Rat hepatocytes in serum-free primary culture elaborate an extensive extracellular matrix containing fibrin and fibronectin

Adult rat hepatocytes cultured on type IV collagen, fibronectin, or laminin and maintained in serum-free medium were examined by indirect immunofluorescence using polyclonal antibodies against extracellular matrix proteins. An extensive fibrillar matrix containing fibronectin and fibrin was detected in all hepatocyte cultures irrespective of the exogenous matrix substratum used to support cell adhesion. Fibrils radiated from the cell periphery and covered the entire culture substratum. In addition, thicker fibers or bundles of fibers were localized on top of hepatocytes. This matrix did not contain laminin or the major types of collagen found in the liver biomatrix (types I, III, and IV). Isolation of the fibrillar matrix and analysis on polyacrylamide gels under reducing conditions demonstrated a major 58-kD polypeptide, derived from beta-fibrinogen as indicated by immunoblotting and two-dimensional peptide mapping. Plasmin rapidly dissolved the matrix. Deposition of the fibrin matrix in hepatocyte cultures was arrested by hirudin, by specific heparin oligosaccharides that potentiate thrombin inhibition by antithrombin III, and by dermatan sulfate, an activator of heparin cofactor II-mediated inhibition of thrombin. The results indicate that hepatocytes in culture synthesize and activate coagulation zymogens. In the absence of inhibitory and fibrinolytic mechanisms, a fibrin clot is formed by the action of thrombin on fibrinogen. Fibronectin attaches to this fibrin clot but fails to elaborate a fibrillar matrix on its own in the presence of coagulation inhibitors.     

Chicken macrophages synthesize and secrete avidin in culture

It was previously shown that avidin, a glycoprotein secreted in vivo by chicken oviduct, is produced by cultured transformed or damaged chicken embryo fibroblasts [27]. This report demonstrates synthesis and secretion of large amounts of avidin by macrophages isolated from chicken yolk sac. Avidin was secreted to the culture medium as shown by immunoprecipitation of metabolically labeled proteins. In the culture medium of macrophages the avidin concentration (up to 47.5 +/- 0.5 microgram/mg cellular protein) exceeded, in agreement with previous findings, that of fibroblasts (up to 7.3 +/- 0.7 microgram/mg) infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature sensitive for transformation and OK 10 virus). No difference between the macrophage avidin and the egg white avidin was detected by both the heat-induced [14C] biotin exchange assay and immunoblotting (subunit Mr = 15600). By immunofluorescence 10 to 20% of the cells were positive for avidin, independent of the time in culture (1-30 days). The staining pattern varied between dense or granular perinuclear and strong reticulo-granular fluorescence throughout the cytoplasm. Double staining for avidin and the Golgi region by wheat germ agglutinin showed that avidin is concentrated, and might be processed, in the Golgi complex. The production of avidin by macrophages supports a role for avidin in host defence mechanisms.     

Human monocytes in culture synthesize and secrete lipoprotein lipase

Within the first day in culture, human monocytes begin to synthesize and secrete a triglyceride lipase. The designation of this activity as lipoprotein lipase is based upon: 1) a requirement of serum or apolipoprotein C-II for full activity; 2) inhibition by 1M NaCl or apolipoprotein C-III₂; 3) a pH optimum of 8; and 4) binding to endothelial cells that is releasable by heparin. The enzyme also exhibits immunological cross reactivity with antibody to purified bovine milk lipoprotein lipase as does human postheparin plasma lipoprotein lipase. Lymphocytes and polymorphonuclear leukocytes do not appear to contain this enzyme.     

Human fibroblast cells synthesize and secrete nerve growth factor in culture.

Using our enzyme immunoassay system developed for recombinant hNGF, we examined the synthesis and secretion of human NGF (hNGF) by human fibroblast (WS-1) cells. The amount of the factor secreted by WS-1 cells increased linearly and a significant amount of NGF was detected in the conditioned medium of WS-1 cultures. WS-1 NGF showed properties identical to those of recombinant human NGF in immunoreactivity and molecular weight. An increase in cell density or the withdrawal of serum from the culture medium caused a drastic decrease in the rate of NGF secretion. These results suggest that WS-1 cells are able to synthesize and secrete hNGF in culture and that the synthesis/secretion is regulated in a growth phase-dependent manner.     

Rat natural killer cells synthesize fibronectin. Possible involvement in the cytotoxic function

The ability of NK cells to synthesize and secrete fibronectin (FN), an extracellular matrix glycoprotein which plays a key role in many biologic processes including cellular adhesion, morphology, cytoskeletal organization, cell migration, and invasiveness, was studied. By using affinity-purified polyclonal antibodies directed against human cellular or plasma FN, the presence of FN was evidentiated on Percoll-purified rat large granular lymphocyte or on a large granular lymphocyte tumor cell line (CRC) by flow cytometry and immunoelectron microscopy. Its expression increased after NK cell activation by poly I:C administration. Biochemical analysis by immunoprecipitation and SDS-PAGE indicated that FN was associated to cell surface and secreted in the supernatant in a molecular form similar to that of FN from L929 fibroblasts. In an attempt to understand the role of FN in the NK cell function, we found that an antibody against human plasma FN and its F(ab')2 fragment inhibited NK cytotoxicity against YAC-1 target at the effector cell level. Inhibition occurred at the postbinding level, because F(ab')2 anti-FN inhibited induction of phosphatidylinositol hydrolysis by YAC-1 target cells, whereas binding to target cells was not affected. The possible role of FN in the NK cytotoxic function is suggested.     

Human breast cancer cells synthesize and secrete an EGF-like immunoreactive factor in culture

A human breast cancer cell line, strain MCF-7, in culture synthesized and secreted a large amount of a polypeptide (designated as MCF-7 EGF) immunologically related to human epidermal growth factor (hEGF). The molecular weight of MCF-7 EGF estimated by gel filtration on Sephadex G-75 was similar to that of hEGF from human urine. On isoelectric focusing analysis, MCF-7 EGF gave a major peak at pH 4.6 and a minor peak at pH 5.0. In our enzyme immunoassay system, however, the dose-response curve of MCF-7 EGF did not show good parallelism with that of standard hEGF. From these results, it is suggested that MCF-7 cells synthesize and secrete a polypeptide immunologically related to hEGF into the culture medium.     

Thawed human hepatocytes in primary culture.

In drug metabolism studies, isolated and cultured human hepatocytes provide a useful model for overcoming the difficulty of extrapolating from animal data. In vitro studies with human hepatocytes are scarce because of the lack of livers and suitable methods of storage. After developing a new method for cryopreservation of human hepatocytes, we evaluated the effects of deep freezing storage on their viability, morphology, and functional and toxicological capabilities in classical culture conditions. Freshly isolated human hepatocytes were cryopreserved in medium containing 10% Me2SO and 20% fetal calf serum, using a Nicool ST20 programmable freezer (-1.9 degrees C/min for 18 min and -30 degrees C/min for 4 min). Cells were stored in liquid nitrogen. Viability of thawed human hepatocytes was 50-65% as assessed by erythrosin exclusion test prior to purification on a Percoll density gradient. Morphological criteria showed that thawed human hepatocytes require an adaptation period to the medium after seeding. Functional assessments showed that human hepatocytes which survive freezing and thawing preserve their protein synthesis capabilities and are able to secrete a specific protein, anionic peptidic fraction, which is involved in the hepatic uptake of bile-destined cholesterol. We then studied Midazolam biotransformation to test metabolic functions, and erythromycin toxicity by Neutral Red test (cell viability) and 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide test (cell metabolism). All of these experiments indicated that thawed human hepatocytes should be used 38 h after seeding for optimum recovery of their functions: membrane integrity, protein synthesis, and stabilization of drug metabolism enzymes.     

Rat mesenteric artery endothelial cells in culture secrete ET-1

Endothelial cells were harvested by the collagenase perfusion of isolated mesenteric arteries of rats and cultured. An endothelin peptide was detected in the supernatant of these cells by an antibody which recognizes ET-1 but not "rat" endothelin (ET-3). Culture media was extracted using a C-8 solid phase column and subjected to reverse phase HPLC using a system that separates all known endothelins and immunoreactive endothelins measured using another antibody which recognizes all endothelins. The main immunoreactive peak co-eluted with ET-1. We could not detect any ET-2, ET-3 or Vasoactive Intestinal Contractor. A smaller immunoreactive peak of unknown structure that eluted earlier than ET-1 was also detected. In conclusion, rat endothelial cells secrete a peptide of similar chromatographic and immunoreactive properties as ET-1.     

Rat liver dimethylglycine dehydrogenase. Flavinylation of the enzyme in hepatocytes in primary culture and characterization of a cDNA clone.

Dimethylglycine dehydrogenase (Me2GlyDH), an enzyme of choline catabolism specifically expressed in the mammalian liver, was analyzed in rat hepatocytes in culture. This mitochondrial enzyme carries the FAD cofactor covalently attached to the polypeptide chain by its riboflavin 8 alpha position to N pi of histidine [Cook, R., Misono, K.S. & Wagner, C. (1980) J. Biol. Chem. 259, 12475-12480]. Subcellular fractionation of [14C]riboflavin-labelled hepatocytes and immunoprecipitation with Me2GlyDH-specific antiserum identified a [14C]riboflavin-labelled polypeptide of the size of mature Me2GlyDH only in the mitochondrial fraction. Immunoprecipitation of extracts from [35S]Met-labelled hepatocytes revealed a putative precursor protein to the mature Me2GlyDH in the cytoplasmic fraction. These Me2GlyDH polypeptides were not expressed in cells of the rat hepatoma cell line FAO. A Me2GlyDH cDNA clone of apparent full length was isolated from a rat liver cDNA bank constructed in the plasmid vector pcD-X [Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T. & Arai, K. (1987) Methods Enzymol. 154, 3-28]. The nucleotide sequence of the cDNA contains an open reading frame encoding a protein of 96059 Da. This molecular mass agrees well with the migration on SDS/PAGE of the assumed Me2GlyDH precursor immunoprecipitated from the cytoplasm of [35S]Met-labelled cells. Proteolytic cleavage at the putative mitochondrial processing protease-recognition site Arg(-2)-Ala(-1)-Glu(+1) would lead to the formation of a protein of 91391 Da, which is in good agreement with the estimated 90 kDa of mature Me2GlyDH [Wittwer, A.J. & Wagner, C. (1981) J. Biol. Chem. 256, 4102-4108], and a 43-amino-acid leader peptide. The N-terminus of Me2GlyDH contains a conserved amino acid sequence which forms the dinucleotide-binding site in many enzymes with noncovalently bound FAD. Close to the modified histidine there is an amino acid sequence resembling a sequence conserved in thymidylate synthases and shown in these enzymes to be involved in the binding of the pteroyl polyglutamate cofactor.     

Sertoli cells synthesize and secrete a ceruloplasmin-like protein

Sertoli cells synthesize and secrete a ceruloplasmin-like protein (testicular ceruloplasmin) that is immunologically similar to serum ceruloplasmin. Rat serum ceruloplasmin was purified and an antiserum was produced to the purified protein which specifically immunoprecipitated a 130,000 dalton protein from rat serum. This ceruloplasmin antiserum was found to also immunoprecipitate a 130,000 dalton protein synthesized and secreted by Sertoli cells. The presence of a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), was required during the immunoprecipitation procedure to prevent the proteolytic degradation of testicular ceruloplasmin. Immunoprecipitation of proteins secreted by Sertoli cells with an antiserum to rat serum proteins was found to precipitate two proteins, testicular ceruloplasmin and testicular transferrin.     

Characterization of sheep hepatocytes in primary culture

• 1.1. Primary cultures of isolated sheep hepatocytes were used to characterize metabolic functions of liver: gluconeogenesis, ureagenesis and protein synthesis. The rates of all three metabolic activities were linear over a 20 hr culture period.
• 2.2. Hepatocytes in the presence of glucagon increased the synthesis of urea by approx 30% (P < 0.05) and increased release of glucose into the medium by 60% (P < 0.05).
• 3.3. In the absence of insulin, significantly more (35%; P < 0.05) glucose was released in the medium than in the presence of insulin.
• 4.4. Results help evaluate the primary culture of sheep hepatocytes as an appropriate experimental model to study nutritional and hormonal regulation of liver in the ruminant species.

     

Vascular endothelial cells synthesize and secrete brain-derived neurotrophic factor

Brain-derived neurotrophic factor (BDNF) is an abundant neurotrophin in brain and peripheral nerves, where it affects neural development, survival and repair after injury. BDNF has been detected in rat and human blood, but the source of circulating BDNF is not established. BDNF messenger and peptide were detected in cultured cells and in the culture medium of human umbilical vein endothelial cells. The expression of BDNF was up-regulated by elevation of intracellular cAMP and down-regulated by Ca(2+) ionophore, bovine brain extract and laminar fluid shear stress. These results suggest that vascular endothelial cells may contribute to circulating BDNF.     

Sympathetic neurons synthesize and secrete pro-nerve growth factor protein

Postmitotic sympathetic neuronal survival is dependent upon nerve growth factor (NGF) provided by peripheral targets, and this dependency serves as a central tenet of the neurotrophic hypothesis. In some other systems, NGF has been shown to play an autocrine role, although the pervasiveness and significance of this phenomenon within the nervous system remain unclear. We show here that rat sympathetic neurons synthesize and secrete NGF. NGF mRNA is expressed in nearly half of superior cervical ganglion sympathetic neurons at embryonic day 17, rising to over 90% in the early postnatal period, and declining in the adult. Neuronal immunoreactivity is reduced when retrograde transport is interrupted by axotomy, but persists in a subpopulation of neurons despite diminished mRNA expression, suggesting that intrinsic protein synthesis occurs. Cultured neonatal neurons express NGF mRNA, which is maintained even when they are undergoing apoptosis. To determine which NGF isoforms are secreted, we performed metabolic labeling and immunoprecipitation of NGF-immunoreactive proteins synthesized by cultured NGF-dependent and -independent neurons. Conditioned medium contained high molecular weight NGF precursor proteins, which varied depending upon the state of NGF dependence. Mature NGF was undetectable by these methods. High molecular weight NGF isoforms were also detected in ganglion homogenates, and persisted at diminished levels following axotomy. We conclude that sympathetic neurons express NGF mRNA, and synthesize and secrete pro-NGF protein. These findings suggest that a potential NGF-sympathetic neuron autocrine loop may exist in this prototypic target-dependent system, but that the secreted forms of this neurotrophin apparently do not support neuronal survival.     

Calf adrenocortical fasciculata cells secrete aldosterone when placed in primary culture

Aldosterone production occurs in the outer area of the adrenal cortex, the zona glomerulosa. The glucocortocoids cortisol and corticosterone, depending upon the species, are synthesized in the inner cortex, the zona fasciculata. Calf zona glomerulosa cells rapidly lose the ability to synthesize aldosterone when placed in primary culture unless they are incubated in the presence of the antioxidants butylated hydroxyanisol and selenous acid, the radioprotectant DMSO, and the cytochrome P-450 inhibitor metyrapone. In the presence of these additives, calf zona fasciculata cells in primary culture synthesize aldosterone at rates which can approach those from cells isolated from the zona glomerulosa. Calf zona glomerulosa and fasciculata cells both responded well to ACTH and angiotensin II, but the zona fasciculata cells respond very poorly compared to glomerulosa cells to increased potassium in the media. Rat zona fasciculata cells in primary culture under similar conditions did not synthesize aldesterone, suggesting that the regulation of the expression of the enzymes responsible for the biosynthesis of aldosterone in the two species is different. Two distinct cytochrome P-450 cDNAs which hydroxylate deoxycorticosterone at the 11β position have been described in the rat, human and mouse. Both cytochrome P-450 cDNAs have been cloned and expressed in non-steroidogenic cells, but only one is expressed in the zona glomerulosa and only this glomerulosa cytochrome P450 can further hydroxylate deoxycorticosterone to generate aldosterone. Two bovine adrenal cDNAs have been described with 11β-hydroxylase activity and their expression products in transiently transfected COS cells can convert deoxycorticosterone into aldosterone. Both enzymes are expressed in all zones of the adrenal cortex. Zonal regulation of aldosterone synthesis in the bovine adrenal gland may be due to an 11β-hydroxylase with aldosterone synthesizing capacity which has not yet been isolated. Alternatively, a single enzyme might be responsible for the several hydroxylations in the pathway between deoxycorticosterone and aldosterone and zonal synthesis might be controlled by unknown factors regulating the expression of C-18 hydroxylation. The incubation of zona fasciculata with antioxidants and metyrapone results in atypical expression of this activity by an unclear mechanism.     

Rat testicular peritubular cells in culture secrete an inhibitor of plasminogen activator activity

Rat testicular peritubular cells in culture secrete an inhibitor of plasminogen activator (PA) activity. Conditioned serum-free medium from secondary cultures of peritubular cells (PcMEM) was concentrated and then fractionated by gel exclusion chromatography. Under native or denaturing conditions, PA inhibitor (PA-I) activity appeared in fractions having a molecular weight of approximately 55,000. The PA-I inhibited the tissue-type plasminogen activator, and also that of the two-chain form of urokinase, but not the one-chain form. Addition of guanidine HCl (4 M) to PcMEM resulted in a large increase of inhibitory activity. The 55,000 molecular weight PA-I band in PcMEM reacted with antibodies against plasminogen activator inhibitors produced by bovine vascular endothelial cells, or by human fibrosarcoma cells, as detected by immunoadsorption experiments, by immunoblotting, and by reverse fibrin autography. We describe other characteristics of the protease inhibitor produced by testicular peritubular cells, and we discuss its possible functions in the control of PA activity in the seminiferous tubule at different stages of spermatogenesis.