Differential glucocorticoid effects on repair mechanisms and NF-kappaB activity in the intestinal epithelium

Glucocorticoids are effective agents in the management of inflammatory bowel diseases. However, information about their effects on repair mechanisms of the intestinal epithelium is incomplete.Therefore, the aim was to analyse in vitro effects of glucocorticoids on proliferation, restitution, and apoptosis as well as their effects on activity and expression of nuclear factor (NF)-kappaB, a known regulator of apoptosis and inflammation, in intestinal epithelial cells.Non-transformed rat jejunum epithelial cells (IEC-6) were cultured in the presence and absence of various concentrations of prednisolone and budesonide. IEC-6 cell proliferation was assessed by [3H]-thymidine incorporation. Restitution was analysed by an IEC-6 in vitro assay. Apoptosis was evaluated by ELISA and fluorescence microscopy. DNA binding activity and nuclear expression of NF-kappaB was determined by electrophoretic mobility shift assays and Western blotting, respectively.Prednisolone and budesonide stimulated IEC-6 cell proliferation at low to medium pharmacologic concentrations (prednisolone: 10(-9) to 10(-6) M; budesonide: 10(-11) to 10(-8) M). In contrast, high concentrations (>5 x 10(-5) M) had inhibitory effects on proliferation. 10(-7) M prednisolone and 10(-8) M budesonide increased restitution of IEC-6 cells, whereas high concentrations (10(-4) M) of prednisolone and budesonide decreased restitution. Apoptosis of IEC-6 cells was substantially enhanced by 10(-4) M budesonide; apoptosis was slightly increased by the highest prednisolone concentration used (5 x 10(-4) M). Furthermore, both glucocorticoids inhibited DNA binding activity and nuclear NF-kappaB expression in IEC-6 cells in a dose- and time-dependent fashion.In conclusion, prednisolone and budesonide modulate repair mechanisms of intestinal epithelial cells in vitro in a dose-dependent manner and profoundly modulate the inflammatory regulator NF-kappaB.     

Cellular mechanisms of glucocorticoid immunosuppression in salmon

Limiting dilution analysis was employed to determine the effect of cortisol and conditioned media on precursor frequency and clone size of antibody-producing cells sensitive to trinitrophenylated-lipopolysaccharide (TNP-LPS). This analysis demonstrated that the restoration of the cortisol-suppressed antibody response by conditioned media occurs through the activation of previously inhibited B cell precursors to that antigen, and not by the recruitment of other antigen-insensitive B cell precursors.     

Oxidative stress disruption of receptor-mediated calcium signaling mechanisms

Background

Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [³H]N-methylscopolamine ([³H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca²⁺]_i and [Ca²⁺]_L, respectively) were measured by fluorescent imaging.

Results

Activation of M3 muscarinic receptors induced a biphasic increase in [Ca²⁺]_i: an initial, inositol trisphosphate (IP3)-mediated release of Ca²⁺ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca²⁺ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca²⁺]_i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca²⁺]_i from 26 to up to 127 nM and decreased [Ca²⁺]_L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca²⁺ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP.

Conclusions

Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca²⁺]_i, [Ca²⁺]_L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.     

Molecular and cellular mechanisms of receptor-mediated endocytosis.

In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors and ligands are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of certain viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Although endocytosis is common to all nucleated eukaryotic cells, the factors that regulate these receptor-mediated endocytic pathways are not fully understood. Defective receptors that are not capable of undergoing normal endocytosis can lead to certain disease states, as in the case of familial hypercholesteremia (FH). This review has three objectives: (i) to describe the different routes that receptors and ligands follow after internaliation; (ii) to describe the potential mechanisms which regulate the initiation and subsequent sorting of receptors and ligands so they reach their final destination; and (iii) to describe the potential functions of receptor-mediated endocytosis.