17beta-estradiol augments 18F-FDG uptake and glycolysis of T47D breast cancer cells via membrane-initiated rapid PI3K-Akt activation.

UNLABELLED: Use of (18)F-FDG uptake as a surrogate marker of therapeutic response requires the recognition of biologic factors that influence cancer cell glucose metabolism. Estrogen is a potent stimulator of breast cancer proliferation, a process that requires sufficient energy, which is likely met by increased glycolysis. We thus explored the effect of estrogen on (18)F-FDG uptake in responsive breast cancer cells and investigated the mediating molecular mechanisms. METHODS: T47D breast cancer cells were stimulated with 17ß-estradiol (E(2)) or bovine serum albumin (BSA)-E(2) and measured for (18)F-FDG uptake, lactate release, and mitochondrial hexokinase activity. The effects of antiestrogens, cycloheximide, and major protein kinase inhibitors were investigated. Immunoblots were performed for membrane glucose transporter type 1, phosphorylated phosphatidylinositol 3-kinase (PI3K), and Akt. RESULTS: E(2) augmented T47D cell (18)F-FDG uptake in a dose- and time-dependent manner that preceded and surpassed its proliferative effect. With exposure to 10 nM E(2), protein content-corrected (18)F-FDG uptake reached 172.7% ± 6.6% and 294.4% ± 9.5% of controls by 24 and 48 h, respectively. Lactate release reached 110.9% ± 7.3% and 145.2% ± 10.5% of controls at 24 and 48 h, and mitochondrial hexokinase activity increased to 187.1% ± 31.6% at 24 h. Membrane glucose transporter type 1 expression was unaltered. The effect was absent in estrogen receptor (ER)-negative breast cancer cells and was abrogated by ICI182780, indicating ER dependence. The E(2) effect was not blocked by tamoxifen and was mimicked by membrane-impermeable BSA-E(2), consistent with nongenomic membrane-initiated E(2) action. Inhibition by cycloheximide demonstrated the requirement of a new protein synthesis. Immunoblots displayed rapid phosphorylation of PI3K and Akt within minutes of E(2) treatment, and the specific PI3K inhibitors wortmannin and LY294002 abolished the ability of E(2) to elevate (18)F-FDG uptake. CONCLUSION: Estrogen augments breast cancer cell (18)F-FDG uptake by stimulating glycolysis and hexokinase activity via membrane-initiated E(2) action that activates the PI3K-Akt pathway. These findings yield important insight into our understanding of the biology of breast cancer metabolism and may have potential implications for (18)F-FDG uptake as a surrogate marker of therapeutic response.

Effects of theophylline on radioiodide uptake in MCF-7 breast cancer and NIS gene-transduced SNU-C5 colon cancer cells.

BACKGROUND: We investigated whether theophylline has the potential to increase radioiodide uptake in nonthyroidal cancer cells. MATERIALS AND METHODS: MCF-7 cells that express endogenous sodium/iodide symporter (NIS) and SNU-C5 cells adenovirally transduced with the human NIS gene (SNU-C5/NIS) were treated with 10(-7)-2x10(-4) mol/L theophylline for 24 hours before incubation with (125)I, and then, radioiodide uptake and retention were measured. NIS expression was assessed by immunohistochemistry and Western blot analysis, using an antihuman NIS monoclonal antibody. RESULTS: Theophylline at 10(-6)-2x10(-4) mol/L significantly and dose dependently augmented radioiodide uptake in MCF-7 cells and at 10(-6)-10(-5) mol/L in SNU-C5/NIS cells, without affecting radioiodide efflux. Abrogation by KClO(4)(-) demonstrated that the effect of theophylline occurred through specific iodide transport. Immunohistochemistry revealed dose-dependent increases of NIS staining in MCF-7 and SNU-C5/NIS cells by 10(-6)-10(-4) and 10(-6)-10(-5) mol/L theophylline, respectively. Western blot analysis demonstrated similar findings, showing increased expression of NIS on the membrane of SNU-C5/NIS and MCF-7 cells by theophylline treatment. CONCLUSIONS: Theophylline can augment radioiodide uptake in breast cancer cells and NIS gene-transduced cancer cells through the upregulation of NIS expression. Therefore, further investigations are warranted to explore the potential utility of this phenomenon for enhancing radioiodide-based imaging and therapies of NIS gene-transduced cancer cells.

Fibronectin stimulates endothelial cell 18F-FDG uptake through focal adhesion kinase-mediated phosphatidylinositol 3-kinase/Akt signaling.

UNLABELLED: There has been recent interest in the relationship between (18)F-FDG uptake and the angiogenic activity of endothelial cells (ECs). The angiogenic process is strongly dependent on the interaction of ECs with matrix fibronectin (FN), a key regulator of EC survival, migration, and proliferation. Therefore, we investigated how FN influences EC glucose uptake and elucidated the signaling pathways that mediate this effect. METHODS: Human umbilical vein ECs were allowed to adhere to FN-coated plates and were compared with control cells for (18)F-FDG uptake, membrane GLUT1 levels, and hexokinase activity. The roles of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), and Akt were evaluated with Western blotting, small interfering RNA (siRNA), and specific inhibitors. RESULTS: FN adhesion significantly enhanced the protein-corrected (18)F-FDG uptake in HUVEC, to 2.1-, 2.7-, and 4.3-fold that in control cells by 2, 3, and 5 d, respectively. This effect was mediated by the upregulation of both membrane GLUT1 expression and hexokinase activity and was accompanied by FAK activation. Silencing of FAK signaling by siRNA completely abrogated both FN-induced FAK phosphorylation and (18)F-FDG uptake. FN also activated PI3K and Akt, well-known angiogenesis mediators, and the inhibition of either pathway totally abolished the effect of FN on (18)F-FDG uptake. Nitric oxide, a downstream Akt effector that stimulates glucose uptake, was not involved in the metabolic effect of FN. CONCLUSION: The results of this study demonstrated that an EC-FN interaction induces strong enhancement of (18)F-FDG uptake through the upregulation of GLUT1 expression and hexokinase activity. The findings also showed that the response occurs through FAK-mediated activation of PI3K and Akt, indicating a role for this pathway in modulating EC glucose metabolism.

Mitogen-activated protein kinase signaling enhances sodium iodide symporter function and efficacy of radioiodide therapy in nonthyroidal cancer cells.

UNLABELLED: Although the success of sodium/iodide symporter (NIS) gene-based cancer therapy is critically dependent on the level of radioiodide accumulation attained, recent evidence indicates that successful therapy relies not solely on NIS amount but also crucially on its functional activity. In this study, we investigated the role of kinase-linked signaling on the regulation of NIS function in cancer cells. METHODS: T47D human breast cancer and PC-12 rat pheochromocytoma cells were transduced with the human NIS genes via an adenoviral vector. Cells were measured for (125)I uptake, and the effects of activation or inhibition of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase pathways were evaluated. Membrane localization of NIS was evaluated by biotinylation-immunoblotting and confocal microscopy. (131)I-mediated cancer cell killing was evaluated by clonogenic assays. RESULTS: NIS function was acutely reduced by short stimulation with the PKC activator phorbol 12-myristate 13-acetate and increased by its inhibition with staurosporine or prolonged phorbol 12-myristate 13-acetate exposure. Surprisingly, epidermal growth factor (EGF) caused a strong dose-dependent augmentation of radioiodide transport, accompanied by extracellular signal-regulated kinase (ERK)-1/2 activation. Both effects were completely abrogated by specific MAP kinase kinase (MEK) inhibitors, which also reduced basal NIS function. Hence, radioiodide uptake levels could differ 24-fold, depending on ERK activity. Biotinylation-immunoblotting and confocal microscopy revealed that EGF increases plasma membrane-localized NIS without affecting total cellular levels. EGF stimulation was sufficient to enhance the killing effect of (131)I on the cancer cells. CONCLUSION: Thus, PKC and ERK signaling play important roles in the regulation of NIS function, and control of these signaling pathways may help enhance the efficacy of radioiodide cancer therapy.

Effects of anesthetic agents on cellular 123I-MIBG transport and in vivo 123I-MIBG biodistribution.

OBJECTIVES: Small animal imaging with meta-iodobenzylguanidine (MIBG) allows characterization of animal models, optimization of tumor treatment strategies, and monitoring of gene expression. Anesthetic agents, however, can affect norepinephrine (NE) transport and systemic sympathetic activity. We thus elucidated the effects of anesthetic agents on MIBG transport and biodistribution. METHODS: SK-N-SH neuroblastoma and PC-12 pheochromocytoma cells were measured for (123)I-MIBG uptake after treatment with ketamine (Ke), xylazine (Xy), Ke/Xy, or pentobarbital (Pb). NE transporters were assessed by Western blots. Normal ICR mice and PC-12 tumor-bearing mice were injected with (123)I-MIBG 10 min after anesthesia with Ke/Xy, Ke, Xy, or Pb. Plasma NE levels and MIBG biodistribution were assessed. RESULTS: Cellular (123)I-MIBG uptake was dose-dependently inhibited by Ke and Xy but not by Pb. Treatment for 2 h with 300 microM Ke, Xy, and Ke/Xy decreased uptake to 46.0 +/- 1.6, 24.8 +/- 1.5, and 18.3 +/- 1.6% of controls. This effect was completely reversed by fresh media, and there was no change in NE transporter levels. In contrast, mice anesthetized with Ke/Xy showed no decrease of MIBG uptake in target organs. Instead, uptakes and organ-to-blood ratios were increased in the heart, lung, liver, and adrenals. Plasma NE was notably reduced in the animals with corresponding decreases in blood MIBG, which partly contributed to the increase in target organ uptake. CONCLUSION: In spite of their inhibitory effect at the transporter level, Ke/Xy anesthesia is a satisfactory method for MIBG imaging that allows favorable target tissue uptake and contrast by reducing circulating NE and MIBG.

Favorable biokinetic and tumor-targeting properties of 99mTc-labeled glucosamino RGD and effect of paclitaxel therapy.

UNLABELLED: Compared with the recent advancements in radiohalogenated Arg-Gly-Asp (RGD) peptides for alpha(v)beta(3)-targeted imaging, there has been limited success with (99m)Tc-labeled RGD compounds. In this article, we describe the favorable in vivo kinetics and tumor-imaging properties of a novel (99m)Tc-RGD compound that contains a glucosamine moiety. METHODS: Glucosamino (99m)Tc-d-c(RGDfK) was prepared by incorporating (99m)Tc(CO)(3) to the glucosamino peptide precursor in high radiochemical yield. Cell-binding characteristics were tested on human endothelial cells. Mice bearing RR1022 fibrosarcoma and Lewis lung carcinoma (LLC) tumors were used for in vivo biodistribution and blocking experiments and for imaging studies. Separate LLC-bearing mice underwent antiangiogenic therapy with 0, 20, or 40 mg of paclitaxel per kilogram of body weight every 2 d. Tumor volume was serially monitored, and tumor glucosamino (99m)Tc-d-c(RGDfK) uptake and Western blots of alpha(v) integrin expression were analyzed at day 14. RESULTS: Glucosamino (99m)Tc-d-c(RGDfK) binding to endothelial cells was dose-dependently inhibited by excess RGD. Biodistribution in mice showed rapid blood clearance of glucosamino (99m)Tc-d-c(RGDfK), with substantially lower liver uptake and higher tumor uptake compared with (125)I-c(RGD(I)yV). Tumor uptake was 1.03 +/- 0.21 and 1.18 +/- 0.26 %ID/g at 1 h and 0.85 +/- 0.05 and 0.89 +/- 0.28 %ID/g at 4 h for sarcomas and carcinomas, respectively. Excess RGD blocked uptake by 76.5% and 70.2% for the respective tumors. gamma-Camera imaging allowed clear tumor visualization, with an increase of sarcoma-to-thigh count ratios from 5.5 +/- 0.7 at 1 h to 10.1 +/- 2.2 at 4 h and sustained carcinoma-to-thigh count ratios from 4 to 17 h. Pretreatment with excess cRGDyV significantly reduced tumor contrast on images. Paclitaxel therapy in LLC tumor-bearing mice significantly retarded tumor growth. This was accompanied by a corresponding reduction of tumor glucosamino (99m)Tc-d-c(RGDfK) uptake, which correlated significantly with tumor alpha(v) integrin expression levels. CONCLUSION: Glucosamino (99m)Tc-d-c(RGDfK) has favorable in vivo biokinetics and tumor-imaging properties and may be useful for noninvasive evaluation of tumor integrin expression and response to antiangiogenic therapeutics. Because of the wide accessibility of gamma-cameras and high availability and excellent imaging characteristics of (99m)Tc, glucosamino (99m)Tc-d-c(RGDfK) may be an attractive alternative to radiohalogenated RGD peptides for angiogenesis-imaging research.

Pharmacokinetics and biodistribution of a small radioiodine labeled nerve growth factor fragment.

Nerve growth factor (NGF) exerts various actions on neuronal and non-neuronal tissues and has potential therapeutic utility, but difficulties in using the whole protein have stimulated interest in small NGF fragments. We radioiodinated a small cyclic peptide derived from NGF using the Bolton-Hunter method [125I-C(92-96)], and confirmed binding to high affinity NGF receptors by cross-linkage analysis. Pharmacokinetic characteristics in intravenously injected mice were T 1/2 alpha 5.2 min, T 1/2beta 121.3 min, clearance 11.8+/-0.5 ml/min, and volume of distribution 69.7+/-4.6 ml. Dose-proportionate increases in areas-under-curve and peak-concentrations indicated linear pharmacokinetics. Biodistribution data revealed that clinically relevant doses allowed C(92-96) accumulation sufficient to elicit biological responses in receptor expressing organs including the lungs, liver, spleen, and pancreas.

Evidence that myocardial Na/I symporter gene imaging does not perturb cardiac function.

UNLABELLED: Adenoviral Na/I symporter (NIS) gene transfer has emerged as a promising method for myocardial gene imaging but concern over possible perturbation of cardiac function persists. In this study, we addressed this issue with cultured cardiac cells and serial echocardiography, creatine kinase (CK) measurements, and histologic examination of rats intramyocardially injected with an adenovirus that expresses both NIS and enhanced green fluorescent protein (EGFP) (Ad.EGFP.NIS) or a control virus (Ad.EGFP). METHODS: H9C2 cardiac myoblasts differentiated into cardiomyocytes were evaluated for the effect of Ad.EGFP.NIS and Ad.EGFP infection on viable cell number and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Rats injected intramyocardially with 3 x 10(8) plaque-forming units of Ad.EGFP.NIS (n = 9) or Ad.EGFP (n = 8) underwent serial echocardiographic measurements of heart rate, left ventricular (LV) dimensions, ejection fraction (EF), and fractional shortening (FS) on the day before and on days 4 and 9 after gene transfer. Five Ad.EGFP.NIS rats also underwent repeated (123)I imaging from which ratios of cardiac to mediastinal counts (C/M ratios) were obtained. Separate rats underwent serial measurements of serum CK, myocardial myeloperoxidase assays, and microscopic assessment of inflammation. RESULTS: Cultured cardiac cells showed no change in cell viability or proliferation at 4 and 9 d after Ad.EGFP.NIS or Ad.EGFP infection compared with controls. (123)I scintigraphy demonstrated high cardiac radiouptake at Ad.EGFP.NIS injection sites by days 2 and 4 (C/M ratios, 5.0 +/- 0.6 and 5.1 +/- 1.0, respectively), followed by a complete loss of uptake by day 9 (C/M ratio, 1.4 +/- 0.0). Serial echocardiography revealed no difference in heart rate, LV dimensions, or functional parameters between Ad.EGFP.NIS and Ad.EGFP groups at any given time. Mild reductions in LVEF and LVFS by day 9 compared with baseline were similar for both Ad.EGFP (88.2% +/- 6.4% vs. 79.6% +/- 5.0% for LVEF and 0.55 +/- 0.10 vs. 0.44 +/- 0.05 for LVFS) and Ad.EGFP.NIS groups (88.0% +/- 5.4% vs. 78.7% +/- 4.6% for LVEF and 0.54 +/- 0.09 vs. 0.42 +/- 0.05 for LVFS). Serial serum CK and myocardial myeloperoxidase activities were not elevated in either group, in contrast to substantial increases found after ischemia-reperfusion injury. Histology revealed similar mild inflammatory cell infiltration restricted to the injection site for both groups. CONCLUSION: The results of this study demonstrate that myocardial NIS gene imaging does not cause significant myocardial injury or perturbed cardiac function, other than mild effects likely due to adenoviral vector-associated host response. Thus, this practical and convenient reporter gene strategy can be used safely for noninvasive myocardial gene imaging in living subjects.

In vitro metabolism studies of (18)F-labeled 1-phenylpiperazine using mouse liver S9 fraction.

The in vitro metabolism of 1-(4-[(18)F]fluoromethylbenzyl)-4-phenylpiperazine ([(18)F]1) and 1-(4-[(18)F]fluorobenzyl)-4-phenylpiperazine ([(18)F]2) was investigated using mouse liver S9 fraction. Results were compared to those of in vivo metabolism using mouse blood and bone and to in vitro metabolism using mouse liver microsomes. Defluorination was the main metabolic pathway for [(18)F]1 in vitro and in vivo. Based on TLC, HPLC and LC-MS data, [(18)F]fluoride ion and less polar radioactive metabolites derived from aromatic ring oxidation were detected in vitro, and the latter metabolites were rapidly converted into the former with time, whereas only the [(18)F]fluoride ion was detected in vivo. Similarly, the in vitro metabolism of [(18)F]2 using either S9 fraction or microsomes showed the same pattern as the in vivo method using blood; however, the radioactive metabolites derived from aromatic ring oxidation were not detected in vivo. These results demonstrate that liver S9 fraction can be widely used to investigate the intermediate radioactive metabolites and to predict the in vivo metabolism of radiotracers.

Characteristics and regulation of 123I-MIBG transport in cultured pulmonary endothelial cells.

UNLABELLED: 123I-Metaiodobenzylguanidine (123I-MIBG) is used for lung scintigraphy to assess pulmonary endothelial cell integrity, but its processing at the cellular level has not been investigated to date. We thus characterized the mechanisms that mediate 123I-MIBG transport in pulmonary endothelial cells and investigated the effects of stimuli associated with pulmonary dysfunction. METHODS: Calf pulmonary artery endothelial (CPAE) cells were examined for 123I-MIBG uptake and efflux rates and evaluated for the presence of norepinephrine (NE) transporters by Western blotting. The specificity of 123I-MIBG uptake was investigated with inhibitors of the uptake 1 and uptake 2 transport systems. In addition, we tested the effects of hypoxia (1% O2), phorbol 12-myristate 13-acetate (PMA, a protein kinase C [PKC] activator), and NG-nitro-L-arginine methyl ester (L-NAME) (a nitric oxide synthase inhibitor) treatments on CPAE cell 123I-MIBG uptake. RESULTS: CPAE cells demonstrated a time-dependent increase in 123I-MIBG uptake that reached a relative plateau (mean +/- SD) at 4 h of 375.6% +/- 5.9% the 30-min level. When the culture medium was changed after 30 min of uptake, 123I-MIBG gradually was eluted from the cells at an efflux rate of 43.8% over 2 h. Western blotting confirmed the presence of NE transporters in CPAE cells. The uptake 1 inhibitors desipramine, imipramine, and phenoxybenzamine at 50 micromol/L reduced 123I-MIBG uptake to 55.3% +/- 2.7%, 62.4% +/- 3.5%, and 48.0% +/- 2.2% control levels, respectively, whereas none of the uptake 2 inhibitors had an effect. Exposure to hypoxia resulted in a reduction in 123I-MIBG uptake to 77.5% +/- 0.2% and 50.0% +/- 3.4% control levels at 0.5 and 4 h, respectively. PMA (10 ng/mL) and L-NAME (2 nmol/L) decreased 123I-MIBG uptake to 76.7% +/- 9.0% and 86.5% +/- 5.6% control levels, respectively. CONCLUSION: Pulmonary endothelial cells express NE transporters and actively take up 123I-MIBG through the specific uptake 1 system. Furthermore, 123I-MIBG transport can be reduced by hypoxia, PKC activation, and nitric oxide deficiency, which may contribute partly to the lower levels of lung uptake observed in diseases that compromise pulmonary endothelial cell integrity.

Effects of anesthetic agents and fasting duration on 18F-FDG biodistribution and insulin levels in tumor-bearing mice.

UNLABELLED: Small-animal PET has opened the way for imaging (18)F-FDG uptake in murine tumor models, but the need for anesthesia raises concern over its potential influence on (18)F-FDG kinetics. We thus investigated such effects on cultured cells and on tumor-bearing mice after short- and long-term fasting. METHODS: Lewis lung carcinoma (LLC) cells and cardiomyoblasts were treated for 2 h with a 100 micromol/L concentration of xylazine, ketamine, xylazine plus ketamine (Xy/Ke), or pentobarbital and were measured for (18)F-FDG uptake. LLC tumor-bearing C57BL6 mice that had been kept fasting for either 4 or 20 h were injected with Xy/Ke, pentobarbital, or saline and were administered 1.8 MBq of (18)F-FDG 15 min later. Biodistribution studies and plasma glucose and insulin assays were performed 45 min after injection. Separate anesthetized and control mice underwent (18)F-FDG PET. RESULTS: (18)F-FDG uptake in LLC cells was unaffected by anesthetic agents, whereas xylazine and ketamine caused a small increase of uptake in cardiomyoblasts. In mice kept fasting 4 h, Xy/Ke induced a marked elevation of (18)F-FDG activity (percentage injected dose [%ID]) in blood (6.8 +/- 0.9%ID/g vs. 1.1 +/- 0.6%ID/g) and kidneys while decreasing myocardial uptake (2.3 +/- 1.3%ID/g vs. 4.7 +/- 1.8%ID/g). Target-to-blood ratios were significantly reduced. Pentobarbital caused a moderate increase in blood activity (2.5 +/- 0.8%ID/g), decreased myocardial uptake (2.8 +/- 0.5%ID/g), and reduced target-to-blood ratios. PET images of mice kept fasting 4 h were consistent with the biodistribution data. Insulin levels were lower with Xy/Ke and higher with pentobarbital. In mice kept fasting 20 h, Xy/Ke and pentobarbital increased blood (18)F-FDG activity (5.5 +/- 2.2 and 4.9 +/- 0.9%ID/g vs. 2.4 +/- 0.3%ID/g) and reduced target-to-blood ratios, but these changes were substantially attenuated, compared with those in mice kept fasting 4 h. In addition, insulin levels were low and unaffected by anesthesia. CONCLUSION: Xy/Ke anesthesia markedly elevates blood (18)F-FDG activity and reduces tumor uptake ratios through inhibition of insulin release in mice kept fasting 4 h, whereas pentobarbital induces a similar but less severe response through insulin resistance. These metabolic effects, however, are substantially attenuated after 20 h of fasting. Hence both the choice of anesthetic and the duration of fasting have important effects on (18)F-FDG kinetics and PET images of tumor-bearing mice and should be considered when such studies are performed.

PMA-enhanced neutrophil [18F]FDG uptake is independent of integrin occupancy but requires PI3K activity.

OBJECTIVE: While respiratory burst enhances neutrophil glucose utilization, many neutrophil functions are critically influenced by extracellular matrix interaction and phosphoinositide-3-OH kinase (PI3K) signaling. We thus evaluated the role of RGD integrin occupancy and PI3K inhibition on respiratory burst and [(18)F]fluorodeoxyglucose ([(18)F]FDG) uptake of stimulated neutrophils. METHODS: Human neutrophils were stimulated by 100 ng/ml phorbol-myristate-acetate (PMA), and respiratory burst was measured by cumulative luminescence with lucigenin. [(18)F]FDG uptake and total hexokinase activity was measured 20 min after PMA stimulation in the presence or absence of soluble RGD peptides (200 microg/ml) and/or the PI3K inhibitor wortmannin (200 nM). RESULTS: Phorbol-myristate-acetate induced a 71.7+/-0.9 fold increase in neutrophil oxygen intermediate generation. [(18)F]FDG uptake was increased to 194.6+/-3.7% and hexokinase activity to 145.0+/-2.0% of basal levels (both P<.0005). RGD peptides attenuated respiratory burst activation to 35.6+/-0.2% (P<.005) but did not inhibit stimulated [(18)F]FDG uptake or hexokinase activity. In contrast, without affecting respiratory burst activation, wortmannin inhibited PMA-stimulated [(18)F]FDG uptake to 66.9+/-1.6% and hexokinase activity to 81.0+/-4.2% (both P<.0005), demonstrating its dependence on PI3K activity. Neither RGD nor wortmannin reversed the other's inhibitory effect on stimulated [(18)F]FDG uptake and hexokinase activity or respiratory burst, which suggests the involvement of distinct signaling pathways. CONCLUSION: Neutrophil [(18)F]FDG uptake is enhanced by PMA through a mechanism that requires PI3K activity but is independent of integrin receptor occupancy or respiratory burst activation.

Nitric oxide stimulates 18F-FDG uptake in human endothelial cells through increased hexokinase activity and GLUT1 expression.

UNLABELLED: The endothelium constitutes a functionally active organ critically involved in angiogenesis. Nitric oxide (NO) is an important regulator of vascular homeostasis and angiogenesis and stimulates glucose metabolism in certain cells. We thus investigated the effect of exogenous NO on (18)F-FDG transport in human endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with the NO donors sodium nitroprusside (SNP) or diethylenetriamine (DETA), in concentrations of 1 micromol/L-1 mmol/L for up to 24 h. (18)F-FDG uptake levels corrected for protein content were determined by cellular radioactivity measured after 30-min incubation. Cells were evaluated for total hexokinase activity and plasma membrane glucose transporter 1 (GLUT1) levels, and involvement of potential signaling pathways was investigated by cotreatment with respective protein kinase inhibitors. RESULTS: Both SNP and DETA stimulated HUVEC (18)F-FDG uptake, which began at 16 h and peaked at 24 h. The increase in (18)F-FDG uptake was dose dependent, reaching 464.0% +/- 49.8% and 254.5% +/- 10.8% of control levels at 24 h with 1 mmol/L SNP and DETA, respectively. Exposure of HUVECs to 1 mmol/L SNP resulted in a 3.5 +/- 0.3-fold elevation in hexokinase activity (P < 0.01) and a significant increase in GLUT1 levels. SNP-stimulated (18)F-FDG uptake was abolished by cotreatment with cycloheximide, the tyrosine kinase inhibitor genistein, the phosphatidylinositol-3 kinase (PI3K) inhibitor wortmannin, or the protein kinase C inhibitor staurosporine. CONCLUSION: NO stimulates (18)F-FDG uptake in HUVECs through an increase in GLUT1 expression and hexokinase activity, which appears to involve both protein kinase C and PI3K pathways.