Role of oxidant stress and iron delocalization in acidosis-induced intestinal epithelial hyperpermeability.

Using Caco-2BBe monolayers as a model of the intestinal epithelium, we tested the hypothesis that reactive oxygen metabolites contribute to lactic acid-induced hyperpermeability. Compared to monolayers incubated at normal pH (i.e., 7.4) monolayers incubated in medium titrated to extracellular pH (pHo) 5.0 with 10 mM lactic acid demonstrated increased permeability to both fluorescein sulfonic acid (FS) and fluorescein isothiocyanate-dextran (average molecular mass = 4000 Da; FD4). Lactic acid-induced hyperpermeability to both FS and FD4 was reduced by adding either 30 microM EUK-8, a superoxide dismutase/catalase mimetic, or catalase (10(4) U/mL). Incubation of monolayers with lactic acid increased cellular malondialdehyde content, a measure of lipid peroxidation. EUK-8 (30 microM) completely abrogated this effect. Incubation with ferrous sulfate (100 microM) exacerbated both lactic acid-induced hyperpermeability to FS and lactic acid-induced lipid peroxidation. Iron chelation with 1 mM diethylene triamine pentaacetic acid (DTPA)-trisodium calcium salt attenuated lactic acid-induced hyperpermeability, whereas iron-loaded DTPA (1 mM) was not protective. Treatment with DTPA-trisodium calcium salt also ameliorated lactic acid-induced lipid peroxidation. Incubation with lactic acid (pHo 5.0) for 16 h increased the cellular content of low molecular weight iron species. Incubation with lactic acid (pHo 5.0) for 24 h significantly increased the percentage of oxidized protein-bound thiols in Caco-2BBe cells. We conclude that lactic acidosis induces hyperpermeability in Caco-2BBe monolayers, in part, via an iron-dependent increase in reactive oxygen metabolite-mediated damage.

Role of oxidant stress in the adult respiratory distress syndrome: evaluation of a novel antioxidant strategy in a porcine model of endotoxin-induced acute lung injury.

Reactive oxygen metabolites (ROMs) are thought to play a key role in the pathogenesis of the adult respiratory distress syndrome (ARDS). Accordingly, the use of ROM scavengers, such as N-acetyl-cysteine or dimethylthiourea, as therapeutic adjuncts to prevent oxidant-mediated damage to the lung have been evaluated extensively in animal models of ARDS. Results with this approach have been quite variable among studies. Another strategy that has been examined in animal models of ARDS is the administration of various enzymes, particularly superoxide dismutase (SOD) or catalase (CAT), in an effort to promote the conversion of ROMs to inactive metabolites. In theory, this strategy should be more effective than the use of ROM scavengers since a single molecule of a catalytically active molecule can neutralize a large number of molecules of a reactive species, whereas most scavengers act in a stoichiometric fashion to neutralize radicals on a mole-for-mole basis. This notion is supported by studies showing that prophylactic treatment with CAT provides impressive protection against acute lung injury induced in experimental animals by the administration of lipopolysaccharide (LPS). Results with SOD have been more variable. Recently, we have utilized a porcine model of LPS-induced ARDS to investigate the therapeutic potential of EUK-8, a novel, synthetic, low molecular salen-manganese complex that exhibits both SOD-like and CAT-like activities in vitro. Using both pre- and post-treatment designs, we have documented that treatment with EUK-8 significantly attenuates many of the features of LPS-induced acute lung injury, including arterial hypoxemia, pulmonary hypertension, decreased dynamic pulmonary compliance, and pulmonary edema. These findings support the view that salen-manganese complexes warrant further evaluation as therapeutic agents for treatment or prevention of sepsis-related ARDS in humans.

Decreased frequency, but normal functional integrity of mesenchymal stromal cells derived from untreated and Imatinib-treated chronic myeloid leukemia patients.

In vitro, Imatinib inhibits the proliferation and stimulates the osteogenic and adipogenic differentiation of mesenchymal stromal cells (MSC). However, it is unknown whether Imatinib affects the biology of MSC in vivo. We asked whether MSC from long-term Imatinib-treated CML patients were affected by the in vivo treatment. MSC from untreated and Imatinib-treated patients displayed normal functional properties (i.e. proliferation, immunophenotype, differentiation and hematopoietic supportive capacity) - but a decreased frequency. In vitro, Imatinib lost its effect when discontinued; which suggest that it has a reversible effect on MSC. Therefore it might lose its effect on MSC after discontinuation in vivo.

Contribution of L-type channels to Ca2+ regulation of neuronal properties in early developing purkinje neurons.

Activity driven Ca2+ signaling is an important regulator of neuronal development. Early developing Purkinje neurons (postnatal day 5-7) prior to the stage of dendritic development express a somatic Ca2+ signaling pathway that is electrically driven and communicates information from the cell membrane to the cytosol and nucleus. In the current studies, we examined the properties and potential functional role of this pathway using acutely isolated Purkinje neurons from postnatal day 5-7 rat pups and brief K+ stimulation to activate the pathway. Results show that the amplitude of the nuclear Ca2+ signal increases as a function of the cytosolic Ca2+ signal but is larger than the cytosolic Ca2+ signal at strong K+ stimulations. Both L-type and P-type Ca2+ channels contribute to the Ca2+ signal. We also show using semiquantitative immunohistochemical methods that activation of this Ca2+ signaling pathway results in activation the transcription factor CREB and that L-type Ca2+ channels play a prominent role in this effect. The level of cfos, a transcription factor whose expression is regulated by CREB, was also increased by K+ stimulation. K+ stimulation also altered the level of the Ca2+ binding protein calbindin, an effect that involved L-type Ca2+ channels. The relationship between increases in Ca2+ and calbindin expression was bell-shaped, with high levels of Ca2+ decreasing calbindin expression. The level of the transmitter GABA was also increased by K+ stimulation but this effect was not dependent on L-type Ca2+ channels. Taken together, these results support a role for L-type channels in the phenotypic expression of Purkinje neuron properties during early development and suggest that the different activity patterns of early developing Purkinje neurons could be one mechanism for signaling the induction of specific genes through differences in cytosolic or nuclear Ca2+.

Nitric oxide dilates tight junctions and depletes ATP in cultured Caco-2BBe intestinal epithelial monolayers.

We tested the hypothesis that nitric oxide (NO) modulates the permeability of tight junctions in a model intestinal epithelium (Caco-2BBe monolayers). Incubation with sodium nitroprusside (SNP) resulted in time- and concentration-dependent decreases in transepithelial resistance. Permeability to fluorescein sulfonic acid increased during incubation for 24 h in the presence of 1.25 mM SNP, 5 mM S-nitroso-N-acetylpenicillamine (SNAP), or 1% NO gas. SNP-induced hyperpermeability was not due to loss of cell viability, as confirmed by intact ultrastructure, unaltered lactate dehydrogenase release, and ability to recover baseline permeability. Incubation with SNP increased permeability but only minimally increased intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP). Incubation with Escherichia coli heat-stable enterotoxin greatly increased cGMP levels with only a minimal effect on permeability. Cellular ATP levels decreased after incubation with SNP, SNAP, or gaseous NO. Incubation with SNP led to diminished fluoresceinphalloidin staining of junctional actin (confocal microscopy) and widened tight junctions (electron microscopy). We conclude that NO reduces ATP levels and reversibly increases the permeability of tight junctions in cultured Caco-2BBe cells.

EUK-8, a synthetic superoxide dismutase and catalase mimetic, ameliorates acute lung injury in endotoxemic swine.

Reactive oxygen metabolites are believed to be important mediators of sepsis- or lipopolysaccharide (LPS)-induced adult respiratory distress syndrome. EUK-8 is a novel, synthetic, low-molecular-weight salen-manganese complex that exhibits both superoxide dismutase and catalase activities in vitro. We hypothesized that treatment with EUK-8 would ameliorate pulmonary dysfunction in a porcine model of LPS-induced adult respiratory distress syndrome. At T = -18 h, pigs received an intravenous priming dose of LPS (20 micrograms/kg). Anesthetized ventilated swine were randomized to receive 1) no further treatment (n = 5); 2) LPS (250 micrograms/kg from T = 0 to 60 min, n = 6); 3) LPS and a low dose of EUK-8 (10-mg/kg bolus at T = -15 min and 1 mg/kg.h from T = 0 to 240 min, n = 6) or 4) LPS and a higher dose of EUK-8 (10-mg/kg bolus and 3 mg/kg.h, n = 6). Treatment with EUK-8, particularly at the higher dose, significantly attenuated many of the features of LPS-induced acute lung injury, including arterial hypoxemia, pulmonary hypertension, decreased dynamic pulmonary compliance and pulmonary edema. LPS caused an increase in lung tissue malondialdehyde content that was abrogated in both EUK-8-treated groups. EUK-8 treatment had no effect on circulating plasma levels of tumor necrosis factor-alpha, thromboxane B2 or 6-keto-prostaglandin F1 alpha. We conclude that EUK-8 prevents many of the manifestations of LPS-induced adult respiratory distress syndrome in pigs by detoxifying reactive oxygen metabolites without affecting the release of other important proinflammatory mediators.