Adaptive cytoprotection against alcohol injury in the rat stomach is not due to increased prostanoid synthesis

This study evaluated the effects of 25% ethanol, a mild irritant, on endogenous prostanoid synthesis in the rat stomach before and after exposure to oral 100% ethanol. Rats received water or 25% ethanol orally. After 15 min, a portion of each group was sacrificed and the remaining animals treated with 100% ethanol prior to sacrifice one minute later. Microsomal membrane fractions were prepared from the glandular gastric mucosa in all groups and incubated with 14C arachidonic acid in the presence of cofactors. Endogenous mucosal prostanoid synthesis was analyzed by radiochromatography and results correlated with the presence or absence of gastric injury macroscopically. Prostanoids measured included PGI2, PGF2 alpha, PGE2, PGD2, PGA2, and thromboxane A2. Additional experiments were performed in like manner to those just described with the exception that indomethacin (5 mg/kg intraperitoneally) pretreatment was rendered. Stomachs exposed to water or 25% ethanol alone demonstrated a modest and equivalent level of synthesis of all prostanoids measured. Exposure to 100% ethanol (with and without mild irritant pretreatment) significantly increased prostanoid synthesis (especially PGI2, PGF2 alpha, and PGE2) compared with stomachs exposed to water or 25% ethanol alone; only mild irritant treated mucosa was protected from injury by 100% ethanol. Indomethacin pretreatment reversed the increased prostanoid synthesis in mucosa exposed to 100% ethanol, with or without mild irritant pretreatment, and partially reversed the protective effect of 25% ethanol. Other experiments using tissue slices in which perturbations in mucosal levels of prostanoids were measured by radioimmunoassay under identical experimental conditions exhibited similar results. These data dispute the notion that adaptive cytoprotection is mediated by increased endogenous prostanoid synthesis. The partial reversal of this process by indomethacin was most likely secondary to some other action of this agent, such as a reduction in gastric blood flow, rather than direct effects on prostanoid synthesis.     

Elevation of hepatic epoxide hydratase activity by ethoxyquin is due to increased synthesis of the enzyme

Feeding of the antioxidant ethoxyquin to rats leads to an increase of epoxide hydratase activity in liver microsomes. The apparent half life of the increase is 3–4 days. Elevation of epoxide hydratase activity is also obtained by intraperitoneal treatment of mice with ethoxyquin. This elevation is prevented by concomitant treatment with cycloheximide. When radiolabelled leucine is incorporated into microsomal protein by liver cell fractions from either ethoxyquin-fed or untreated rats, gel electrophoresis reveals that ethoxyquin feeding increases incorporation into epoxide hydratase. These results suggest that the elevation of epoxide hydratase activity by ethoxyquin is due to increased biosynthesis of the enzyme, i.e. enzyme induction.     

Alkaline ribonuclease activity is increased in rat sympathetic ganglia after nerve injury

Ribonuclease activity at pH 7.1 (alkaline ribonuclease) was determined in homogenates of rat superior cervical ganglion up to 5 days after postganglionic nerve injury under optimal conditions of assay. Measurements were performed in the presence and absence of the sulfhydryl blocking agent, N-ethylmaleimide, to assess the proportion of alkaline ribonuclease apparently bound to endogenous inhibitor. Total ribonuclease activity per ganglion was stimulated 1.3 fold by 1 day after injury and remained elevated over the 5 day period. Free ribonuclease activity accounted for about 60% of the observed increase in total activity at day 1, but had returned to control level by day 3. At day 3 the entire 90% increase in total activity was attributable to ribonuclease bound to endogenous inhibitor (i.e. latent activity). These changes are occurring at times after nerve injury when marked alterations in RNA turnover have been observed, implicating alkaline ribonucleases in the control of RNA metabolism during nerve regeneration.     

GSK-3beta activity is increased in skeletal muscle after burn injury in rats

Previous reports suggest that burn-induced muscle proteolysis can be inhibited by treatment with GSK-3beta inhibitors, suggesting that burn injury may be associated with increased GSK-3beta activity. The influence of burn injury on muscle GSK-3beta activity, however, is not known. We determined the effect of a 30% total body surface full-thickness burn injury in rats on muscle GSK-3beta activity by measuring GSK-3beta activity and tissue levels of serine 9 phosphorylated GSK-3beta, p(Ser9)-GSK-3beta, by Western blot analysis and immunohistochemistry. Because burn-induced muscle wasting is, at least in part, mediated by glucocorticoids, we used dexamethasone-treated cultured muscle cells in which GSK-3beta expression was reduced with small interfering RNA (siRNA) to further assess the role of GSK-3beta in muscle atrophy. Burn injury resulted in a seven-fold increase in GSK-3beta activity in skeletal muscle. This effect of burn was accompanied by reduced tissue levels of p(Ser9)-GSK-3beta, suggesting that burn injury stimulates GSK-3beta in skeletal muscle secondary to inhibited phosphorylation of the enzyme. In addition, burn injury resulted in inhibited phosphorylation and activation of Akt, an upstream regulatory mechanism of GSK-3beta activity. Reducing the expression of GSK-3beta in cultured muscle cells with siRNA inhibited dexamethasone-induced protein degradation by approximately 50%. The results suggest that burn injury stimulates GSK-3beta activity in skeletal muscle and that GSK-3beta may, at least in part, regulate glucocorticoid-mediated muscle wasting.     

Staphylococcal delta toxin stimulates endogenous phospholipase A2 activity and prostaglandin synthesis in fibroblasts

Delta toxin, one of at least four toxins produced by pathogenic strains of the skin bacterium Staphylococcus aureus, is an amphipathic polypeptide possessing hemolytic and cytolytic activity. Delta toxin stimulates high levels of phospholipase A₂ activity in 3T3 mouse fibroblasts with concomitant synthesis and release of prostaglandins. Alpha toxin, another hemolytic toxin produced by strains of S. aureus, did not stimulate phospholipase A₂ or prostaglandin release in these cells. Analysis of the release of lactate dehydrogenase and β-galactosidase (cytoplasmic and lysosomal marker enzymes, respectively) from delta-toxin-treated cells indicated that cytolytic concentrations of the toxin damage the cell-surface membrane more extensively than lysosomal membranes. During a 30 min exposure, delta toxin stimulated 3T3 cells to hydrolyze up to 32% of the lipids biosynthetically labeled by incorporation of [³H]arachidonic acid. A relatively high percentage of the free arachidonic acid formed in delta-toxin-treated 3T3 cells was converted to prostaglandins (up to 41.3% and 8.3% converted to chromatographically identifiable prostaglandins E₂ and F_2α, respectively, in 30 min), with optimal conversion occurring at sublytic toxin concentrations. The degree of activation of phospholipase A₂ in 3T3 cells by a range of concentrations of delta toxin correlates with cytotoxicity assessed by failure to exclude trypan blue dye. Analysis of the calcium dependency of the toxin-activated phospholipase A₂ was consistent with a cell-surface, Ca²⁺-dependent enzyme. The phospholipase A₂ exhibits a degree of specificity for substrate lipids containing polyunsaturated fatty acid residues which can serve as precursors for prostaglandin formation. Enzymatic activity was not inhibited by diisopropylfluorophosphate (5 mM), N-ethylmaleimide (5 mM) or p-bromophenacylbromide (0.1 mM). Delta toxin did not activate detectable phospholipase A₂ in subcellular preparations containing plasma membrane.     

Oxidative stress mediates synthesis of cytosolic phospholipase A2 after UVB injury

UVB irradiation has previously been shown to significantly increase phospholipase activity and prostaglandin synthesis. Because UVB irradiation is a potent oxidative stress, the role of active oxygen species in regulating UV-induced cPLA₂ synthesis and phosphorylation was examined. In the present study, irradiation produced a 3-fold increase in synthesis within 6 h following irradiation. Phosphorylation of cPLA₂ was also increased to a similar extent. UVB-induced synthesis and phosphorylation of cPLA₂ could be inhibited by pretreatment with the antioxidants 2,2,5,7,8-pentamethyl-6-hydroxychromane (50 μM) or N-acetylcysteine (10 mM). Treatment of unirradiated cultures with the potent oxidant tert-butyl hydroperoxide (500 μM) also increased cPLA₂ synthesis and phosphorylation, suggesting that oxidative injury is an important regulator of cPLA₂ synthesis. Increased synthesis of cPLA₂ correlated well with increased [³H]arachidonic acid release, PGE₂ synthesis and lipid peroxidation in epidermis after oxidant or UVB treatment. The results indicate that UVB-induced upregulation of cPLA₂ synthesis is mediated by UVB-induced formation of free radicals.     

Enhanced regulatory T cell activity is an element of the host response to injury

CD4+CD25+ regulatory T cells (Tregs) play a critical role in suppressing the development of autoimmune disease, in controlling potentially harmful inflammatory responses, and in maintaining immune homeostasis. Because severe injury triggers both excessive inflammation and suppressed adaptive immunity, we wished to test whether injury could influence Treg activity. Using a mouse burn injury model, we demonstrate that injury significantly enhances Treg function. This increase in Treg activity is apparent at 7 days after injury and is restricted to lymph node CD4+CD25+ T cells draining the injury site. Moreover, we show that this injury-induced increase in Treg activity is cell-contact dependent and is mediated in part by increased cell surface TGF-beta1 expression. To test the in vivo significance of these findings, mice were depleted of CD4+CD25+ T cells before sham or burn injury and then were immunized to follow the development of T cell-dependent Ag-specific immune reactivity. We observed that injured mice, which normally demonstrate suppressed Th1-type immunity, showed normal Th1 responses when depleted of CD4+CD25+ T cells. Taken together, these observations suggest that injury can induce or amplify CD4+CD25+ Treg function and that CD4+CD25+ T cells contribute to the development of postinjury immune suppression.     

Cellular confluence determines injury-induced prostaglandin E2 synthesis by human keratinocyte cultures

When keratinocyte cultures become confluent, their prostaglandin E2 synthesis is suppressed. To determine whether the injury response is characterized by increased prostaglandin E2 synthesis, an in vitro injury model was developed. When confluent keratinocyte cultures were focally lethally irradiated using ultraviolet light B, a dose-dependent increase in prostaglandin E2 synthesis was induced by the injury. After irradiation, confluent cultures' prostaglandin E2 synthesis increased for 2 days to 8-fold more than controls, then decreased to control values by day 6. Increased prostaglandin E2 synthesis was first detected 8 h after injury. Focal irradiation of non-confluent cultures (killing isolated colonies) caused no change in prostaglandin E2 synthesis, indicating that culture continuity must be disrupted before synthesis increases. In addition, partial irradiations of petri dishes demonstrated that enhanced metabolism was confined to cells adjacent to the injury site and was not mediated by a soluble factor. When confluent and injured cultures were incubated with [14C]arachidonic acid, and the products formed analyzed by thin layer chromatography, 10-fold more prostaglandin E2 microgram protein was seen in irradiated cultures relative to confluent controls. The products formed by each group were the same, and no consistent increases in metabolites other than prostaglandin E2 were observed. The increased synthesis of prostaglandin E2 by injured cultures was apparently due to an increase in cyclooxygenase activity as determined by kinetic experiments. These data indicate that the pattern of metabolism of arachidonic acid seen in non-confluent cultures is similar to that seen in injury, and that cell-cell contact modulates enhanced prostaglandin E2 synthesis.     

Intracellular depletion of insulin by oleate is due to an inhibited synthesis and not to an increased secretion

In the polyclonal rat pancreatic beta-cell line INS-1, immunoreactive insulin (IRI, insulin and its precursors) and C-peptide (surrogate marker for mature insulin) were quantified after a 1-h incubation at 16.7 mM glucose with or without oleate. Oleate caused a 20% decrease (P 相似文献   

Mitogenic phospholipase D activity is restricted to caveolin-enriched membrane microdomains

Phospholipase D (PLD) activity is elevated in response to the oncogenic stimulus of several signaling oncogenes. PLD activity is also elevated in response to peptide growth factors, indicating that PLD likely plays an important role in mitogenic signaling. Many proteins that mediate mitogenic signaling are localized in caveolin-enriched membrane microdomains (CEMMs). We report here that the elevated PLD activity in NIH 3T3 cells transformed by activated oncogenic forms of Src, Ras, and Raf is largely restricted to the CEMMs. Likewise, the PLD activity stimulated by epidermal growth factor is also restricted to the CEMMs. Although both PLD1 and PLD2 were found in CEMMs, neither was particularly enriched in the CEMMs of the transformed relative to the parental cells, indicating that it is the specific activity of PLD that is increased in the CEMMs. An apparent PLD substrate specificity in transformed cells for phosphatidylcholine lacking arachidonate acyl groups is also explained by the localization of activity in the CEMMs where [(3)H]arachidonate-labeled PC was excluded. These data indicate that mitogenic signals through PLD are initiated in CEMMs where many signaling molecules colocalize.     

Prostaglandin synthesis is increased in selenium supplemented human mesangial cells despite suppression of phospholipase A2-activity

Antioxidants play an important role in the regulation of phospholipid hydrolysis and arachidonate metabolism. Supplementation of cultured human mesangial cells with selenium resulted in suppression of phospholipase A2-activity and significantly increased production of three major prostaglandins. However, prostacyclin synthesis benefits most from selenium supplementation, suggesting that there is a specific action of selenium-dependent glutathione peroxidase on this pathway. Like in endothelial cells, production of platelet activating factor is significantly inhibited by selenium supplementation.     

Increased glomerular Vwf after kidney irradiation is not due to increased biosynthesis or endothelial cell proliferation.

Kuin, A., Citarella, F., Oussoren, Y. G., Van der Wal, A. F., Dewit, L. G. H. and Stewart, F. A. Increased Glomerular Vwf after Kidney Irradiation is not due to Increased Biosynthesis or Endothelial Cell Proliferation. Radiat. Res. 156, 20-27 (2001).Irradiation of the kidney induces dose-dependent, progressive renal functional impairment, which is partly mediated by vascular damage. It has previously been demonstrated that reduced renal function is preceded by an increased amount of von Willebrand factor (Vwf) in the glomerulus. The underlying mechanism and significance of this observation are unknown but, since it is an important mediator of platelet adhesion, Vwf in increased amounts could be implicated in glomerular thrombosis, resulting in impairment of renal function. Increased Vwf could be the result of increased biosynthesis by endothelial cells, or from increased numbers of endothelial cells after compensatory proliferation induced by irradiation, or it could be secondary to other events. In the present study, expression levels of mRNA for glomerular Vwf and glomerular cell proliferation rates were measured in control mouse kidneys and after irradiation with a single dose of 16 Gy. There were no significant changes in mRNA ratios for Vwf/beta-actin at 10 to 30 weeks after irradiation compared with unirradiated samples, whereas increased amounts of Vwf protein were seen in the glomeruli at these times. Labeling studies with IdU or staining for Ki67 demonstrated that glomerular proliferation was increased from 10 to 30 weeks after irradiation. Despite the increased proliferation rates, there was an absence of glomerular hyperplasia and no increase in the endothelial cell surface coverage in the glomeruli. Staining with antibodies against smooth muscle actin (SMAalpha) revealed that the observed proliferation mainly involved mesangial cells. These results indicate that the increased presence of glomerular Vwf after irradiation is not due to an increased number of endothelial cells per glomerulus, or to an increased production of Vwf. It is presumably secondary to other events, such as increased release of Vwf by damaged endothelial cells or entrapment of Vwf in the irradiated mesangial matrix.     

Ribulose diphosphate carboxylase synthesis in euglena: increased enzyme activity after transferring regreening cells to darkness

The transfer of dark-grown cultures of Euglena gracilis Klebs strain Z regreening in the light back into darkness resulted in a dramatic increase in ribulose diphosphate carboxylase activity. On a culture volume basis activity increased 4-fold over a 24-hour dark period, although on a protein basis activity declined because of rapid cell division. Mixed assays with light- and dark-growing cell extracts provided no evidence for the removal of an inhibitor of ribulose diphosphate carboxylase upon transferring regreening cells back to darkness. Although ribulose diphosphate carboxylase activity increased over a 24-hour dark period, there was no concomitant increase in the potential of the cells for photosynthetic carbon dioxide fixation.     

Jatropha is vulnerable to cold injury due to impaired activity and expression of plasma membrane H+-ATPase

Cold stress is one of the major environmental factors limiting the amount of plant mass for bioenergy production. A chilling-sensitive Jatropha (Jatropha curcas L.) as a bioenergy crop was used to investigate the cold injury process at the physiological and biochemical levels. Various physiological parameters such as leaf length, width, stomatal conductance, chlorophyll fluorescence, and electrolyte leakage were measured to determine the growth rate of leaves cold-treated (7 and 2 °C) for 5 days. These parameters of cold-treated Jatropha were significantly reduced from day 1 compared with control (23 °C). Using the pH indicator bromocresol purple, it was shown that surface pH of Jatropha root in control was strongly acidified by time only from the starting pH 6, while H⁺-efflux of the surface of cold-treated roots did not change. H⁺-ATPase activity of plasma membrane (PM) isolated from leaves and roots of cold-treated Jatropha was decreased in a time-dependent manner. The expression of PM H⁺-ATPase and 14-3-3 protein, which participates in phosphorylation of PM H⁺-ATPase was reduced in the presence of cold stress. Interestingly, fusicoccin, an activator of the PM H⁺-ATPase, alleviated cold-injury by stimulating the enzyme in leaves. These results may suggest that the activity and expression of PM H⁺-ATPase in Jatropha is closely related to the overcoming of cold stress.