimulates PAI production in human synovial fibroblasts. These results suggest that activation of PKC may play an important role in regulating increased PA production associated with joint destruction in rheumatoid arthritis (RA).     

Heparin requirement for the quantitation of fibrinogen production by primary hepatocyte cultures

We have reported a rapid method for the quantitation of proteins secreted in culture media ([12.]). Using the same method, we observe that serum-free rat hepatocyte cultures exhibited a 100% increase in detectable secreted fibrinogen-antigen in the presence of 1 unit/ml heparin or greater at 24 h of culture. The amount of transferrin, haptoglobin, and albumin detected was unaltered by the presence of heparin. Since heparin is known to affect certain cellular functions, the fates of [³⁵S]methonine-labeled fibrinogen in cell extracts and culture media were examined employing pulse-chase experiments. Labeled intracellular fibrinogen disappeared at similar rates and was initially released into the media in similar amounts in the presence or absence of heparin. At 8 h during the chase, there was a 40–50% reduction in fibrinogen-antigen in spent culture medium lacking heparin. The presence of heparin did not alter the proteolytic degradation of secreted fibrinogen as determined by immunoblotting of spent culture media proteins separated by polyacrylamide gel electrophoresis. In vitro experiments indicate that clotting of fibrinogen by thrombin reduces the amount of immunodetectable fibrinogen. The results indicate that heparin increases the amount of detectable fibrinogen secreted by cultured hepatocytes by preventing clotting and not by stimulating synthesis or secretion or by inhibiting degradation. Hence, it is critically important to include heparin when secreted fibrinogen is quantitated by the method that we have developed.     

Clonal growth of primary cultures of rabbit ear chondrocytes in a lipid-supplemented defined medium

Clonal growth of primary cultures of rabbit ear chondrocytes in a defined medium without serum or other undefined additives has been achieved. The clonal inoculum is a suspension of fully differentiated chondrocytes prepared by collagenase digestion of rabbit ear cartilage and used with no prior adaptation or selection in culture. When inoculated into medium MCDB 104 supplemented with 100 ng/ml fibroblast growth factor (FGF), 1 microgram/ml insulin, and 5 micrograms/ml of a lipid supplement previously developed for human fibroblasts, the isolated chondrocytes undergo clonal multiplication to form large colonies of epithelial-like cells. Colonies grown in the defined medium for 14 days accumulate at their centers refractile cartilage-like matrix that is stained by acidified Alcian green, although the amount is significantly less than with undefined additives. This system opens the way for detailed studies, in a defined background medium, of factors that regulate phenotypic expression of cartilage-like differentiated properties.     

Growth and myelination of explant cultures in defined medium

Summary The purpose of this study was to compare the development of organotypic cultures in defined medium versus nutrient containing serum and embryo extract (EE). Explant cultures of cerebellum with or without locus ceruleus were grown in the Maximow system and monitored in the living state and with histological stains. Thinner explants, fibronectin and a more frequent feeding schedule were required to overcome the growth differences encountered using a defined medium. The final medium formulation was arrived at by evaluation of living cultures and consisted of a basal medium (Dulbecco's minimal essential medium), a number of hormones and other supplements, and a final glucose concentration of 750 mg %. Using a Golgi stain and histofluorescence, it was shown that the three major types of neurons—Purkinje, deep nuclear, and locus ceruleus—developed similarly in the defined medium and in serum-EE cultures. Myelination occurred in virtually all cerebellar cultures in defined medium and the onset was earlier than in serum-EE cultures. These results indicate that differentiation of oligodendroglia and maturation of neurons occur in a defined medium. Elimination of thyroid hormone delayed the maturation of the cultures, both neurons and myelin, by 3–4 days. This project was supported by a grant from Supply and Services (Canada) and from the Department of Health and Welfare (Canada). The findings and opinions are the sole responsibility of the authors. EDITOR'S STATEMENT This article describes adaptations of serum-free cell culture methods previously developed by other laboratories to the organ culture of central nervous system tissues. Although it is difficult to develop reliable procedures for quantitative analyses in cultures of this type, organ cultures provide unique advantages in the study of development, regeneration and response to damage, organismal and cellular senescence and genetic abnormalities of the nervous system. Observations reported here regarding effects of thyroid hormone on cellular maturation in this culture system may be valuable in future studies in these areas.     

The role of plasminogen activator in ovulation

The role of plasminogen activator in ovulation was investigated using the inhibitor, trans-aminomethylcyclohexane carboxylic acid (t-AMCHA). In the regular cycle rat, the plasminogen activator activity of the follicles increased from the diestrus to the estrus phase. In the latter phase, a proteolytic enzyme which was not inhibited by t-AMCHA appeared. After ovulation, the plasminogen activator activity decreased. When ovulation was induced in immature rats by pregnant mare serum gonadotrophin and human chorionic gonadotrophin, remarkable fibrinolytic activity appeared in the ovaries immediately before ovulation. When t-AMCHA was given in the ovulation-induced rats, the fibrinolytic activity of the ovaries was suppressed, the number of ovulated ova decreased and the timing of ovulation was delayed. When t-AMCHA solution was given to rats in the proestrus phase, ovulation was almost completely suppressed, but aprotinin solution exerted no effect on ovulation. These results suggest that plasminogen activator is a key enzyme in ovulation, and that the chain reaction from plasminogen activator to proteolytic enzyme (including collagenase) is of greater importance than that of plasminogen activator to plasmin.     

Mechanisms of cyclosporine A toxicity in defined cultures of renal tubule epithelia: a role for cysteine proteases.

The mechanisms of toxicity of cyclosporine A (CsA) were studied in primary cultures of individually microdissected rabbit and human renal tubules of proximal and distal regions of the nephron. A direct toxic effect of CsA on renal tubule epithelia was demonstrated using nigrosine uptake and LDH release as indicators of cell death. Proximal convoluted tubules (PCT) and proximal straight tubules (PST) were shown to be highly sensitive, while thick ascending limbs of Henle (TAL) were much less sensitive and cortical collecting tubules (CCT) relatively resistant. The effects of CsA were time and dose dependent over the range 50 ng/ml to 100 micrograms/ml. Protection against CsA-induced PST cell death was afforded by reduction in extracellular calcium levels in the media or addition of the calcium entry antagonists: verapamil, nifedipine or diltiazem. In addition, treatment with the cysteine protease inhibitor, E64, attenuated CsA-induced cell damage. A role for the lysosomal cysteine proteases (cathepsins), however, was ruled out on the basis of identical activity levels in all cell types; no beneficial effects of lysosomal enzyme depletion and no evidence of lysosomal rupture prior to death. By contrast, a role for the cytoplasmic, calcium-dependent cysteine protease calpain was suggested since activity levels were significantly higher in PST than CCT cultures and were inducible by CsA.     

Invasion of brain tissue by primary glioma: evidence for the involvement of urokinase-type plasminogen activator as an activator of type IV collagenase.

The immunocharacterization of a metalloproteinase isolated from rat glioma cell conditioned medium is described and confirms that the enzyme is identical to type IV collagenase. Free, active plasminogen activator (PA) and PA-PAI complexes were identified as being secreted by the same cells. Using affinity-purified metalloproteinase we demonstrate that the enzyme can be partially activated by u-PA but not by plasmin in vitro. On the basis of these findings and previous published work we propose a scheme for the proteolytic degradation of normal brain tissue during tumour invasion.     

Retinoids and synovial factor(s) stimulate the production of plasminogen activator by cultured human chondrocytes. A possible role for plasminogen activator in the resorption of cartilage in vitro

Agents such as retinol, interleukin 1 and catabolin stimulate resorption of cultured cartilage. This process seems to be mediated by chondrocytes, but the mechanism by which breakdown occurs remains unknown. We have found that (10(-6)-10(-8) M) retinoic acid and (1 X 10(-6) M) retinol, in the presence or absence of a factor derived from cultured synovium (synovial factor), stimulate the degradation of fibrin by human chondrocytes in culture. Plasminogen was required for the enhancement of fibrinolysis, suggesting that the breakdown depended upon the production of plasminogen activators and subsequent liberation of plasmin. However, the chondrocytes did not release significant amounts of plasminogen activator, and the effects of the synovial factor and retinoids resulted from augmentation of the production or activity of enzymes which remained bound to the cell layer. The role of plasminogen in the resorption of cultured cartilage was also investigated. In the presence of plasminogen, (1 X 10(-8) M) retinoic acid or synovial factor stimulated the breakdown of cultured bovine nasal cartilage, but in the absence of plasminogen, the effect of synovial factor was abolished and that of retinoic acid reduced. However, in cultures containing both retinoic acid and synovial factor the resorption process was not affected by removal of plasminogen. Thus, the resorption of cartilage matrix in vitro may be partially mediated by plasminogen activators and plasmin.     

Characteristics of human chondrocyte cultures in completely defined medium

Summary Chondrocytes derived from normal human adult articular cartilage were established and maintained for over 5 months in a completely defined medium without the addition of serum or any other growth factors. At the end of 5 months, these cells were still metabolically active. The cells incorporated [³H]thymidine into DNA, incorporated [³⁵S]sulfate into proteoglycans, and exhibited lysosomal enzyme activities. The³⁵S-labeled proteoglycans isolated from the culture medium had elution profiles on high pressure liquid chromatography (HPCL) similar to those observed from proteoglycans from other mammalian sources. This self-contained growth competence may reflect a need produced by the unusual avascular and alymphatic character of articular cartilage. This research was supported, in part, by Grant AM22057 from the National Institutes of Health, Bethesda, MD.     

Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium

Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone- deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha- methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.     

Vitronectin governs the interaction between plasminogen activator inhibitor 1 and tissue-type plasminogen activator.

The "serpin" plasminogen activator inhibitor 1 (PAI-1) is the fast acting inhibitor of plasminogen activators (tissue-type (t-PA) and urokinase type-PA) and is an essential regulatory protein of the fibrinolytic system. Its P1-P1' reactive center (R346 M347) acts as a "bait" for tight binding to t-PA/urokinase-type PA. In vivo, PAI-1 is encountered in complex with vitronectin, an interaction known to stabilize its activity but not to affect the second-order association rate constant (k1) between PAI-1 and t-PA. Nevertheless, by using PAI-1 reactive site variants (R346M, M347S, and R346M M347S), we show that the binding of vitronectin to the PAI-1 mutant proteins improves plasminogen activator inhibition. In the absence of vitronectin the PAI-1 R346M mutants are virtually inactive toward t-PA (k1 less than 1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin the rate of association increases about 1,000-fold (k1 of 6-8 x 10(5) M-1 s-1). This inhibition coincides with the formation of serpin-typical, sodium dodecyl sulfide-stable t-PA.PAI-1 R346M (R346M M347S) complexes. As evidenced by amino acid sequence analysis, the newly created M346-M/S347 peptide bond is susceptible to attack by t-PA, similar to the wild-type R346-M347 peptide bond, indicating that in the presence of vitronectin M346 functions as an efficient P1 residue. In addition, we show that the inhibition of t-PA and urokinase-type PA by PAI-1 mutant proteins is accelerated by the presence of the nonprotease A chains of the plasminogen activators.     

Rat liver epithelial cell cultures in a serum-free medium: primary cultures and derived cell lines expressing differentiated functions

Rat liver epithelial cells explanted in a serum-free medium (SFM) composed of Ham's F10 basal medium plus free fatty acids adsorbed on bovine albumin gave successful rise to primary cultures and then to long-term cell lines that expressed liver functions; induction of L-tyrosine aminotransferase by glucocorticoids, hepatic pattern of progesterone metabolism, and biosynthesis of murine primary bile acids; chenodeoxycholic and cholic acid common to higher vertebrates and alpha-muricholic acid specific of the rat bile.