Expression of the ectoenzyme γ-glutamyl transpeptidase (GGT) is regulated on T lymphocytes. It is present at a low level on naive T cells, at a high level on activated T cells, and at an intermediate level on resting memory T cells. GGT cleaves the glutamyl group from glutathione, which is the first step in the uptake of extracellular glutathione. In vitro, purified GGT also metabolizes the naturally occurring nitrosothiol, S-nitrosoglutathione (GSNO). Because of this relationship, the effects of cellular GGT on the metabolism of and cellular response to GSNO were tested. The GGT-negative lymphoblasts Ramos and SupT1 were transfected with cDNA for human GGT. In the presence of cells lacking GGT, GSNO is extremely stable. In contrast, GGT-expressing cells rapidly metabolize GSNO leading to nitric oxide release. The nitric oxide causes a rapid (<2-h) inhibition of DNA synthesis. There is a concomitant decrease in the concentration of intracellular deoxyribonucleotides, suggesting that one effect of the nitric oxide generated from GSNO is the previously described inactivation of the enzyme ribonucleotide reductase. GSNO also caused a rapid, GGT-dependent cytostatic effect in Hut-78, a human T cell lymphoma, as well as in activated peripheral blood T cells. Although DNA synthesis was decreased to 16% of control values in anti-CD3-stimulated Hut-78, the production of IL-2 was unchanged by GSNO. These data show that GGT, a regulated ectoenzyme on T cells, controls the rate of nitric oxide production from GSNO and thus markedly affects the physiological response to this biologically active nitrosothiol.

The cellular ectoenzyme γ-glutamyl transpeptidase (GGT,3 EC 2.3.2.2) is capable of hydrolyzing γ-glutamyl peptide bonds and transferring the glutamyl group to a suitable acceptor (1). A major substrate for GGT is glutathione (GSH). This tripeptide is the most abundant intracellular nonprotein thiol and is thus the primary determinant of the cellular redox state. The cleavage of GSH by GGT is the first step in the glutamyl cycle, whereby extracellular GSH, which cannot directly enter cells, is hydrolyzed into glutamate (or a glutamyl-amino acid), cysteine, and glycine. These components are then transported back into the cell for resynthesis of GSH (reviewed in 2). The functional importance of GGT in the maintenance of intracellular glutathione levels has recently been documented in mice in which the GGT gene has been inactivated (3). These animals have intracellular levels of GSH that are ∼50% of wild-type levels whereas plasma and urine GSH levels are markedly elevated.

GGT is found on many cell types, primarily in secretory or absorptive tissues such as the proximal convoluted tubule of the kidney, the liver, seminal vesicles/prostate, small intestine, choroid plexus, and mammary gland. Resting and activated human mononuclear cells, as well as many transformed lymphoblastoid cell lines, have been shown to express GGT by enzymatic assays (4, 5, 6, 7). Recently, we have developed a novel mAb to human GGT (3A8). With this Ab, it was possible to demonstrate several unrecognized features of GGT expression (8).4 First, although the level of GGT protein is low on resting lymphocytes, it begins to be up-regulated 2–3 days after stimulation with mitogens or superantigens. Second, the level of GGT on resting memory T cells is significantly greater than that seen on naive T cells. The highest levels are seen on those memory T cells that have the capacity to migrate across an endothelial barrier. This population of memory T cells with high GGT levels is expanded in patients with rheumatoid arthritis. Third, GGT was found to associate on both T and B cell surfaces with a subset of tetraspan proteins. These proteins, with characteristic four-membrane-spanning domains, include CD81, CD82, and CD53. Associations between GGT and the B cell complement receptor/signaling molecules CD21 and CD19 were also demonstrated, likely mediated by the coassociation of these molecules and CD81. These molecular associations were demonstrated by coimmunoprecipitation of GGT by Abs to the tetraspan proteins and by cocapping of GGT with the other proteins on live cells. Finally, in a survey of common T and B lymphoblastoid cell lines, it was demonstrated that GGT expression is not uniform. Although most cell lines expressed the Ag, pre-B cells such as Nalm-6, mature B cells such as Daudi and Ramos, and T cells such as SupT1 and HSB-2 did not express immunoreactive or enzymatically functional protein. Together, these data show that the expression of GGT on lymphocytes is regulated not only by cellular activation but also by virtue of cellular differentiation. Therefore, cells that express high levels of GGT may respond differently in an environment containing substrates for this enzyme.

It has recently been shown that the nitrosothiol, S-nitrosoglutathione (GSNO) is a substrate for purified GGT in vitro (9, 10). Unlike free NO, which has a short half-life in aerobic solutions, nitrosothiols such as S-nitrosocysteine, GSNO, and S-nitrosoalbumin are more stable (11). For example, the half-life for the decomposition of S-nitrosocysteine is on the order of seconds to minutes, for S-nitroso-N-acetylpenicillamine it is minutes to hours, and for GSNO it has been estimated to be over 100 h. This makes nitrosothiols useful experimental tools for study of the effects of nitric oxide. For example, they cause relaxation of smooth muscle (12) and inhibit platelet activation (13). In addition, nitrosothiols have been detected in plasma and other biological fluids (reviewed in 14) where, for example, the reversible nitrosylation of hemoglobin controlled by oxygen tension regulates blood flow in peripheral tissues (15).

GSNO has been used to study the effects of nitric oxide on mitogen-activated human peripheral blood T cells (16). It inhibited proliferation at 72 h and caused up-regulation of cellular cGMP levels. Because the spontaneous release of nitric oxide from GSNO in vitro is greatly accelerated by hydrolysis of the γ-glutamyl bond (10), we considered that the effect of GSNO on activated T cells would be GGT dependent. GSNO has been detected in culture supernatants of activated macrophages in vitro (17). The increased expression of GGT on T cell subsets would make these T cells particularly sensitive to nitric oxide in the form of GSNO produced in an inflammatory microenvironment. Herein, we show that GGT expression by lymphocytes is required for the rapid decomposition of GSNO. This leads to the liberation of nitric oxide from the resultant S-nitrosocysteinylglycine, which then rapidly down-regulates de novo DNA synthesis of the GGT-expressing cells. These results demonstrate a novel role for this tightly regulated enzyme in controlling the cellular response to physiological forms of NO.

SupT1 (human T cell lymphoma), Hut-78 (human T cell lymphoma), Ramos (human B cell lymphoma), and PA317 (murine amphipathic packaging cell line) were all obtained from the American Type Tissue Collection (Manassas, VA). The lymphoma cells were routinely cultured in RPMI 1640 supplemented with 10% FBS and antibiotics (RPMI/10). PA317 was cultured in DMEM supplemented with 5% FBS. Resting human T cells were purified from peripheral blood of healthy adult volunteers as previously described (18).

3A8, an IgG2a mAb to human GGT, was produced as described (8) and conjugated to FITC by standard techniques. 64.1 (anti-human CD3) was a kind gift from Dr. Ellen Vitetta (University of Texas Southwestern Medical Center).

GSNO, spermine, and 1-N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino-diazen-1-ium-1,2-diolate (spermine NONOate) were from Alexis Biochemicals (San Diego, CA). Reduced glutathione and l-γ-glutamyl-p-nitroanilide were from Calbiochem (San Diego, CA). G418 was from Life Technologies (Grand Island, NY), and Polybrene and acivicin were from Sigma (St. Louis, MO).

The plasmid phGGT (19) was obtained from Dr. Henry Pitot (University of Wisconsin, Madison, WI). A 2331-bp EcoRI fragment containing the GGT coding region flanked by 492 bp of 5′- and 203 bp of 3′-untranslated region was cloned into the EcoRI site of the amphipathic retroviral expression vector, pLXSN (gift of Dr. A. Dusty Miller, Fred Hutchison Cancer Research Center, Seattle, WA). The resulting pLGGTSN recombinant plasmid was transfected into subconfluent PA317 cells by the calcium phosphate technique. After transfection, the cells were selected in medium containing 1 mg/ml G418. Drug-resistant cells were analyzed for human GGT expression by staining with FITC-3A8 and analysis by flow cytometry. Whereas G418-resistant cells transfected with the parental pLXSN vector (control) were negative, the pLGGTSN-transfected packaging cells were uniformly positive for expression of GGT (not shown). The supernatants of confluent cultures of control and GGT-transfected PA317 were used as a source of retrovirus stock. They were diluted 1:10 with RPMI 1640, and Polybrene (800 μg/ml) was added. SupT1 and Ramos cells were cultured in the diluted viral stock at a concentration of 106/ml for 4 h at 37°C. They were then washed and recultured in RPMI/10 overnight. The bulk cell cultures were then diluted 1:1 with RPMI/10 containing 1 mg/ml G418. After 3 days, the cells were again split 1:1 with G418-containing medium. Drug-resistant cells were allowed to grow to near confluence and then tested for GGT expression by flow cytometry. The GGT expressing cells (∼50–75% of the G418-resistant cells) were then sorted on the FACStarPlus for a uniform population of GGT-expressing cells, termed SupT1/GGT and Ramos/GGT. G418-resistant cells that grew from SupT1 or Ramos transfected with empty retroviral vector were used without further manipulation and termed SupT1/X and Ramos/X.

GGT enzyme activity on intact cells was assayed by resuspending 106 washed, GGT-expressing Ramos or SupT1 cells in 1 ml PBS containing 60 mM glycylglycine, pH 7.4, and 2.5 mM l-γ-glutamyl-p-nitroanilide (Calbiochem). All solutions were treated with Chelex resin (Bio-Rad, Richmond, CA) to remove trace metal contamination, because this has been shown to accelerate the decomposition of nitrosothiols. In addition, 106 GGT-expressing cells were treated with a 200 μM concentration of the irreversible GGT inhibitor acivicin (20) for 1 h at 37°C and washed before assay. After 90 min at 37°C, the reaction was stopped by the addition of 2 ml 1.5 M acetic acid. The cells were pelleted by centrifugation, and the absorbance of the supernatants was read at 410 nm.

The intact S-nitroso bond of GSNO was measured by spectrophotometry (10). intact Ramos or SupT1 cells (106) were resuspended in 1 ml PBS plus glycylglycine containing 500 μM GSNO at 37°C in the dark. GGT-expressing cells were also tested after acivicin treatment as described above. At the indicated intervals, the absorbance of the supernatant at 336 nm was measured. Control incubations contained PBS, glycylglycine, and GSNO, but no cells.

The production of nitrite after the incubation of cells with GSNO in vitro was used as a surrogate for NO production. Nitrite was determined by the Greiss reaction. Supernatants (50 μl) from the incubation of control and GGT-expressing Ramos and SupT1 cells with GSNO in PBS plus glycylglycine, as above, or from control or GGT-expressing cells incubated in RPMI 1640 with 10% FCS containing 500 μM GSNO were incubated at room temperature with 100 μl of 1% sulfanilamide in 2.5% phosphoric acid for 5 min in flat-bottom 96-well plates. Naphthylethylenediamine, 0.5% (100 μl) in 2.5% phosphoric acid were added for an additional 5 min. The absorbance at 550 nm was measured, and the nitrite concentration was determined by comparison to sodium nitrite standards. In some experiments, 1 mM EDTA, pH 7.4, or 4 μM CuSO4 was added to the GSNO solution during incubation with the Ramos and SupT1 cells before nitrite determination. All enzyme assays were done at least twice.

The effect of GSNO on lymphoblast proliferation was determined by thymidine incorporation. Control or GGT-expressing Ramos or SupT1 cells (105) were washed and resuspended in 50 μl serum-free DMEM lacking cysteine and methionine (to prevent trans-nitrosylation by GSNO), and plated in 96-well U-bottom plates. Glutathione, GSNO, spermine, and spermine NONOate were added at 0, 125, 250, and 500 μM. After 2 h at 37°C, 1 μCi [methyl-3H]thymidine (New England Nuclear, Boston, MA, 6.7 Ci/mmol) was added for an additional 4 h. The cells were harvested onto glass microfiber filters, and incorporation of thymidine into DNA determined by liquid scintillation counting. All measurements were done in triplicate. Similar assays were performed on human peripheral blood T lymphocytes. T cells were purified from venous blood as described and incubated with 10 ng/ml PMA and 0.5 μM ionomycin for 72 h. The cells were then incubated with GSNO as described and assayed for thymidine incorporation.

Hut-78 cells were incubated with GSNO as above for 2 h and then transferred in the GSNO-containing medium to flat-bottom 96-well microtiter plates that had been coated with 1 μg/ml anti-CD3 (64.1). The cultures were pulsed with [3H]thymidine, and incorporation into DNA was determined after a 6-h incubation. The supernatants of replicate cultures were collected for determination of IL-2 content by ELISA (PharMingen, San Diego, CA).

The concentrations of intracellular deoxyribonucleotides in cells treated with GSNO were determined by modification of the primer extension method of Sherman and Fyfe (21). SupT1/X or SupT1/GGT cells were incubated for 4 h in medium containing various concentrations of GSNO, washed, and extracted overnight in 60% methanol at −20°C. After the methanol-insoluble material was pelleted, the extracts were dried in a Speed-Vac (Savant Instruments, Farmingdale, NY) and resuspended in water at a concentration of 106 cell equivalents per 20 μl. Oligonucleotides specific for the measurement of dATP, dCTP, dGTP, and TTP were synthesized on an ABI 394 DNA/RNA synthesizer and annealed to form double-stranded primers as described (21). Reactions were set up in 25 μl containing 32 mM Tris-HCl, pH 7.5, 16 mM MgCl2, 40 mM NaCl, 4 mM DTT, 2 mM unlabeled dATP, 1.25 μCi [35S]dATP (1250 Ci/mmol, DuPont/NEN, Boston, MA) 100 pmol primer, 0.4 unit Sequenase T7 DNA polymerase (Amersham, Cleveland, OH) and 2.5 μl of either deoxyribonucleotide standards or cell extract. For the measurement of dATP, TTP and [35S]TTP were substituted for the dATP in the reaction mixtures. The reactions were incubated at 37°C for 1 h, and then aliquots were spotted in duplicate on Whatman DE81 filter paper (Fisher Scientific, Pittsburgh, PA). The filters were washed three times in 5% Na2HPO4, once in water, and once in 100% ethanol and dried. The radioactivity on the dried filters was then quantified by liquid scintillation counting.

To facilitate the study of GGT expression on cell physiology, human GGT cDNA was expressed in two GGT lymphoblastoid cell lines. Many T and B cell lines tested express high levels of GGT (8), but the B cell lymphoma, Ramos, and the T cell lymphoma, SupT1, do not. A defective retrovirus capable of expressing GGT from the viral LTR was constructed and used to infect these cells. Cells infected with virus created from the empty vector served as controls. The expression of GGT on the control (/X) and transfected (/GGT) cell lines is shown in Fig. 1. The levels of GGT expressed normally on the surface of the T lymphoblastoid cell line, Hut-78, and on PMA-stimulated peripheral blood T cells are shown for comparison. To avoid artifacts arising from clonal selection, the cell lines were not subcloned but were selected for high levels of GGT expression by fluorescence activated cell sorting. Despite this, the SupT1/GGT line continued to have heterogeneous expression. The level of GGT enzyme activity correlated with the level of antigenic expression (Fig. 2). Acivicin (AT-125), an irreversible inhibitor of GGT in vitro (20) was able to completely prevent the cleavage of the synthetic GGT substrate, γ-glutamyl paranitroanilide.

FIGURE 1.

Expression of GGT on human lymphoid cells. Ramos B lymphoma cells and SupT1 T lymphoma cells were transfected with control LXSN retrovirus or LGGTSN virus containing the human GGT cDNA. Hut-78 is a T cell lymphoma that naturally expresses GGT at levels comparable with those of other lymphoid cell lines. T cells were purified from peripheral blood and stimulated with PMA and ionomycin for 72 h before staining. The cells were stained with FITC-conjugated control (thin line) or 3A8 mAb (thick line). A, Ramos/X; B, Ramos/GGT; C, SupT1/X; D, SupT1/GGT; E, Hut-78; F, activated peripheral blood T cells.

FIGURE 1.

Expression of GGT on human lymphoid cells. Ramos B lymphoma cells and SupT1 T lymphoma cells were transfected with control LXSN retrovirus or LGGTSN virus containing the human GGT cDNA. Hut-78 is a T cell lymphoma that naturally expresses GGT at levels comparable with those of other lymphoid cell lines. T cells were purified from peripheral blood and stimulated with PMA and ionomycin for 72 h before staining. The cells were stained with FITC-conjugated control (thin line) or 3A8 mAb (thick line). A, Ramos/X; B, Ramos/GGT; C, SupT1/X; D, SupT1/GGT; E, Hut-78; F, activated peripheral blood T cells.

Close modal
FIGURE 2.

GGT enzymatic activity in transfected human lymphoblasts. Cells were incubated in PBS containing γ-glutamyl-p-nitroanilide as a glutamyl donor and glycylglycine as the glutamyl acceptor. The hydrolysis of the substrate was monitored by the change in absorbance of the solution at 410 nm over time. Left, Ramos/X (▪), Ramos/GGT (•), Ramos/GGT treated with 200 μM acivicin for 1 h (○). Right, SupT1/X (▪), SupT1/GGT (•), SupT1/GGT treated with acivicin (○). The results are representative of three separate experiments.

FIGURE 2.

GGT enzymatic activity in transfected human lymphoblasts. Cells were incubated in PBS containing γ-glutamyl-p-nitroanilide as a glutamyl donor and glycylglycine as the glutamyl acceptor. The hydrolysis of the substrate was monitored by the change in absorbance of the solution at 410 nm over time. Left, Ramos/X (▪), Ramos/GGT (•), Ramos/GGT treated with 200 μM acivicin for 1 h (○). Right, SupT1/X (▪), SupT1/GGT (•), SupT1/GGT treated with acivicin (○). The results are representative of three separate experiments.

Close modal

Although glutathione is the major substrate for GGT, other glutamyl-containing molecules are cleaved by purified enzyme in vitro. One of these is GSNO, a natural adduct of nitric oxide and glutathione. The stability of GSNO in the presence of live cells was tested. The control and GGT-expressing Ramos and SupT1 cells were incubated with GSNO. The presence of intact nitrosothiol was measured by monitoring the absorbance of the solution at 336 nm (10). The cell lines containing the control vector did not degrade GSNO (Fig. 3). When GGT was expressed on the cell surface, there was loss of the nitrosothiol absorbance. The rate of disappearance of the nitrosothiol was correlated with the level of GGT on the cell surface. That is, the SupT1/GGT cells degraded GSNO more rapidly that the Ramos/GGT cells. Inclusion of acivicin prevented cleavage of GSNO in the incubations with Ramos/GGT or SupT1/GGT cells. These data emphasize the stability of GSNO in aqueous solutions even when exposed to living cells which could potentially destroy the nitrosothiol through disulfide interchange with cell surface sulfhydryl-containing proteins or low m.w. thiols secreted from the cells.

FIGURE 3.

Degradation of GSNO by transfected human lymphoblasts. Cells were incubated in PBS containing glycylglycine and 500 μM GSNO for the indicated times. The presence of an intact S-nitroso bond was measured by the absorbance of the solution at 336 nm. Left, no cells (×), Ramos/X (□), Ramos/GGT (•), Ramos/GGT treated with 200 μM acivicin for 1 h (○). Right, no cells (×), SupT1/X (□), SupT1/GGT (•), SupT1/GGT treated with acivicin (○). Data are representative of three experiments.

FIGURE 3.

Degradation of GSNO by transfected human lymphoblasts. Cells were incubated in PBS containing glycylglycine and 500 μM GSNO for the indicated times. The presence of an intact S-nitroso bond was measured by the absorbance of the solution at 336 nm. Left, no cells (×), Ramos/X (□), Ramos/GGT (•), Ramos/GGT treated with 200 μM acivicin for 1 h (○). Right, no cells (×), SupT1/X (□), SupT1/GGT (•), SupT1/GGT treated with acivicin (○). Data are representative of three experiments.

Close modal

It has been proposed that the release of nitric oxide from nitrosothiols is catalyzed by metals, particularly Cu+ ions (10, 22). Both enzymatic and nonenzymatic mechanisms have been postulated. The Ramos and SupT1 transfectants were incubated with GSNO, and the appearance of nitrite was determined as an indicator of nitric oxide production (Fig. 4, top). In the incubations with the control transfectants, there was little or no accumulation of nitrite in the supernatant. In the presence of GGT-expressing cells, there was prompt production of nitrite that increased during the 2-h incubation. The presence of serum had only a small effect on the stability of GSNO in the absence of GGT. Therefore, trans-nitrosylation of BSA or other proteins is not a significant mechanism for the release of nitric oxide from GSNO. When 1 mM EDTA was included in the incubation (Fig. 4, bottom), there was an almost total abrogation of nitrite production. Conversely, when copper was added to the incubation, there was an increase in the level of nitrite produced. The buffers used in these experiments were treated with ion-exchange resins to remove trace metals. In vitro, therefore, the metal needed to effect the conversion of GSNO to nitric oxide presumably comes from the cells themselves. In vivo, the metal ions are likely to be present in the extracellular fluid as well as secreted by the cells.

FIGURE 4.

Production of nitrite from SNO by transfected human lymphoblasts. Top, SupT1 cells were incubated in either PBS with glycylglycine and GSNO, as in Fig. 3, or in RPMI 1640 containing 10% FCS and 500 μM GSNO. After 90 min, the supernatants were collected and analyzed for nitrite content. Bottom, Ramos and SupT1 transfectants were incubated in PBS/glycylglycine with 500 μM GSNO alone or with either copper sulfate or EDTA. □, No additions; ▪, addition of 4 μM CuSO4; ▨, addition of 1 mM EDTA. Data are means ± SEM of triplicate determinations and are representative of two different experiments.

FIGURE 4.

Production of nitrite from SNO by transfected human lymphoblasts. Top, SupT1 cells were incubated in either PBS with glycylglycine and GSNO, as in Fig. 3, or in RPMI 1640 containing 10% FCS and 500 μM GSNO. After 90 min, the supernatants were collected and analyzed for nitrite content. Bottom, Ramos and SupT1 transfectants were incubated in PBS/glycylglycine with 500 μM GSNO alone or with either copper sulfate or EDTA. □, No additions; ▪, addition of 4 μM CuSO4; ▨, addition of 1 mM EDTA. Data are means ± SEM of triplicate determinations and are representative of two different experiments.

Close modal

The release of nitric oxide (measured as nitrite) by primary human T cells was also enhanced by GGT. Resting T cells express a low level of GGT on their surface that increases 5- to 10-fold after stimulation.4 This is paralleled by an increase in GGT enzymatic activity. T cells were purified from venous blood and either used immediately (“resting T cells”) or stimulated for 72 h with phorbol ester and ionomycin (“activated T cells”). GGT enzymatic activity in the two populations was measured spectrophotometrically (Fig. 5, open bars). There was an ∼7-fold increase in GGT activity per cell after activation that was inhibited by >90% after a 1-h exposure to 200 μM acivicin. Nitrite production from GSNO in the presence of resting T cells was only slightly over background. In the presence of activated T cells, there was a significant amount of nitrite produced from GSNO that was inhibited by acivicin. Thus, the increased expression of GGT on human T cells after activation facilitates the delivery of nitric oxide from this physiological nitrosothiol.

FIGURE 5.

Production of nitrite from GSNO by resting and activated human T cells. T cells were purified from venous blood and stimulated with PMA and ionomycin. GGT activity was determined by hydrolysis of γ-glutamyl-p-nitroanilide as in Fig. 2 and expressed as the absorbance of the solution at 410 nm after 90 min. Nitrite production was determined as in Fig. 4. GGT activity was inhibited in a sample of activated T cells by a 1-h incubation with 200 μM acivicin.

FIGURE 5.

Production of nitrite from GSNO by resting and activated human T cells. T cells were purified from venous blood and stimulated with PMA and ionomycin. GGT activity was determined by hydrolysis of γ-glutamyl-p-nitroanilide as in Fig. 2 and expressed as the absorbance of the solution at 410 nm after 90 min. Nitrite production was determined as in Fig. 4. GGT activity was inhibited in a sample of activated T cells by a 1-h incubation with 200 μM acivicin.

Close modal

Nitrite accumulation is a surrogate marker for nitric oxide production. To demonstrate a physiological effect of the NO produced after hydrolysis of GSNO by GGT, the transfected cell lines were exposed to different concentrations of GSNO and then tested for the incorporation of [3H]thymidine into DNA. After a 2-h pretreatment with GSNO in tissue culture medium, there was a concentration-dependent inhibition of DNA synthesis only in the GGT-expressing cell lines (Fig. 6). This effect was not caused by GSNO inhibition of initial thymidine uptake into the cells, in that no difference was seen in the accumulation of radiolabeled thymidine by mitomycin C-treated cells (data not shown).

FIGURE 6.

GSNO inhibits the proliferation of GGT-expressing lymphoblasts. Cells were cultured with the indicated concentrations of GSH, GSNO, spermine, or spermine NONOate for 2 h, pulsed with [3H]thymidine for an additional 6 h, and then assayed for incorporation of tritium into DNA. To control for differences between cell types, the ordinate for each graph represents DNA synthesis normalized to the values obtained from cells cultured in medium alone. The absolute baseline values were: Ramos/X, 17,792 ± 1,544 cpm; Ramos/GGT, 17,234 ± 1,278 cpm; SupT1/X, 73,208 ± 8,588 cpm; SupT1/GGT, 75,389 ± 13,999 cpm. Data are the means ± SEM of triplicate determinations and represent the results of five separate experiments.

FIGURE 6.

GSNO inhibits the proliferation of GGT-expressing lymphoblasts. Cells were cultured with the indicated concentrations of GSH, GSNO, spermine, or spermine NONOate for 2 h, pulsed with [3H]thymidine for an additional 6 h, and then assayed for incorporation of tritium into DNA. To control for differences between cell types, the ordinate for each graph represents DNA synthesis normalized to the values obtained from cells cultured in medium alone. The absolute baseline values were: Ramos/X, 17,792 ± 1,544 cpm; Ramos/GGT, 17,234 ± 1,278 cpm; SupT1/X, 73,208 ± 8,588 cpm; SupT1/GGT, 75,389 ± 13,999 cpm. Data are the means ± SEM of triplicate determinations and represent the results of five separate experiments.

Close modal

GSNO had no effect on the proliferation of Ramos/X cells. The SupT1/X cells showed a small but reproducible increase in DNA synthesis when exposed to GSNO. The mechanism for this is unclear but is presumably not the result of NO production or significant trans-nitrosylation of surface proteins, because we were unable to detect either the production of nitrite or the destruction of the S-nitroso bond in solutions of GSNO incubated with these cells (see above). However, a catalytic level of modification of SupT1-specific membrane proteins cannot be ruled out. Importantly, the Ramos/X and SupT1/X cells are as sensitive to the effects of NO as the GGT-transfected cells. This was shown by the observation that DNA synthesis in all cells was inhibited equally in the GGT and GGT+ cells by spermine NONOate, a NO donor that does not require GGT for activity. These effects were caused by the NO liberated from GSNO and spermine NONOate, because neither of the parent compounds glutathione and spermine had an effect on thymidine incorporation by any of the cells.

Although the transfected cell lines provide the opportunity to test the effect of GGT on GSNO degradation directly, they are limited in their functional capacity. For example, SupT1 cells have low levels of TCR expression and do not respond to anti-CD3 stimulation (D. Karp, unpublished observations). To obsrve the GGT-dependent effects of GSNO metabolism on T cell activation, other cells were analyzed. The inhibition of DNA synthesis by short term exposure to GSNO was tested in activated peripheral blood T cells and in the Hut-78 lymphoma line (Table I). T cells were activated by a 72-h exposure to phorbol ester-ionomycin and then incubated with 250, 500, and 1000 μM GSNO, resulting in 23, 70, and 91% inhibition of thymidine incorporation, respectively. Hut-78 cells express moderate levels of GGT quantified by flow cytometry using 3A8 (Fig. 1) and secrete IL-2 in response to stimulation with anti-CD3. When exposed to GSNO for 4 h, there is almost total inhibition of DNA synthesis as expected from the experiments with GGT-transfected cell lines. Addition of 200 μM acivicin to either the peripheral blood or Hut-78 cultures 1 h before the incubation with GSNO prevented the decrease in thymidine incorporation. This acivicin treatment irreversibly inhibits >90% of the GGT enzyme activity without altering the expression of GGT on the cell surface determined by 3A8 staining (data not shown). Therefore, the inhibition of DNA synthesis caused by exposure to GSNO depends on its metabolism by cell surface GGT.

Table I.

Short-term exposure to GSNO inhibits proliferation of peripheral blood T cells and Hut-78 lymphoblasts

Cell TypeAcivicina (200 μM)GSNO (μM)
1252505001000
PBL-Ta − ND 77 ± 12b 30 ± 3 9 ± 1 
PBL-T ND 111 ± 22 100 ± 14 78 ± 8 
Hut-78 − 82 ± 21 29 ± 6 16 ± 7 ND 
Hut-78 113 ± 16 110 ± 6 99 ± 8 ND 
Cell TypeAcivicina (200 μM)GSNO (μM)
1252505001000
PBL-Ta − ND 77 ± 12b 30 ± 3 9 ± 1 
PBL-T ND 111 ± 22 100 ± 14 78 ± 8 
Hut-78 − 82 ± 21 29 ± 6 16 ± 7 ND 
Hut-78 113 ± 16 110 ± 6 99 ± 8 ND 
a

Peripheral blood T lymphocytes stimulated with PMA and ionomycin.

b

Thymidine incorporation expressed as the percentage of control cultures not exposed to GSNO.

Mean ± SEM of triplicate determinations.

Various nitric oxide donors have been shown to cause a decrease in the activity of ribonucleotide reductase (23, 24, 25). This enzyme catalyzes the rate-limiting step in the pathway leading to DNA synthesis. The catalytic reduction of ribonucleotides involves a tyrosyl free radical that is subject to reversible nitrosylation by nitric oxide (26, 27). The intracellular pools of the four deoxyribonucleotides were measured in cells expressing GGT and exposed to GSNO as an indicator of ribonucleotide reductase activity (Table II). Methanol extracts were prepared from Hut-78 cells after a 4-h exposure to GSNO. The concentrations of the individual deoxyribonucleotides in the extracts were determined by separate primer-extension reactions, each dependent on the presence of a single dNTP (21). Exposure of the Hut-78 cells to GSNO was associated with concentration-dependent decreases in all four deoxyribonucleotides. At the highest concentration tested (1 mM), dATP was decreased to 67% of control, dCTP to 40% of control, dGTP to 30% of control, and TTP to 56% of control.

Table II.

Reduction in cellular deoxyribonucleotides in Hut-78 by exposure to GSNO

GSNO (μM)
062.52501,000
dATP 18.9a 18.0 14.0 12.7 
dCTP 5.0 4.9 2.1 2.0 
dGTP 4.7 3.4 2.2 1.4 
TTP 12.0 12.6 9.4 6.7 
GSNO (μM)
062.52501,000
dATP 18.9a 18.0 14.0 12.7 
dCTP 5.0 4.9 2.1 2.0 
dGTP 4.7 3.4 2.2 1.4 
TTP 12.0 12.6 9.4 6.7 
a

Picomols of deoxyribonucleotide/106 cells.

Hut-78 cells were used to test the effect of GSNO on T cell activation (Table III). After stimulation with anti-CD3, these cells secrete IL-2. In the presence of increasing concentrations of GSNO without exposure to plastic-immobilized anti-CD3, there was a slight increase in baseline IL-2 production that was statistically significant (p < 0.01 for 0 vs 500 μM GSNO, Student’s t test). Exposure of the cells to anti-CD3 caused significant IL-2 to be secreted during the 6-h culture that was not altered by exposure to GSNO. This indicates that in contrast to the profound inhibition of DNA synthesis caused by GSNO, signaling from the TCR and IL-2 gene transcription is not decreased by the NO produced from GSNO. This provides further evidence that GSNO exposure does not globally suppress cell functions but, like NO itself, results in specific alterations in cell capacities.

Table III.

Short-term exposure to GSNO does not alter anti-CD3-stimulated secretion of IL-2 by Hut-78 T lymphoblasts

Anti-CD3aGSNO (μM)
031.25125500
− 0.12 ± 0.02b 0.10 ± 0.02 0.13 ± 0.02 0.17 ± 0.02 
2.6 ± 0.3 2.8 ± 0.34 2.7 ± 0.32 2.3 ± 0.24 
Anti-CD3aGSNO (μM)
031.25125500
− 0.12 ± 0.02b 0.10 ± 0.02 0.13 ± 0.02 0.17 ± 0.02 
2.6 ± 0.3 2.8 ± 0.34 2.7 ± 0.32 2.3 ± 0.24 
a

Culture for 6 h with or without immobilized 64.1 after 2 h of pretreatment with GSNO.

b

IL-2 produced in 6 h of culture, ng/ml. Mean ± SEM of triplicate determinations.

Nitrosothiols have been used experimentally as convenient sources of nitric oxide, have been detected in vivo, and demonstrate biological activities. They are easy to prepare and relatively stable. GSNO is unique among these compounds in that it has a long half-life (t1/2 ∼160 h (11) in metal-free aqueous solution) and is subject to enzymatic modification. In vitro, the removal of the γ-glutamyl group from GSNO markedly increases the rate of release of nitric oxide into the medium (9, 10). The enzyme that catalyzes this reaction is GGT, which initiates the cleavage of extracellular GSH for transport of the component amino acids into the cell and resynthesis as intracellular GSH. Here we demonstrate that GGT naturally expressed at the surface of lymphocytes is required for the rapid breakdown of GSNO. This GGT-dependent delivery of NO causes cytostasis without leading to a change in T cell function, as determined by IL-2 secretion.

The level of GGT expression differs on subpopulations of human T cells. Both the enzymatic activity (28) and cell surface expression4 of GGT increase markedly with T cell activation. Moreover, the expression of GGT remains elevated on T cells that express CD45RO and intermediate levels of CD45RB. These isoforms of the CD45 protein phosphatase are markers for memory T cells. A subset of memory T cells has the highest capacity for transendothelial migration into sites of tissue inflammation such as rheumatoid synovium (29, 30). Moreover, the relative proportion of memory T cells is enhanced in the peripheral blood of patients with rheumatoid arthritis (31). We have recently documented that both the CD4+ T cells that exhibit rapid transendothelial migration and the expanded memory T cell population in patients with rheumatoid arthritis have very high levels of GGT compared with resting, naïve T cells.4 Therefore, the population of T cells that is found outside the vasculature, in inflamed tissue, will be able to metabolize GSNO and be subject to the effects of the released nitric oxide. The fact that GGT is expressed at the highest level on activated T cells explains the previous observation that GSNO is cytostatic for human peripheral blood T cells 3 to 5 days after exposure to mitogens (16). This corresponds to the time required for the expression of high levels of GGT on the cell surface.

GGT metabolism of GSNO is generally thought of as a mechanism for cells to recapture GSH as outlined above. However, there is also experimental evidence that under certain conditions, the cysteinylglycine produced from GSH by GGT has a prooxidant effect. This has been seen in cultured hepatocytes as well human monoblastic leukemia cell lines. In the latter system, an decrease in peroxide released from cells was seen when GGT was inhibited by acivicin. Also, an increase in exogenous lipid peroxidation could be seen when the cells were incubated with chelated iron and GSH. These effects were thought to be caused by the facilitated generation of reactive oxygen intermediates by the presence of the more basic thiolate anion of cysteinylglycine. It is unknown whether such a prooxidant effect of GGT occurs in lymphocytes as well. In the short term assays that have been performed in this study, we have been unable to see any effect of either cysteinylglycine at concentrations of up to 500 μM, or of exogenous bovine heart catalase on the proliferation of lymphocytes (data not shown). It is unlikely that the generation of reactive oxygen intermediates from the metabolism of either GSH or GSNO accounts for the cytostasis observed here.

GSNO has been documented in vivo in a number of systems. Low m.w. nitrosothiols including GSNO have been demonstrated in human (14) and murine (32) bronchoalveolar lavage fluid. The concentration of GSNO is highest in fluid from inflamed lungs, as a result of either infection or orthotopic lung transplantation. It is also present in human erythrocytes (15). Finally, GSNO has been produced by murine macrophages activated by LPS in the presence of GSH (17). The concentrations of GSNO found in these different fluids range from several hundred nanomolar to several micromolar (33). Although these are below the levels studied here, it is likely that higher concentrations are obtained in tissue than in plasma or other body fluids, where the GSNO is produced in response to induction of nitric oxide synthase. The expression of GGT on activated T cells at sites of inflammation would therefore increase the local concentration of NO leading to control of lymphocyte proliferation.

The observations that nitric oxide release from GSNO is accelerated by cellular GGT and is dependent on the presence of metals in the extracellular environment support a biochemical mechanism derived from in vitro studies. The homolytic cleavage of the S-nitroso bond is catalyzed by transition metals (22). In particular, reduced metals such as Cu+ are more efficient than oxidized metals such as Cu2+. The products of this cleavage are nitric oxide and thiyl radicals, which then form disulfides. Different nitrosothiols are more susceptible to metal-dependent cleavage than others (22). The product of GGT metabolism of GSNO is S-nitrosocysteinylglycine, which can bind metals at both the sulfur and free amino nitrogen (which is not present in GSNO). S-Nitrosocysteinylglycine rapidly decomposes to nitric oxide and the disulfide of cysteinylglycine (10). The overall scheme for the release of nitric oxide from GSNO is shown in Fig. 6. In phosphate buffers or tissue culture medium, GSNO is stable before cleavage by GGT. Once the nitrosocysteinylglycine is formed, there is sufficient metal contamination from the buffer solution, or from the cells themselves, to cause homolytic cleavage. Chelation of the metals in solution by EDTA does not affect the degree of cleavage of GSNO by GGT (10), whereas the resulting nitrosothiol is still stable even in the presence of cells. Lastly, it is possible that the products of GGT hydrolysis of GSNO have direct effects on cells, unrelated to their release of NO or the transfer of NO to another molecule. Certain tissues have been shown to have specific and stereoselective receptors for intact nitrosothiols, such as l-s-nitrosocysteine (34). The similar effects of spermine NONOate and GSNO in GGT-expressing cells argue against a direct effect in this system.

FIGURE 7.

Mechanism of GGT-mediated release of NO from GSNO. GGT transfers the γ-glutamyl group from GSNO (1 ) to another amine (amino acid, dipeptide, or protein) or hydrolyzes it to glutamate (2 ). The resulting GSNO (3 ) can more easily coordinate transition metal cations which catalyze the release of nitric oxide. The cysteinylglycine (4 ) forms a disulfide under normal conditions via a thiyl radical (not shown).

FIGURE 7.

Mechanism of GGT-mediated release of NO from GSNO. GGT transfers the γ-glutamyl group from GSNO (1 ) to another amine (amino acid, dipeptide, or protein) or hydrolyzes it to glutamate (2 ). The resulting GSNO (3 ) can more easily coordinate transition metal cations which catalyze the release of nitric oxide. The cysteinylglycine (4 ) forms a disulfide under normal conditions via a thiyl radical (not shown).

Close modal

There are several physiological effects of nitric oxide on lymphocytes. As in other cell types, nitric oxide donors cause the accumulation of cGMP through the modification of guanyl cyclase (16). Nitric oxide exposure inhibits apoptosis in human B and T lymphoblastoid cell lines caused by either arginine depletion or exposure to a calcium ionophore (35) or by anti-Fas Ab (36). The inhibition of apoptosis is caused, in part, by the inhibition of caspase-3 activity by both direct and indirect effects of nitric oxide (37). The work described here demonstrates that exposure to nitric oxide causes rapid cytostasis in both T lymphoblastoid cell lines and activated peripheral blood T cells. DNA synthesis was inhibited >90% by GSNO in a concentration-dependent and GGT-dependent manner. This effect was seen as early as 2 h after exposure to GSNO. Although nitric oxide has been shown to affect the levels of cell cycle-regulatory proteins such as p53 (38) or proapoptotic protein, bax (39), no changes in the level of p53 or G1 cyclins could be seen in GGT-expressing cells exposed to GSNO for short times (data not shown). There was a concentration-dependent decrease in the levels of deoxyribonucleotides in cells exposed to GSNO. The activity of purified ribonucleotide reductase, the enzyme responsible for regulation of the dNTP pools, is inhibited by nitric oxide (24, 27). Nitrosylation of both cysteinyl residues and a catalytic tyrosyl radical has been shown to occur (27). Exposure of intact murine adenocarcinoma cells to LPS, TNF, and IFN-γ for 24 h activated endogenous nitric oxide synthesis and caused a 45% decrease in ribonucleotide reductase activity that was associated with an 86% inhibition of DNA synthesis (23). In the experiments described here, DNA synthesis in Hut-78 cells was shown to be inhibited by exposure to GSNO. The cytostatic effect was reversed by inhibition of GGT. The different dNTP pools were decreased 32–70%. These results are similar to level of dNTP pool depletion resulting from inhibition of ribonucleotide reductase caused by other cytostatic agents (40).

The role of nitric oxide in T cell signaling is not clear. When examined in isolation, the MAP kinase pathways are up-regulated by nitric oxide. Nitrosylation of a specific cysteine residue of p21ras enhances GDP/GTP exchange (41). This is reflected by the 2.5- to 3.5-fold increase in ERK1/2 activity seen in Jurkat cells exposed to nitric oxide donors (42). Interestingly, the kinetics of nitric oxide-induced activation of the other MAP kinases, JNK, and p38 was different from that seen for ERK1/2. In resting Hut-78 T cells, an increase in ERK1/2 phosphorylation is seen after exposure to GSNO (D. Karp, unpublished observations). Whether nitric oxide has other effects on the intermediate stages of this pathway is unknown. The precise role of nitric oxide on T cell-signaling pathways in the presence or absence of T cell activation is currently under investigation. Finally, it is possible that the liberation of NO from GSNO by GGT expressed on memory/effector T cells may act in trans on neighboring cells, perhaps those that are producing the GSNO.

In conclusion, the data presented here show that GSNO is stable and does not decompose to form nitric oxide species even in the presence of B or T lymphocytes unless those cells express GGT. GGT is present in serum and, presumably, other extracellular fluids. However, the low level of the enzyme in serum is insufficient to degrade GSNO in short term assays (data not shown). The activity of GGT on the cell surface is both necessary and, in the presence of metal ions, sufficient to cause release of nitric oxide from GSNO. The nitric oxide released from GSNO by cell surface GGT causes rapid cytostasis without preventing T cell activation. This pathway could represent a mechanism for GSNO produced at sites of inflammation to regulate the proliferation of previously activated T cells that have up-regulated their expression of GGT. For example, synovial T cells from patients with rheumatoid arthritis proliferate poorly. These T cells also express very high levels of GGT.4 The synovium also contains cells with up-regulated nitric oxide synthase. Although it remains to be proved that these cells produce GSNO, it is possible that this represents a mechanism whereby the regulated expression of an ectoenzyme by T cells controls their function.

We thank Drs. Jonathan Stamler and Nicolai Van Oers for their critical reading of the manuscript.

1

This work was supported by a Biomedical Science Grant from the Arthritis Foundation (D.R.K.), a grant from the Rocky Mountain Chapter of the Arthritis Foundation (V.M.H.), and National Institutes of Health Grant AI42772 (D.R.K.).

3

Abbreviations used in this paper: GGT, γ-glutamyl transpeptidase; GSH, glutathione; spermine NONOate, 1-N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino-diazen-1-ium-1,2-diolate; GSNO, S-nitrosoglutathione.

4

D. R. Karp, T. C. Nichols, N. Oppenheimer-Marks, R. I. Brezinschek, and V. M. Holers. Submitted for publication.

1
Meister, A., S. S. Tate, O. W. Griffith.
1981
. γ-glutamyl transpeptidase.
Methods Enzymol.
77
:
237
2
Lieberman, M. W., R. Barrios, B. Z. Carter, H. G. M. R. M. Lebovitz, S. Rajagopalan, A. R. Sepulveda, Z.-Z. Shi, D.-F. Wan.
1995
. γ-Glutamyl transpeptidase.
Am. J. Pathol.
147
:
1175
3
Lieberman, M. W., A. L. Wiseman, Z. Z. Shi, B. Z. Carter, R. Barrios, C. N. Ou, P. Chevez-Barrios, Y. Wang, G. M. Habib, J. C. Goodman, S. L. Huang, R. M. Lebovitz, M. M. Matzuk.
1996
. Growth retardation and cysteine deficiency in γ-glutamyl transpeptidase-deficient mice.
Proc. Natl. Acad. Sci. USA
93
:
7923
4
Novogrodsky, A., S. S. Tate, A. Meister.
1976
. γ-Glutamyl transpeptidase, a lymphoid cell-surface marker: relationship to blastogenesis, differentiation, and neoplasia.
Proc. Natl. Acad. Sci. USA
73
:
2414
5
Grisk, O., U. Küster, S. Ansorge.
1993
. The activity of γ-glutamyl transpeptidase (γ-GT) in populations of mononuclear cells from human peripheral blood.
Biol. Chem. Hoppe Seyler
374
:
287
6
Marathe, G. V., N. S. Damle, R. H. Haschemeyer, S. S. Tate.
1980
. Localization of γ-glutamyl transpeptidase in lymphoid cells.
FEBS Lett.
115
:
273
7
Tager, M., A. Ittenson, A. Franke, A. Frey, H. G. Gassen, S. Ansorge.
1995
. γ-Glutamyl transpeptidase: cellular expression in populations of normal human mononuclear cells and patients suffering from leukemias.
Ann. Hematol.
70
:
237
8
Nichols, T. C., J. M. Guthridge, D. R. Karp, H. Molina, D. R. Fletcher, V. M. Holers.
1998
. γ-Glutamyl transpeptidase, an ectoenzyme regulator of intracelluar redox potential, is a component of TM4 signal transduction complexes.
Eur. J. Immunol.
28
:
4123
9
Hogg, N., R. J. Singh, E. Konorev, J. Joseph, B. Kalyanaraman.
1997
.
S-Nitrosoglutathione as a substrate for γ-glutamyl transpeptidase. Biochem. J.
323
:
477
10
Askew, S. C., A. R. Butler, F. W. Flitney, G. D. Kemp, I. L. Megson.
1995
. Chemical mechanisms underlying the vasodilator and platelet anti-aggregating properties of S-nitroso-N-acetyl-dl-penicillamine and S-nitrosoglutathione.
Bioorg. Med. Chem.
3
:
1
11
Mathews, W. R., S. W. Kerr.
1993
. Biological activity of S-nitrosothiols: the role of nitric oxide.
J. Pharmacol. Exp. Ther.
267
:
1529
12
Gibson, A., R. Babbedge, S. R. Brave, S. L. Hart, A. J. Hobbs, J. F. Tucker, P. Wallace, P. K. Moore.
1992
. An investigation of some S-nitrosothiols, and of hydroxy-arginine, on the mouse anococcygeus.
Br. J. Pharmacol.
107
:
715
13
Radomski, M. W., D. D. Rees, A. Dutra, S. Moncada.
1992
.
S-nitroso-glutathione inhibits platelet activation in vitro and in vivo. Br. J. Pharmacol.
107
:
745
14
Stamler, J. S..
1995
. S-Nitrosothiols and the bioregulatory actions of nitrogen oxides through reactions with thiol groups.
Curr. Top. Microbiol. Immunol.
196
:
19
15
Stamler, J. S., L. Jia, J. P. Eu, T. J. McMahon, I. T. Demchenko, J. Bonaventura, K. Gernert, C. A. Piantadsosi.
1997
. Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient.
Science
276
:
2034
16
Merryman, P. F., R. M. Clancy, X. Y. He, S. B. Abramson.
1993
. Modulation of human T cell responses by nitric oxide and its derivative, S-nitrosoglutathione.
Arthritis Rheum.
36
:
1414
17
Akaike, T., K. Inoue, T. Okamoto, H. Nishino, M. Otagiri, S. Fujii, H. Maeda.
1997
. Nanomolar quantification and identification of various nitrosothiols by high performance liquid chromatography coupled with flow reactors of metals and Griess reagent.
J. Biochem.
122
:
459
18
Karp, D. R., R. N. Jenkins, E. O. Long.
1992
. Distinct binding sites on HLA-DR for invariant chain and staphylococcal enterotoxins.
Proc. Natl. Acad. Sci USA
89
:
9657
19
Goodspeed, D. C., T. J. Dunn, C. D. Miller, H. C. Pitot.
1989
. Human γ-glutamyl transpeptidase cDNA: comparison of hepatoma and kidney mRNA in the human and rat.
Gene
76
:
1
20
Stole, E., T. K. Smith, J. M. Manning, A. Meister.
1994
. Interaction of γ-glutamyl transpeptidase with acivicin.
J. Biol. Chem.
269
:
21435
21
Sherman, P. A., J. A. Fyfe.
1989
. Enzymatic assay of deoxyribonucleotide triphosphates using synthetic oligonucleotides as template primers.
Anal. Biochem.
180
:
222
22
Singh, R. J., N. Hogg, J. Joseph, B. Kalyanaraman.
1996
. Mechanism of nitric oxide release from S-nitrosothiols.
J. Biol. Chem.
271
:
18596
23
Lepoivre, M., B. Chenais, A. Yapo, G. Lemaire, L. Thelander, J. P. Tenu.
1990
. Alterations of ribonucleotide reductase activity following induction of the nitrite-generating pathway in adenocarcinoma cells.
J. Biol. Chem.
265
:
14143
24
Kwon, N. S., D. J. Stuehr, C. F. Nathan.
1991
. Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide.
J. Exp. Med.
174
:
761
25
Lepoivre, M., F. Fieschi, J. Coves, L. Thelander, M. Fontecave.
1991
. Inactivation of ribonucleotide reductase by nitric oxide.
Biochem. Biophys. Res. Commun.
179
:
442
26
Lepoivre, M., J. M. Flaman, Y. Henry.
1992
. Early loss of the tyrosyl radical in ribonucleotide reductase of adenocarcinoma cells producing nitric oxide.
J. Biol. Chem.
267
:
22994
27
Roy, B., M. Lepoivre, Y. Henry, M. Fontecave.
1995
. Inhibition of ribonucleotide reductase by nitric oxide derived from thionitrites: reversible modifications of both subunits.
Biochemistry
34
:
5411
28
Novogrodsky, A., S. S. Tate, A. Meister.
1976
. γ-glutamyl transpeptidase, a lymphoid cell-surface marker: relationship to blastogenesis, differentiation, and neoplasia.
Proc. Natl. Acad. Sci. USA
73
:
2414
29
Brezinschek, R. I., P. E. Lipsky, P. Galea, R. Vita, N. Oppenheimer-Marks.
1995
. Phenotypic characterization of CD4+ T cells that exhibit a transendothelial migratory capacity.
J. Immunol.
154
:
3062
30
Pietschmann, P., J. J. Cush, P. E. Lipsky, N. Oppenheimer-Marks.
1992
. Identification of subsets of human T cells capable of enhanced transendothelial migration.
J. Immunol.
149
:
1170
31
Kohem, C. L., R. I. Brezinschek, H. Wisbey, C. Tortorella, P. E. Lipsky, N. Oppenheimer-Marks.
1996
. Enrichment of differentiated CD45RBdim,CD27 memory T cells in the peripheral blood, synovial fluid, and synovial tissue of patients with rheumatoid arthritis.
Arthritis Rheum.
39
:
844
32
MacMicking, J. D., R. J. North, R. LaCourse, J. S. Mudgett, S. K. Shah, C. F. Nathan.
1997
. Identification of nitric oxide synthase as a protective locus against tuberculosis.
Proc. Natl. Acad. Sci. USA
94
:
5243
33
Butler, A. R., P. Rhodes.
1997
. Chemistry, analysis and biological roles of S-nitrosothiols.
Anal. Biochem.
249
:
1
34
Ohta, H., J. N. Bates, S. J. Lewis, W. T. Talman.
1997
. Actions of S-nitrosocysteine in the nucleus tractus solitarii are unrelated to release of nitric oxide.
Brain Res.
746
:
98
35
Mannick, J. B., K. Asano, K. Izumi, E. Kieff, J. S. Stamler.
1994
. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation.
Cell
79
:
1137
36
Mannick, J. B., X. Q. Miao, J. S. Stamler.
1997
. Nitric oxide inhibits Fas-induced apoptosis.
J. Biol. Chem.
272
:
24125
37
Kim, Y.-M., R. V. Talanian, T. R. Billiar.
1997
. Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms.
J. Biol. Chem.
272
:
31138
38
Messmer, U. K., B. Brune.
1996
. Nitric oxide-induced apoptosis: p53-dependent and p53-independent signalling pathways.
Biochem. J.
319
:
299
39
Messmer, U. K., J. C. Reed, B. Brüne.
1996
. Bcl-2 protects macrophages from nitric oxide-induced apoptosis.
J. Biol. Chem.
271
:
20192
40
Xie, K. C., W. Plunkett.
1996
. Deoxynucleotide pool depletion and sustained inhibition of ribonucleotide reductase and DNA synthesis after treatment of human lymphoblastoid cells with 2-chloro-9-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)adenine.
Cancer Res.
56
:
3030
41
Lander, H. M., D. P. Hajjar, B. L. Hempstead, U. A. Mirza, B. T. Chait, S. Campbell, L. A. Quilliam.
1997
. A molecular redox switch on p21(ras): structural basis for the nitric oxide-p21(ras) interaction.
J. Biol. Chem.
272
:
4323
42
Lander, H. M., A. T. Jacovina, R. J. Davis, J. M. Tauras.
1996
. Differential activation of mitogen-activated protein kinases by nitric oxide-related species.
J. Biol. Chem.
271
:
19705