ABSTRACT
BACKGROUND: Polyamines (putrescine, spermidine and spermine) are ubiquitous molecules indispensable for cell proliferation. In the intestinal lumen they are present in high amounts. Polyamine accumulation in proliferating cells of the intestinal mucosa is high, and it occurs both by enhanced synthesis and by increased uptake from the lumen. AIMS: To study mitogen-induced polyamine accumulation in the gut, we treated proliferating Caco-2 cells with epidermal growth factor (EGF) and measured the activity of ornithine decarboxylase (ODC) and putrescine uptake. Furthermore, we investigated whether EGF-induced changes in the apical membrane could be responsible for the effect of EGF on polyamine uptake in Caco-2 cells. METHODS: Putrescine uptake, ODC activity and intracellular polyamine content were evaluated in the presence of 100 ng/ml EGF. To study the mechanisms of EGF-stimulated polyamine uptake, apical membrane vesicles were isolated, and putrescine uptake into the vesicles measured. Possible enrichment in brush border membrane cytoskeleton proteins (ezrin and villin) was assessed by Western blot. RESULTS: Treatment with EGF induced an increase in ODC activity, which occurred within the first minutes of treatment and reached peak values after 3 h. In contrast, an increase in putrescine uptake was more sustained, with peak levels at 12 h. Both synthesis and uptake contributed to an over 60% increase in intracellular putrescine and spermidine after EGF treatment. There were no detectable changes in apical membrane cytoskeleton (as concluded by the absence of ezrin and villin enrichment in EGF-treated Caco-2 cells). However, in apical membrane vesicles isolated from EGF-pretreated cells, putrescine uptake was enhanced twofold. CONCLUSIONS: EGF stimulates both synthesis and uptake of polyamines in Caco-2 cells. Enhanced synthesis seems to ensure rapid supply with polyamines in the earliest stages of growth, while the uptake is responsible for the maintenance of high polyamine intracellular levels during late growth phases. EGF-stimulated polyamine uptake is apparently not a consequence of structural changes in the apical membrane, but is likely to occur by a distinct EGF-induced alteration of the polyamine transporter itself.
Subject(s)
Caco-2 Cells/metabolism , Epidermal Growth Factor/pharmacology , Polyamines/metabolism , Biological Transport/drug effects , Blotting, Western , Caco-2 Cells/drug effects , Caco-2 Cells/pathology , Cell Division/drug effects , Cell Membrane Permeability/drug effects , Humans , Intracellular Fluid/metabolism , Ornithine Decarboxylase/metabolism , Putrescine/metabolismABSTRACT
Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. In this study, for the first time, members of this family of proteins in two functionally different intestinal epithelial cell lines are identified and characterized. [alpha-32P]GTP blot overlay assays of cytosolic and membranous fractions revealed the presence of specific GTP-binding proteins in the range of 20-30 kDa (small GTPases) in both fractions, with considerably higher amounts in the membranous insoluble fraction. Analysis by two-dimensional electrophoresis, immunoprecipitation using monoclonal and sequence-specific polyclonal antibodies, and C3 exoenzyme-mediated ADP ribosylation demonstrated the presence of Ras, Rap, Rho, Rac, Rab, and several other small GTPases. The pattern of small GTP-binding proteins corresponded to the characteristics of the cell lines. Caco-2 cells showed a Rab5 protein that is known to be involved in endocytosis but was not found in T84 cells. On the contrary Rab3 has been shown to participate in secretory processes. It is highly expressed in T84 cells (sixfold compared to Caco-2 cells).
Subject(s)
Epithelial Cells/chemistry , Epithelial Cells/metabolism , Intestinal Mucosa/chemistry , Intestinal Mucosa/metabolism , Monomeric GTP-Binding Proteins/analysis , Monomeric GTP-Binding Proteins/metabolism , Caco-2 Cells , Electrophoresis, Gel, Two-Dimensional , Guanosine Triphosphate/metabolism , Guanosine Triphosphate/pharmacology , Humans , Intestinal Mucosa/cytology , Isoelectric Focusing , Molecular Weight , Phosphorus Radioisotopes , rab GTP-Binding Proteins/analysis , rab GTP-Binding Proteins/metabolism , rac GTP-Binding Proteins/analysis , rac GTP-Binding Proteins/metabolism , rap GTP-Binding Proteins/analysis , rap GTP-Binding Proteins/metabolism , ras Proteins/analysis , ras Proteins/metabolism , rho GTP-Binding Proteins/analysis , rho GTP-Binding Proteins/metabolismSubject(s)
Epidermal Growth Factor/pharmacology , Epithelial Cells/metabolism , Intestinal Mucosa/metabolism , Polyamines/pharmacology , Caco-2 Cells , Carrier Proteins/metabolism , Cytoskeletal Proteins , Epithelial Cells/cytology , Epithelial Cells/drug effects , Humans , Intestinal Mucosa/cytology , Microfilament Proteins/metabolism , Phosphoproteins/metabolismABSTRACT
The activity spectrum of 4 polyvalent Staph. aureus-phages, of 22 phages from coagulase negative staphylococci and of 64 micrococcal phages was established on 20 Staph. aureus-strains, 116 coagulase-negative staphylococci and 142 micrococci. Staphylococcal phages showed to be only active on strains of the genus Staphylococcus and on cocci related to this genus. Micrococcal phages on the other hand lysed only micrococci.
