Glucose-6-phosphate Dehydrogenase mRNA Levels Research Articles

Efficacy and mechanism of combination of oxaliplatin with PKM2 knockdown in colorectal cancer.

M2 isomer of pyruvate kinase (PKM2), a key enzyme in aerobic glycolysis, is closely related to cancer development and progression. Suppression of PKM2 exhibits synergistic effects with docetaxel in lung cancer, but the therapeutic potential in colorectal cancer (CRC) is unclear. The aim of the present study was to explore the synergic effects and mechanism of knocking down PKM2 combined with oxaliplatin (a chemosensitizer) treatment in two CRC cell lines (HCT116 and DLD1). The PKM2 gene was initially knocked down using small interfering (si)RNAs (si155 and si156). Subsequently, the effects of PKM2-siRNAs and oxaliplatin, on CRC cells were determined using MTS, cell cycle analysis and apoptosis assays. The mechanism of targeting PKM2 was explored by detecting glucose uptake, lactate secretion fluxes, and the levels of glucose-6-phosphate dehydrogenase (G6PD) mRNA, glutathione (GSH) and reactive oxygen species (ROS). Cell viability in the experimental groups (PKM2-siRNAs, oxaliplatin, PKM2-siRNAs + oxaliplatin) was significantly reduced compared with the control group, and combination treatments (PKM2-siRNAs + oxaliplatin) were more effective than single treatments (PKM2-siRNAs and oxaliplatin only groups). Similar results were observed with the apoptosis assay. The combination groups showed synergistic effects compared with both single treatment groups. Furthermore, glucose uptake and lactate secretion and mRNA levels of G6PD and PKM2 were decreased after PKM2 knockdown in the PKM2-siRNAs and PKM2-siRNAs + oxaliplatin groups. The GSH levels in the PKM2-siRNAs group was significantly lower compared with the negative control group. The ROS levels in the PKM2-siRNAs groups were also significantly increased. The combination of PKM2-siRNAs and oxaliplatin had synergistic effects on CRC cells (HCT116 and DLD1). PKM2 silencing may alter energy metabolism in cancer cells and initiate ROS-induced apoptosis after downregulation of the pentose phosphate pathway by PKM2-siRNAs.

Thioredoxin-interacting protein regulates glucose metabolism and improves the intracellular redox state in bovine oocytes during in vitro maturation.

The extent of glucose metabolism during oocyte maturation is closely related to oocyte developmental potential. Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that negatively regulates glucose uptake into cells. However, little information is available regarding the function of TXNIP in bovine oocytes. Accordingly, the present study was performed to investigate the influence of TXNIP on glucose metabolism in bovine oocytes during in vitro maturation. Pharmacological inhibition of TXNIP by azaserine enhanced glucose uptake and imparted a specific metabolic effect on glycolysis and pentose phosphate pathway (PPP). RNA interference (RNAi) was adopted to further determine the biological significance of TXNIP in regulating glucose metabolism. The maturation rate and the developmental competence of TXNIP siRNA-treated oocytes were significantly improved. Knockdown of TXNIP in bovine oocytes significantly increased glycolysis by increasing the activities of phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase; pyruvate and lactate production; and intracellular ATP level, as well as mitochondrial activity. Furthermore, glucose metabolism through PPP was also enhanced by TXNIP depletion, as TXNIP siRNA treatment promoted glucose-6-phosphate dehydrogenase (G6PDH) activity and NADPH content, and helped maintain a high level of glutathione and a low level of reactive oxygen species within the oocytes. Further studies revealed that inhibition of TXNIP resulted increases in glucose transporter 1 (GLUT1) expression, as well as PFK1 platelet isoform (PFKP) and G6PDH mRNA levels. These results reveal that TXNIP depletion promotes oocyte maturation by enhancing both glycolysis and the PPP. During in vitro maturation of bovine oocytes, TXNIP serves as a key regulator of glucose uptake by controlling GLUT1 expression.

Synergistic effects of dietary carbohydrate and taurine on growth performance, digestive enzyme activities and glucose metabolism in juvenile turbot Scophthalmus maximus L.

A 10-week feeding trial was conducted to evaluate the effects of taurine on growth performance, digestive enzyme activities and glucose metabolism of juvenile turbot Scophthalmus maximus (initial body weight, 3.66 ± 0.02 g) fed diets with different carbohydrate level. With a 2 × 3 two factoral experimental design, two levels of dietary carbohydrate (15% and 21%, respectively) with three levels of taurine (0.4%, 1.2%, and 2.0%, respectively) were used. In addition, the diet with 15% carbohydrate and without taurine supplementation was used as the control. The results showed that, compared with the control group, 1.2% of dietary taurine supplementation significantly increased the specific growth rate, the activities of amylase and lipase in intestine, glycogen content and the mRNA levels of glucokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (G6PD), glycogen synthase (GS) and glucose transporter 2 in liver at 15% of dietary carbohydrate level. Meanwhile, it significantly decreased the blood glucose concentration and the mRNA levels of cytosolic phosphoenolpyruvate carboxykinase (cPEPCK) in liver. In addition, significant interaction between dietary carbohydrate and taurine were observed in blood glucose level. Compared to 15% dietary carbohydrate, 21% dietary carbohydrate significantly increased the blood glucose level at 1.2% and 2.0% dietary taurine level, whereas it had no significant effect at 0.4% dietary taurine supplementation level. At 21% of dietary carbohydrate level, although 1.2% and 2.0% of dietary taurine supplementation had no significant effects on blood glucose level, it significantly decreased the advanced glycationend products level in plasma and the fructose-1, 6-bisphosphatase mRNA level in liver. Moreover, compared to that fed diet with 15% carbohydrate, fish fed diet with 21% carbohydrate had lower activities of amylase and lipase in intestine and lower mRNA levels of G6PD, mitochondrial phosphoenolpyruvate carboxykinase and cPEPCK in liver. Furthermore, significant interactions between dietary carbohydrate and taurine were also observed in the expression of the genes involved in liver glucose metabolism above in addition to GS and cPEPCK. Overall, this study indicated that taurine had a beneficial effect on glucose homeostasis through enhancement of glycolysis, glucose transport, glycogen synthesis and the inhibition of gluconeogenesis in liver of turbot, thus decreased blood glucose level. However, the beneficial effect of taurine on glucose metabolism was affected by dietary carbohydrate level.

Phosphofructokinase and glucose-6-phosphate dehydrogenase in response to drought and bicarbonate stress at transcriptional and functional levels in mulberry

Drought and bicarbonate stress are the most common abiotic stresses limiting plant growth and crop production all over the world. Glycolytic and pentose phosphate pathways have important effects on the resistance response of plants to abiotic stress. Phosphofructokinase (PFK) and glucose-6-phosphate dehydrogenase (G6PDH) are the rate limiting enzymes of the two pathways which control carbon flow through the two pathways, respectively. In Morus alba L. leaves, the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and the proline content increased under drought and bicarbonate stress at first, and then decreased. At the functional level, drought and bicarbonate stress exert an activating effect on the G6PDH activity and an inhibitory effect on the PFK activity in M. alba leaves. At the transcriptional level ATP-PFK, PPi-PFK and G6PDH genes showed no significant change under 80 g/L PEG 6000 treatment. The changes were observed at the ATP-PFK, PPi-PFK and G6PDH mRNA levels under 30 mM NaHCO3 treatment. Although M. alba stopped growing under two different treatments and the changes of G6PDH and PFK are different suggesting that may be additional factors playing important roles in glucose and energy metabolism in plant responses to stresses.

Cortisol 유발 세포독성에 대한 아연 관련 항산화 유전자 발현 증가에 의한 세포보호 효과

The protective effect of zinc against cortisol-induced cell injury was examined in rainbow trout gill epithelial cells. Cells exposed to cortisol for 24 h showed increased leakage of lactate dehydrogenase (LDH) as well as decreased cell viability in a dose-dependent manner. Treatment with zinc (100 μM ZnSO₄) reduced the severity of both LDH release and cell death as well as protected cells against cortisol-induced caspase-3 activation, indicating reduction of apoptosis. Cortisol-induced cell death, leakage of LDH, and caspase-3 activation were blocked by the glucocorticoid receptor antagonist Mifepristone (RU-486), suggesting that cell injury was cortisol-dependent. In addition, we studied the effect of zinc on the expression of antioxidant genes such as metallothionein A (MTA), metallothionein B (MTB), glutathione-S-transferase (GST), and glucose-6-phosphate dehydrogenase (G6PD) during cortisol-induced cell injury. MTA, MTB, GST, and G6PD mRNA levels increased after treatment with zinc or cortisol, separately or in combination. Higher mRNA levels of MTA, MTB, GST, and G6PD were detected when cells were treated with 100 μM ZnSO₄ and 1 μM cortisol in combination at the same time compared to treatment with zinc or cortisol separately. Cells treated with zinc showed increased intracellular free zinc concentrations, and this response was significantly enhanced in cells treated with cortisol and zinc. In conclusion, zinc treatment inhibited cortisol-induced cytotoxicity and apoptosis through indirect antioxidant action.

Nicotinamide, a glucose-6-phosphate dehydrogenase non-competitive mixed inhibitor, modifies redox balance and lipid accumulation in 3T3-L1 cells

AimsExcessive energy uptake of dietary carbohydrates results in their storage as fat and requires glucose-6-phosphate dehydrogenase (G6PD)-mediated NADPH production. We sought to assess whether the nicotinamide-induced reduction of G6PD activity might modulate redox balance and lipid accumulation in 3T3-L1 cells. Main methods3T3-L1 preadipocytes (days 4 and 6 of differentiation) and adipocytes were cultured in the presence of 5 or 25mM glucose. The cells cultured in 25mM glucose were supplemented with nicotinamide (5–15mM). Next, we evaluated the following parameters: cell viability, apoptosis, lipid accumulation, lipolysis, reducing power, reactive oxygen species (ROS), NAD(P)H and NAD(P)+, isocitrate dehydrogenase (IDP), malic enzyme and G6PD, as well as the protein and mRNA levels of G6PD. We also analysed the kinetics of the nicotinamide-induced inhibition of G6PD. Key findingsG6PD mRNA levels increased at day 4 of adipocyte differentiation, whereas G6PD activity progressively increased at days 4 and 6 of differentiation and was reduced in adipocytes. Concomitantly, ROS, reducing power and lipid accumulation increased gradually as the preadipocytes matured into adipocytes. High glucose increased the activity of G6PD, which coincided with an increase in ROS, reducing power and lipid accumulation. All of these changes are prevented by nicotinamide, with the exception of lipid accumulation in adipocytes. Nicotinamide increased IDP activity without affecting NADPH levels. Lastly, nicotinamide inhibited G6PD in a non-competitive mixed way. SignificanceNicotinamide modulates G6PD via a non-competitive mixed inhibition and decreases high glucose-dependent oxidative stress and lipid accumulation. Nicotinamide maintains NADPH levels by increasing the activity of IDP.

Puerarin enhances adipocyte differentiation, adiponectin expression, and antioxidant response in 3T3‐L1 cells

Puerarin, a major isoflavone glycoside from Kudzu root (Pueraria lobata), has been reported to exert antihyperglycemic and antioxidant effects and thus have pharmacological actions in the treatment of diabetes and cardiovascular diseases. We investigated the effects of puerarin on the changes of key gene expression associated with adipocyte differentiation and insulin sensitivity and link to cellular antioxidant response pathways. Puerarin treatment significantly enhanced differentiation of 3T3-L1 preadipocytes accompanying increased lipid accumulation and glucose-6-phosphate dehydrogenase (G6PDH) activity. At a molecular level, puerarin upregulated mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes, an adipocyte-specific fatty acid binding protein (aP2) and GLUT4. Puerarin also caused a significant increase in mRNA level of adiponectin, an important insulin-sensitizing adipocytokine that is downregulated in insulin-resistant and diabetic states. In addition, treatment with puerarin was found to upregulate mRNA levels of G6PDH, glutathione reductase, and catalase, all of which are important for endogenous antioxidant responses. These data suggest that the hypoglycemic effects of puerarin can be attributed to the upregulation of PPARγ and its downstream target genes, GLUT4 and adiponectin expression, leading to increased glucose utilization. Puerarin may also be effective in preventing the rise of oxidative stress during adipocyte differentiation by increasing endogenous antioxidant responses.

Glucose-6-phosphate-dehydrogenase Deficiency as a Risk Factor for Pterygium

Glucose-6-phosphate dehydrogenase (G6PD) is an important site of metabolic control in the pentose phosphate pathway (PPP), providing reducing power (NADPH) and pentose phosphates. The purpose of this study was to investigate the possible involvement of G6PD deficiency (G6PD-) in the pathogenesis of pterygium. Erythrocyte G6PD activity was evaluated in 123 pterygium patients and in 112 age-matched control patients. Enzyme activity, mRNA, rate of growth, green autofluorescence, response to oxidative stress, and cholesterol metabolism were determined in pterygium fibroblasts (PFs) and in normal conjunctival fibroblasts (NCFs) isolated from G6PD normal (NCFs+ and PFs+) and G6PD- (NCFs- and PFs-) patients. Higher prevalence of G6PD- was found in patients affected by primary pterygium than in control subjects, both men and women, suggesting that this enzymatic defect may be a predisposing factor for pterygium. G6PD activity was significantly lower in NCFs- than in NCFs+, but not in PFs- than in PFs+. In PFs-, G6PD mRNA levels were significantly higher than in PFs+. Growth-stimulated NCFs- grew at half the rate of NCFs+, although PFs- and PFs+ grew at the same rate. Increased green autofluorescence and susceptibility to oxidative stress were observed in PFs (+/-) and in NCFs-, but not in NCFs+. Moreover, ex vivo PFs (+/-) accumulated more lipids than corresponding NCFs. The results of this study, although restricted to a limited group of subjects (i.e., those of Sardinian ancestry), suggest that G6PD- not only does not protect against pterygium, but may even be considered a risk factor for the development of this disorder.

인간 HepG2 Cell에서 항산화 효소의 mRNA 발현에 대한 잔대 에틸아세테이트 추출물 효과

잔대 뿌리는 우리나라에서 예로부터 민간약으로 이용되어 오고 있다. 본 연구에서는 인간 간세포인 HepG2에 잔대 뿌리의 에틸아세테이트 추출물을 처리했을 때 sodium nitroprusside(SNP)에 의해 유도된 세포 독성 및 항산화 유전자 발현에 미치는 영향력을 알아보았다. 먼저, 잔대 에틸아세테이트 추출물이 NO에 의해 유도된 세포 사멸을 저해할 수 있는지를 알아보기 위하여 HepG2 세포에 잔대 에틸아세 테이트 추출물(각각 50과 100 ㎍/mL)을 24시간 먼저 처리한 후 세포내에서 NO을 생성시킬 수 있는 0.5 mM SNP를 처리하였다. NO에 의한 세포독성이 에틸아세테이트 추출물에 의해 저해되었다는 것을 mitochondrial dehydrogenase 활성을 알아보는 MTT assay를 실시하여 알아보았다. 더하여 우리는 잔대 에틸아세테이트 추출물이 세포내 항산화 방어 시스템인 Cu,Zn superoxide dismutase(SOD 1), Mn SOD(SOD 2), glutathione peroxidase(GPx), catalase와 glutathione metabolism과 관련되어져 있는 glutathione reductase(GR), γ-glutamyl-cystein synthetase(GCS), glutathione-S-transferase(GST), γ-glutamyltranspeptidase (γ-GT), glucose-6-phosphate dehydrogenase(G6PD)의 mRNA 발현에 미치는 영향을 RT-PCR로 알아보았다. CAT, GCS 그리고 G6PD mRNA 수준이 잔대 에틸아세테이트 추출물 처리 후 증가하였으나, SOD 1, SOD 2, GPx, GST 그리고 γ-GT mRNA 수준은 변화지 않았다. 따라서 잔대 에틸아세테이트 추출물이 간접적 항산화 효과가 있고, 이들 효과는 아마 CAT, GCS, GR 그리고 G6PD 유전자 발현 증가에 의한 것이라고 추정되었다.

Cytotoxicity of Nitric Oxide Is Alleviated by Zinc-Mediated Expression of Antioxidant Genes

Metallothioneins (MTs) are small, cysteine-rich zinc binding proteins that are powerful antioxidants. In this study, we investigated the interaction between zinc, MTs, and other components of the antioxidant defense system in HepG2 cells. Cells were preincubated with zinc and then exposed to sodium nitroprusside (SNP), a nitric oxide (NO) donor. Both zinc pretreatment and SNP exposure separately induced transcription of MT genes (MT1A, MT2A, MT1E, MT1X), as measured using real time-polymerase chain reaction (PCR) after reverse transcription (RT). Pretreatment of HepG2 cells with zinc sulfate (ZnSO4) followed by SNP exposure caused MT and glucose-6-phosphate dehydrogenase (G6PD) mRNA levels to increase more than in cells only exposed to SNP. However, when cells were incubated with N,N,N',N'-tetrakis(2-pyridylmethyl)ethyl-enediamine (TPEN), a membrane-permeant Zn2+ chelator, the stimulation of MT transcription by SNP was blocked, suggesting that SNP-induced upregulation of these genes is zinc-dependent. Human glutathione-S-transferase (hGSTA1) and G6PD mRNA levels in the cells treated with 5 microM TPEN decreased. Additionally, the induction of MT by SNP after zinc pretreatment appears to be mediated by metal-activated transcription factor-1 (MTF-1), which is induced by labile zinc in the cytosol. SNP cytotoxicity was inhibited by preincubation with zinc. Taken together, these results suggest that NO plays an important role in regulation of cellular zinc homeostasis and that NO-mediated release of protein-bound Zn2+ may be an important signal in antioxidant defense.

ZINC-mediated gene expression offers protection against H 2O 2-induced cytotoxicity

The ability of zinc to mobilize defense against reactive oxygen species (ROS) and H 2O 2-induced apoptosis was studied using a primary culture of rainbow trout gill cells. Gill cells were pretreated for 24 h with 100 μM ZnSO 4 followed by 24-h exposure to 100 or 200 μM H 2O 2, or were subjected to 100 μM ZnSO 4 together with 100 or 200 μM H 2O 2. Metallothionein-A (MTA) and metallothionein-B (MTB) mRNA levels were increased after treatment with zinc or H 2O 2, separately or in combination. Similarly, mRNA for glutathione S-transferase (GST) and glucose 6-phosphate dehydrogenase (G6PD) were increased in response to either zinc or H 2O 2, or after sequential treatments with zinc followed by H 2O 2. The stimulatory effects of zinc or H 2O 2 on MTA, MTB, GST, and G6PD mRNA levels could be blocked by addition of the membrane permeable zinc chelator, N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), suggesting that H 2O 2-induced upregulation of these genes is zinc-dependent. Pretreatment with zinc protected the cells from subsequent cell damage and apoptosis, as assessed by lactate dehydrogenase leakage, mitochondrial dehydrogenase activity (MTT assay), caspase-3 activity, and DNA fragmentation. In contrast, when gill cells were coincubated with zinc and H 2O 2 at the same time, H 2O 2 toxicity was higher than after treatment with H 2O 2 alone. It is concluded that zinc had a direct pro-oxidant effect when administered together with H 2O 2, but that pretreatment of zinc inhibited cytotoxicity and apoptosis through an indirect antioxidant action. We propose that the antioxidant action is manifested through zinc-dependent expression of several genes encoding antioxidant proteins (e.g., MTA, MTB, G6PD, and GST). Furthermore, the apparent zinc-dependency of H 2O 2-induced expression of antioxidant genes suggests that zinc might act as a physiological signal to mediate the response to oxidative stress.

Differential effect of clofibrate on acetyl-coa carboxylase mrna level in rat white and brown adipose tissue

Regulation of some lipogenic enzyme gene expression by clofibrate was studied in rat white and brown adipose tissue. In white adipose tissue the drug administration for 14 days to rats resulted in the increase in acetyl-CoA carboxylase, ATP-citrate lyase, and glucose 6-phosphate dehydrogenase mRNA levels. Opposing effect of clofibrate on the acetyl-CoA carboxylase, ATP-citrate lyase, and glucose 6-phosphate dehydrogenase mRNA levels was found in brown adipose tissue. These data indicate a tissue specificity of clofibrate action on lipogenic enzyme gene expression. The results presented in this paper provide further evidence that hypolipidaemia caused by the treatment with clofibrate cannot be related to the inhibition of fatty acid synthesis in white adipose tissue in rat.

A Carbohydrate-Rich Diet Stimulates Glucose-6-Phosphate Dehydrogenase Expression in Rat Hepatic Sinusoidal Endothelial Cells

A carbohydrate-rich diet induces glucose-6-phosphate dehydrogenase (G6PD) in liver parenchymal cells, which supports fatty acid synthesis de novo. Bacterial endotoxins stimulate G6PD expression in hepatic sinusoidal endothelial and Kupffer cells but not in parenchymal cells. This study was designed to elucidate whether G6PD expression is regulated uniformly by dietary carbohydrates in hepatic sinusoidal and parenchymal cells. Freshly isolated cells from five groups of Sprague-Dawley rats were analyzed for G6PD activity and mRNA abundance. The rats were grouped as follows: 1) food deprived for 24 h; 2) food deprived for 24 h followed by consumption of the standard diet for 48 h; 3) food deprived for 24 h followed by consumption of a carbohydrate-rich diet for 48 h; 4) fed standard diet; and 5) fed standard diet followed by consumption of a carbohydrate-rich diet for 48 h. In endothelial cells, G6PD activity was 150% greater in group 3 than in group 1 and 125% greater in group 5 than in group 4. Steady-state G6PD mRNA levels were elevated by 300% in endothelial cells from group 3 compared with those from group 1. In Kupffer cells, G6PD activity and mRNA abundance were not different among the groups. As expected, G6PD expression was 700–1200% greater in parenchymal cells from rats fed a carbohydrate diet (groups 3 and 5) than from controls. Our results indicate that short-term consumption of a carbohydrate-rich diet stimulates G6PD expression in endothelial and parenchymal cells. Because G6PD supports reactive oxygen metabolism, the response may represent a preconditioning of antioxidant pathways in the hepatic cell populations that are targets of sinusoid-born reactive oxygen species during infections.

INTRAUTERINE GROWTH RETARDATION ALTERS MUSCLE GENE EXPRESSION OF ENZYMES INVOLVED IN ENERGY METABOLISM. • 1867

The factors causing intrauterine growth retardation (SGA) affect fetal metabolism and may alter neonatal metabolism. Growth retarded neonates have increased oxygen consumption and energy demands. The molecular mechanisms for this are not known. We hypothesized that gene expression of enzymes involved in energy production, NADH dehydrogenase, mitochondrial malate dehydrogenase(MMD), and glucose-6-phosphate dehydrogenase (G6PD) might be altered in SGA muscle, a metabolically active organ. We performed bilateral uterine artery ligation to induce intrauterine growth retardation (SGA) and sham surgery (C) in pregnant rats on day 19 of gestation (term = 21.5 days), and harvested muscle on day 20 of fetal life, and day 1 (N1), 4 (N4), and 14 (N14) of neonatal life. To measure mRNA levels, we used co-reverse transcription and amplification with bovine rhodopsin as an internal control. Muscle RNA (doped with bovine retinal RNA) was co-reverse transcribed and amplified with PCR using primers to ND, MMD, and G6PD as well as primers for the internal control rhodopsin. PCR products were quantified by phosphorimaging and standardized to controls. mRNA of all 3 enzymes were significantly increased in SGA fetal rat muscle compared to control (C = 1.00; ND = 8.54, MMD = 3.01, G6PD = 3.36)(p<0.01). These findings in muscle are similar to our previous observations of increased expression of these enzymes in SGA fetal liver. After birth (N1, N4, and N14), MMD and G6PD mRNA levels did not differ from controls with the exception of G6PD, which was still elevated at N1 (C = 1.0, G6PD = 2.67). ND mRNA levels in SGA muscle also did not differ from C at N1 and N4. However, ND mRNA levels were significantly decreased in N14 SGA muscle compared to controls (C = 1.0; ND = 0.20). We speculate that increased gene expression of ND, MMD, and G6PD in SGA fetal muscle are a consequence of limited metabolic fuel and oxygen provision during fetal life. With removal from this intrauterine milieu, muscle MMD and G6PD normalize. Interestingly, ND gene expression declines. This may contribute to the altered metabolic state of the SGA neonate.

Potentiation of retinoic acid-induced U-937 differentiation into respiratory burst-competent cells by nitric oxide donors

All- trans retinoic acid (tretinoin) is a known inducer of differentiation of the human monoblastic cell line, U-937. We now report that the ability of retinoic acid (RA) to induce differentiation of U-937 cells into cells possessing respiratory burst activity is enhanced by the known nitric oxide-donating drugs glyceryl trinitrate, molsidomine and CAS 936, and by tetranitromethane in combination with cysteine. RA alone was a strong inducer of U-937 differentiation as indicated by the following responses to 12- O-tetradecanoylphorbol-13-acetate (TPA) stimulation: (1) increase in the percentage of cells staining with nitroblue tetrazolium (NBT); (2) increase in the total amount of formazan (the product of NBT reduction by O 2 −) as determined spectrophotometrically; (3) increase in hexose monophosphate shunt (HMPS) activity as assessed by [ 14C]CO 2 released from d-[1- 14C]glucose. RA was also able to increase mRNA levels for two respiratory burst-related genes and for glucose-6-phosphate dehydrogenase (G6PD), an HMPS enzyme. Other indications of differentiation were reduced cell proliferation, increased adherence and altered nuclear morphology. The observed increase in formazan production and HMPS activity and the reduction of cell proliferation due to RA were augmented by co-treatment with either glyceryl trinitrate, molsidomine, CAS 936 or tetranitromethane plus cysteine. Glyceryl trinitrate alone increased HMPS activity and G6PD mRNA levels and also reduced cell proliferation. Glyceryl trinitrate, molsidomine and CAS 936 are presumed to release nitric oxide and increase intracellular cGMP levels by stimulation of soluble guanylate cyclase. The mechanism of action of tetranitromethane is less certain, although it may also generate reactive nitrogen intermediates. These data suggest that a NO·/ cGMP pathway may augment a retinoic acid-mediated pathway to enhance maturation of U-937 cells with respect to the respiratory burst. Glyceryl trinitrate may act additionally by another pathway.