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H J Forman

Publications and source records attributed to H J Forman.

At least 19 recordsLinked to original sources
PubMed2001

ADP stimulates the respiratory burst without activation of ERK and AKT in rat alveolar macrophages.

Alveolar macrophages (AM) are the first line of defense against infection in the lungs. We previously showed that the production of superoxide and hydrogen peroxide, i.e., the respiratory burst, is stimulated by adenine nucleotides (ADP >> ATP) in rat AM through signaling pathways involving calcium and protein kinase C. Here, we further show that ADP induces a rapid increase in the tyrosine phosphorylation of several proteins that was reduced by the tyrosine kinase inhibitor genistein, which also inhibited the respiratory burst. Interestingly, ADP did not trigger the activation of the mitogen-activated protein kinases ERK1 and ERK2, or that of protein kinase B/AKT, a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway. This is in contrast to another stimulus of the respiratory burst, zymosan-activated serum (ZAS), which activates both the ERK and PI3K pathways. Thus, this study demonstrates that the receptor for ADP in rat AM is not coupled to the ERK and AKT pathways and, that neither the ERK pathway nor AKT is essential to induce the activation of the NAPDH oxidase by ADP in rat AM while tyrosine kinases appeared to be required. The rate and amount of hydrogen peroxide released by the ADP-stimulated respiratory burst was similar to that produced by ZAS stimulation. The absence of ERK activation after ADP stimulation therefore suggests that hydrogen peroxide is not sufficient to activate the ERK pathway in rat AM. Nonetheless, as hydrogen peroxide was necessary for ERK activation by ZAS, this indicates that, in contrast to ADP, ZAS stimulates a pathway that is targeted by hydrogen peroxide and leads to ERK activation.

Adenosine Diphosphate↗
PubMed2001

Signaling by the respiratory burst in macrophages.

During phagocytosis or stimulation with a wide variety of agents, macrophages and other phagocytic cells produce reactive oxygen species (ROS) through activation of a multicomponent NADPH oxidase. ROS production through related NADPH oxidases has recently been demonstrated in several other cell types. Furthermore, the physiological generation of ROS production has now been clearly implicated in activating signaling pathways resulting in a broad array of physiological responses from cell proliferation to gene expression and apoptosis. This brief review suggests that: 1) hydrogen peroxide and superoxide, but not the hydroxyl radical, function as second messengers; 2) antioxidant enzymes function in the "turn-off" phase of signal transduction; 3) the chemistry of thiols is critical in redox signaling; and 4) the primary physiological role of the respiratory burst in macrophages may be in redox signaling rather than microbicidal activity.

Animals↗
PubMed2001

Synthetic chloride channel restores glutathione secretion in cystic fibrosis airway epithelia.

Cystic fibrosis (CF), an inherited disease characterized by defective epithelial Cl- transport, damages lungs via chronic inflammation and oxidative stress. Glutathione, a major antioxidant in the epithelial lung lining fluid, is decreased in the apical fluid of CF airway epithelia due to reduced glutathione efflux (Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999). The present study examined the question of whether restoration of chloride transport would also restore glutathione secretion. We found that a Cl- channel-forming peptide (N-K4-M2GlyR) and a K+ channel activator (chlorzoxazone) increased Cl- secretion, measured as bumetanide-sensitive short-circuit current, and glutathione efflux, measured by high-performance liquid chromatography, in a human CF airway epithelial cell line (CFT1). Addition of the peptide alone increased glutathione secretion (181 +/- 8% of the control value), whereas chlorzoxazone alone did not significantly affect glutathione efflux; however, chlorzoxazone potentiated the effect of the peptide on glutathione release (359 +/- 16% of the control value). These studies demonstrate that glutathione efflux is associated with apical chloride secretion, not with the CF transmembrane conductance regulator per se, and the defect of glutathione efflux in CF can be overcome pharmacologically.

Cell Line, Transformed↗
PubMed2001

Chlorzoxazone or 1-EBIO increases Na(+) absorption across cystic fibrosis airway epithelial cells.

Previous studies demonstrated that chlorzoxazone or 1-ethyl-2-benzimidazolinone (1-EBIO) enhances transepithelial Cl(-) secretion by increasing basolateral K(+) conductance (G(K)) (Singh AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, and Bridges RJ. J Pharmacol Exp Ther 292: 778-787, 2000). Hence these compounds may be useful to treat cystic fibrosis (CF) airway disease. The goal of the present study was to determine whether chlorzoxazone or 1-EBIO altered ion transport across Delta F508-CF transmembrane conductance regulator homozygous CFT1 airway cells. CFT1 monolayers exhibited a basal short-circuit current that was abolished by apical amiloride (inhibition constant 320 nM) as expected for Na(+) absorption. The addition of chlorzoxazone (400 microM) or 1-EBIO (2 mM) increased the amiloride-sensitive I(sc) approximately 2.5-fold. This overlapping specificity may preclude use of these compounds as CF therapeutics. Assaying for changes in the basolateral G(K) with a K(+) gradient plus the pore-forming antibiotic amphotericin B revealed that chlorzoxazone or 1-EBIO evoked an approximately 10-fold increase in clotrimazole-sensitive G(K). In contrast, chlorzoxazone did not alter epithelial Na(+) channel-mediated currents across basolateral-permeabilized monolayers or in Xenopus oocytes. These data further suggest that alterations in basolateral G(K) alone can modulate epithelial Na(+) transport.

Amiloride↗
PubMed2001

Biphasic effects of 15-deoxy-delta(12,14)-prostaglandin J(2) on glutathione induction and apoptosis in human endothelial cells.

The lipid products derived from the cyclooxygenase pathway can have diverse and often contrasting effects on vascular cell function. Cyclopentenone prostaglandins (cyPGs), such as 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)), a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist, have been reported to cause endothelial cell apoptosis, yet in other cell types, cyPGs induce cytoprotective mediators, such as heat shock proteins, heme oxygenase-1, and glutathione (GSH). Herein, we show in human endothelial cells that low micromolar concentrations of 15d-PGJ(2) enhance GSH-dependent cytoprotection through the upregulation of glutamate-cysteine ligase, the rate-limiting enzyme of GSH synthesis, as well as GSH reductase. The effect of 15d-PGJ(2) on GSH synthesis is attributable to the cyPG structure but is independent of PPAR, inasmuch as the other cyPGs, but not PPARgamma or PPARalpha agonists, are able to increase GSH. The increase in cellular GSH is accompanied by abrogation of the proapoptotic effects of 4-hydroxynonenal, a product of lipid peroxidation present in atherosclerotic lesions. However, higher concentrations of 15d-PGJ(2) (10 micromol/L) cause endothelial cell apoptosis, which is further enhanced by depletion of cellular GSH by buthionine sulfoximine. We propose that the GSH-dependent cytoprotective pathways induced by 15d-PGJ(2) contribute to its antiatherogenic effects and that these pathways are distinct from those leading to apoptosis.

Aldehydes↗
PubMed2001

4-Hydroxy-2-nonenal increases gamma-glutamylcysteine synthetase gene expression in alveolar epithelial cells.

Previous studies from this laboratory demonstrated that 4-hydroxy-2-nonenal (4HNE), a lipid peroxidation product, induces expression of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo glutathione (GSH) synthesis, in rat alveolar epithelial L2 cells. The present study demonstrates that 4HNE also induces GCS in primary cultured alveolar epithelial type II (AT2) cells. Enzyme activity, protein content, and messenger RNA levels of both the catalytic (GCS-HS) and regulatory (GCS-LS) subunits were significantly increased in AT2 cells treated with 5 or 10 microM 4HNE, the same concentrations that induced GCS expression in L2 cells. As in L2 cells, 4HNE induced a greater AT2-cell increase in GCS-LS than in GCS-HS, suggesting that modulation of GCS-LS may play a dominant role in regulating GSH concentration in response to oxidative stress. Additional studies using mitogen-activated protein kinase pathway inhibitors showed that induction by 4HNE of GCS-HS, but not GCS-LS, was mediated through activation of the extracellular regulated kinase pathway in L2 cells. The results demonstrate that L2 cells maintain the same responsiveness to oxidant challenge as do primary cultured AT2 cells in terms of increasing GSH synthetic capacity, and that different pathways are involved in the induction of two GCS subunits by 4HNE.

Aldehydes↗
PubMed2000

Glutathione depletion in PC12 results in selective inhibition of mitochondrial complex I activity. Implications for Parkinson's disease.

Oxidative stress appears to play an important role in degeneration of dopaminergic neurons of the substantia nigra (SN) associated with Parkinson's disease (PD). The SN of early PD patients have dramatically decreased levels of the thiol tripeptide glutathione (GSH). GSH plays multiple roles in the nervous system both as an antioxidant and a redox modulator. We have generated dopaminergic PC12 cell lines in which levels of GSH can be inducibly down-regulated via doxycycline induction of antisense messages against both the heavy and light subunits of gamma-glutamyl-cysteine synthetase, the rate-limiting enzyme in glutathione synthesis. Down-regulation of glutamyl-cysteine synthetase results in reduction in mitochondrial GSH levels, increased oxidative stress, and decreased mitochondrial function. Interestingly, decreases in mitochondrial activities in GSH-depleted PC12 cells appears to be because of a selective inhibition of complex I activity as a result of thiol oxidation. These results suggest that the early observed GSH losses in the SN may be directly responsible for the noted decreases in complex I activity and the subsequent mitochondrial dysfunction, which ultimately leads to dopaminergic cell death associated with PD.

Animals↗
PubMed2000

Modulation of glutathione synthetic enzymes by acidic fibroblast growth factor.

Increasing evidence suggests that glutathione (GSH) synthesis is a regulated process. Documented increases in gamma-glutamylcysteine synthetase (GCS) occur in response to oxidants, in tumors, on plating cells at a low cell density, and with nerve growth factor stimulation, suggesting that GSH synthesis may be related to the cell growth and transformation. Previously, extracellular acidic fibroblast growth factor (FGF-1) has been demonstrated to cause transformation and aggressive cell growth in murine embryonic fibroblasts. In the present investigation, we sought to determine whether FGF-1, with its growth inducing properties, resulted in the modulation of GSH biosynthetic enzymes, GCS and GSH synthetase. Murine fibroblasts transduced with (hst/KS)FGF-1, a chimeric human FGF-1 gene containing a signal peptide sequence for secretion, displayed elevated gene expression of both heavy and light subunits of GCS. Activity of GSH synthetase was also elevated in these cells compared with control cells. Nonetheless, GSH was decreased in the FGF-1-transduced cells along with high energy phosphates, adenine nucleotides, NADH, and the redox poise. However, GSSG was not elevated in these cells. Fibroblasts stably expressing human immunodeficiency virus type 1 Tat, which induces intrinsic FGF-1 secretion, resulted in similar changes in GCS, GS, and GSH. The results suggest that although increases in the enzymes of GSH synthesis are a common response to growth factors, an increase in GSH content per se is not required for altered cell growth.

Adenosine Triphosphate↗
PubMed2000

Dominant-negative Jun N-terminal protein kinase (JNK-1) inhibits metabolic oxidative stress during glucose deprivation in a human breast carcinoma cell line.

Signal transduction pathway involved in glucose deprivation-induced oxidative stress were investigated in human breast carcinoma cells (MCF-7/ADR). In MCF-7/ADR, glucose deprivation-induced prolonged activation of c-Jun N-terminal kinase (JNK1) as well as cytoxicity and the accumulation of oxidized glutathione. Glucose deprivation also caused significant increases in total glutathione, cysteine, gamma-glutamylcysteine, and immunoreactive proteins corresponding to the catalytic as well as regulatory subunits of gamma-glutamylcysteine, and immunoreactive proteins corresponding to the catalytic as well as regulatory subunits of gamma-glutamylcysteine synthetase, suggesting that the synthesis of glutathione increased as an adaptive response. Expression of a catalytically inactive dominant negative JNK1 in MCF-7/ADR inhibited glucose deprivation- induced cell death and the accumulation of oxidized glutathione as well as altered the duration of JNK activation from persistent (> 2 h) to transient (30 min). In addition, stimulation of glutathione synthesis during glucose deprivation was not observed in cells expressing the highest levels of dominant negative protein. Finally, a linear dose response suppression of oxidized glutathione accumulation was noted for clones expressing increasing levels of dominant negative JNK1 during glucose deprivation. These results show that expression of a dominant negative JNK1 protein was capable of suppressing persistent JNK activation as well as oxidative stress and cytotoxicity caused by glucose deprivation in MCF-7/ADR. These findings support the hypothesis that JNK signaling pathways may control the expression of proteins contributing to cell death mediated by metabolic oxidative stress during glucose deprivation. Finally, these results support the concept that JNK signaling-induced shifts in oxidative metabolism may provide a general mechanism for understanding the diverse biological effects seen during the activation of JNK signaling cascades.

Breast Neoplasms↗
PubMed2000

Molecular mechanism of decreased glutathione content in human immunodeficiency virus type 1 Tat-transgenic mice.

Human immunodeficiency virus (HIV) progressively depletes GSH content in humans. Although the accumulated evidence suggests a role of decreased GSH in the pathogenesis of HIV, significant controversy remains concerning the mechanism of GSH depletion, especially in regard to envisioning appropriate therapeutic strategies to help compensate for such decreased antioxidant capacity. Tat, a transactivator encoded by HIV, is sufficient to cause GSH depletion in vitro and is implicated in AIDS-associated Kaposi's sarcoma and B cell lymphoma. In this study, we report a decrease in GSH biosynthesis with Tat, using HIV-1 Tat transgenic (Tat+) mice. A significant decline in the total intracellular GSH content in liver and erythrocytes of Tat+ mice was accompanied by decreased gamma-glutamylcysteine synthetase regulatory subunit mRNA and protein content, which resulted in an increased sensitivity of gamma-glutamylcysteine synthetase to feedback inhibition by GSH. Further study revealed a significant reduction in the activity of GSH synthetase in liver of Tat+ mice, which was linearly associated with their GSH content. Therefore, Tat appears to decrease GSH in vivo, at least partially, through modulation of GSH biosynthetic enzymes.

Animals↗
PubMed2000

Phospholipase D and priming of the respiratory burst by H(2)O(2) in NR8383 alveolar macrophages.

Previous investigation showed that preincubation within a range of nontoxic H(2)O(2) concentrations enhanced subsequently stimulated superoxide production by rat alveolar macrophages in response to various stimuli. In the present study, the NR8383 rat alveolar macrophage cell line was used to further investigate the priming effect of H(2)O(2). Using nitroblue tetrazolium, which formed an insoluble formazan when reduced by superoxide, modulation of the respiratory burst was visualized in a cell population exposed to a concentration gradient of H(2)O(2) before stimulation. This model system illustrates how H(2)O(2) may constitute a signaling molecule for a feed-forward regulation of the respiratory burst during inflammation. n-Butanol, which allows consumption of phosphatidic acid by the transphosphatidylation reaction, and propanolol, which inhibits phosphatidic acid phosphohydrolase, were used to investigate the possible involvement of phospholipase D in this phenomenon. These two agents were found to inhibit the basal adenosine diphosphate-stimulated respiratory burst. Inhibition of the H(2)O(2)-enhanced respiratory burst was equally or slightly less effective when expressed as percentage of controls. Furthermore, phospholipase D was not activated by H(2)O(2) concentrations that enhance superoxide production. Thus, phospholipase D does not mediate the enhancement of the respiratory burst by H(2)O(2), although it may be activated by high concentrations of this hydroperoxide.

1-Butanol↗
PubMed1999

Activation of several MAP kinases upon stimulation of rat alveolar macrophages: role of the NADPH oxidase.

Zymosan-activated serum (ZAS), a source of C5a, stimulates the rat alveolar macrophages (AM) to release superoxide anion. Here we show that treatment of rat AM with ZAS induced a time-dependent increase in the tyrosine phosphorylation of several proteins (116, 105-110, 82-78, 66-72, 62, 45, 42, and 38 kDa). This increase was sensitive to genistein, a tyrosine kinase inhibitor. ZAS stimulated the tyrosine phosphorylation and activation of three members of a family of serine/threonine kinases known as the mitogen-activated protein kinases (MAPK), i.e., ERK1 and ERK2, as assessed by immunoblotting, immunoprecipitation, and phosphotransferase activity, and p38 MAPK, as determined by immunoblotting with phospho-specific antibodies. In addition, ZAS induced the tyrosine phosphorylation of the SHC proteins and their association with GRB2, suggesting a role for this complex in the activation of the ERK pathway. Addition of extracellular catalase during ZAS stimulation significantly reduced the tyrosine phosphorylation response and the activation of ERK1 and ERK2 and their activator MEK1/2 while it did not affect that of p38 MAPK and MKK3/MKK6. Superoxide dismutase marginally increased the response to ZAS, supporting a role for hydrogen peroxide. In contrast to the results with AM, stimulation of human neutrophils with ZAS in the presence of catalase minimally altered the activation of ERK1 and ERK2. These data show that, in ZAS-stimulated rat AM, activation of the respiratory burst and production of hydrogen peroxide via superoxide dismutation are largely responsible for the activation of the ERK pathway through an upstream target.

Adaptor Proteins, Signal Transducing↗
PubMed1999

The induction of GSH synthesis by nanomolar concentrations of NO in endothelial cells: a role for gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase.

Nitric oxide protects cells from oxidative stress through a number of direct scavenging reactions with free radicals but the effects of nitric oxide on the regulation of antioxidant enzymes are only now emerging. Using bovine aortic endothelial cells as a model, we show that nitric oxide, at physiological rates of production (1-3 nM/s), is capable of inducing the synthesis of glutathione through a mechanism involving gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase. This novel nitric oxide signalling pathway is cGMP-independent and we hypothesize that it makes an important contribution to the anti-atherosclerotic and antioxidant properties of nitric oxide.

Animals↗
PubMed1999

Depletion of glutathione by buthionine sulfoxine is cytotoxic for human neuroblastoma cell lines via apoptosis.

Buthionine sulfoximine (BSO) selectively inhibits glutathione (GSH) synthesis and has been used to sensitize tumor cells to alkylating agents, but has minimal single-agent cytotoxicity for most cell types. We determined the cytotoxicity of BSO for 18 (12 MYCN amplified; 6 MYCN nonamplified) human neuroblastoma cell lines using DIMSCAN, a digital image microscopy cytotoxicity assay. D-L(R:S) BSO was highly cytotoxic (>3 logs of cell kill) for most neuroblastoma cell lines, with 17/18 cell lines having IC90 values (range 2. 1->1000 microM) below equivalent steady state plasma levels of L(R:S) BSO reported in adult human trials. Cell lines with genomic amplification of MYCN were more sensitive to BSO than MYCN nonamplified cell lines (P = 0.04). D-L(R:S) BSO (500 microM for 72 h) induced apoptosis as detected by DNA laddering, nuclear morphology, and TUNEL staining of DNA fragments using flow cytometry. Maximal cell killing occurred within 48 h and was antagonized byic value in neuroblastoma.

Antioxidants↗
PubMed1999

Abnormal glutathione transport in cystic fibrosis airway epithelia.

Glutathione (GSH) is a potentially important component of antioxidant defense in the epithelial lung lining fluid. Cystic fibrosis (CF) patients have chronic inflammation in which oxidative stress can be a factor. To examine the hypothesis that the transport of GSH content was defective in CF patients, intracellular and extracellular GSH were measured by HPLC. Four cell lines were used: CFT1 cells [with defective CF transmembrane conductance regulator (CFTR), DeltaF508 homozygous, two clones] and one of the CFT1 clones transfected with either normal CFTR (CFTR repleted) or beta-galactosidase. GSH content in the apical fluid was 55% lower in CFTR-deficient cultures than in CFTR-repleted cells (P < 0.001). In contrast, intracellular GSH content was similar in CFT1 cells and CFTR-repleted cells. gamma-Glutamyl transpeptidase activity, which degrades extracellular GSH, did not account for differences in apical GSH. Rather, GSH efflux of CFTR-deficient cells was lower than that of CFTR-repleted cells. These studies suggested that decreased GSH content in the apical fluid in CF resulted from abnormal GSH transport associated with a defective CFTR.

Biological Transport↗
PubMed1998

Nitric oxide-dependent induction of glutathione synthesis through increased expression of gamma-glutamylcysteine synthetase.

The nitric oxide (NO) donors S-nitrosopenicillamine or DetaNONOate, which release NO at a rate of 0-15 nM sec-1, were exposed to rat aortic vascular smooth muscle cells for a period of 0-24 h. This treatment resulted in an increase in total glutathione levels of two- to threefold under conditions where no cytotoxicity was detected. The signaling pathways do not involve activation of protein kinase G Ialpha nor are they cGMP dependent. Oxidation of reduced glutathione (GSH) was found after exposure to NO for 3-4 h at rates of formation at or above 8 nM sec-1. Increased intracellular GSH was due to enhanced expression of the rate-limiting enzyme for GSH synthesis, gamma-glutamylcysteine synthetase. Since NO has been shown previously to protect cells against oxidative stress, we propose that the increase in GSH by NO is a potential mechanism for enhancing the antioxidant defenses of the cell. This result also has important implications for identifying redox-sensitive cell signaling pathways that can be activated by NO.

Animals↗
PubMed1998

Role of protein kinase C in basal and hydrogen peroxide-stimulated NF-kappa B activation in the murine macrophage J774A.1 cell line.

In macrophages, hydrogen peroxide appears to be a physiological activator of the transcription factor, nuclear factor kappa B (NF-kappa B); however, the molecular basis of H2O2-stimulated NF-kappa B activation is not well defined. The observations that NF-kappa B can be activated in cells by phorbol 12-myristate 13-acetate and in vitro by addition of protein kinase C (PKC) are suggestive of a role of PKC in NF-kappa B activation, which was investigated in the J774A.1 murine macrophage cell line. Basal NF-kappa B DNA-binding activity and nuclear localization were decreased by PKC inhibitors. Although PKC activity was modified by H2O2 with a similar time course as H2O2 activation of NF-kappa B, the H2O2-stimulated increase in NF-kappa B DNA binding and translocation to the nucleus was unaffected by PKC inhibitors. Furthermore, PKC down-regulation (through preincubation with phorbol esters) also affected only baseline NF-kappa B DNA binding but not H2O2-stimulated NF-kappa B activation. Buffering of changes in intracellular free calcium concentration also had no effect upon H2O2-stimulated NF-kappa B activation. Thus, classical PKC activity may modulate basal NF-kappa B activity but does not participate in H2O2-stimulated NF-kappa B activation.

Animals↗