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H J Forman

Publications and source records attributed to H J Forman.

At least 37 records · Page 2Linked to original sources
PubMed1998

Transmembrane redox signaling activates NF-kappaB in macrophages.

In macrophages, NF-kappaB can be activated by H2O2 generated by the respiratory burst or added exogenously. The mechanism of H2O2 signaling may involve changes in the cellular redox state or a redox reaction at the plasma membrane; however, the site of H2O2 action cannot be readily ascertained because of its membrane permeability. Ferricyanide, a nonpermeable redox active anion, activated NF-kappaB in the macrophage cell line, J774A.1. In contrast with exogenous H2O2, activation by ferricyanide did not correlate with net oxidation of NAD(P)H or glutathione, suggesting that a transplasma membrane redox reaction itself was the first signaling process in NF-kappaB activation.

Animals↗
PubMed1998

Quinones increase gamma-glutamyl transpeptidase expression by multiple mechanisms in rat lung epithelial cells.

gamma-Glutamyl transpeptidase (GGT) plays an important role in glutathione (GSH) metabolism. GGT expression is increased in oxidant-challenged cells; however, the signaling mechanisms involved are uncertain. The present study used 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a redox cycling quinone that continuously produced H2O2 in rat lung epithelial L2 cells. It was found that DMNQ increased GGT mRNA content by increasing transcription, as measured by nuclear run-on. This was accompanied by increased GGT specific activity. Cycloheximide, a protein synthesis inhibitor, blocked neither the increased GGT mRNA content nor the increased GGT transcription rate caused by DMNQ, suggesting that increased GGT transcription was a direct rather than secondary response. Previous data from this laboratory (R.-M. Liu, H. Hu, T. W. Robinson, and H. J. Forman. Am. J. Respir. Cell Mol. Biol. 14: 186-191, 1996) showed that tert-butylhydroquinone (TBHQ) increased GGT mRNA content by increasing its stability. TBHQ differs markedly from DMNQ in terms of its conjugation with GSH and H2O2 generation. Together, the data suggest that quinones upregulate GGT through multiple mechanisms, increased transcription and posttranscriptional modulation, which are apparently mediated through generation of reactive oxygen species and GSH conjugated formation, respectively.

Animals↗
PubMed1998

gamma-glutamylcysteine synthetase: mRNA stabilization and independent subunit transcription by 4-hydroxy-2-nonenal.

gamma-Glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo glutathione (GSH) synthesis, is composed of one catalytic (heavy) and one regulatory (light) subunit. Although both subunits are increased at the mRNA level by oxidants, it is not clear whether they are regulated through the same mechanism. 4-Hydroxy-2-nonenal (4HNE), a lipid peroxidation product, may act as a mediator for the induction of gene expression by oxidants. In the present study, 4HNE was used to study the mechanism of induction of the two GCS subunits in rat lung epithelial L2 cells. 4HNE increased both the transcription rates and the stability of mRNA for both GCS subunits, resulting in an increased mRNA content for both subunits. Both GCS subunit proteins and enzymatic activities also increased. Emetine, a protein synthesis inhibitor, blocked the increase in GCS light subunit mRNA but not the increase in GCS heavy subunit mRNA. This suggested that although 4HNE increased transcription and stabilization of both GCS subunit mRNAs, the signaling pathways involved in the induction of the two GCS subunits differed.

Aldehydes↗
PubMed1998

Modulation of the alveolar macrophage superoxide production by protein phosphorylation.

Stimulation of alveolar macrophages (AM) with adenosine-5-diphosphate (ADP) results in transient production of superoxide anion radical (O2.-; superoxide) and H2O2 in a metabolic event known as the respiratory burst. Initiation of the respiratory burst appears to depend on activation of protein kinase activity, whereas protein phosphatases might involved in termination of the burst. The involvement of protein kinase C was suggested by inhibition by bisindolylmaleimide I (GF 109203X), a relatively specific inhibitor. KN-62, an inhibitor of calcium-calmodulin protein kinase II, also partly inhibited the respiratory burst stimulated by ADP and phorbol esters. The role of protein phosphatases in termination of the ADP-stimulated respiratory burst of AM was examined with calyculin A (CA) (25-75 nM) or okadaic acid (OA) (1-5 microM), two inhibitors of protein phosphatase 1 and 2a (PP1;PP2a). A dose-dependent prolongation of the respiratory burst was observed in the presence of these inhibitors. CA and OA also markedly enhanced the rate of superoxide production stimulated by ADP, consistent with involvement of PP1/PP2a in regulating both the rate of activation and timing of termination. Treatment of AM with cyclosporin A (CsA) (1-50 microM), an inhibitor of the calcium-dependent protein phosphatase 2b (PP2b), stimulated superoxide production by itself and significantly prolonged the duration of ADP-stimulated superoxide production. CsA, however, did not increase the ADP-stimulated rate of superoxide production. Thus, PP1/PP2a appear to be the primary phosphatases for controlling the intensity of the respiratory burst during receptor-elicited superoxide production in AM, whereas PP1/PP2a and PP2b play a role in turning off the respiratory burst.

Adenosine Diphosphate↗
PubMed1997

Increased transcription of the regulatory subunit of gamma-glutamylcysteine synthetase in rat lung epithelial L2 cells exposed to oxidative stress or glutathione depletion.

gamma-Glutamylcysteine synthetase (GCS) is the initial and rate-limiting enzyme in the glutathione (GSH) de novo synthesis pathway. GCS is composed of a heavy (73-kDa) catalytic subunit and a light (30-kDa) regulatory subunit, which maintains the Km for glutamate near physiologic concentrations. Previous studies have shown that the steady-state mRNA level and gene transcription for the catalytic subunit increased in response to the redox-cycling quinone 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) in rat lung epithelial L2 cells (M. M. Shi, et al., 1994, J. Biol. Chem. 269,26512-26517). The ratio of the catalytic to regulatory subunit mRNAs varies among tissues, and the anticancer drug cisplatin appears to induce only the catalytic subunit, suggesting independent gene regulation of the two subunits. Nonetheless, the present study found that the steady-state mRNA level and the transcription rate of the GCS regulatory subunit also increased under DMNQ-induced oxidative stress. Changes in mRNA followed a pattern similar to that for the catalytic subunit. The mRNA levels of the two subunits of GCS also both increased above the baseline levels in cells treated with BSO, an inhibitor of GCS enzymatic activity. These data suggest that, under conditions of oxidative stress or glutathione depletion, the regulatory subunit is upregulated at the level of mRNA transcription. Along with the elevation of the catalytic subunit, this increase in GCS regulatory subunit transcription contributes to increases in GCS enzymatic activity and cellular GSH content.

Animals↗
PubMed1997

Adaptation to oxidative stress: quinone-mediated protection of signaling in rat lung epithelial L2 cells.

Cells can respond to a sublethal oxidative stress by up-regulating their intracellular glutathione (GSH) pool. Such increased GSH concentration is likely to be protective against further oxidative challenge, and, in fact, pre-exposure to low levels of oxidants confers increased cellular resistance to subsequent greater oxidative stress. Previously, we have shown that pretreatment of rat lung epithelial L2 cells with sublethal concentrations of tert-butylhydroquinone (TBHQ) increases intracellular GSH concentration in a concentration- and time-dependent manner. This increase resulted from up-regulation of both gamma-glutamyltranspeptidase (GGT) and gamma-glutamylcysteine synthetase (GCS). Therefore, we investigated whether such increased GSH concentration protected these cells against a subtle loss in function caused by a subsequent challenge with sublethal concentrations of tert-butyl hydroperoxide (tBOOH) (< or = 200 microM), mimicking a physiological oxidative stress. Activation of L2 cell purinoreceptors with 100 microM ADP caused an elevation of intracellular Ca2+. This response was suppressed by a brief pre-exposure to tBOOH. The inhibition, however, was alleviated dramatically by a 16-hr pretreatment with 50 microM TBHQ. The same TBHQ pretreatment also protected the cells from ATP-depletion induced by tBOOH. L-Buthionine S,R-sulfoximine (BSO), an irreversible inhibitor of GCS, prevented the increase in intracellular GSH and also completely removed the protection by TBHQ in maintaining the ATP level. Thus, pre-exposure to a sublethal level of TBHQ results in protection of cell functions from hydroperoxide toxicity. This protection appears to depend on alteration of the intracellular GSH pool, the modulation of which constitutes an adaptive response to oxidative stress.

Adenosine Diphosphate↗
PubMed1997

Oxidants as stimulators of signal transduction.

Redox (oxidation-reduction) reactions regulate signal transduction. Oxidants such as superoxide, hydrogen peroxide, hydroxyl radicals, and lipid hydroperoxides (i.e., reactive oxygen species) are now realized as signaling molecules under subtoxic conditions. Nitric oxide is also an example of a redox mediator. Reactive oxygen species induce various biological processes such as gene expression by stimulating signal transduction components such as Ca(2+)-signaling and protein phosphorylation. Various oxidants increase cytosolic Ca2+; however, the exact origin of Ca2+ is controversial. Ca2+ may be released from the endoplasmic reticulum, extracellular space, or mitochondria in response to oxidant-influence on Ca2+ pumps, channels, and transporters. Alternatively, oxidants may release Ca2+ from Ca2+ binding proteins. Various oxidants stimulate tyrosine as well as serine/threonine phosphorylation, and direct stimulation of protein kinases and inhibition of protein phosphatases by oxidants have been proposed as mechanisms. The oxidant-stimulation of the effector molecules such as phospholipase A2 as well as the activation of oxidative stress-responsive transcription factors may also depend on the oxidant-mediated activation of Ca(2+)-signaling and/or protein phosphorylation. In addition to the stimulation of signal transduction by oxidants, the observations that ligand-receptor interactions produce reactive oxygen species and that antioxidants block receptor-mediated signal transduction led to a proposal that reactive oxygen species may be second messengers for transcription factor activation, apoptosis, bone resorption, cell growth, and chemotaxis. Physiological significance of the role of biological oxidants in the regulation of signal transduction as well as the mechanisms of the oxidant-stimulation of signal transduction are discussed.

Animals↗
PubMed1997

Buthionine sulphoximine alone and in combination with melphalan (L-PAM) is highly cytotoxic for human neuroblastoma cell lines.

Buthionine sulphoximine (BSO) selectively inhibits glutathione (GSH) synthesis and may enhance the antineuroblastoma activity of melphalan (L-PAM). We determined the cytotoxicity of BSO (dose range 0-1000 microM) alone and in combination with L-PAM (dose range 0-0 microM) in a panel of 18 human neuroblastoma cell lines. BSO alone was highly cytotoxic with 16/18 neuroblastoma cell lines having IC90 values (range 2.1- > 1000 microM) below the clinically achievable steady-state plasma level of 500 microM BSO. Maximal cell killing correlated with GSH levels decreased to less than 10% baseline, and was partially reversed by the addition of exogenous anti-oxidants (GSH, vitamin E and ascorbate). Fluorocytometric analysis of DNA fragments by the Tunnel method detected 92% of a BSO sensitive cell line in apoptosis after a 48 h exposure to 500 microM BSO. The combination of L-PAM and BSO synergistically enhanced the cell killing of L-PAM alone by > 1-3 logs (combination index < 1). We conclude that BSO has significant single-agent cytotoxicity against neuroblastoma and enhances cell killing when combined with L-PAM.

Antineoplastic Agents↗
PubMed1997

Ca(2+)-dependent p47phox translocation in hydroperoxide modulation of the alveolar macrophage respiratory burst.

Oxidative stress produces dual effects on the respiratory burst of rat alveolar macrophages. Preincubation with hydroperoxide concentrations [H2O2 or tert-butyl hydroperoxide (t-BOOH); < 50 microM] enhances stimulation of the respiratory burst, whereas higher concentrations inhibit stimulation. Both the enhancement and inhibition are markedly attenuated by buffering t-BOOH-induced changes in intracellular Ca2+ concentration ([Ca2+]i). Phosphorylation of the NADPH oxidase component p47phox and its translocation from cytoplasm to plasma membrane are essential in respiratory burst activation. Phorbol 12-myristate 13-acetate (PMA)-stimulated p47phox phosphorylation was negligibly affected by 25 or 100 microM t-BOOH. Nonetheless, 25 microM t-BOOH increased PMA-stimulated p47phox translocation, whereas 100 microM t-BOOH decreased PMA-stimulated translocation. In unstimulated cells, however, neither phosphorylation nor translocation of p47phox was affected by t-BOOH. Buffering of the t-BOOH-mediated changes of [Ca2+]i abolished the effects of t-BOOH on PMA-stimulated translocation in parallel to effects upon the respiratory burst. The results suggest that the dual effects of hydroperoxides are mediated, in part, by Ca(2+)-dependent processes affecting the assembly of the respiratory burst oxidase at steps that are separate from p47phox phosphorylation.

Animals↗
PubMed1996

Induction of p21 mediated by reactive oxygen species formed during the metabolism of aziridinylbenzoquinones by HCT116 cells.

Aziridinylbenzoquinones are a group of antitumor agents that elicit cytotoxicity by generating either alkylating intermediates or reactive oxygen species. The mechanism of toxicity may not always, however, involve profound damage of cellular constituents, but may involve a cytostatic effect through interference with the cell cycle. In this context, we have examined the induction of the cell cycle inhibitor p21 (WAF1, CIP1, or sdi1), whose overexpression suppresses the growth of various tumor cells, in human tumor cells metabolizing 3,6-diaziridinyl-1,4-benzoquinone (DZQ) and its C2,C5-substituted derivatives: 2,5-bis-(carboethoxyamino) (AZQ) and 2, 5-bis-2(-hydroxyethylamino) (BZQ). Both DZQ and AZQ were effectively activated by HCT116 human colonic carcinoma cells; the activation of the former involved largely a dicoumarol-sensitive activity, whereas that of the latter appeared to be accomplished primarily by one-electron transfer reductases. BZQ was not a substrate for the dicoumarol-sensitive enzyme in HCT116 cells. Cellular activation of the first two quinones was associated with formation of oxygen-centered radicals as detected by EPR in conjunction with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide. The redox transitions of DZQ involved hydroxyl radical formation and were strongly inhibited by catalase, whereas those of AZQ showed a strong superoxide anion component sensitive to superoxide dismutase. These signals were suppressed by N-acetylcysteine with concomitant production of a thiyl radical adduct. This suggests an effective electron transfer between the thiol and free radicals formed during the activation of these quinones. DZQ and AZQ induced significantly the expression of p21 in HCT116 cells, but a 10-fold higher concentration of AZQ was required to achieve the level of induction elicited by DZQ. BZQ had little effect on p21 expression. p21 induction at both mRNA and protein levels correlated with the inhibition of either cyclin-dependent kinase activity or cell proliferation. p21 induction elicited by the above quinones was inhibited by N-acetylcysteine, whereas the non-sulfur analog, N-acetylalanine, was without effect. Catalase and superoxide dismutase did not effect p21 induction by aziridinylbenzoquinones in HCT116 cells, thus suggesting that extracellular sources of oxygen radicals generated by plasma membrane reductases have no influence in the expression of this gene. Hydrogen peroxide, a product of quinone redox cycling, elicited an increase of p21 mRNA levels in HCT116 and K562 human chronic myelogenous leukemia cells. The latter lacks p53, one of the activators of p21 transcription, thus suggesting that p21 expression can be accomplished in a p53-independent manner in these cells. This study suggests that p21 induction is mediated by an increase in the cellular steady-state concentration of oxygen radicals and that the greater effectiveness in p21 induction by DZQ may be related to its efficient metabolism by NAD(P)H:quinone oxidoreductase activity in HCT116 cells.

Acetylcysteine↗
PubMed1996

Hydroperoxide-induced increases in intracellular calcium due to annexin VI translocation and inactivation of plasma membrane Ca2+-ATPase.

Oxidative stress can cause changes in intracellular free calcium concentration ([Ca2+]i) that resemble those occurring under normal cell signaling. In the alveolar macrophage, hydroperoxide-induced elevation of [Ca2+]i modulates the respiratory burst and other important physiologic functions. The source of Ca2+ released by hydroperoxide is intracellular but separate from the endoplasmic reticulum pool released by receptor-mediated stimuli (Hoyal, C. R., Gozal, E., Zhou, H., Foldenauer, K., and Forman, H. J. (1996) Arch. Biochem. Biophys. 326, 166-171). Previous studies in other cells have suggested that mitochondria are a potential source of oxidant-induced [Ca2+]i elevation. In this study we have identified another potential source of hydroperoxide-releasable intracellular calcium, that bound to annexin VI on the inner surface of the plasma membrane. Translocation of annexin VI from the membrane during exposure to t-butyl hydroperoxide matched elevation of [Ca2+]i as a function of time and t-butyl hydroperoxide concentration. The translocation was possibly due to a combination of ATP depletion and oxidative modification of membrane lipids and proteins. A sustained increase in [Ca2+]i occurring > 50 pmol/10(6) cells (50 microM under these conditions) appeared to be a consequence of membrane Ca2+-ATPase dysfunction. These results suggest that exposure to oxidative stress results in early alterations to the plasma membrane and concomitant release of Ca2+ into the cytosol. In addition it suggests a mechanism for participation of annexin VI translocation that may underlie the alterations in macrophage function by oxidative stress.

Adenosine Diphosphate↗
PubMed1996

Modulation of the rat alveolar macrophage respiratory burst by hydroperoxides is calcium dependent.

Sublethal concentrations of hydroperoxides (H2O2 or tert-butylhydroperoxide) produce a dual effect upon the respiratory burst of rat alveolar macrophages in which low concentrations (< 50 microM) enhance and higher concentrations (> 50 microM) produce inhibition (J. K. Murphy, et al., Free Radical. Biol. Med. 18, 37-45, 1995). These effects correlate with transient versus sustained elevation of [Ca2+]i caused by exposure to hydroperoxides prior to stimulation of the respiratory burst. In the present study changes in [Ca2+]i caused by exposure to sublethal levels of hydroperoxide were buffered by incubating macrophages with the acetoxy-methyl ester of BAPTA, an intracellular Ca2+ chelator. The enhancement of the phorbol ester-stimulated respiratory burst by tBOOH was abolished by BAPTA, while the inhibition was attenuated. Thus, the modulation by tBOOH appears to be largely dependent upon the changes in [Ca2+]i. Receptor mediated stimulation of the respiratory burst (ADP stimulation) involves release of Ca2+ from the inositol-1,4,5-triphosphate (IP3)-sensitive pool in the endoplasmic reticulum. Comparisons were made of the effects of thapsigargin (TG), an endoplasmic reticulum Ca-ATPase inhibitor, with tBOOH on release of intracellular Ca2+ and the respiratory burst. Treatment with TG did not affect changes in [Ca2+]i caused by tBOOH or vice versa. Although TG decreased the ADP-stimulated respiratory burst, it had no effect upon tBOOH modulation. Thus, the effect of tBOOH upon the respiratory burst is dependent upon the release of Ca2+ and the release of Ca2+ occurs from a non-IP3-dependent pool. This aberrant mimicry of normal signal transduction underlies oxidative modulation of the respiratory burst.

Animals↗
PubMed1996

Activation of NF kappa B by the respiratory burst of macrophages.

H2O2 and other reduced oxygen species have been proposed as activators of the transcription factor, NF Kappa B. Stimulated macrophages produce superoxide and H2O2 (the respiratory burst). We tested the hypothesis that production of these species could serve as part of the NF Kappa B activation pathway in rat alveolar macrophages and the J774A.1 mouse monocyte/macrophage cell line. Phorbol myristate acetate (PMA) and ADP, which stimulate the respiratory burst, caused NF Kappa B activation in both cells. Catalase abolished NF kappa B activation, while superoxide dismutase produced little inhibition. Thus, H2O2 was the principal agent of respiratory burst-associated NF kappa B activation. Abolition of NF kappa B activation by catalase also suggested that intermediate signaling pathways, such as protein kinase C activation or intracellular free calcium elevation must not be involved. Exogenous H2O2 added as a bolus > or = 50 microM (> or = 50 nmol/10(6) macrophages) also activated NF kappa B in macrophages. Nevertheless, the maximum endogenous production of H2O2 by stimulated alveolar macrophages during a 30-min incubation was < or = 1.3 nmol H2O2/10(6) cells for PMA stimulation and < or = 0.2 nmol H2O2/10(6) cells for ADP stimulation. Thus, relatively little endogenous H2O2 generation was required to produce NF kappa B activation compared to the required amount of exogenous H2O2. As H2O2 rapidly diffuses and is consumed, these results suggest that the site of action for endogenously generated H2O2 is probably close to its origin, the plasma membrane.

Adenosine Diphosphate↗
PubMed1996

Differential enhancement of gamma-glutamyl transpeptidase and gamma-glutamylcysteine synthetase by tert-butylhydroquinone in rat lung epithelial L2 cells.

Sublethal quinone-mediated oxidative stress stimulates increases in the activities and mRNA levels of gamma-glutamyl transpeptidase (GGT) and gamma-glutamylcysteine synthetase (GCS) in rat lung epithelial L2 cells [Kugelman, A. et al. 1994. Am. J. Respir. Cell Mol. Biol. 11:586-592; Shi, M. M. et al. 1994. J. Biol. Chem. 269:26512-26517]. The present study demonstrated that the quinone-induced increases in these two enzymes were differentially regulated. L2 cells were exposed to various concentrations of tertiary-butylhydroquinone (TBHQ) for different periods of times. TBHQ increased the activities and the mRNAs for GGT and the catalytic subunit of GCS; however, the time- and concentration-dependencies differed. With 50 microM TBHQ, GCS activity increased significantly by 6 h whereas the activity of GGT was not increased until later. Under the same conditions, the highest GCS-mRNA level observed was at 6 h whereas the mRNA level of GGT increased after 6 h, reached a higher level at 12 h, and then returned to the control level by 24 h. Differences were also observed in the concentration-dependence of mRNA increases between the GGT and GCS. Actinomycin D (an inhibitor of RNA synthesis) abolished the increase of GCS-mRNA but not the increase in GGT-mRNA, suggesting a difference in regulation by TBHQ between these two genes. Nuclear run-on experiments confirmed that the increase of GCS-mRNA, but not GGT-mRNA was due to increased transcription. The increase in GGT-mRNA probably results from a decreased degradation rate. The differences between these two enzymes demonstrate how cells can use multiple mechanisms for regulating gene expression in response to oxidative stress.

Animals↗
PubMed1996

Increased gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase activities enhance resistance of rat lung epithelial L2 cells to quinone toxicity.

Tert-butylhydroquinone (TBHQ) is a monofunctional Phase II enzyme inducer, which produces reactive oxygen species. Incubation with a sublethal concentration of TBHQ increased the activities of both gamma-glutamyl transpeptidase (GGT) and gamma-glutamylcysteine synthetase (GCS), although the mechanisms are different (Liu and colleagues, accompanying manuscript). In this study, we found that TBHQ increased intracellular glutathione (GSH) content in rat lung epithelial L2 cells. L2 cells pretreated with a nontoxic concentration of TBHQ (50 microM) acquired resistance to a subsequent challenge with a normally lethal concentration of TBHQ (200 microM). Pretreatment with L-buthionine S,R-sulfoximine (BSO), an inhibitor of GCS, prevented the TBHQ-induced increase in GSH and markedly diminished resistance to 200 microM TBHQ. Similarly, pretreatment with acivicin, an inhibitor of GGT, also prevented the TBHQ-induced increase in GSH and markedly diminished resistance to 200 microM TBHQ. Nevertheless, blockage of GGT by acivicin could be bypassed using 2-oxothiazolidine-4-carboxylate (procysteine) to provide the cell with a source of cysteine. This allowed an increase in GSH and restored resistance in the TBHQ-pretreated cells. The results suggest that elevation of GCS and GGT activities participated in acquired resistance to quinone toxicity.

Animals↗
PubMed1995

Release of aldehydes from rat alveolar macrophages exposed in vitro to low concentrations of nitrogen dioxide.

This study demonstrated that aldehydes are released into the extracellular medium when alveolar macrophages (AM) are exposed to nitrogen dioxide (NO2) at concentrations that impair cell function but do not cause cell death. Butanal, glycolaldehyde, 4-hydroxynonenal, pentanal, pentenal, and hexanal were found. Dinitrophenylhydrazine (DNP) derivitization, thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry were used to identify the products. Some of the aldehydes have potential toxicity and may be responsible, in part, for altered AM function observed following NO2 exposure.

Aldehydes↗
PubMed1995

Modulation of ADP-stimulated inositol phosphate metabolism in rat alveolar macrophages by oxidative stress.

The present study examined alterations of adenosine-5-diphosphate (ADP)-stimulated inositol phosphate (IP3) metabolism and the respiratory burst of rat alveolar macrophages (AM) under oxidant stress using the model oxidant, tert-butyl hydroperoxide (tBOOH). Isolated AM were maintained in a system with an air-liquid interface, which approximates the lung environment. tBOOH produced a dual effect on the ADP-stimulated respiratory burst of these AM. Pretreatment of these AM with 50 microM tBOOH for 15 min led to a significant enhancement of the respiratory burst while significant inhibition was observed with 200 microM tBOOH. Treatment of AM with 100 microM ADP led to a significant increase in the generation of IP3, reaching a maximum at 30 s. In contrast, treatment of AM with 50 or 200 microM tBOOH did not stimulate IP3 generation during a 15-min period. Pretreatment of AM with 50 microM tBOOH for 15 min had no effect on ADP-stimulated IP3 generation. Preincubation with 200 microM tBOOH significantly inhibited generation of IP3 in response to ADP stimulation; however, this probably did not contribute to inhibition of the respiratory burst. The results suggest that production of IP3 may participate in stimulation of the respiratory burst by ADP but that enhancement of the respiratory burst by 50 microM tBOOH probably did not involve alteration of IP3 production.

Adenosine Diphosphate↗