Bradykinin-induced release of prostacyclin and thromboxanes from bovine pulmonary artery endothelial cells. Studies with lower homologs and calcium antagonists

Bovine pulmonary artery endothelial cells, in serum-free culture medium, release small quantities of prostacyclin and thromboxane A2 (3-10 and 0.1-0.3 ng/ml; measured as immunoreactive 6-ketoprostaglandin F1 alpha and thromboxane B2, respectively). The release of these substances is stimulated by up to 20-fold during a 3 min incubation with the vasodilator, bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9). Endothelial cells incubated with [3H]arachidonic acid for 24 h and then exposed to bradykinin for 3 min release 3H into the medium, approximately 65% of which co-chromatographs with 6-ketoprostaglandin F1 alpha and 3% with thromboxane B2. The effects of bradykinin are dose-related and are often discernible when the hormone is used at concentrations believed to occur physiologically (10 pg/ml; approximately 10 pM). Furthermore, the bradykinin molecule must be intact: none of its lower homologs affects the release of prostacyclin, thromboxane A2, or 3H unless used at concentrations (1 microM or higher) unlikely to be achieved in vivo. The release appears to involve calcium uptake and calmodulin: it is abolished by EGTA (5 mM) and inhibited by the 'slow channel' calcium antagonists, verapamil and nifedipine (10-100 microM), and by the calmodulin inhibitor, trifluoperazine (3-30 microM). Our findings suggest that bradykinin exerts some of its hormonal effects by acting on specific receptors possessed by vascular endothelial cells; receptor activation is associated with calcium transport, arachidonate mobilization, and a selective synthesis of prostacyclin, a vasodilator in its own right.     

Inhibition by angiotensin converting enzyme inhibitors of endothelin secretion from cultured human endothelial cells.

We conducted a study to determine whether angiotensin converting enzyme inhibitors (ACEIs) inhibit endothelin secretion from cultured human endothelial cells. Confluent umbilical vein endothelial cells were incubated in multi-well plates with culture medium containing either captopril (10(-6), 10(-5), 10(-4) M) or enalaprilat (10(-7), 10(-6), 10(-5) M) for 6 hours. Immunoreactive endothelin in the medium was measured by radioimmunoassay. Calf serum (CS) stimulated endothelin release in a concentration-dependent manner, and both ACEIs inhibited 5% CS-stimulated endothelin release in a concentration-dependent manner. To explore the mechanisms of ACEI-induced suppression of endothelin release, the effects of angiotensin II (10(-8), 10(-7), 10(-6) M), angiotensin converting enzyme (0.1, 1, 10 mU/ml), bradykinin (10(-8), 10(-7), 10(-6) M), and sodium nitroprusside (10(-6), 10(-5), 10(-4) M) on endothelin release were also examined. Although angiotensin II and angiotensin converting enzyme had no significant effect on endothelin release, concentration-dependent suppression occurred with bradykinin and sodium nitroprusside. These results indicate that ACEIs inhibit the stimulated release of endothelin from human endothelial cells, and provide indirect evidence that ACEI-induced ET suppression may be mediated via potentiation of autacoid formation from the cells.     

Super-CHO—A cell line capable of autocrine growth under fully defined protein-free conditions

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10⁶ cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10⁶ cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10⁶cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.     

Angiotensin converting enzyme in cultured endothelial cells and growth medium. Relationships to enzyme from kidney and plasma

We have previously reported that cultured cells from swine aorta possess angiotensin converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) and release it into serum-free culture medium. The present work compares enzyme from these two sources, and from swine kidney and serum, with respect to antibody and lectin binding. Purified enzyme from swine kidney, and the activity in swine serum, cultured endothelial cells and culture medium bind similarly to rabbit antibodies prepared against the kidney converting enzyme. Enzyme from each of these sources was allowed to bind to an immobilized lectin (Ricinus communis), which binds to terminal galactose residues of glycoproteins. Increasing concentrations of galactose were used to remove enzyme from the lectin column and the distribution of enzyme activity in the galactose eluates was determined. The elution pattern was similar for kidney and endothelial cell enzyme, and different from the pattern found for both serum and medium enzymes. Neuraminidase treatment of either serum or medium enzyme altered the distribution of activity eluted to that found for endothelial cell or kidney enzymes. The effects of neuraminidase suggest that the difference in lectin binding between cell and medium enzyme reflects differences in the number of terminal sialic acid residues that cover galactose residues.     

Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines

Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID₅₀) titers of the two cell lines reached 4.51×10⁶ cells/mL, 2.94Log₁₀(HAU/50 μL) and 8.49Log₁₀(virions/mL), and 5.97×10⁶ cells/mL, 3.88Log₁₀(HAU/50 μL), and 10.34Log₁₀(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.     

Cytochrome P4501A induced differentially in endothelial cells cultured from different organs of Anguilia rostrata

Summary Endothelial cells are a structural barrier and an active regulator of many bodily processes. Cytochrome P4501A (CYPIA) activity is induced in the endothelium of teleosts and mammals exposed to lipophilic xenobiotics, such as polycyclic aromatic hydrocarbons, and can have significant consequences for endothelial functions. We exposed cultures of characterized endothelial cells from the heart, kidney, and rete mirabile of the eel, Anguilla rostrata, to aryl hydrocarbon receptor (AhR) agonists. In heart endothelial cells, the maximum response (based on O-deethylation of 7-ethoxyresorufin to resorufin [EROD] activity) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 113 pmol/mg/min, was at 1 nM TCDD and the peak response to β-napthoflavone (βNF), 135 pmol/mg/min, was at 3 μM βNF. The maximum response to TCDD in the kidney endothelial cells is 12 pmol/mg/min at 0.3 nM TCDD. The rete mirabile capillary endothelial cells responded minimally or not at all to exposure to TCDD and βNF. Both the heart and kidney endothelial cells (but not the rete mirabile capillary cells) have a low level of EROD activity (12.7 and 5.2 pmol/mg/min, respectively) in untreated or dimethylsulfoxide-treated cells. The robust response of the heart endothelial cells to induction and the lack of response in the rete mirabile capillary endothelial cells indicate that these cells are a good resource to use to investigate the physiological consequences of AhR agonist exposure and CYP1A induction in different areas of the vasculature.     

Sulfated glycoproteins and extracellular matrix of cultured human pulmonary endothelial cells

Endothelial cells derived from human pulmonary arteries incorporate (³H)-glucosamine and ³⁵SO₄ into glycosaminoglycans and into the carbohydrate side chains of glycoproteins. These ³H/³⁵S-carbohydrate chains were isolated from cells and culture medium after Pronase digestion. The ³H/³⁵S-glycosaminoglycans were separated from the ³H/³⁵S glycopeptides by chromatography on Sephadex G-50. The distribution of cellular glycosaminoglycans and glycopeptides indicated that 30–60% of the cellular ³⁵S-glycopeptides may be associated with the matrix components that are synthesized by the cell and attached to a plastic substratum. Human pulmonary arterial endothelial cells were grown on collagen or on a matrix derived from vascular smooth muscle cells in order to investigate how smooth muscle cell extracellular matrix components may regulate the synthesis of endothelial cell glycoconjugates. Endothelial cells grown on plastic release various proportions of the glycoconjugates they synthesize into the culture medium. However, these same cells, when grown on substratum composed of extracellular matrix materials, synthesized altered proportions of cell-associated glycosaminoglycans and reduced the levels of total glycosaminoglycans they released into the culture medium. Thus the growth of endothelial cells on a matrix of smooth muscle cell components indicates that the glycosaminoglycan materials released into the culture medium by cells grown on a plastic substratum may not be an accurate reflection of the levels or composition of extracellular matrix materials made by endothelial cells in vivo.     

Selective isolation of rat aortic wall layers and their cell types in culture—Application to converting enzyme activity measurement

The rat aorta, whose three wall layers can be separated by microdissection offers the rare possibility of comparing physiological characteristics of in vivo tissular cell components and corresponding cells after culture.We developed a technique allowing the dissociation of the three tunicae (intima, media and adventitia) of the rat aorta and the culture of their main cell types i.e: endothelial cells (EC) from intima, smooth muscle cells (SMC) from media and fibroblasts (Fib) from adventitia. Comparison between selected tunicae in vivo and their corresponding cells in vitro was performed via arterial angiotensin converting enzyme (ACE) activity measurements in Wistar rats.In vivo microsomial ACE activity for each tunica was as follows: 368.9 ± 34.3 (endothelium), 10.5 ± 1.9 (media) and 10.2 ± 4.9 (adventitia) pmol/mg protein/min. Corresponding cell primary culture values were 1.2 ± 0.1 (EC), 0.06 ± 0.02 (SMC) and 0.24 ± 0.01 (Fib) pmol/mg protein/min. Incubation of serum-deprived cells with Dexamethasone (10⁻⁷M) over 48 hr induced a statistically significant shift of total ACE activity from controls to stimulated cells of 2.9 ± 0.3 to 9.7 ± 1.0 in EC, 0.8 ± 0.1 to 32.1 ± 4.9 in SMC and 1.03 ± 0.65 to 57.2 ± 2.1 pmol/ mg prot/min in fibroblasts.In the rat aorta, ACE was present not only in the intimal endothelial cell lining, but also in the media and the adventitia. ACE activity levels in primary cultured vascular cells were about 100-fold less than those found in the ex vivo tissues. Nevertheless, ACE expression seems to be more constitutive in endothelial cells and more inducible in smooth muscle cells and fibroblasts. This methodological approach should be of interest in studying environmental or genetic regulation of protein expression in the three layers/three cell types of the vascular wall.     

Transferrin is an autocrine growth factor secreted by reuber H-35 cells in serum-free culture

Summary We have previously reported that Reuber H-35 rat hepatoma cells secrete an autocrine growth-stimulating activity in serum-free culture. To characterize this activity, conditioned serum-free medium from dense H-35 donor cultures was collected in the absence and presence of [³⁵S]methionine. A 1∶4 dilution of conditioned medium into fresh serum-free medium resulted in an increase in mean H-35 cell numbers per assay dish from 1.59±0.12×10⁵ to 3.35±0.34×10⁵ after 44 h of incubation. Control, unconditioned medium, resulted in significantly (P<0.05) less growth (2.14±0.41×10⁵ cells per dish). Trypsin digestion eliminated the growth-promoting effect of conditioned medium but had no effect on unconditioned medium. Dialysis did not diminish the growth-promoting activity of conditioned medium. The immunoprecipitate of [³⁵S]methionine-containing conditioned medium with antisera against rat serum transferrin contained a dominant radioactive doublet of molecular weight equal to purified rat serum transferrin. A rat transferrin radioimmunoassay was devised and used to quantitate that 29.1±1.2 ng of transferrin was secreted per 10⁶ cells per hour in serum-free culture. Addition of antitransferrin antibody resulted in a significant (P<0.025) decrease in H-35 cell growth after 48 h. Thus, a portion of the autocrine growth-promoting activity secreted by H-35 cells into serum-free culture is due to transferrin. This work was funded by a feasibility grant from the American Diabetes Association, as well as by grants CA 24604-09 and CA 16463-14 from The National Institutes of Health, Bethesda, MD.     

Tumors skew endothelial cells to disrupt NK cell, T-cell and macrophage functions

Introduction Patients and mice with solid tumors, such as Lewis lung carcinoma (LLC), have defects in functions of immune effector cells. Endothelial cells, a component of the tumor vasculature, are potential regulators of immune cell functions. Therefore, these studies examined the impact of exposure to LLC tumor on the ability of endothelial cells to modulate immune cell functions. Materials and methods Endothelial cells were pre-treated with LLC tumor-conditioned medium (Endo^T-sup) for 24 h. Control endothelial cells that were exposed to medium (Endo^Media) or epithelial cell-conditioned medium (Endo^Epi-sup). After the initial 24 h incubation, endothelial cells were washed and fresh media was added. Cells were allowed to incubate for an additional 24 h. Supernatants from Endo^Media, Endo^Epi-sup or Endo^T-sup were collected and assayed for immune modulatory products and for immune modulatory activity. Results Supernatant from Endo^T-sup contained increased levels of PGE₂, IL-6 and VEGF as compared to Endo^Media and Endo^Epi-sup controls. NK cell activity, as measured by TNF-α and IFN-γ secretion, was increased following exposure to media conditioned by Endo^Media and Endo^Epi-sup. Exposure of NK cells to supernatants of Endo^T-sup, also increases TNF-α and IFN-γ secretion, but to a lesser extent than by Endo^Media and Endo^Epi-sup. Examination of macrophage functions demonstrated that supernatant from Endo^T-sup decreased microbead phagocytosis and increased production of the immune suppressive mediators, IL-10 and PGE₂. Lastly, T-cell responses to stimulation with anti-CD3 in the presence of supernatants from Endo^T-sup were examined. IFN-γ production by CD8⁺ T-cells was reduced after exposure to Endo^T-sup-conditioned medium, as compared to cells treatments with medium or control conditioned medium. Production of IFN-γ by CD4⁺ T-cells exposed to Endo^T-sup was not altered. Conclusions Taken together, these studies demonstrate that tumors skew endothelial cells to disrupt NK cell, T-cell and macrophages functions, and represents a novel mechanism of tumor-induced immune suppression.     

Morphological and proliferative responses of endothelial cells to hydrostatic pressure: Role of fibroblast growth factor

Subconfluent bovine pulmonary artery endothelial cells on rigid substrates were exposed to 1.5–15 cm H₂O sustained hydrostatic pressure for up to 7 days and exhibited elongation, cytoskeletal rearrangement, increased cell proliferation, and bilayering. The role of basic fibroblast growth factor (bFGF) in the mechanism(s) of these endothelial cell responses to sustained hydrostatic pressure was investigated. Evidence that bFGF was released from endothelial cells exposed to sustained hydrostatic pressure or compression was provided by the following experimental results: (1) Cells exposed to control (3 mm H₂O) pressure displayed intense nuclear and cytoplasmic bFGF staining by immunocytochemical techniques; this staining was absent in cells exposed to 10 cm H₂O for 7 days. (2) Conditioned medium from endothelial cells exposed to 10 cm H₂O for 7 days contained at ansferable, growth-promoting activity exhibiting heparin-Sepharose affinity, lability to both heat and freeze/thawing, and neutralization by anti-bovine bFGF. (3) Suramin (0.1 mM), a growth-factor receptor inhibitor, abrogated the proliferative and morphological responses of endothelial cells exposed to sustained hydrostatic pressure. Endothelial cells exposed to elevated hydrostatic pressure demonstrated no detectable decrement in cell viability as assessed by Trypan blue exclusion. The results of the present study indicate that hydrostatic pressure or compression can induce bFGF release from endothelial cells independent of cell injury or death; bFGF is subsequently responsible for the morphological, proliferative, and bilayering responses of endothelial cells to hydrostatic pressure. © 1993 Wiley-Liss, Inc.     

Adenylate cyclase and phosphodiesterase activities in relation to a rapid increase in cellular cAMP concentration by hypotonic shock indunaliella viridis

An adenylate cyclase activity of 16.02±1.03 pmol cAMP produced min⁻¹ (mg protein)⁻¹ was detected in a cell homogenate ofDunaliella viridis, a unicellular halotolerant green alga. It was present in both the membrane fraction and soluble fraction separated from the homogenate. Adenylate cyclase activity in the homogenate was activated by 1μM GTPγS but not by Ca²⁺+calmodulin, suggesting this enzyme to be regulated by a G-protein. A phosphodiesterase activity of 23.12±15.03 pmol cAMP decomposed min⁻¹ (mg protein)⁻¹ was found in the homogenate. These activities suggest the presence of a cAMP mediated signal transduction system inDunaliella. Cells, transferred from 1.7 M NaCl medium to 1 M NaCl, showed rapid increase in cAMP within 2 min to about 1.5 times the original concentration (from 2.4±0.2 to 3.9±0.2 pmol per 10⁸ cells) which was recovered in 30 min.     

Apparent inhibition of in vitro juvenile hormone biosynthesis by the corpus cardiacum of adult crickets (Gryllus bimaculatus and Acheta domesticus) is due to juvenile hormone esterase

When measuring the in vitro JH III-biosynthesis by corpora allata (CA) from adult female crickets in the presence of corpora cardiaca (CC), the amount of JH III in the medium decreased in a dose dependent manner. The CC of a 4-day-old female Gryllus bimaculatus contain 42 pmol.pair CC⁻¹ Grb-AKH, 0.62 pmol.pair CC⁻¹ octopamine, and a JH-esterase activity of 9.8 pmol JH.h⁻¹.pair CC⁻¹. Comparable values for Acheta domesticus are 21 pmol.pair CC⁻¹ Grb-AKH, 0.53 pmol.pair CC⁻¹ octopamine, and 6.5 pmolJH.h⁻¹.pair CC⁻¹ of JH-esterase activity. Even if the entire octopamine content of the CC were released into the medium, the concentration would be below the 10⁻⁵ M threshold for octopamine inhibition of JH synthesis. An in vitro AKH inhibition of JH III synthesis was observed, but only at a relatively high concentration (10⁻⁵ M). If the entire AKH content (10⁻⁶ M) of the CC were released into the medium, the AKH concentration would approach JH synthesis inhibiting levels. However, the rate of release of AKH in vitro was very low, and, therefore, AKH from the CC could not affect JH synthesis. In contrast, a specific JH-esterase, released by isolated CC into the medium, was sufficiently high in both cricket species to account for the observed decrease in JH III present. OTFP-sulfone (10⁻⁵ M) restored apparent JH synthesis of the CA to the control level. There was no reduction in the amount of JH released when CA were incubated with heat treated CC. The CA themselves contained almost no JH-esterase activity. © 1997 Wiley-Liss, Inc.     

Regulation of fructose-1,6-bisphosphatase in yeast by phosphorylation/dephosphorylation

Fructose-1,6-bisphosphatase was precipitated with purified rabbit antiserum from extracts of ³²P-orthophosphate labelled yeast cells, submitted to SDS polyacrylamide gel electrophoresis, extracted from the gels and counted for radioactivity due to ³²P incorporation. Fructose-1,6-bisphosphatase from glucose starved yeast cells contained a very low ³²P label. During 3 min treatment of the glucose starved cells with glucose the ³²P-label increased drastically. Subsequent incubation of the cells in an acetate containing, glucose-free medium led to a label which was again low. Analysis for phosphorylated amino acids in the immunpprecipitated fructose-1,6-bisphosphatase protein from the 3 min glucose-inactivated cells exhibited phospho-serine as the only labelled phosphoamino acid. These data demonstrate a phosphorylation of a serine residue of fructose-1,6-bisphosphatase during this 3 min glucose treatment of glucose starved cells. A concomitant about 60 % inactivation of the enzyme had been shown to occur. The data in addition show a release of the esterified phosphate from the enzyme upon incubation of cells in a glucose-free medium, a treatment which leads to peactivation of enzyme activity. A protein kinase and a protein phosphatase catalysing this metabolic interconversion of fructose-1,6-bisphosphatase are postulated. It is assumed that metabolites accumulating after the addition of glucose exert a positive effect on the kinase activity and/or have a negative effect on the phosphatase activity. A role of the enzymic phosphorylation of fructose-1,6-bisphosphatase in the initiation of complete proteolysis of the enzyme during “catabolite inactivation” is discussed.     

Paraquat depresses the activity of angiotensin converting enzyme expressed by human umbilical vein endothelial cells in culture

The activity of angiotensin converting enzyme (ACE) in cell lysate of cultured human umbilical vein endothelial cells (HUVEC) after a 24-hour incubation with 10(-3) and 10(-4)M of paraquat (PQ) was decreased. However, LDH released into the culture medium of HUVEC during the 24-hour incubation with PQ was not increased. Many investigators show that the change in serum ACE activity reflects the impairment of vascular endothelial cells. We showed in this report that ACE was decreased even at an early stage of endothelial injury induced by PQ, when LDH release is not yet increased.     

Estrogen synthetase in choriocarcinoma cell culture. stimulation by dibutyryl cyclic adenosine monophosphate and theophylline

The stimulation of estrogen biosynthesis by N⁶, O² -dibutyryl adenosine 3':5'-cyclic monophosphate and theophylline (dbT) in cultures of the JAr line of choriocarcinoma cells was investigated by measuring the specific activity and kinetic constants of estrogen synthetase (aromatase) in the various subcellular fractions after differential centrifugation of homogenized cells in isotonic sucrose. The low speed (900x$\text{g}$) pellet,from cells grown with or without dbT and homogenized in isotonic sucrose,contains the majority of the aromatase activity and the highest aromatase specific activity. The aromatase specific activity in the homogenate of cells grown with dbT and in the various subcellular fractions is 4- to 10-fold higher than in cells grown without dbT. The Vmax of androstenedione (4-androstene-3,17-dione) aromatization in homogenates from dbT-stimulated cells (6.9 pmol estrogen/min per mg protein) is significantly increased over that measured in the absence of dbT (1.5 pmol estrogen/min per mg protein); the Km values, however, are not significantly different (average of 43.8nM in dbT-stimulated fractions; 53.2nM in control fractions). These results suggest that the increased aromatase specific activity in dbT-stimulated cells results from an increase in amount of active enzyme, rather than from an increase in affinity of the enzyme for its substrate.     

Growth characterizations of a human monocytic leukemia cell line in a serum-free medium

A clone, AH-01S, derived from a human monocytic leukemia cell line, THP-1, grew rapidly in a serum-free medium containing insulin, transferrin, ethanolamine, and sodium selenite. In batch culture using the serum-free medium, the AH-01S cells proliferated at a specific growth rate (μ) of 0.30 to 0.50 (1/day) from a cell concentration of 1 × 10⁴ cells/ml to 1.6 × 10⁶ cells/ml, an increase of 160 times. A higher cell concentration of 0.45 × 10⁷ cells/ml (cell volume ratio was 0.5%) was obtained in spinner flask culture using the serum-free medium. A mean specific growth rate 0.50 (1/day) was also observed in a culture in a fully instrumented cell culture fermentor. However, μ decreased drastically after the cell concentration reached 1.5 × 10⁶ cells/ml. Analyses of medium composition during cultivation revealed that under lower cell concentration, l-glutamine was the main carbon source while glucose was converted to lactate almost stoichiometrically, and that the production of lactate from glucose decreased at higher cell concentrations. To obtain cultures of 1 × 10⁹ cells, 1,200 to 1,300 mg of a carbon source (glucose) and 400 to 500 of amino acids were consumed during high cell concentration cultivation of the AH-01S cells in the serum-free medium.     

Factors influencing the pattern of dopamine secretion in Tetrahymena pyriformis

Dopamine production and secretion by the unicellular eukaryote Tetrahymena pyriformis were examined through the use of high performance liquid chromatography (HPLC) with electrochemical detection and through labeling studies with radioactive precursors. Growing cultures maintained a steady state intracellular level of 1.6 ± 0.3 pmol dopamine/10⁶ cells while secreting dopamine into the medium at a rate of 0.2–0.3 pmol/10⁶ cells per min. Incorporation of [¹⁴C]tyrosine and l-[³H]dihydroxyphenylalanine (DOPA) into dopamine was most successful in a basal medium (1.3 mM Tris-HCl, 1 mM citric acid, and 1 mM Ca(OH)₂, (pH 6.5)). A rapid conversion of added l-[³H]DOPA into dopamine confirmed the dynamic pattern of dopamine synthesis and secretion first indicated by the quantitative chromatographic analyses. The intracellular concentration of dopamine dropped sharply after cells were resuspended in the basal medium at 10⁶ cells/ml, so that by approx. 1 h after resuspension, dopamine dropped below the level detectable by HPLC (0.15 pmol/10⁶ cells). Under these conditions, dopamine secretion continued at a high rate for some time, finally leading to a maximal extracellular concentration of 8.71 ± 1.73 pmol/ml by 1 h. At this concentration, the rate of secretion appears to match that of degradation. Pulse chase experiments confirmed the rapid 3urnover of intracellular dopamine. Approx. 90% of [³H]dopamine and l-[³H]DOPA disappeared from l-[³H]DOPA-prelabeled cells during a 5 min chase, with approx. 50% of this being recovered as [³H]dopamine in the cells' medium. Dopamine secretion could be increased by nearly 100-fold by adding high levels (15 nmol/ml) of l-DOPA to the medium. In contrast, NSD-1015, a potent inhibitor of dopamine synthesis, completely blocked dopamine production. 0.15 mM dibucaine and 0.02 mM reserpine reduced dopamine secretion by approx. 65% over a 25-min incubation, but 5 mM EGTA had no noticeable effect.     

Direct measurement of vitamin K-dependent enzymes in various isolated and cultured tumor and non-tumor cells

A modification of the assay for vitamin K-dependent carboxylase is described with which the enzyme could be detected in relatively low amounts of cells (n = 10⁶). Using this assay, we could demonstrate vitamin K-dependent carboxylase activity in hepatocytes, renal tubular cells, osteoblasts, endothelial cells and macrophages, but not in lymphocytes or platelets. The cultured tumor cells UMR-106, B16 and 5583 also contained vitamin K-dependent carboxylase activity. Vitamin K epoxide reductase activity was demonstrated only in cells where vitamin K-dependent carboxylase activity was present. The tumor cells possessed remarkably less K epoxide reductase activity than the normal cells. When cells were cultured in medium containing warfarin, the K epoxide reductase activity was found to be decreased and the amount of non-carboxylated precursor protein and increased, suggesting an analogous vitamin K mechanism as in liver.