Zinc homeostasis in C6 glioma cells

Zinc homeostasis in mammalian cells is precisely regulated by cellular signal transduction mechanisms. The main result of this study is the finding that modulators of phospholipase C (PLC) activity affect cellular zinc export. Two different PLC inhibitors caused an increase of the total cellular zinc level whereas two different PLC activators caused a decrease. Furthermore, both the inhibition of cyclic nucleotide phosphodiesterases as well as the administration of 8-bromo-cAMP evoked a drop in the intracellular zinc level, indicating the involvement of cAMP in the control of cellular zinc export. It is concluded that the activity of PLC controls cellular zinc transport and that the effect of elevated zinc concentrations on PLC activity might be mediated by cAMP. However, modulation of other major signaling enzymes did not affect the cellular zinc homeostasis. These include activation and inhibition of guanylate cyclase, activation of protein kinase G, activation of protein kinase A, and activation or inhibition of protein kinase C. Furthermore there was no evidence for the existence of a zinc-sensing receptor in C6 glioma cells, which would stimulate PLC activity and evoke a mobilization of intracellular free-calcium levels.     

Glutamine transport in C6 glioma cells

Glutamine transport across the cell membranes of a variety of mammalian tissues is mediated by at least four transport systems: a sodium-independent system L, and sodium-dependent systems A, ASC and N, the latter occurring in different tissue-specific variants. In this study we assessed the contribution of these systems to the uptake of [(3)H]glutamine in C6 rat glioma cells. The sodium-dependent uptake, which accounted for more than 80% of the total uptake, was not inhibited by 2-methylaminoisobutyric acid (MeAIB), indicating that system A was inactive, possibly being depressed by glutamine present in the culture medium. About 80% of the sodium-dependent uptake was mediated by system ASC, which differed from system ASC common to other CNS- and non-CNS tissues by its pH-dependence and partial lithium tolerance. The residual 20% of sodium-dependent uptake appeared to be mediated by system N, which was identified as a component resistant to inhibition by MeAIB+threonine. The system N in C6 cells appeared to be neither fully compatible with the neuronal system Nb, nor with the N system described in astrocytes: it differed from the former in being strongly inhibited by histidine and showing fair tolerance for lithium, and from the latter in its pH-insensitivity and strong inhibition by glutamate. The sodium-independent glutamine uptake differed from the astrocytic or neuronal uptake in its relatively weak inhibition by system L substrates and a strong inhibition by system ASC substrates, indicating a possible contribution of a variant of the ASC system.     

Association of glycolytic enzymes with the cytoplasmic side of the plasma membrane of glioma cells

A latex phagocytosis technique was used to prepare relatively pure plasma membranes with inside-out orientation. This method was adapted through a number of modifications in order to evaluate the association of glycolytic enzymes with the cytoplasmic side of the plasma membrane of C6 glial cells. As phosphorylation is strictly coupled with transport in these cells, glycolytic enzymes, especially hexokinase, could metabolize glucose in close vicinity to its transporter. Of the enzymes tested, hexokinase is present in considerable quantities on these membranes (nearly 40% of homogenate specific activity), followed by D-glyceraldehyde-3-phosphate dehydrogenase (10%), pyruvate kinase (8%), and 3-phosphoglycerate kinase (1%). Except for hexokinase, the enzyme pattern presented here is different from that published for other membrane preparations.     

The DNA methylation system in proliferating and differentiated cells

The human melanoma cell line M21 can be induced to differentiate into oligodendrocyte-like cells with concommitant cessation of cell division. Cytosine-arabinoside, 5-aza-2'-deoxycytidine, hydroxyurea, aphidicolin, and phorbol-12-myristate-13-acetate were found to be potent differentiation inducers. We have analyzed the changes of methylation of DNA cytosines that occur after treatment of M21 cells with these compounds. Although DNA methylation levels remain unchanged in the presence of aphidicolin and phorbol ester, 5-aza-2'-deoxycytidine-induced differentiation of these cells results in a 40% DNA demethylation. On the other hand, hydroxyurea and cytosine-arabinoside treatment causes DNA hypermethylation, which, in the case of the cytidine analogue is of only transient nature. These results show that the differentiation of human melanoma cells can be accompanied by variable changes of DNA methylation levels. In another set of experiments, the DNA methylation levels have been analyzed during cytosine-arabinoside-induced differentiation of human K562 erythroleukemia cells. In this system, a transient DNA demethylation precedes the establishment of the differentiated phenotype. Since DNA replication is inhibited, this demethylation cannot be explained by inhibition of the maintenance activity of DNA methyltransferase, but is more likely caused by an active excision of 5-methylcytosine from DNA.     

Microcompartmentation of glycolytic enzymes in cultured cells.

The microcompartmentation of aldolase and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was investigated in four different cell types (3T3 cells, SV 40 transformed 3T3 cells, mouse fibroblasts, chick embryo cardiomyocytes) combining cell permeabilization and indirect immunofluorescence technique. Permeabilization of the cells prior to fixation released the soluble fractions, whilst the total amount of enzymes was preserved in nonpermeabilized cells. Both enzymes exist in a soluble as well as in a structure-bound form. The soluble fraction of aldolase and GAPDH is distributed homogeneously throughout the cytoplasm, excluding the nucleus and vesicles. The permeabilization-resistant form is associated with the actin cytoskeleton. A considerable amount of both enzymes is located in the perinuclear region and cannot be attributed to a definite structure. Comparing the staining patterns of aldolase and GAPDH in four different cell types we found that the distribution of the enzymes corresponds with diverse forms of actin cytoskeletal organization of these cells. The codistribution is maintained in cells treated with cytochalasin D.     

RNA in the periphery of rapidly proliferating mouse lymphoid cells

RNA in the peripheries of various populations of lymph node cells (LNC) has been evaluated by measuring the electrophoretic mobilities of cells, before and after treatment with active or inactivated ribonucleases. Three different populations of LNC were studied: (1) “resting” normal age control LNC; (2) “syngeneic” LNC from irradiated (C3H × C57BL)F₁ or C3H mice four to six days following transplantation of syngeneic spleen cells; such cells were progeny of lymphopoietic progenitor cells of the spleen; and (3) “allogeneic” LNC from irradiated (C3H × C57BL)F₁ mice four to six days after grafting C3H (parental) spleen cells; such cells were progeny of lymphopoietic progenitor cells, but also alloantigen-sensitive cells of the spleen which proliferate in response to the host's alloantigens (a “graft-versus-host” immunological reaction). Whereas the normal LNC had no detectable peripheral RNA, the allogeneic and syngeneic LNC did, i.e., ribonuclease reduced their mean electrophoretic mobilities by 13.6 and 9.2 per cent, respectively. Since both allogeneic and syngeneic LNC had peripheral RNA, no specific correlation could be made with immunological activity. ³H-uridine and ¹⁴C-thymidine incorporation into lymph nodes was greatest in allogeneic, intermediate in syngeneic and least in age control lymph nodes, indicating a “population shift” in the spleen cell chimeras toward relatively immature, rapidly proliferating cells, which had a relatively high rate of RNA synthesis. Thus, rapidly proliferating lymphoid cells do have RNA in their peripheries, but its relation to specific immunological function has yet to be ascertained.     

Intracellular zinc distribution and transport in C6 rat glioma cells

In mammalian cells, the intracellular availability of zinc influences numerous crucial processes. Its distribution has previously been visualized with several fluorescent probes, but it was unclear how these probes are compartmentalized within the cell. Here, we show that in C6 cells the zinc-specific probe Zinquin is evenly distributed. Thus, the significantly lower level of fluorescence in the nucleus and a punctuate vesicular staining are real differences in the concentrations of zinc. Chemical perturbation of the steady state by releasing intracellular protein-bound zinc with the sulfhydryl-reactive N-ethylmaleimide (NEM) resulted in a vanadate sensitive transport of zinc out of the nucleus and into zincosomes. If the zinc-release was performed with the histidine-reactive diethylpyrocarbonate, sequestration was reduced compared to treatment with NEM, indicating the importance of histidine within membrane zinc transporters. Another major factor regulating the zinc homeostasis is ion export. As determined by atomic absorption spectroscopy, up to 50% of the cellular zinc was exported by a mechanism sensitive to lanthanum ions. We conclude that different concentrations of labile zinc exist in different cellular compartments, which are maintained by export and intracellular transport of zinc.     

Effect of leptin in proliferating and differentiated HC11 mouse mammary cells

Leptin and its receptors have been shown to be expressed in several tissues thus suggesting that this protein might be effective not only at the CNS level, but also peripherically. We demonstrated by RT-PCR analysis that leptin and its long isoform receptor are expressed in the mouse mammary epithelial cell line HC11, an in vitro cell model considered suitable to study the regulation of the functional development of the mammary epithelium. Furthermore, leptin secretion by HC11 cells was demonstrated by heterologous ELISA. Neither mRNA expression nor protein secretion changed throughout the different phases of differentiation of the cell line. Receptor mRNA was not modified when cells were induced to express beta-casein. High concentrations of leptin (between 1.5 and 15 microM) significantly (p<0.05) reduced cell growth as measured by MTT test. HC11 cells were transfected with pbetacCAT, a chimeric rat-beta casein gene promoter-CAT gene construct and CAT ELISA was used to determine gene expression. Leptin, from 1.5 nM to 15 microM, was shown to positively (p<0.05) influence beta-casein expression both in the presence or in the absence of prolactin. These data provide evidence that leptin, through its receptor, may be an important mediator in regulating mammary gland growth and development.     

The nuclear matrix of slowly and rapidly proliferating liver cells.

The nuclear matrix of slowly proliferating rat liver is compared with rapidly proliferating regenerating liver and Zajdela ascites hepatoma cells. While no differences are detected in overall ultrastructure, composition or polypeptide profiles of normal liver versus regenerating liver matrices, significant alterations are observed in the polypeptides of Zajdela hepatoma nuclear matrices.     

Chlorpromazine induces accumulation of inositol phosphates in C6 glioma cells

The capacity to modify the incorporation of [2-3H]myo-inositol into inositides and inositol phosphates was different for three psychotropic cationic amphiphilic drugs. Chlorpromazine, desmethylimipramine and propranolol were able to increase the labeling of inositol-containing lipids, but only chlorpromazine dramatically increased the incorporation into inositol phosphate, -bisphosphate and -trisphosphate. The increase was 10- to 50-fold in 60 min as compared with controls. This effect is not due to stimulation of lipid labeling, because in chase experiments radioactivity in inositol phosphates increased to a greater extent than in their parent lipids. It is possible that the alteration of phosphoinositide catabolism is related to the neuroleptic activity of the drug.     

Comparative proteomic analysis of proliferating and functionally differentiated mammary epithelial cells

Proliferation and differentiation of mammary epithelial cells are governed by hormonal stimuli, cell-cell, and cell-matrix interactions. Terminal differentiation of mammary epithelial cells depends upon the action of the lactogenic hormones, insulin, glucocorticoids, and prolactin that enable them to synthesize and secrete milk proteins. These differentiated cells are polarized and carry out vectorial transport of milk constituents across the apical plasma membrane. To gain additional insights into the mechanisms governing differentiation of mammary epithelial cells, we identified proteins whose expression distinguishes proliferating from differentiated mammary epithelial cells. For this purpose we made use of the HC11 mammary epithelial line, which is capable of differentiation in response to lactogenic hormones. Using two-dimensional gel electrophoresis and mass spectrometry, we found about 60 proteins whose expression levels changed in between these two differentiation states. Bioinformatic analysis revealed differential expression of cytoskeletal components, molecular chaperones and regulators of protein folding and stability, calcium-binding proteins, and components of RNA-processing pathways. The actin cytoskeleton is asymmetrically distributed in differentiated epithelial cells, and the identification of proteins involved in mRNA binding and localization suggests that asymmetry might in part be achieved by controlling cellular localization of mRNAs. The proteins identified provide insights into the differentiation of mammary epithelial cells and the regulation of this process.     

Ca regulation of connexin 43 hemichannels in C6 glioma and glial cells

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca²⁺ ([Ca²⁺]_i) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca²⁺]_i was in the 500 nM range but the responses disappeared with larger [Ca²⁺]_i transients. Ca²⁺-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca²⁺-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca²⁺]_i triggers hemichannel opening, not by a direct action of Ca²⁺ on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca²⁺]_i and acting to restrain cellular ATP loss.     

Activities of enzymes of lipid metabolism in Morris hepatoma 7800 C1 cells

(1) The rate of palmitate oxidation in the 7800 C1 Morris hepatoma cells was about 60% of the activity observed in hepatocytes. The stimulatory effect of glucagon in hepatocytes was not observed in the hepatoma cells. The rate of fatty acid synthesis from [2-14C]acetate in the hepatoma cells was 1/20 of the activity in hepatocytes. The conversion of [2-14C]acetate to cholesterol was not different in the two kinds of cell. (2) Acetyl-CoA carboxylase and fatty acid synthetase were significantly decreased in the hepatoma cells. The hepatoma cells had, however, raised activities of malate dehydrogenase (decarboxylating), and glucose-6-phosphate and 6-phosphogluconate dehydrogenases. (3) The activities of the enzymes were not affected by different concentrations of glucose or palmitate in the culture medium. Insulin, dexamethasone, triiothyronine and glucagon had no effect on the enzyme activities. This is in contrast to the adaptation of the peroxisomal beta-oxidation system, which is induced by fatty acids and modified by hormones.