Dexamethasone induces increased catecholamine biosynthesis in cultured neuroblastoma

The effect of dexamethasone on morphological and biochemical parameters of catecholamine biosynthesis in cultured murine neuroblastoma cells was determined. Treatment for 7 days with 25 μM dexamethasone produced a more intense and uniform catecholamine fluorescence as determined by fluorescence histochemistry using the glyoxylic acid technique. Treatment with dexamethasone also produced a two-fold increase in dopamine content and a threefold increase in tyrosine hydroxylase activity compared with controls. These results suggest that this potent glucocorticoid induces differentiation in cultured murine neuroblastoma, as measured by both biochemical and morphological techniques.     

Helicobacter pylori induces an increase in inositol phosphates in cultured epithelial cells

Abstract Helicobacter pylori is a bacterial pathogen of humans that infects the gastric mucosa. This infection has been associated with gastritis, peptic ulcers, and gastric carcinomas. Diverse in vitro studies have described efficient adherence of H. pylori to different types of epithelial cells. Because of its varied effects on host cells, we have analysed signal transduction events in H. pyfori -infected epithelial cells. Our results show that H. pylori induces an increase in inositol phosphates in all cultured epithelial cells used, including HeLa, Henle 407, Hep-2, and the human gastric adenocarcinoma cell line AGS. Bacterial growth medium supernatants induce a similar response in the host cell. The increase in inositol phosphates is not related to redistribution of cytoskeletal proteins such as actin or α-actinin nor tyrosine-phosphorylation of host cell proteins. The inositol phosphate increase is also observed in cells infected with low or non-adherent H. pylori mutants or mutants defective in the vacuolating toxin or urease holoenzyme. These results indicate that inositol phosphate release in H. pytori -infected cells is not dependent on bacterial adherence, and that a soluble bacterial factor, but not the vacuolating toxin or urease holoenzyme, mediates such an effect.     

Expression of the glucose transporter isoform GLUT 4 is insufficient to confer insulin-regulatable hexose uptake to cultured muscle cells.

Glucose transporter isoform expression was studied in the skeletal muscle-like cell line, C2C12. Northern and Western blot analysis showed that the insulin-responsive muscle/fat glucose transporter isoform, GLUT 4, was expressed in these cells at very low levels, whereas the erythrocyte isoform, GLUT 1, was expressed at readily detectable levels. Insulin did not stimulate glucose transport in this cultured muscle cell line. The C2C12 cells were then transfected separately with either GLUT 1 or GLUT 4, and stable cell lines expressing high levels of mRNA and protein were isolated. GLUT 1-transfected cells exhibited a 3-fold increase in the amount of the GLUT 1 transporter protein which was accompanied by a 2- to 3-fold increase in the glucose uptake rate. However, despite at least a 10-fold increase in GLUT 4 mRNA and protein detected after GLUT 4 cDNA transfection, the glucose uptake of these cells was unchanged and remained insulin-insensitive. By laser confocal immunofluorescence imaging, it was established that the transfected GLUT 4 protein was localized almost entirely in cytoplasmic compartments. In contrast, the GLUT 1 isoform was detected both at the plasma membrane as well as in intracellular compartments. These results suggest that acute insulin stimulation of glucose transport is not solely dependent on the presence of the insulin receptor and the GLUT 4 protein, and that the presence of some additional protein(s) must be required.     

Effects of the anticancer agent VM-26 on hexose uptake in Ehrlich cells

The chemotherapeutic agent VM-26 is a membrane-interactive drug which we have previously demonstrated to be a potent inhibitor of nucleoside transport. Since the carriers mediating nucleoside and hexose transport are structurally and functionally similar, we have further characterized the membrane related properties of this agent by examining its effect on the transport and phosphorylation of hexoses in Ehrlich ascites cells. Under conditions in which only the transport component of hexose uptake was measured, VM-26 had no effect on the influx of 2-deoxyglucose, 3-0-methylglucose, or D-glucose. Glucose-sensitive cytochalasin B binding was only weakly inhibited by the drug. However, VM-26 was an apparent non-competitive inhibitor of the net uptake of 2-deoxyglucose (transport and phosphorylation). Measurement of hexokinase activity in cell extracts failed to demonstrate any significant effect of VM-26 on enzyme activity. In summary, although VM-26 is a potent inhibitor of the transport of nucleosides, it has no apparent effect on the transmembrane flux of hexoses indicating a differential effect on nucleoside and hexose transporters. The ability of the drug to decrease the net accumulation of hexoses in the absence of any detectable effect on hexokinase activity warrants further investigation.     

2-Deoxy-D-glucose uptake in cultured human muscle cells

Hexose uptake was studied with cultured human muscle cells using 2-deoxy-D-[1-3H]glucose. At a concentration of 0.25 and 4 mM, phosphorylation rather than transport was the rate-limiting step in the uptake of 2-deoxy-D-glucose. This was not due to inhibition of the hexokinase activity by either ATP depletion or 2-deoxyglucose 6-phosphate accumulation. In cellular homogenates, hexokinase showed a lower Km value for glucose as compared to 2-deoxyglucose. Intact cells preferentially phosphorylated glucose instead of 2-deoxyglucose. Therefore, transport instead of phosphorylation may be rate limiting in the uptake of glucose by cultured human muscle cells. These data suggest caution in using 2-deoxyglucose for measuring glucose transport.     

Triggering by thyrotropin of an increased synthesis of triglycerides in cultured human thyroid cells

Addition of thyrotropin to cultured human thyroid cells induces a marked increase of the incorporation of (1,3-³H)-glycerol and (1,2-¹⁴C)-acetate in the triglycerides. The presence of thyrotropin in the medium does not modify the synthesis of phospholipids from glycerol; however, it may perhaps slightly decrease the incorporation of radioactive acetate in the phospholipids and in cholesterol. The specific radioactivity of the triglycerides remains unchanged after thyrotropin stimulation and the triglycerides'cell content is accordingly greatly increased.     

Taurine uptake by cultured human lymphoblastoid cells

Cultured human lymphoblastoid cells take up taurine from the medium by two processes: 1) a temperature-dependent, Na⁺-dependent, saturable “active”-transport system and 2) diffusion. The active transport has properties similar to those reported for taurine transport by other tissues. Apparent K_m is about 25 μM and V_max about 7.2 pmol/min/10⁶ cells; saturation occurs at 100 μM taurine. Uptake is competitively inhibited by the β-amino acids hypotaurine (50% inhibition at 44 μM) and β-alanine (50% at 152 μM), as measured at 50 μM taurine. Taurocyamine inhibits 50% at 260 μM. Chlorpromazine and imipramine are strong uncompetitive inhibitors, giving 50% inhibition at 26 μM and 115 μM, respectively; at these concentrations cellular viability per se is not affected. Ouabain inhibits 40–50% over a concentration range of 4–500 μM. Diffusion of taurine into the cells is proportional to concentration up to 20 mM. However, at the concentration of taurine in human plasma, 40–100 μM, active transport would provide 90% of the taurine taken up.     

Agmatine uptake by cultured hamster kidney cells

Agmatine, the product of arginine decarboxylation, has been recently found in a wide variety of animal tissues. In spite of the emergent interest on agmatine in animals, the mechanism of agmatine uptake in mammalian cells has been scarcely studied. An analysis of radiolabeled agmatine uptake was carried out by using a classical, kinetic approach with BHK-21 hamster kidney cells in culture. A high affinity, temperature- and energy-dependent agmatine transport system in BHK-21 kidney cells is here kinetically characterized which seems to be a "general" transporter shared by di- and triamines and different to a highly specific carrier for the tetraamine spermine.     

Active hexose uptake in Lemna gibba G1

Growth of autotrophically growing duck-weeds (Lemna gibba L., G1) was stimulated by sucrose. The rate of respiration increased when plants had been grown on sucrose (8.7 mol O₂ g^-1 fresh weight (FW) h^-1) and was reduced after growth without sucrose in the dark or under longday conditions (2.5 mol O₂ g^-1 FW h^-1). Photosynthesis was induced already by low light intensities (0.1 klx).Short-time application of glucose or sucrose stimulated respiration in proportion to the hexose uptake rate. Sucrose is probably not taken up as the disaccharide. The transported sugar species after addition of sucrose are its hexose moieties produced by the high activity of the cell wall invertase. Fructose stimulated to a lesser extent; mannitol induced no enhancement; 2-deoxyglucose slightly inhibited O₂ uptake. After mild carbon starvation of the plants the uptake of glucose and 3-O-methylglucose proceeded without any lag phase, with similar saturation kinetics in both cases. The initial uptake rate at substrate saturation was 2.6 mol glucose g^-1 FW h^-1 in the dark. Light stimulated hexose uptake by 2 to 3 times. The results show that Lemna gibba has an energy-dependent constitutive system for hexose uptake.Abbreviation FW fresh weight - LD long day - SD short day     

Beta blockade induces apoptosis in cultured capillary endothelial cells

Continuous beta blockade stimulates deposition of collagen in the pulmonary alveolar interstitium of adult rats. It also causes changes to the capillary endothelial cell compartment reminiscent of programmed cell death. To test whether beta blockade results in endothelial cell apoptosis, cultures of capillary endothelial cells were treated with both a wide-spectrum beta blocker and a beta-2-specific antagonist. Apoptosis was measured in these cultures using both terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling and annexin-V assays. Both forms of beta blockade stimulated programmed cell death in these cultures. To test whether the apoptotic effect of beta blockade was related to interstitial collagen deposition, capillary endothelial cells were cocultured with beta-blocked pulmonary fibroblast monolayers. Cocultured endothelial cells were substantially protected from apoptosis after beta blockade; coculture over plain tissue culture plastic or over exogenous collagen films had no effect on programmed cell death in endothelial cells. These results suggest that both pulmonary endothelial and interstitial cells are vulnerable to injury from beta blockade but that paracrine interactions between these cells may protect the peripheral lung from substantive damage.     

Concanavalin A induces endoreduplication in cultured mammalian cells.

Concanavalin A (Con A) induced endoreduplication in an established cell line, Don, of the Chinese hamster. The inducibility of Con A was inhibited by α-methyl-D-mannoside. When a secondary culture of kidney cells (CHK), which showed the contact-inhibition of growth, was used, there was an increase in spontaneous endoreduplication. CHK cells or some of them were more sensitive to Con A than Don cells, in which few spontaneous endoreduplications were observed. Mitotic shake-off after Con A treatment led to the higher ratio of endoreduplicated cells to normal mitoses, suggesting that endoreduplicating cells do not “round-up” and probably do not condense chromosomes through the cell cycle until M is reached.     

3-O-methyl-D-glucose uptake in cultured bovine adrenal chromaffin cells

The membrane transport of glucose was studied in bovine adrenal chromaffin cell cultures by following the cell/medium distribution of the nonmetabolizable glucose analog, 3-O-methyl-D-glucose. Uptake of this sugar in day-1 cultures that are undergoing rapid morphological change and differentiation had a Vmax of 138 nmol/(mg protein.min) and Km of 15 mM, and was only slightly increased by 50 mU/mL insulin. In day-5 cultures where morphological changes were essentially completed, Vmax and Km decreased to 51 nmol/(mg protein.min) and 9.5 mM, respectively, and the response to insulin was restored to the level found in freshly isolated cells; this effect was abolished in the nominal absence of Ca2+. Thus, saturation kinetics and insulin and Ca2+ sensitivity of 3-methylglucose uptake observed in freshly isolated cells were maintained in culture. However, the insulin response was almost absent during the initial period of rapid morphological change when sugar transport was strongly stimulated. Culture of chromaffin cells in the presence of dexamethasone did not inhibit the formation of processes, but decreased 3-methylglucose uptake in day-5 cultures by an apparently competitive effect.     

Heat shock induces protein tyrosine phosphorylation in cultured cells

We examined the effect of heat shock on protein tyrosine phosphorylation in cultured animal cells using antiphosphotyrosine antibodies in immunoblotting and immunofluorescence microscopy experiments. Heat shock significantly elevated the level of phosphotyrosine in proteins in most of the cultured cells examined, including fibroblasts, epithelial cells, nerve cells, and muscle cells, but not in Rous sarcoma virus-transformed fibroblasts. The increase in protein tyrosine phosphorylation induced by heat shock occurred in proteins with a wide range of molecular masses and was dependent on the temperature and duration of the heat shock.     

Ovarian substance induces steroid production in cultured granulosa cells

Summary The rat ovary produces an apparently low molecular weight substance that mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induces temporal cell rounding and, later on, intensive progestin production. However, unlike FSH, OS does not induce accumulation of cyclic AMP (cAMP) in the granulosa cells. The ovarian factor cannot be cAMP as its action is not abolished by phosphodiesterase (PDE) treatment. Neither is it a possible PDE inhibitor, as it does not augment cAMP accumulation in granulosa cells or Friend erythroleukemic cells induced by FSH or PGE₁, respectively. The factor is still active after heating for 20 min at 90° C but is rapidly inactivated by alkali treatment. In addition, treatment with various proteases did not abolish the steroidogenic activity. These findings suggest a possible novel intraovarian regulator of the granulosa cell function. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This work was supported by the United States-Israel Binational Science Foundation, Grant 2656/81. This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.     

Inositol uptake by cultured isolated rat Schwann cells

The uptake of radiolabeled myo-inositol by Schwann cells isolated from the sciatic nerve of 2–4 day old rats was found to occur by a saturable, sodium-dependent phlorizin-inhibited mechanism with an estimated K_m of 30μM. The system was inhibited by galactose and glucose but not by galactitol. At high concentrations of myo-inositol, a diffusion-like process appeared to be functional. The characteristics of the saturable system are very similar to those of myo-inositol uptake by the endoneural fascicle preparation of sciatic nerve.