Hexose Monophosphate Shunt Activity Research Articles

65 HEXOSEMONOPHOSPHATE (HMP) SHUNT ACTIVITY AND GLUCOSE TRANSPORT IN POLYMORPHONUCLEAR (PMNL) AND LYMPHOCYTE CELLS OF PATIENTS WITH GLYCOGENOSIS (GSD) I

GSD I is characterized by deficiency of liver glucose-6-phosphatase (Ia) or of a microsomal transporter for G6P (Ib). Both variants present with similar clinical features; however, GSD Ib patients suffer from neutropenia and impaired functions of their PMNL cells. This study measures HMP shunt activity and glucose transport in PMNL cells and lymphocytes of patients with GSD la and Ib, as well as of controls. The HMP shunt activity decreased significantly in intact PMNL cells of GSD Ib patients as compared to GSD Ia patients and to controls (Ib: 15.5±2.5, la: 47, control: 44.7±5.0 nmole/mg prot/h; P control:Ib<0.001). The reduced HMP shunt activity rose to above normal levels in disrupted GSD Ib PMNL cells (Ib: 117±18, control 75±10; P<0.005). HMP shunt activity of intact lymphocytes was the same in all 3 groups studied. The rate of deoxyglucose transport into GSD Ib PMNL cells was 30% of normal (0.86±0.38 as compared to control of 3.1±0.7 nmole/mg prot/min). This abnormal transport was present neither in GSD Ib lymphocytes nor in GSD Ia PMNL cells and lymphocytes. The striking limitation of glucose transport via the cell membrane of PMNL cells of GSD Ib patients can account for the impairment of leucocyte function which is characteristic of GSD Ib but not found in GSD Ia.

Dynamic assessment of hexose monophosphate shunt activity in the intact rabbit lens by proton NMR spectroscopy

A proton nuclear magnetic resonance technique is demonstrated for ascertaining the real-time contribution of the hexose monophosphate shunt to glucose metabolism in the intact incubated rabbit lens. This measurement requires incubation of the tissue in medium supplemented with [1-13C]glucose, and depends on the presence of the 13C label in the methyl position of lactate which creates satellite resonances by way of 13C - 1H spin-spin scalar coupling. The assumptions required to make the measurement are presented. For lenses maintained under control conditions, a basal level corresponding to 5% hexose monophosphate shunt activity was determined. An eight-fold increase in activity was observed under conditions known to stimulate the shunt.

Neutrophil-endothelial cell interaction. Evidence for and mechanisms of the self-protection of bovine microvascular endothelial cells from hydrogen peroxide-induced oxidative stress.

Bovine microvascular endothelial cells (MEC) were able to degrade the H2O2 generated by phorbol myristate acetate-activated bovine neutrophils or by glucose oxidase with a maximal capacity of 4.0 +/- 1.2 (SD) nmol/10(6) cells/min, corresponding to the H2O2 released by about 3 X 10(6) neutrophils. H2O2 degradation occurred via the glutathione redox cycle and catalase. Degradation via the glutathione redox cycle was coupled with a marked stimulation of the hexose monophosphate shunt activity. The effect of H2O2 on ethidium bromide exclusion and on succinate oxidation was studied. Neither parameter was altered when MEC were exposed to H2O2 produced at rates within their degradative capacity. As soon as this was exceeded, impairment of both functions occurred. It is concluded that endothelial cells can protect themselves from H2O2-induced injury in a well-defined range of environmental H2O2 concentrations by actively degrading the peroxide.

Corynebacterium parvum-elicited hepatic macrophages demonstrate enhanced respiratory burst activity compared with resident kupffer cells in the rat

We have recently demonstrated that release of oxygen-derived free radicals by activated hepatic macrophages may be involved in the pathogenesis of a rat model of liver injury induced by Corynebacterium parvum and endotoxin. In the present study we have compared the respiratory burst activity of isolated normal rat Kupffer cells with that of hepatic macrophages elicited by C. parvum. Superoxide production (O2·−) and glucose oxidation via the hexose monophosphate shunt (HMPS) were low in normal Kupffer cells, but were significantly increased (O2·− 2.1-fold, HMPS 1.7-fold) by phorbol myristate acetate, a stimulant of the respiratory burst. Corynebacterium parvum-elicited hepatic macrophages demonstrated significantly enhanced Superoxide production and HMPS activity compared with normal Kupffer cells, both in the absence of specific stimuli (O2·− 3.3-fold, HMPS 5.3-fold) and after exposure to phorbol myristate acetate (O2·− 4.5-fold, HMPS 5.3-fold). These results demonstrate that normal Kupffer cells are capable of exhibiting respiratory burst activity, but this is markedly increased for hepatic macrophages elicited by an inflammatory stimulus.

Effects of nine synthetic putative metabolites of primaquine on activity of the hexose monophosphate shunt in intact human red blood cells in vitro

Suspensions of washed human red blood cells were treated with nine synthetic putative metabolic derivatives of primaquine (PQ'), and their individual effects on activity of the hexose monophosphate shunt (HMS) were quantitated by radiometric analysis of 14CO 2 from [ 14C]glucose. The most potent HMS stimulant was 5-hydroxy-6-methoxy-8-aminoquinoline (5H6MQ), which caused 10-fold elevation of HMS activity at an estimated concentration of 0.004 mM. Ten millimolar primaquine (PQ) was required to achieve the same effect. Thus, 5H6MQ was approximately 2500-fold more reactive with the HMS than PQ. Other analogs achieved <0.4- to 154-fold increases in HMS reactivity. Patterns of effects on HMS activity indicated that 5-hydroxylation and/or N-dealkylation of PQ strongly enhanced HMS reactivity. In contrast, none of the putative metabolites of PQ activated the proteolytic system known to degrade oxidized protein in red cells, indicating that stimulation of the HMS by the PQ analogs was not related to an injurious oxidative stress. Red cells pretreated with 1.0 mM N-ethylmaleimide (NEM) or with 1.0% (w/v) sodium nitrite to cause glutathione sulfhydryl blockage and conversion of red cell hemoglobin to methemoglobin (metHb), respectively, also showed elevation of HMS activity when exposed to 5H6MQ. These observations suggested that 5H6MQ-induced elevation of HMS activity was at least partially independent of glutathione redox reactions, hydrogen peroxide accumulation and reaction with oxyhemoglobin. The relevance of these observations to proposed mechanisms of hemolytic toxicity of PQ is discussed.

Oxidative activity of hydroxylated primaquine analogs: Non-toxicity to glucose-6-phosphate dehydrogenase-deficient human red blood cells in vitro

The individual effects of two putative metabolites of primaquine (5,6-dihydroxyprimaquine and 5,6-dihydroxy-8-aminoquinoline) on the hexose monophosphate shunt (HMS) and on the ATP-dependent proteolytic system which rapidly degrades oxidized erythrocyte protein were measured in intact red blood cells in vitro from two blood donors. In red cells treated with nitrite (1–40 mM) or phenylhydrazine (0.01–10 mM), proteolytic activity was detected only with concentrations (7.5 mM NaNO 2 and 0.25mM phenylhydrazine) causing >15-fold elevation of HMS activity, and glucose-6-phosphate dehydrogenase (G6PD)-deficient (25% of normal activity) red cell suspensions thus treated showed approximatly 30% greater proteolysis. G6PD-normal and deficient red cells treated with the primaquine analogs, however, did not experience proteolysis with concentrations (0.25 mM) in excess of those causing 17-fold elevation of HMS activity. Stimulation of the HMS by the primaquine analogs thus appears unrelated to an erythrotoxic oxidative stress. Methylene blue is known to cause an elevation of HMS activity through direct and diaphorase II-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) which is independent of injurious oxidative stress. It was found that the putative primaquine metabolites also caused direct and diaphorase II-dependent oxidation of NADPH in dilute hemolysate, thus suggesting that the putative primaquine metabolites have a methylene blue-like redox disposition in red blood cells. Results obtained in this study suggest that the hemolytic toxicity of primaquine may be unrelated to processes which lead to oxidative deterioration of red cell protein.

Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis.

Neutrophil functions of phagocytosis and intracellular killing of bacteria were examined in 40 patients with alcoholic cirrhosis of whom 18 had a superimposed acute alcoholic hepatitis. In 65% of these, defective neutrophil phagocytosis was demonstrable, and in 62.5% there was a defect of intracellular killing of either Staphylococcus aureus or Escherichia coli. Studies of the patients' serum failed to reveal inhibitors of neutrophil function. Additional assays of superoxide (O2-) and hydrogen peroxide production, hexose monophosphate shunt activity, degranulation and cellular levels of granule enzymes and glutathione revealed that these neutrophil defects are caused by both reduced production of superoxide and defects of degranulation. The hydrogen peroxide/superoxide molar ratio was raised in patients' neutrophils, and the strong inverse correlation found between the value of this ratio and intracellular levels of reduced glutathione would be consistent with the hypothesis that the neutrophils from patients with cirrhosis are unable to detoxify hydrogen peroxide effectively and that this is a result of reduced levels of glutathione in the cells. The consequent increase in oxidant stress, both intra- and extracellularly, may be the cause of phagocytic and degranulation defects. The reduced responses of patients' neutrophils may be caused by previous exposure of the cells to activating stimuli in circulation, as evidenced by depleted intracellular levels of granule enzymes and glutathione. Neutrophils from the patients with a superimposed acute alcoholic hepatitis had depressed phagocytosis in the early stages of incubation but, on the whole, neutrophils from these patients had a greater capacity for ingestion and killing of bacteria than neutrophils from patients with cirrhosis alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Subpopulations of neutrophils with increased oxidative product formation in blood of patients with infection.

Stimulated human polymorphonuclear leukocytes (PMNL) have a marked increase in oxidative metabolism, producing reduced oxygen species (e.g., H2O2) that mediate bacterial killing. Previously, quantitation of metabolic responses of PMNL from patients with acute infections employed assays that measure mean activity of the entire PMNL population; such studies reported a modest and highly variable increase in oxidative metabolic responses of such "toxic" PMNL compared with normal cells. To assess metabolic capability of PMNL from 51 patients with acute bacterial infection, we employed a quantitative flow cytometric assay of H2O2-dependent oxidative product formation, the intracellular oxidation of 2',7'-dichlorofluorescin (DCFH). After stimulation by phorbol myristate acetate, the PMNL of patients demonstrated an increase in mean DCFH oxidation (315 +/- 14 and 180 +/- 4.5 amol/cell, patients and controls). Hexose monophosphate shunt activation was similarly increased in stimulated PMNL from bacteremic patients. These data are comparable with previous studies of mean metabolic activities of toxic PMNL. However, these mean values underestimate the quantitative responses of the hyperresponsive ("primed") PMNL within a mixture of normal and primed PMNL in the patients' blood. The flow cytometric assay demonstrated that the PMNL of the patients were composed of two populations. One population of PMNL had normal oxidative responses; the other "primed" population had up to 4.6 times the oxidative product formation of normal cells. Similar priming of circulating PMNL was caused by infection with gram-positive or gram-negative staining bacteria or by Candida species. The proportion and oxidative ability of the primed PMNL occurred independently of the number of juvenile neutrophil forms and independently of "toxic" morphologic changes of Wright's-stained PMNL. On the average, 40% of the PMNL of patients were primed, but the size of the primed PMNL population varied widely between patients (range 0 to 80%). This variable subpopulation may explain the variability of mean responsiveness of the PMNL of patients reported previously. Moreover, the marked increase in oxidative metabolic capability of the primed PMNL may be a significant component of the host response to acute infection. It could also contribute to the damage to host tissues such as pulmonary vascular endothelium during bacteremia.

Lithium Carbonate Therapy in Severe Felty's Syndrome

Lithium carbonate was administered to six patients with severe Felty's syndrome, five of whom had problems with infection. Two patients had granulocyte increments that persisted after therapy was discontinued; in one of them problems with infections resolved. In another patient a transient granulocyte rise accompanied treatment. There was no response in three patients. Granulocyte function was measured in three patients during treatment. It was normal except for subnormal hexose monophosphate shunt activity in two patients. Although serum lithium levels were less than 1.5 mmole/L, serious toxic effect occurred in one patient and significant side effects in the other five. These results support a short trial of lithium carbonate therapy in patients with severe symptomatic Felty's syndrome. Potentially beneficial granulocyte increases occur in a minority of patients only, however, and side effects and toxic effects are common.

Influence of the poly (ADP-ribose) polymerase inhibitor 3-aminobenzamide on macrophage and granulocyte differentiation of HL-60 cells.

We investigated the influence of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (ABA) on induction of phenotypic markers of granulocyte differentiation by retinoic acid and markers of macrophage differentiation by TPA in HL-60 cells. The differentiation of HL-60 cells towards the granulocyte lineage was assessed by hexose monophosphate shunt activity, proportion of cells capable of reducing NBT dye, and the appearance of recognizable neutrophils and bands. The effect of ABA and retinoic acid on NBT dye reduction and appearance of mature neutrophils and bands was synergistic, whereas the effects of these agents on hexose monophosphate shunt activity were additive. The differentiation inducing capacity of ABA in the presence of retinoic acid was dose-related. The influence of ABA on TPA-induced markers of macrophage differentiation was assessed by determining the proportion of adherent cells produced after treatment and by measuring acid phosphatase activity in the adherent cell fraction. In the presence of ABA, the number of cells adhering to plastic declined after day 2 of exposure to TPA, and acid phosphatase activity in adherent cells was inhibited fourfold (p = 0.01). The influence of ABA on the phenotypic markers of granulocyte and macrophage differentiation was detectable at concentrations that were not cytotoxic. The influence of ABA on HL-60 differentiation is similar to that previously reported for human bone marrow CFU-GM. Our data suggest that poly(ADP-ribose) polymerase plays a role in differentiation of HL-60 cells and that HL-60 might provide a useful model for evaluating control mechanisms involved in the differentiation of CFU-GM.

Serum factor from patients with chronic renal failure enhances polymorphonuclear leukocyte oxidative metabolism.

Sera from patients with chronic renal failure (CRF) contain a factor(s) which enhances the oxidative metabolism of polymorphonuclear leukocytes (PMN) as assessed by chemiluminescence (CL), superoxide anion generation, and hexose monophosphate shunt activity. PMN oxidative metabolic activity was higher in CRF sera than in sera from hospitalized patients with normal renal function or in sera from normal healthy subjects. The enhancement occurred regardless of whether PMN were unstimulated or were stimulated by a nonspecific soluble membrane stimulant (phorbol myristate acetate), or by opsonized Candida albicans. The enhanced CL was significantly reduced in their sera after normal renal function was restored with successful renal transplantation. This CL-enhancing factor was also detected in dialysate fluids from CRF patients and in urine from normal healthy subjects. When serum, urine, dialysate fluids of these CRF patients were fractionated by Sephadex G-25 column chromatography, the specific fraction responsible for enhanced CL was found in the molecular weight range less than 1,000 daltons, and is an ethanol extractable substance with natural fluorescence. Our findings suggest that the enhanced PMN stimulatory activity in CRF serum is specifically associated with renal dysfunction and can be useful, along with other conventional parameters, for monitoring the progression of CRF.

ＨＬ‐６０ヒト前骨髄球性白血病細胞の１，２５‐ｄｉｈｙｄｒｏｘｙｖｉｔａｍｉｎＤ３による分化 活性酸素産生能の発現

HL-60 human promyelocyic leukemia cells were treated with 10-7M 1, 25-dihydroxy vitamin (VD3) for 3-4 days. VD3-treated cells expressed a NADPH-oxidase activity as evidenced by chemiluminescence, cytochrome C reduction and hexose monophosphate shunt activity. Furthermore, cytochrome b that is an essential component of electron transport chain in the respiratory burst appeared in the VD3-treated cells. However, reduced-minus-oxidized spectrum of VD3-treated cells was somewhat different from that of human blood neutrophils. The particulate fraction was prepared from the VD3-treated cells that had been activated with phorbol myristate acetate. NADPH induced luminol mediated chemiluminescence (LMCL) in the fraction. The LMCL required halide and was susceptible to NaN3. No luminescence occurred in the particule fraction of untreated HL-60 cells. It was suggested that NADPH-oxidase dependent H2O2 formaion and H2O2-halide-MPO system were involved in LMCL of the particulate fraction.

Proton NMR spectroscopy of glucose consumption by cultured lens epithelial cells.

Proton NMR spectroscopy was performed on media collected from cultured lens epithelial cells of the rabbit eye incubated with Krebs-Ringer's solution containing 5.5mM 13C-glucose (labeled at the C-1 position). Comparing proton resonance intensities of the lactate-methyl group of the C-3 carbon (both 12C and 13C) enabled us to quantify the hexose monophosphate shunt (HMPS) activity. Our results showed that the epithelial cells remained stable in both lactate production and HMPS activity for at least 8 hours. In addition, although tBHP (1mM) resulted in an increase of glucose flow through the HMPS, the rate of lactate production was not affected. In contrast, KCN (2mM) caused a 72% increase of lactate production and a slight (6%) decrease of glucose consumption through the HMPS.

Inhibition of the respiratory burst of human neutrophils by the polyamine oxidase-polyamine system

The addition of the polyamines, spermine and spermidine, to human neutrophils caused a depression of the hexose-monophosphate (HMP) shunt activity of neutrophils stimulated with latex particles but not of unstimulated cells. The effect was dependent on the presence of bovine serum and was not observed when normal human serum was substituted for bovine serum. The polyamine oxidase (PAO) in bovine serum was probably responsible for generating the activity since normal human serum lacks PAO. A role for PAO was further supported by the finding that partially purified bovine PAO in the presence of polyamines similarly mediated inhibition of HMP shunt activity in stimulated neutrophils. Catalase failed to prevent the inhibitory effects of the PAO-polyamine system suggesting that H 2O 2 is not the responsible product. In addition, our results show that human pregnancy serum known to contain PAO activity in the presence of polyamines mediated a similar inhibition of the respiratory burst.

Clofazimine-mediated regulation of human polymorphonuclear leukocyte migration by pro-oxidative inactivation of both leukoattractants and cellular migratory responsiveness

The effects of clofazimine (0.15 – 20 μg/ml) on the spontaneous and N- formyl- L-methionyl- L- leucyl- L-phenylalanine (FMLP)-stimulated migration, membrane-associated oxidative metabolism, degranulation and production of prostaglandin (PG) E 2 by human polymorphonuclear leukocytes (PMNL) in vitro have been investigated. Clofazimine at concentrations of 0.3 μg/ml and greater significantly increased both the spontaneous and FMLP-stimulated chemiluminescence (CL), hexose monophosphate shunt (HMS) activity, myeloperoxidase-mediated protein iodination, auto-iodination, degranulation and PGE 2 production by PMNL. At the same concentrations clofazimine inhibited both random and leukoattractant-induced migration of PMNL. Inhibition of PMNL migration by clofazimine was due to both a cell-directed auto-oxidative mechanism and by functional inactivation of FMLP. Clofazimine mediated inhibition of PMNL migration was prevented by the anti-oxidants cysteine and dapsone but not by the potent inhibitors of PG synthetase indomethacin and piroxicam. Anti-oxidants also protected FMLP from functional inactivation by clofazimine-exposed PMNL. Clofazimine increased both the spontaneous and FMLP-stimulated production of PGE 2 by PMNL from four children with chronic granulomatous disease (CGD). Clofazimine is not an oxidising agent nor did it stimulate membrane-associated oxidative metabolism in CGD or NaF-pulsed normal PMNL. These data show that clofazimine-mediated inhibition of PMNL migration is dependent on intact cellular membrane-associated oxidative metabolism. Clofazimine is therefore a pro-oxidative anti-inflammatory agent.

The protective effect of glucose on soluble rat lens hexokinase in the presence of oxidative stress.

An in vitro animal model was used to characterize the protective effect of glucose on lenses subjected to oxidative stress. Paired rat lenses were incubated in TC-199 medium for six hours in the presence of an oxidant (0.06 mM H2O2, superoxide produced from 5 mM purine, or hydroxyl radical) and 2 mM glucose (control) or no glucose (experimental). Soluble hexokinase (HK) specific activity and lactate production were measured. 0.06 mM H2O2 inactivates 48% of the hexokinase in the absence of glucose; with glucose present hexokinase activity is reduced only 26%. Control experiments without oxidants show a statistically insignificant difference between hexokinase activities in the 0 and 2 mM groups, suggesting that the changes observed are not simply due to the presence or absence of glucose. Hexosemonophosphate shunt activity increases nearly 2.5-fold in the presence of 0.06 mM H2O2 and 2.0, 4.0 or 5.5 mM glucose. This suggests that the loss of hexokinase (a -SH enzyme) in the presence of H2O2 and 0 mM glucose is due to NADPH production inadequate to offset the oxidative stress on enzyme -SH groups. FPLC analysis suggests that type II HK is more susceptible to oxidative inactivation than type I, and further studies have shown that this inactivation is localized to the capsule/epithelium. Lactate levels were measured and controls (without oxidants) were run, to obtain a baseline value for fresh lenses and assess the contribution of endogenous glucose to lactate production. H2O2 levels in superoxide and hydroxyl radical media were measured, and the protective effects of mannitol and catalase were also determined.

Inhibition of human neutrophil function by 6-aminonicotinamide: The role of the hexose monophosphate shunt in cell activation

Stimulation of polymorphonuclear leukocytes with phorbol myristate acetate (PMA) or chemotactic factors such as f-Met-Leu-Phe (fMLP) activates a membrane oxidase which results in the generation of the superoxide anion (O2−) and the oxidation of NADPH to NADP+. The subsequent reduction of NADP+ to NADPH is believed to be directly dependent upon activation of the hexose monophosphate shunt (HMPS). To further understand the role of the HMPS in the oxidative burst, we examined the kinetics of HMPS activation by fMLP and PMA. Both of these agents stimulate an increase in HMPS activity that parallels their production of O2−. To examine the role of the HMPS in cell activation, we treated polymorphonuclear leukocytes with the specific HMPS inhibitor, 6-aminonicotinamide. This pretreatment inhibited fMLP- and PMA-stimulated HMPS activity and O2− release by 80% and 60% respectively with a 50% inhibitory dose (ID50) of 5 × 10−7 M. Measurement of reduced NADPH using 350 nm ultraviolet light-stimulated fluorescence and flow cytometry indicated that 6-aminonicotinamide had no effect on resting levels of NADPH fluorescence but significantly inhibited the fluorescence recovery following stimulation with fMLP or PMA. In contrast, PMA- and fMLP-stimulated membrane depolarization measured with the carbocyanine dye 3,3′-dihexyloxacarbocyanine iodide and chemotaxis to fMLP were unaffected by 6-aminonicotinamide treatment. On the contrary, fMLP- or PMA-stimulated myeloperoxidase release by fMLP or PMA was enhanced by 30% and 150%, respectively, following treatment with 6-aminonicotinamide, suggesting a decreased oxidative inactivation of myeloperoxidase. These findings show that 6-aminonicotinamide can be used to preferentially inhibit the oxidative burst in neutrophils by inhibiting the HMPS and suggest that the HMPS functions primarily to reduce NADP+ for the production of O2− and has minimal involvement in neutrophil chemotaxis and enzyme release.

Interactive Effects of Acute Changes in Temperature and pH on Metabolism in Hepatocytes from the Sea Raven Hemitripterus americanus

Sea raven (Hemitripterus americanus) liver metabolism is similar to liver metabolism in other teleost fishes, with the following two notable exceptions: (1) lactate dehydrogenase levels and lactate utilization rates are extremely low, and (2) fatty-acid oxidation rates are much lower than those in other fish. The interaction of variations in temperature and extracellular pH on sea raven hepatocyte metabolism and intracellular pH were examined by measuring their effects on (1) both the rates of release of 14CO2 and production of 14C-glucose from labeled precursors (glucose, alanine, lactate, and oleate) and (2) the distribution ratios of 14C-5,5 dimethyloxazolidine-2,4dione (DMO). Temperature increases (from 10 to 18.5 C) resulted in increased metabolic rates (Q10 ≅ from 2 to 4). However, temperature decreases (from 10 to 2 C) led to marked deactivation of most pathways examined (Q10 ≅ from 5 to 20). Hexose monophosphate shunt activity was less temperature sensitive than glycolysis (as indicated by ratios of CO₂ release rates from C₁- vs. C₆-labeled glucose) and thus exhibited a relative increase in activity at 2 C. Temperature shifts produced small, transient changes in pHi (<0.15 units), which returned to control values by 1–2 h posttransfer. Decreases in pHe (0.4 units) had no significant effect on rates of CO₂ release and glucose production, and this appears to be due to the ability of the hepatocytes to rapidly return pHi to control values. These patterns of adjustment of pHi following temperature and extracellular pH changes observed in sea raven hepatocytes are different than those observed in other fish species (American eel and rainbow trout).

Hexose monophosphate shunt-stimulated reduction of methemoglobin by divicine

Reduced divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism, reduces methemoglobin efficiently in intact erythrocytes and in hemolysates. Oxidized divicine produces the same effect when glucose or an NADPH-generating system is added to intact erythrocytes or to hemolysates. Although NADPH, NADH, and GSH have no direct methemoglobin-reducing activity in vitro, they convert oxidized divicine to the reduced hydroquinone species, which is responsible for the electron transfer to methemoglobin. Reduction of methemoglobin is optimally observed under nitrogen since, in the presence of oxygen, reduced divicine undergoes autoxidation. Several lines of evidence rule out the reduction of methemoglobin by divicine through an enzyme-catalyzed process, although it is certainly sustained by the hexose monophosphate shunt activity of erythrocytes through the generation of both NADPH and GSH. Thus, the strong enhancing effect that glucose produces on the divicine-dependent methemoglobin reduction within intact normal erythrocytes is completely absent in erythrocytes from glucose-6-phosphate dehydrogenase-deficient subjects. This distinctive behavior might account for the enhanced methemoglobin levels that are found both in vitro in glucose-6-phosphate dehydrogenase-deficient erythrocytes exposed to divicine and in vivo as a typical feature of the acute hemolytic crisis of favic patients.

Glutathione cycle activity and pyridine nucleotide levels in oxidant-induced injury of cells.

Exposure of target cells to a bolus of H2O2 induced cell lysis after a latent period of several hours, which was prevented only when the H2O2 was removed within the first 30 min of injury by addition of catalase. This indicated that early metabolic events take place that are important in the fate of the cell exposed to oxidants. In this study, we described two early and independent events of H2O2-induced injury in P388D1 macrophagelike tumor cells: activation of the glutathione cycle and depletion of cellular NAD. Glutathione cycle and hexose monophosphate shunt (HMPS) were activated within seconds after the addition of H2O2. High HMPS activity maintained glutathione that was largely reduced. However, when HMPS activity was inhibited--by glucose depletion or by incubation at 4 degrees C--glutathione remained in the oxidized state. Total pyridine nucleotide levels were diminished when cells were exposed to H2O2, and the breakdown product, nicotinamide, was recovered in the extracellular medium. Intracellular NAD levels fell by 80% within 20 min of exposure of cells to H2O2. The loss of NADP(H) and stimulation of the HMPS could be prevented when the glutathione cycle was inhibited by either blocking glutathione synthesis with buthionine sulfoximine (BSO) or by inhibiting glutathione reductase with (1,3-bis) 2 chlorethyl-1-nitrosourea. The loss of NAD developed independently of glutathione cycle and HMPS activity, as it also occurred in BSO-treated cells.