scholarly journals Microinjection of ADP-ribosylated actin inhibits actin synthesis in hepatocyte-hepatoma hybrid cells

1996 ◽  
Vol 319 (3) ◽  
pp. 843-849 ◽  
Author(s):  
Karl H. REUNER ◽  
Anke van der DOES ◽  
Petra DUNKER ◽  
Ingo JUST ◽  
Klaus AKTORIES ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Culture Medium ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Hybrid Cells ◽  
Filamentous Actin ◽  
Clostridium Botulinum C2 Toxin ◽  
Actin Mrna ◽  
Immortalized Hepatocytes ◽  
C2 Toxin

Treatment of hepatocyte-hepatoma hybrid cells with Clostridium botulinum C2 toxin led to a 167% increase in monomeric globular actin (G-actin) and to a 57% decrease in filamentous actin (F-actin) within 2 h. Simultaneously, the level of actin mRNA was specifically decreased to 49% and actin synthesis was significantly diminished. In contrast, treatment of hybrid cells with phalloidin led to a decrease in G-actin to 55% and to a reciprocal increase in actin mRNA to 244% and an increase in actin synthesis. These alterations of actin synthesis depending on the G-actin/F-actin ratio corresponded to the autoregulation of actin synthesis observed in primary cultures of rat hepatocytes. Microinjection of C2 toxin or of phalloidin into hepatocyte-hepatoma hybrid cells had the same effects on actin synthesis as incubation with either toxin in the culture medium. Microinjection of non-polymerizable ADP-ribosylated G-actin into hepatocyte-hepatoma hybrid cells specifically decreased the incorporation of [35S]methionine into newly synthesized actin within 1 h. This decrease continued for at least 19 h. Microinjection of ADP-ribosylated actin led to rounding of cells and obvious disaggregation of actin filaments, which might be due to capping of actin filaments by the ADP-ribosylated actin. Because stabilization of actin filaments by phalloidin before microinjection of ADP-ribosylated actin also resulted in decreased actin synthesis, the concentration of monomeric G-actin seems to be responsible for the regulation of actin synthesis in hepatocyte-hepatoma hybrid cells, which can be regarded as immortalized hepatocytes.

Induction and regulation of connexin26 by glucagon in primary cultures of adult rat hepatocytes

1995 ◽  
Vol 108 (8) ◽  
pp. 2771-2780 ◽  
Author(s):  
T. Kojima ◽  
T. Mitaka ◽  
Y. Shibata ◽  
Y. Mochizuki
Keyword(s):  
Culture Medium ◽  
Primary Cultures ◽  
Minor Component ◽  
Rat Hepatocytes ◽  
Primary Hepatocytes ◽  
Adult Rat ◽  
Adult Rat Hepatocytes ◽  
Epidermal Growth ◽  
A Minor ◽  
Junction Proteins

In the adult rat hepatocyte, the gap junction proteins consist of a major component, connexin32 (Cx32) and a minor component, connexin26 (Cx26). Although we recently reported our success in inducing and maintaining Cx32 in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor and 2% dimethyl sulfoxide, it was very difficult to induce Cx26 in the primary hepatocytes. In the present study, we found that the addition of 10(−7) M glucagon into the culture medium could dramatically induce Cx26 mRNA and protein. Although the expression of Cx32 mRNA was also influenced by glucagon, the increase of the expression was small. Immunocytochemically, Cx26-positive spots were observed between most adjacent cells and were co-localized with the Cx32-positive spots. We also examined whether 0.5 mM dibutyl cyclic AMP could induce expression of Cx26 in the cells. The effect of dexamethasone on the expression of Cx26 mRNA compared to that of Cx32 mRNA was examined. For the induction and maintenance of Cx26 mRNA, more than 10(−7) M dexamethasone was necessary in this culture. These results suggest that expression of Cx26 in hepatocytes may be regulated by the concentrations of glucagon and glucocorticoid hormones.

Effect of tri-iodothyronine on protein turnover in rat hepatocyte primary cultures

1987 ◽  
Vol 113 (2) ◽  
pp. 173-177 ◽  
Author(s):  
G. Gallo ◽  
A. Voci ◽  
P. E. Schwarze ◽  
E. Fugassa
Keyword(s):  
Protein Synthesis ◽  
Protein Turnover ◽  
Culture Medium ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Cellular Proteins ◽  
Cultured Hepatocytes ◽  
Rat Hepatocyte ◽  
Cells Cultured ◽  
Stimulation Of

ABSTRACT The effect of tri-iodothyronine (T3) on protein turnover was studied using primary cultures of rat hepatocytes. Protein synthesis was significantly stimulated in cells cultured for 6 days in the presence of T3 (1 μmol/l). Protein secretion into the culture medium was not affected by the hormone. Breakdown of long-lived proteins, the bulk of cellular proteins which are preferentially degraded through the autophagic lysosomal pathway, was significantly stimulated by the hormone. It is concluded that T3 elicits a general stimulation of protein turnover in cultured hepatocytes. J. Endocr. (1987) 113, 173–177

scholarly journals Glucagon and ammonia influence the long-term regulation of phosphate-dependent glutaminase activity in primary cultures of rat hepatocytes

1991 ◽  
Vol 274 (1) ◽  
pp. 103-108 ◽  
Author(s):  
J D McGivan ◽  
K Boon ◽  
F A Doyle
Keyword(s):  
Culture Medium ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Extracellular Medium ◽  
High Concentrations ◽  
Continuous Presence ◽  
Protein Free Diet ◽  
Glutaminase Activity

1. Glutaminase activity was measured in primary cultures of hepatocytes. 2. Enzyme activity decreased markedly after 24-40 h in culture, and this loss of activity was accompanied by loss of enzyme protein. 3. The loss of activity was delayed by high concentrations of glutamine, and was abolished by the continuous presence of NH4Cl in the culture medium. 4. In cells from rats fed on high-carbohydrate protein-free diet, glutaminase activity was increased by glucagon, but not by dexamethasone. This induction was observed only in the continuous presence of NH3 or high concentrations of glutamine. 5. It is concluded that NH3 and glutamine are essential for the stabilization and induction of glutaminase activity in hepatocytes. The inactivation of glutaminase in hepatocytes and in vivo under certain conditions may be due to lack of NH3 in the extracellular medium.

Polyamine deficiency causes reorganization of F-actin and tropomyosin in IEC-6 cells

1994 ◽  
Vol 267 (3) ◽  
pp. C715-C722 ◽  
Author(s):  
S. A. McCormack ◽  
J. Y. Wang ◽  
L. R. Johnson
Keyword(s):  
Actin Filaments ◽  
Cytoskeletal Proteins ◽  
Stress Fibers ◽  
Filamentous Actin ◽  
Rhodamine Phalloidin ◽  
Tropomyosin Isoforms ◽  
Actin Cortex ◽  
Beta Actin ◽  
Actin Mrna ◽  
As Stress

In earlier work we have shown that polyamine-deficient IEC-6 cells lose most of their ability to migrate. In this report we describe the effect of polyamine deficiency on the cytoskeleton of migrating IEC-6 cells. Cells were grown on cover slips for 4 days. One-third of the monolayer was removed, and the remainder was incubated for 6 h. The monolayers were fixed and stained with rhodamine phalloidin for actin filaments and by immunocytochemistry for tropomyosin. In control cells, actin filaments were found as stress fibers traversing the cell, in a thin actin cortex often visible on only one edge of the cell, and in fine fibers extending into the lamellipodia. Tropomyosin was found in the same distribution. A Western blot showed that tropomyosin was present as 35- and 37-kDa isoforms. In polyamine-deficient cells, actin stress fibers were less dense, whereas the actin cortex was greatly increased in density and lamellipodia were less extensive. Tropomyosin distribution was similar and included a 30-kDa isoform not seen previously. In spite of the obvious changes in the distribution of these cytoskeletal proteins, the concentrations of filamentous actin, beta-actin mRNA, and the higher molecular weight tropomyosin isoforms did not change. In all cases the addition of putrescine to polyamine-deficient cells prevented the changes described. We conclude that polyamines are essential for migration in this system because of their effects on the organization of cytoskeletal actin, tropomyosin, and perhaps other proteins as well.

scholarly journals Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets.

1994 ◽  
Vol 5 (11) ◽  
pp. 1199-1213 ◽  
Author(s):  
G Li ◽  
E Rungger-Brändle ◽  
I Just ◽  
J C Jonas ◽  
K Aktories ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
Insulin Release ◽  
Actin Filaments ◽  
Clostridium Botulinum ◽  
Receptor Activation ◽  
Second Phase ◽  
Dependent Manner ◽  
Clostridium Botulinum C2 Toxin ◽  
C2 Toxin

To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin. Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin. Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time- and dose-dependent manner. The toxin did not affect cellular insulin content or nonstimulated secretion. In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase). In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by approximately 80%. Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin. Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed. In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G- and F-actin and by staining F-actin with fluorescent phallacidin. C2 toxin dose-dependently reduced cellular F-actin content. Stimulation of insulin secretion was not associated with changes in F-actin content and organization. Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content. In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of microfilaments with C2 toxin, most notably during the first phase. This effect was, however, diminished, and the second phase became slightly inhibited when the islets were degranulated. These results indicate an important role for AFs in insulin secretion. In the poorly granulated HIT-T15 cells actin-myosin interactions may participate in the recruitment of secretory granules to the releasable pool. In native islet beta-cells the predominant function of AFs appears to be the limitation of the access of granules to the plasma membrane.

scholarly journals The state of actin assembly regulates actin and vinculin expression by a feedback loop

1995 ◽  
Vol 108 (3) ◽  
pp. 1183-1193 ◽  
Author(s):  
A.D. Bershadsky ◽  
U. Gluck ◽  
O.N. Denisenko ◽  
T.V. Sklyarova ◽  
I. Spector ◽  
...  
Keyword(s):  
Cell Shape ◽  
Actin Polymerization ◽  
The State ◽  
Cell Junctions ◽  
Actin Assembly ◽  
3T3 Cells ◽  
Clostridium Botulinum C2 Toxin ◽  
Actin Mrna ◽  
C2 Toxin ◽  
In Cells

Actin filaments are major determinants of cell shape, motility and adhesion, which control important biological processes including embryonic development and wound healing. These processes are associated with changes in actin assembly, which is regulated by controlling the balance between polymerized and non-polymerized actin. To maintain a significant pool of non-polymerized actin, mechanism(s) linking actin synthesis to its state of polymerization were proposed. We have studied this relationship between actin synthesis and organization by modulating actin assembly using different drugs. Unassembled actin was increased in 3T3 cells using either the Clostridium botulinum C2 toxin, which ADP-ribosylates actin, or by latrunculin A, a Red Sea sponge product, which binds monomeric actin. The synthesis of actin was dramatically reduced in these cells owing to a concomitant decrease in actin RNA level. Similar results were obtained with HeLa cells grown in both monolayer and in suspension, suggesting that cell shape changes associated with drug treatment are not the primary cause for the effect on actin synthesis. In contrast, the scrape-loading of 3T3 cells with phalloidin, a stabilizer of polymerized actin that increased the level of assembled actin, resulted in elevated actin synthesis and RNA content. The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, which is involved in actin-membrane associations, was altered in parallel with that of actin in cells treated with these drugs. The decrease in actin RNA resulted from destabilization of actin mRNA in cells where unassembled actin level was elevated. This is suggested by the unchanged transcription of actin in isolated nuclei from drug-treated cells, and by demonstrating that actin mRNA was degraded faster in cells after C2 toxin treatment than in control cells. This feedback control mechanism is mainly confined to the cytoplasm, as it remained active in enucleated cells. The results suggest the existence of an autoregulatory pathway for the expression of actin and other microfilament-associated proteins which is linked to the state of actin polymerization in the cell.

scholarly journals Hormonally produced changes in caeruloplasmin synthesis and secretion in primary cultured rat hepatocytes. Relationship to hepatic copper metabolism

1983 ◽  
Vol 212 (2) ◽  
pp. 297-304 ◽  
Author(s):  
A L Weiner ◽  
R J Cousins
Keyword(s):  
Culture Medium ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Copper Metabolism ◽  
Rat Hepatocytes ◽  
Serum Copper ◽  
Copper Accumulation ◽  
Glucocorticoid Treatment ◽  
Hepatic Copper ◽  
Adrenergic Blocker

Hormonally produced changes in the synthesis and secretion of the serum copper-containing protein caeruloplasmin were studied in primary cultures of rat liver parenchymal cells isolated by the collagenase-perfusion technique. A rabbit antibody directed against rat caeruloplasmin was used to immunoprecipitate labelled caeruloplasmin. Isolated liver cells synthesized and secreted caeruloplasmin over a period of 3 days. Synthesis and secretion of this protein was enhanced when cells were treated with dexamethasone. The accumulation of copper was also moderately enhanced with glucocorticoid treatment. Inclusion of adrenaline in the culture medium resulted in elevated incorporation of copper into newly synthesized caeruloplasmin as well as an increase in 64Cu-labelled caeruloplasmin in the culture medium. However, adrenaline did not seem to increase the secretion of 3H-labelled protein, despite the elevation in secreted 64Cu-caeruloplasmin. This may be due to a large increase in the intracellular pool of 64Cu caused by enhanced accumulation of this metal when adrenaline is included in the incubation medium. Enhanced copper accumulation was also seen when cells were treated with glucagon. Adrenaline-stimulated accumulation of 64Cu could be inhibited by including phenoxybenzamine, an alpha-adrenergic blocker, in the culture medium. Elevation of extracellular copper caused enhancement in the detection of labelled caeruloplasmin in the medium of cultured cells, probably owing to the ability of this metal to stabilize the protein.

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