Involvement of Na+-Ca2+ exchanger in intracellular Ca2+ increase and neuronal injury induced by polychlorinated biphenyls in human neuroblastoma SH-SY5Y cells

In SH-SY5Y, a human neuroblastoma cell line, Aroclor 1254 (A1254), induced a dose-dependent (10-50 microg/ml) intracellular calcium concentration ([Ca2+]i) increase. Two rather specific sodium-calcium (Na+-Ca2+) exchanger (NCX) inhibitors, bepridil (10 microM) and KB-R7943 [2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea methanesulfonate] (10 microM), reduced A1254-induced [Ca2+]i increase. A 24-h exposure to 30 microg/ml A1254 caused remarkable SH-SY5Y neuroblastoma cell damage. It is noteworthy that both bepridil and KB-R7943 counteracted A1254-induced neuronal injury. These results indicate that NCX contributes to [Ca2+]i increase and neuronal injury induced by A1254. RT-PCR experiments revealed in SH-SY5Y neuroblastoma cells the expression of NCX1 and NCX3 isoforms. To investigate which isoform was involved in [Ca2+]i increase and neuronal damage induced by A1254, we used specific antisense oligodeoxynucleotides (ODNs) to reduce NCX1 or NCX3 protein expression. The results showed that only NCX1 ODN reduced [Ca2+]i increase and neuronal injury induced by A1254. In conclusion, these results indicate that NCX1 may participate to [Ca2+]i increase and neurotoxicity evoked by A1254 in SH-SY5Y neuroblastoma cells.     

Cytosolic Ca2+ and protein kinase Calpha couple cellular metabolism to membrane K+ permeability in a human biliary cell line.

Cholangiocytes represent an important target of injury during the ischemia and metabolic stress that accompanies liver preservation. Since K+ efflux serves to minimize injury during ATP depletion in certain other cell types, the purpose of these studies was to evaluate the effects of ATP depletion on plasma membrane K+ permeability of Mz-ChA-1 cells, a model human biliary cell line. Cells were exposed to dinitrophenol (50 microM) and 2-deoxyglucose (10 mM) as the standard model of metabolic injury. Whole-cell and single K+ channel currents were measured using patch clamp techniques; and intracellular [Ca2+] ([Ca2+]i) was estimated by calcium green-1 fluorescence. Metabolic stress increased [Ca2+]i, and stimulated translocation of the alpha isoform of protein kinase C (PKCalpha) from cytosolic to particulate cell fractions. The same maneuver increased membrane K+ permeability 40-70-fold as detected by (a) activation of K+selective whole cell currents of 2,176+/-218 pA (n = 34), and (b) opening of apamin-sensitive K+ channels with a unitary conductance of 17.0+/-0.2 pS. PKCalpha translocation and channel opening appear to be related since stress-induced K+ efflux is inhibited by chelation of cytosolic Ca2+, exposure to the PKC inhibitor chelerythrine (25 microM) and downregulation of PKC by phorbol esters. Moreover, K+ currents were activated by intracellular perfusion with recombinant PKCalpha in the absence of metabolic inhibitors. These findings indicate that in biliary cells apamin-sensitive K+ channels are functionally coupled to cell metabolism and suggest that cytosolic Ca2+ and PKCalpha are selectively involved in the response.     

alpha1-adrenergic receptor activation of c-fos expression in transfected rat-1 fibroblasts: role of Ca2+.

alpha1-Adrenergic receptors mediate mitogenic responses and increase intracellular free Ca2+ ([Ca2+]i) in vascular smooth muscle cells. Induction of c-fos is a critical early event in cell growth; expression of this gene is regulated by a number of signaling pathways including Ca2+. We wondered whether Ca2+ signaling plays a critical role in the induction of c-fos gene by alpha1-adrenergic receptors. Using stably transfected rat-1 fibroblasts, we confirmed that PE induced c-fos mRNA expression in a time- and dose-dependent manner, and also increased [Ca2+]i (measured with Fura-2 AM). These responses were blocked by the alpha1-adrenergic receptor antagonist doxazosin. Both intracellular Ca2+ chelation (using BAPTA/AM) and extracellular Ca2+ depletion (using EGTA) significantly inhibited PE-induced c-fos expression by alpha1A and alpha1B receptors. Brief (1-min) stimulation of alpha1A and alpha1B receptors with PE did not maximally induce c-fos expression, suggesting that a sustained increase in [Ca2+]i due to Ca2+ influx is required. The calmodulin (CaM) antagonists, R24571, W7, and trifluoperazine, but not the CaM-dependent protein kinases inhibitor KN-62, significantly inhibited c-fos induction by alpha1A and alpha1B receptors. Neither inhibition of protein kinase C nor inhibition of adenylyl cyclase modified c-fos induction by PE. These results suggest that alpha1-adrenergic receptor-induced c-fos expression in rat-1 cells is dependent on a Ca2+/CaM-associated pathway.     

Altered aldose reductase gene regulation in cultured human retinal pigment epithelial cells.

Aldose reductase (AR2), a putative "hypertonicity stress protein" whose gene is induced by hyperosmolarity, protects renal medullary cells against the interstitial hyperosmolarity of antidiuresis by catalyzing the synthesis of millimolar concentrations of intracellular sorbitol from glucose. Although AR2 gene induction has been noted in a variety of renal and nonrenal cells subjected to hypertonic stress in vitro, the functional significance of AR2 gene expression in cells not normally exposed to a hyperosmolar milieu is not fully understood. The physiological impact of basal AR2 expression in such cells may be limited to hyperglycemic states in which AR2 promotes pathological polyol accumulation, a mechanism invoked in the pathogenesis of diabetic complications. Since AR2 overexpression in the retinal pigment epithelium has been associated with diabetic retinopathy, the regulation of AR2 gene expression and associated changes in sorbitol and myo-inositol were studied in human retinal pigment epithelial cells in culture. The relative abundance of aldehyde reductase (AR1) and AR2 mRNA was quantitated by filter hybridization of RNA from several human retinal pigment epithelial cell lines exposed to hyperglycemic and hyperosmolar conditions in vitro. AR2 but not AR1 mRNA was significantly increased some 11- to 18-fold by hyperosmolarity in several retinal pigment epithelial cell lines. A single cell line with a 15-fold higher basal level of AR2 mRNA than other cell lines tested demonstrated no significant increase in AR2 mRNA in response to hypertonic stress. This cell line demonstrated accelerated and exaggerated production of sorbitol and depletion of myo-inositol upon exposure to 20 mM glucose. Therefore, abnormal AR2 expression may enhance the sensitivity of cells to the biochemical consequences of hyperglycemia potentiating the development of diabetic complications.     

Cell death, calcium mobilization, and immunostaining for phosphorylated eukaryotic initiation factor 2-alpha (eIF2alpha) in neuronally differentiated NB-104 cells: arachidonate and radical-mediated injury mechanisms.

These experiments examine the effects of arachidonate with respect to cell death, radical-mediated injury, Ca2+ mobilization, and formation of ser-51-phosphorylated eukaryotic initiation factor 2alpha [eIF2alpha(P)]. It is known that during brain ischemia the concentration of free arachidonate can reach 180 microM, and during reperfusion oxidative metabolism of arachidonate leads to generation of superoxide that can reduce stored ferric iron and promote lipid peroxidation. During early brain reperfusion, we have shown an approximately 20-fold increase in eIF2alpha(P) which maps to vulnerable neurons that display inhibition of protein synthesis. Here in neuronally differentiated NB-104 cells, equivalent cell death (assessed by LDH release) was induced by 40 microM arachidonate and 20 microM cumene hydroperoxide (CumOOH, a known alkoxyl radical generator). In these injury models (1) radical inhibitors (BHA, BHT, and the lipophilic iron chelator EMHP) block CumOOH-induced cell death but do not block arachidonate-induced death; (2) 40 microM arachidonate (but not up to 40 microM CumOOH) rapidly induces Ca2+ release from intracellular stores; (3) both 40 microM arachidonate and 20 microM CumOOH induce intense immunostaining for eIF2alpha(P); and (4) the elF2alpha(P) immunostaining induced by CumOOH but not that induced by arachidonate is completely blocked by anti-radical intervention with EMHP. Arachidonate-induced formation of eIF2alpha(P) and cell death do not require iron-mediated radical mechanisms and are associated with Ca2+ release from intracellular stores; however, radical-mediated injury also induces both eIF2alpha(P) and cell death without release of intracellular Ca2+. Our data link eIF2alpha(P) formation during brain reperfusion to two established injury mechanisms that may operate concurrently.     

Protection from reoxygenation injury by inhibition of rac1.

We demonstrate that adenoviral-mediated gene transfer of a dominant negative rac1 gene product (N17rac1) inhibits the intracellular burst of reactive oxygen species (ROS) that occurs after reoxygenation of vascular smooth muscle cells. In contrast, expression of a dominant negative ras gene (N17ras) had no effect. Challenge of control cells and cells expressing N17rac1 with a direct oxidant stress produced an equivalent increase in intracellular ROS levels and subsequent cell death. This suggests that N17rac1 expression appears to block production of harmful oxygen radicals and does not act directly or indirectly to scavenge ROS generated during reoxygenation. Expression of N17rac1 results in protection from hypoxia/reoxygenation-induced cell death in a variety of cell types including vascular smooth muscle cells, fibroblasts, endothelial cells, and ventricular myocytes. These results suggest that reoxygenation injury requires the activation of rac proteins, and that inhibition of rac-dependent pathways may be a useful strategy for the prevention of reperfusion injury in ischemic tissues.     

Endothelin-1 stimulates contraction of rat glomerular mesangial cells and potentiates beta-adrenergic-mediated cyclic adenosine monophosphate accumulation.

The newly isolated peptide, endothelin-1 (ET-1), is a potent pressor agent that reduces GFR and the glomerular ultrafiltration coefficient. Recent evidence demonstrates that ET-1 mobilizes intracellular Ca2+ [( Ca2+]i) in glomerular mesangial cells by activating the phosphoinositide cascade. The present experiments were designed to examine whether ET-1 stimulates mesangial cell contraction and regulates the synthesis of PGE2 and cAMP, which dampen vasoconstrictor-induced mesangial contraction. ET-1 (greater than or equal to 1 nM) reduced the cross-sectional area of rat mesangial cells cultured on three-dimensional gels of collagen type I. ET-1 also caused complex rearrangements of F-actin microfilaments consistent with a motile response. Contraction in response to ET-1 occurred only at concentrations that activate phospholipase C, and contraction was unaffected by blockade of dihydropyridine-sensitive Ca2+ channels. Elevation of [Ca2+]i with ionomycin, to equivalent concentrations of [Ca2+]i achieved with ET-1, also reduced mesangial cell cross-sectional area. ET-1 (0.1 microM) also evoked [3H]arachidonate release and a fivefold increase in PGE2 synthesis as well as increased synthesis of PGF2 alpha and small changes of TXB2. ET-1 caused a minor increase in intracellular cAMP accumulation only in the presence of 3-isobutyl-1-methylxanthine. ET-1 also amplified cAMP production in response to isoproterenol. TPA and ionomycin, alone and in combination, failed to mimic the potentiating effect of ET-1; however, indomethacin blocked ET-1-induced potentiation of isoproterenol-stimulated cAMP, which was restored by addition of exogenous 10 nM PGE2. Thus the present data demonstrate that ET-1 stimulates mesangial cell contraction via pharmacomechanical coupling and activates phospholipase A2 to produce PGE2, PGF2 alpha, and TXB2. ET-1 also amplified beta adrenergic-stimulated cAMP accumulation by a PGE2-dependent mechanism.     

External bioenergy increases intracellular free calcium concentration and reduces cellular response to heat stress.

BACKGROUND: External bioenergy (energy emitted from the body) can influence a variety of biological activities. It has been shown to enhance immunity, promote normal cell proliferation, increase tumor cell death, and accelerate bone fracture recovery. In this study, we investigated whether external bioenergy could alter intracellular calcium concentration ([Ca2+]i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. METHODS: A practitioner emitted bioenergy toward tubes of cultured Jurkat cells for one 15-minute period. [Ca2+]i was measured spectrofluorometrically using the fluorescent probe indo-1. The heat shock protein 72 kd was detected using 35S-methionine prepulse and Western blot analysis. RESULTS: The resting [Ca2+]i in Jurkat T cells was 90+/-3 nM in the presence of external calcium. The removal of external calcium decreased the resting [Ca2+]i to 54+/-2 nM, indicating that Ca2+ entry from the external source is important for maintaining the basal level of [Ca2+]i. In the presence of external Ca2+, treatment of Jurkat T cells with external bioenergy for 15 minutes increased [Ca2+]i by 22+/-3%. [Ca2+]i remained elevated in these cells for 2 hours. Surprisingly, we also observed that [Ca2+]i increased by 11+/-1% if cells were simply placed in the area where external bioenergy had been used. This lingering effect of external bioenergy dissipated within 24 hours. Treatment with external bioenergy reduced cellular responses to heat stress and did not induce the production of heat shock protein 72 kd, which is known to provide cytoprotection. CONCLUSIONS: These results suggest that externally applied bioenergy can upregulate [Ca2+]i and downregulate the cellular response to stress. The association between the external bioenergy and increases in [Ca2+]i may be a good index for detecting presence of bioenergy.     

Interaction of insulin and exercise on glucose transport in muscle.

Glucose transport is the rate-limiting step for glucose utilization in muscle. In muscle and adipose tissue, glucose transport is acutely regulated by such factors as insulin and exercise. Translocation of glucose transporters (GLUT4) from an intracellular domain to the cell surface is the major mechanism for this regulation. Using immunocytochemistry, the intracellular distribution of GLUT4 under resting conditions is similar in adipocytes and myocytes. GLUT4 is concentrated in tubulovesicular structures either in the trans-Golgi region or in the cytosol, often close to the cell surface but not on the cell surface. After stimulation, cell surface GLUT4 labeling is increased by as much as 40-fold. GLUT4 is chronically regulated by altered gene expression. Neural and/or contractile activity regulates GLUT4 expression in muscle: 1) GLUT4 levels differ among muscles of different fiber type; 2) GLUT4 levels in muscle are increased with exercise training and decreased with denervation; and 3) cultured muscle cells, which lack an intact nerve supply, express very low levels of GLUT4. GLUT4 expression appears to be regulated in parallel with many oxidative enzymes in muscle, suggesting that there may be a unified developmental program that determines the overall metabolic properties of a particular muscle. Preliminary evidence suggests that impaired GLUT4 expression in muscle is not the primary defect associated with insulin resistance. Nevertheless, it is conceivable that the adaptive increase in muscle GLUT4 that is found with exercise training may have beneficial effects in insulin-resistant states such as non-insulin-dependent diabetes.     

Calcium dependency and free calcium concentrations during insulin secretion in a hamster beta cell line.

Using a glucose-responsive beta cell line, we tested the hypothesis that the free cytosolic Ca2+ concentration ([Ca2+]i) is the primary signal that couples a stimulus to insulin secretion, and examined the involvement of the extracellular Ca2+ pool in this process. Glucose or depolarization of the beta cell with 40 mM K+ stimulated a monophasic release of insulin directly proportional to the extracellular Ca2+ concentration. 40 mM K+ increased 45Ca2+ uptake and increased [Ca2+]i, which was measured with quin 2, 4.7-fold, from 56 +/- 3 to 238 +/- 17 nM. With high glucose, 45Ca2+ uptake did not increase, and [Ca2+]i was unchanged or fell slightly. There was a striking correlation between inhibitory effects of verapamil, the Ca2+ channel blocker, on insulin secretion and the rise in [Ca2+]i evoked by K+. Higher concentrations of verapamil were required to inhibit glucose- than K+-stimulated insulin secretion (dose giving half-maximal effect of 1.4 X 10(-4) M vs. 6.0 X 10(-7) M). Incubation in Ca2+-free, 1 mM EGTA buffer for 30 min lowered [Ca2+]i to 14 +/- 2 nM, and inhibited acute insulin release to both secretagogues. If high glucose was present in the Ca2+-free period, reintroduction of 2.5 mM Ca2+ in high glucose restored insulin secretion only to the basal rate. However, if low glucose was present during the Ca2+-free period, high glucose and 2.5 mM Ca2+ triggered a full first-phase insulin response. These data suggest that high glucose generates a non-Ca2+ signal that turns over rapidly and provide direct evidence that K+ triggers insulin release by drawing extracellular Ca2+ into the beta cell through verapamil-sensitive Ca2+ channels. However, an increase [Ca2+]i is not the primary signal that evokes glucose-stimulated insulin release in this beta cell line.     

Differential regulation of adipose tissue glucose transporters in genetic obesity (fatty rat). Selective increase in the adipose cell/muscle glucose transporter (GLUT 4) expression.

Adipocytes from young obese Zucker rats exhibit a hyperresponsive insulin-mediated glucose transport, together with a marked increase in cytochalasin B binding as compared with lean rat adipocytes. Here, we examined in these cells the expression of two isoforms of glucose transporter, the erythroid (GLUT 1) and the adipose cell/muscle (GLUT 4) types, in rats aged 16 or 30 d, i.e., before and after the emergence of hyperinsulinemia. GLUT 1 protein and mRNA levels were identical in the two genotypes at both ages. In contrast, the levels of GLUT 4 protein in obese rat adipocytes were 2.4- and 4.5-fold those of lean littermates at 16 and 30 d of age, respectively, in perfect agreement with the genotype effect on insulin-stimulated glucose transport activity. The levels of GLUT 4 mRNA per fat pad were increased 2.3- and 6.2-fold in obese vs. lean rats 16- and 30-d-old, indicating a pretranslational level of regulation. The obese phenotype was not associated with overexpression of GLUT 4 mRNA in gastrocnemius muscle. This work indicates that the fa gene exerts a differential control on the expression of GLUT 1 and GLUT 4 in adipose tissue and provides evidence that independent of hyperinsulinemia, genotype is a major regulatory factor of GLUT 4 expression in this tissue.     

Stimulation by leukotriene D4 of increases in the cytosolic concentration of calcium in dimethylsulfoxide-differentiated HL-60 cells.

The C6-sulfidopeptide leukotrienes C4 (LTC4) and D4 (LTD4) evoked increases in the cytosolic concentration of intracellular calcium ([Ca+2]i) in dimethylsulfoxide-differentiated HL-60 cells, as assessed by the fluorescence of quin-2. The increases in [Ca+2]i reached a peak within 15-90 s, attained 50% of the maximum level at 1.2 nM LTD4 and 60 nM LTC4, were greater in maximal magnitude for LTD4 than LTC4, and subsided in 5-7 min. Flow cytometric evaluation of the LTD4-induced increases in [Ca+2]i, reflected in increases in the fluorescence of intracellular indo-1, revealed that a mean of 77% of differentiated HL-60 cells responded, as contrasted with lesser increases in only 50% of undifferentiated HL-60 cells. The capacity of pretreatment of HL-60 cells with LTD4 to prevent subsequent responses of [Ca+2]i to LTC4 and LTD4, and the finding that the serine-borate inhibitor of conversion of LTC4 to LTD4 suppressed concurrently both LTC4-induced rises in [Ca+2]i and increases in adherence to Sephadex G-25 indicated that the responses of HL-60 cells to LTC4 required conversion to LTD4. That pertussis toxin and a chemical antagonist of LTD4 reduced the [Ca+2]i response suggested a dependence on LTD4 receptors. The LTD4-induced increases in [Ca+2]i were dependent on extracellular calcium and diminished by lanthanum, but not affected by nifedipine nor associated with changes in membrane potential, as measured with the fluorescent probe 3,3'-dipentyloxacarbocyanine. Thus, the increase in [Ca+2]i in HL-60 cells, which is coupled to an increase in adherence, appears to involve LTD4 receptor-specific and voltage-independent calcium channels in the plasma membrane.     

Endogenously generated nitric oxide by nitric-oxide synthase gene transfer inhibits cellular proliferation

We investigated whether endothelial nitrite oxide synthase (NOS) gene transfer inhibited cellular proliferation. Endothelial NOS and endothelin type A receptor genes were transferred into 293 cells, a human embryonic kidney cell line, by calcium-phosphate coprecipitation. The cytosolic free Ca(2+) levels ([Ca(2+)](i)) of transfected cells were estimated with fura-2 fluorescence. Thymidine incorporation was increased by endothelin-1 in type A receptor-transfected cells. The endothelial NOS gene transfer did not affect endothelin-1-induced increase in [Ca(2+)](i) of type A receptor-transfected cells, but markedly inhibited mitogen-activated protein kinase and c-fos promoter activities. The endothelial NOS gene transfer also inhibited thymidine incorporation into type A receptor-transfected cells in response to endothelin-1, which was abolished in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine acetate. The endothelin-1-induced increase in cell number was significantly suppressed by endothelial NOS gene transfer as well as by the mitogen-activated protein kinase inhibitor PD98059. These results indicate that endothelial NOS gene transfer inhibits cellular proliferation via inhibition of the mitogen-activated protein kinase cascade.     

Enhanced stimulus-secretion coupling in polyamine-depleted rat insulinoma cells. An effect involving increased cytoplasmic Ca2+, inositol phosphate generation, and phorbol ester sensitivity.

To extend previous observations on the role of polyamines in insulin production, metabolism, and replication of insulin-secreting pancreatic beta cells, we have studied the role of polyamines in the regulation of the stimulus-secretion coupling of clonal rat insulinoma cells (RINm5F). For this purpose, RINm5F cells were partially depleted in their polyamine contents by use of the specific ornithine decarboxylase inhibitor difluoromethylornithine (DFMO), which led to an increase in cellular insulin and ATP contents. Analysis of different parts of the signal transduction pathway revealed that insulin secretion and the increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) after K(+)-induced depolarization were markedly enhanced in DFMO-treated cells. These effects were paralleled by increased voltage-activated Ca2+ currents, as judged by whole-cell patch-clamp analysis, probably reflecting increased channel activity rather than elevated number of channels per cell. DFMO treatment also rendered phospholipase C in these cells more sensitive to the muscarinic receptor agonist carbamylcholine, as evidenced by enhanced generation of inositol phosphates, increase in [Ca2+]i and insulin secretion, despite an unaltered ligand binding to muscarinic receptors and lack of effect on protein kinase C activity. In addition, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate, at concentrations suggested to be specific for protein kinase C activation, evoked an increased insulin output in polyamine-deprived cells compared to control cells. The stimulatory effects of glucose or the cyclic AMP raising agent theophylline on insulin release were not increased by DFMO treatment. In spite of increased binding of sulfonylurea in DFMO-treated cells, there was no secretory response or altered increase in [Ca2+]i in response to the drug in these cells. It is concluded that partial polyamine depletion sensitizes the stimulus-secretion coupling at multiple levels in the insulinoma cells, including increased voltage-dependent Ca2+ influx and enhanced responsiveness to activators of phospholipase C and protein kinase C. In their entirety, our present results indicate that the behavior of the stimulus-secretion coupling of polyamine-depleted RINm5F insulinoma cells changes towards that of native beta cells, thus improving the usefulness of this cell line for studies of beta cell insulin secretion.     

The loss of GLUT2 expression by glucose-unresponsive beta cells of db/db mice is reversible and is induced by the diabetic environment.

Glucose-induced insulin secretion by beta cells of diabetic db/db mice was studied by a pancreas perfusion technique, and the levels of GLUT2 protein in pancreatic islets were assessed by immunofluorescence microscopy and protein blot analysis. Beta cells from diabetic mice had a high basal rate of insulin secretion; they did not respond to glucose stimulation but displayed a normal secretory response to arginine. At the same time, GLUT2 expression by db/db islets was lost whereas beta cells from nondiabetic db/+ mice expressed high levels of this transporter. GLUT2 levels in liver or kidney of diabetic mice were, however, mostly unaltered. Transplanting islets from db/db mice under the kidney capsule of db/+ mice restored normal GLUT2 levels. Conversely, transplantation of db/+ islets into db/db mice induced the disappearance of GLUT2 expression. When islets from db/+ mice were transplanted under the kidney capsule of streptozocin-diabetic mice, the immunodetection of GLUT2 also disappeared. We conclude that: (a) GLUT2 expression is decreased in glucose-unresponsive beta cells from db/db mice; (b) the decreased expression of GLUT2 is reversible; (c) the loss of GLUT2 expression is induced by the diabetic environment of db/db and streptozocin-induced diabetic mice. These observations together with previously published data suggest that a factor different from glucose or insulin regulates the beta cell expression of GLUT2.     

Bcl-2-related protein A1 is an endogenous and cytokine-stimulated mediator of cytoprotection in hyperoxic acute lung injury

Hyperoxic acute lung injury (HALI) is characterized by a cell death response with features of apoptosis and necrosis that is inhibited by IL-11 and other interventions. We hypothesized that Bfl-1/A1, an antiapoptotic Bcl-2 protein, is a critical regulator of HALI and a mediator of IL-11-induced cytoprotection. To test this, we characterized the expression of A1 and the oxygen susceptibility of WT and IL-11 Tg(+) mice with normal and null A1 loci. In WT mice, 100% O(2) caused TUNEL(+) cell death, induction and activation of intrinsic and mitochondrial-death pathways, and alveolar protein leak. Bcl-2 and Bcl-xl were also induced as an apparent protective response. A1 was induced in hyperoxia, and in A1-null mice, the toxic effects of hyperoxia were exaggerated, Bcl-2 and Bcl-xl were not induced, and premature death was seen. In contrast, IL-11 stimulated A1, diminished the toxic effects of hyperoxia, stimulated Bcl-2 and Bcl-xl, and enhanced murine survival in 100% O(2). In A1-null mice, IL-11-induced protection, survival advantage, and Bcl-2 and Bcl-xl induction were significantly decreased. VEGF also conferred protection via an A1-dependent mechanism. In vitro hyperoxia also stimulated A1, and A1 overexpression inhibited oxidant-induced epithelial cell apoptosis and necrosis. A1 is an important regulator of oxidant-induced lung injury, apoptosis, necrosis, and Bcl-2 and Bcl-xl gene expression and a critical mediator of IL-11- and VEGF-induced cytoprotection.     

Prevention of hemorrhagic shock-induced intestinal tissue injury by glutamine via heme oxygenase-1 induction

Hemorrhagic shock (HS) is an oxidative stress that causes intestinal tissue injury. Heme oxygenase 1 (HO-1) is induced by oxidative stress and is thought to play an important role in the protection of tissues from oxidative injury. We previously reported the ileum to be the most susceptible to HS-induced tissue injury site in the intestine because HO-1 induction is the lowest at this site. We also previously demonstrated that glutamine (GLN) significantly induced HO-1 in the lower intestinal tract. In the present study, we investigated whether GLN pretreatment improves HS-induced intestinal tissue injury in the ileum by HO-1 induction. Treatment of rats with GLN (0.75 g/kg, i.v.) markedly induced functional HO-1 protein in mucosal epithelial cells in the ileum. Glutamine treatment before HS (MAP of 30 mmHg for 60 min) significantly ameliorated HS-induced mucosal inflammation and apoptotic cell death in the ileum, as judged by significant decreases in gene expression of TNF-alpha, iNOS, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1, myeloperoxidase activity, the number of infiltrated neutrophils, DNA fragmentation by in situ oligo ligation assay, and activated caspase-3 expression, and by increases in gene expression of IL-10 and Bcl-2. In contrast, treatment with tin mesoporphyrin, a specific inhibitor of HO activity, abolished the beneficial effect of GLN pretreatment. These findings indicate that GLN pretreatment significantly ameliorated tissue injury in the ileum after HS by inducing HO-1. Glutamine treatment may thus protect mucosal cells from HS-induced oxidative damage via the anti-inflammatory and antiapoptotic properties of HO-1.     

Distinct patterns of transmembrane calcium flux and intracellular calcium mobilization after differentiation antigen cluster 2 (E rosette receptor) or 3 (T3) stimulation of human lymphocytes.

We evaluated CD2 (E rosette) and CD3 (T3)-triggered activation of resting lymphocytes by measuring the intracellular free calcium concentration ([Ca2+]i) of individual cells. The [Ca2+]i of indo-1-loaded cells was measured by flow cytometry and responses were correlated with cell surface phenotype. Stimulation with anti-CD3 antibody caused an increase in [Ca2+]i in greater than 90% of CD3+ cells within 1 min, and furthermore, the response was restricted to cells bearing the CD3 marker. In contrast, stimulation of cells with anti-CD2 antibodies produced a biphasic response pattern with an early component in CD3- cells and a late component in CD3+ cells. Thus, the CD2 response does not require cell surface expression of CD3. In addition, stimulation of a single CD2 epitope was sufficient for activation of CD3- cells, whereas stimulation of two CD2 epitopes was required for activation of CD3+ cells. Both the CD2 and CD3 responses were diminished in magnitude and duration by EGTA. However, approximately 50% of T cells still had a brief response in the presence of EGTA, indicating that the increased [Ca2+]i results in part from intracellular calcium mobilization, and furthermore demonstrates that extracellular calcium is required for a full and sustained response. Our results support the concept that CD2 represents the trigger for a distinct pathway of activation both for T cells that express the CD3 molecular complex and for large granular lymphocytes that do not.     

Chondroitin sulfate protects SH-SY5Y cells from oxidative stress by inducing heme oxygenase-1 via phosphatidylinositol 3-kinase/Akt

We investigated the mechanism of the neuroprotective properties of chondroitin sulfate (CS), an endogenous perineuronal net glycosaminoglycan, in human neuroblastoma SH-SY5Y cells subjected to oxidative stress. Preincubation with CS for 24 h afforded concentration-dependent protection against H2O2-induced toxicity (50 microM for 24 h) measured as lactic dehydrogenase released to the incubation media; cell death was prevented at the concentrations of 600 and 1000 microM. Cell death caused by a combination of 10 microM rotenone plus 1 microM oligomycin-A (Rot/oligo) was also reduced by CS at concentrations ranging from 0.3 to 100 microM; in this toxicity model, maximum protection was achieved at 3 microM (48%). No significant protection was observed in a cell death model of Ca2+ overload (70 mM K+, for 24 h). H2O2 and Rot/oligo generated reactive oxygen species (ROS) measured as an increase in the fluorescence of dichlorofluorescein diacetate-loaded cells. CS drastically reduced ROS generation induced by both H2O2 (extracellular ROS) and Rot/oligo (intracellular ROS). CS also increased the expression of phosphorylated Akt and heme oxygenase-1 by 2-fold. The protective effects of CS were prevented by chelerythrine, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), cycloheximide, and Sn(IV)-protoporphyrin IX. Taken together, these results show that CS can protect SH-SY5Y cells under oxidative stress conditions by activating protein kinase C, which phosphorylates Akt that, via the phosphatidylinositol 3-kinase/Akt pathway, induces the synthesis of the antioxidant protein heme oxygenase-1.