Outcomes and toxicities in oligometastatic patients treated with stereotactic body radiotherapy for adrenal gland metastases: A multi-institutional retrospective study.

BACKGROUND: Studies reporting SBRT outcomes in oligometastatic patients with adrenal gland metastases (AGM) are limited. Herein, we present a multi-institutional analysis of oligometastatic patients treated with SBRT for AGM. MATERIAL/METHODS: The Consortium for Oligometastases Research (CORE) is among the largest retrospective series of patients with oligometastases. Among CORE patients, those treated with SBRT for AGM were included. Clinical and dosimetric data were collected. Adrenal metastatic burden (AMB) was defined as the sum of all adrenal GTV if more than one oligometastases is present.Competing risk analysis was used to estimate actuarial cumulative local recurrence (LR) and widespread progression (WP). Kaplan-Meier method was used to report overall survival (OS), local recurrence-free survival (LRFS), and progression-free survival (PFS). Treatment related toxicities were also reported. RESULTS: The analysis included 47 patients with 57 adrenal lesions. Median follow-up was 18.2 months. Median LRFS, PFS, and OS were 15.3, 5.3, and 19.1 months, respectively. A minimum PTV dose BED10 > 46 Gy was associated with an improved OS and LRFS. A prescribed BED10 > 70 Gy was an independent predictor of a lower LR probability. AMB>10 cc was an independent predictor of a lower risk for WP. Only one patient developed an acute Grade 3 toxicity consisting of abdominal pain. CONCLUSION: SBRT to AGM achieved a satisfactory local control and OS in oligometastatic patients. High minimum PTV dose and BED10 prescription doses were predictive of improved LR and OS, respectively. Prospective studies are needed to determine comprehensive criteria for patients SBRT eligibility and dosimetric planning.

Aspirin induces apoptosis in mesenchymal stem cells requiring Wnt/beta-catenin pathway.

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSC) are multipotent progenitor cells that are have found use in regenerative medicine. We have previously observed that aspirin, a widely used anti-inflammatory drug, inhibits MSC proliferation. Here we have aimed to elucidate whether aspirin induces MSC apoptosis and whether this is modulated through the Wnt/beta-catenin pathway. MATERIALS AND METHODS: Apoptosis of MSCs was assessed using Hoechst 33342 dye and an Annexin V-FITC/PI Apoptosis Kit. Expression of protein and protein phosphorylation were investigated using Western blot analysis. Caspase-3 activity was detected by applying a caspase-3/CPP32 Colorimetric Assay Kit. RESULTS: In these MSCs, aspirin induced morphological changes characteristic of apoptosis, cytochrome c release from mitochondria, and caspase-3 activation. Stimulating the Wnt/beta-catenin pathway by both Wnt 3a and GSK-3beta inhibitors (LiCl and SB 216763), blocked aspirin-induced apoptosis and protected mitochondrial function, as demonstrated by decreased cytochrome c release and caspase-3 activity. Aspirin initially caused a time-dependent decrease in COX-2 expression but subsequently, and unexpectedly, elevated the latter. Stimulation of COX-2 expression by aspirin was further enhanced following stimulation of the Wnt/beta-catenin pathway. Application of the COX-2 inhibitor NS-398 suppressed elevated COX-2 expression and promoted aspirin-induced apoptosis. CONCLUSION: These results demonstrate that the Wnt/beta-catenin pathway is a key modulator of aspirin-induced apoptosis in MSCs by regulation of mitochrondrial/caspase-3 function. More importantly, our findings suggest that aspirin may influence MSC survival under certain conditions; therefore, it should be used with caution when considering regenerative MSC transplantation in patients with concomitant chronic inflammatory diseases such as arthritis.

y+ LAT-1 mediates transport of the potent and selective iNOS inhibitor, GW274150, in control J774 macrophages.

This study has characterised the transport mechanism(s) for the novel and selective inhibitor of inducible nitric oxide synthase (iNOS), GW274150, in murine macrophage J774 cells. Transport of GW274150 was saturable (K(m) = 0.24 +/- 0.01 mM and V(max) of 8.5 +/- 0.12 pmol.microg protein(-1) min(-1)), pH-insensitive and largely Na(+)-independent. Transport was also susceptible to trans-stimulation and was significantly inhibited by a 10-fold excess of L-arginine, L-lysine, L-leucine, L-methionine, L-glutamine and 6-diazo-5-oxo-L-norleucine but not by other amino acids or by N-ethylmaleimide. More importantly, the inhibitions caused by the neutral amino acids were critically dependent on Na(+). These results strongly implicate system y(+)L in the transport of GW274150. Northern blot analysis confirmed this by revealing the presence of transcripts for y(+)LAT-1 but not y(+)LAT-2. Thus, taken together, our data show for the first time that J774 macrophages express y(+)LAT-1 transporters and that these carriers mediate transport of GW2741500 at least in these cells.

Signal transduction of cannabinoid CB1 receptors in a smooth muscle cell line.

1. The effects of cannabinoid (CB) receptor stimulation on membrane currents in single cells from the Syrian hamster vas deferens cell line DDT1MF-2 were investigated using the whole cell patch clamp technique. 2. The CB receptor agonist CP55,940 evoked a concentration-dependent transient outward current. The selective CB1 receptor ligand SR141716 (1 microM), but not the selective CB2 receptor ligand SR144528 (1 microM), inhibited the outward current. Pertussis toxin (100 ng ml-1 for 20 h) completely abolished the outward current. 3. Western blotting with an antibody against the rat (r)CB1 receptor showed a band characteristic for the CB1 receptor around 63 kDa in DDT1MF-2 cells. 4. The reversal potential for the outward current measured using a voltage ramp protocol was -84 +/- 5 mV. The current was inhibited by the Ca2+-dependent K+ channel blockers iberiotoxin (10 nM) and charybdotoxin (10 nM). 5. Removal of Ca2+ from the bathing solution, or the addition of 0.1 mM Cd2+ completely abolished the outward current evoked by 10 microM CP55,940. 6. The sarcoplasmic Ca2+ pump inhibitor thapsigargin reduced the outward current evoked by 10 microM CP55,940 in a concentration-dependent manner. 7. The mitogen-activating protein kinase (MAP kinase) inhibitor PD98059, but not the phospholipase C inhibitor U73122, inhibited the outward current evoked by 10 microM CP55,940. 8. The adenylyl cyclase inhibitor SQ22,536 (100 microM) and 8-Br-cyclic AMP (10 microM) significantly reduced the outward current evoked by 10 microM CP55,940. 9. Our data suggest that CB1 receptor stimulation in DDT1MF-2 cells leads to activation of a large conductance Ca2+-dependent K+ channel through a Gi/Go protein-mediated rise in [Ca2+]i, for which both inhibition of adenylyl cyclase and activation of MAP kinase are required. In addition, the cannabinoid-induced increase in [Ca2+]i is likely to arise from capacitive Ca2+ entry.

Nitric oxide-induced cardioprotection in cultured rat ventricular myocytes.

The aim of this study was to investigate the role of nitric oxide (NO) in a cellular model of early preconditioning (PC) in cultured neonatal rat ventricular myocytes. Cardiomyocytes "preconditioned" with 90 min of stimulated ischemia (SI) followed by 30 min reoxygenation in normal culture conditions were protected against subsequent 6 h of SI. PC was blocked by N(G)-monomethyl-L-arginine monoacetate but not by dexamethasone pretreatment. Inducible nitric oxide synthase (NOS) protein expression was not detected during PC ischemia. Pretreatment (90 min) with the NO donor S-nitroso-N-acetyl-L,L-penicillamine (SNAP) mimicked PC, resulting in significant protection. SNAP-triggered protection was completely abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) but was unaffected by chelerythrine or the presence of glibenclamide and 5-hydroxydecanoate. With the use of RIA, SNAP treatment increased cGMP levels, which were blocked by ODQ. Hence, NO is implicated as a trigger in this model of early PC via activation of a constitutive NOS isoform. After exposure to SNAP, the mechanism of cardioprotection is cGMP dependent but independent of protein kinase C or ATP-sensitive K(+) channels. This differs from the proposed mechanism of NO-induced cardioprotection in late PC.

Transmembrane signalling mechanisms regulating expression of cationic amino acid transporters and inducible nitric oxide synthase in rat vascular smooth muscle cells.

The signalling mechanisms involved in the induction of nitric oxide synthase and l-arginine transport were investigated in bacterial lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-stimulated rat cultured aortic smooth muscle cells (RASMCs). The expression profile of transcripts for cationic amino acid transporters (CATs) and their regulation by LPS and IFN-gamma were also examined. Control RASMCs expressed mRNA for CAT-1, CAT-2A and CAT-2B. Levels of all three transcripts were significantly elevated in activated cells. Stimulated CAT mRNA expression and l-arginine transport occurred independently of protein kinase C (PKC), protein tyrosine kinase (PTK) and p44/42 mitogen-activated kinases (MAPKs), but were inhibited by the p38 MAPK inhibitor SB203580, which at 3 microM caused maximum inhibition of both responses. Induction of NO synthesis was independent of p44/42 MAPK activation and only marginally dependent on PKC, but was attenuated markedly by the PTK inhibitors genistein and herbimycin A. SB203580 differentially regulated inducible NO synthase expression and NO production, potentiating both processes at low micromolar concentrations and inhibiting at concentrations of >/=1 microM. In conclusion, our results suggest that RASMCs constitutively express transcripts for CAT-1, CAT-2A and CAT-2B, and that expression of these transcripts is significantly enhanced by LPS and IFN-gamma. Moreover, stimulation of l-arginine transport and induction of NO synthesis by LPS and IFN-gamma appear to be under critical regulation by the p38 MAPK, since both processes were significantly modified by SB203580 at concentrations so far shown to have no effect on other signalling pathways. Thus, in RASMCs, the p38 MAPK cascade represents an important signalling mechanism, regulating both enhanced l-arginine transport and induced NO synthesis.

In vitro and in vivo TNFalpha synthesis modulation by methylguanidine, an uremic catabolyte.

This study was performed in order to examine whether the uraemic toxin, methylguanidine (MG), can modulate tumor necrosis factor alpha (TNF alpha) release by activated macrophages. In this study we have evaluated the ability of MG to influence TNF alpha release in vitro, in Escherichia coli lypopolysaccharide- (LPS)-stimulated J774 cells preincubated overnight with MG, and in vivo in rats treated with MG before and after LPS challenge. Parallel experiments employing N(G)-nitro-L-arginine methyl esther (L-NAME) were also carried out for comparison. The effect of LPS (6 x 10(3) u/ml) on TNF alpha release by J774, following overnight incubation with MG or L-NAME (1 mM), was examined 3 hours after LPS challenge. LPS-stimulated J774 released 287.83+/-88 u/ml TNF alpha into the culture medium. MG (1 mM) significantly inhibited TNF alpha release by 73% (P<0.05). L-NAME (1 mM) significantly inhibited TNF alpha release too by 72.88% (P<0.05). The effect of MG and L-NAME have been also studied in vivo. Serum TNF alpha levels in LPS treated rats 2 h after LPS challenge were 88.33+/-31.7 u/ml as compared to the serum TNF alpha levels of control rats (undetectable). Treatment of rats with MG (30 mg/kg, i.p.) strongly and significantly reduced TNF alpha release (98.71% inhibition; with P<0.001); in the same experimental setting L-NAME (10 mg/kg, i.p.) also significantly reduced TNF alpha serum levels (76.47% inhibition; with P<0.01). These results could indicate that immune disfunction related to uremia may be related to the inhibitory capability of uremic catabolyte, MG, on TNF alpha synthesis and release.

Mechanisms of acute vasodilator response to bacterial lipopolysaccharide in the rat coronary microcirculation.

1. In this study the mechanisms of the acute vasodilator action of bacterial lipopolysaccharide (LPS) were investigated in the rat Langendorff perfused heart. 2. Infusion of LPS (5 microg ml(-1)) caused a rapid and sustained fall in coronary perfusion pressure (PP) of 59 +/- 4 mmHg (n = 12) and a biphasic increase in NO levels determined in the coronary effluent by chemiluminescent detection. Both the fall in PP and the increase in NO release were completely abolished (n = 3) by pretreatment of hearts with the NO synthase inhibitor L-NAME (50 microM). 3. LPS-induced vasodilatation was markedly attenuated to 5 +/- 4 mmHg (n 3) by pretreatment of hearts with the B2 kinin receptor antagonist Hoe-140 (100 nM). 4. Vasodilator responses to LPS were also blocked by brief pretreatment with mepacrine (0.5 microM, n = 3) or nordihydroguaiaretic acid (0.1 microM, n = 4) and markedly attenuated by WEB 2086 (3 microM, n = 4). 5. Thirty minutes pretreatment of hearts with dexamethasone (1 nM), but not progesterone (1 microM), significantly modified responses to LPS. The action of dexamethasone was time-dependent, having no effect when applied either simultaneously with or pre-perfused for 5 min before the administration of LPS but inhibiting the response to LPS by 91 +/- 1% (n = 4) when pre-perfused for 15 min. The inhibition caused by dexamethasone was blocked by 15 min pretreatment with the glucocorticoid receptor antagonist RU-486 (100 nM) or by 2 min pre-perfusion of a 1:200 dilution of LCPS1, a selective antilipocortin 1 (LC1) neutralizing antibody. 6. Treatment with the protein synthesis inhibitor, cycloheximide (10 microM, for 15 min) selectively blunted LPS-induced vasodilatation, reducing the latter to 3 +/- 5 mmHg (n = 3), while having no effect on vasodilator responses to either bradykinin or sodium nitroprusside. 7. These results indicate that LPS-induced vasodilatation in the rat heart is dependent on activation of kinin B2 receptors and synthesis of NO. In addition, phospholipase A2 (PLA2) is activated by LPS resulting in the release of platelet-activating factor (PAF) and lipoxygenase but not cyclo-oxygenase products. These effects are dependent on de novo synthesis of an intermediate protein which remains to be identified.

Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells.

We have examined whether modulation of the polyamine biosynthetic pathway, through inhibition by alpha-difluoromethylornithine (DFMO) of the rate limiting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J774 macrophages. DFMO potentiated LPS-stimulated nitrite production in both a concentration- and time-dependent manner, increasing nitrite levels by 48+/-5% at 10 mM. This effect was observed in cells pre-treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS-induced nitrite production. Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS-induced nitrite production or in the ability of DFMO (10 mM) to potentiate LPS-induced NO synthesis. Metabolism of L-[3H]arginine to L-[3H]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by either DFMO (1-10 mM) or by putrescine (0.001-1 mM), spermidine (0.001-1 mM) or spermine (0.001-1 mM). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70+/-0.07%) by L-NMMA (100 microM). Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with LPS resulted in enhanced (approximately 2 fold) iNOS protein expression. These results show that DFMO potentiates LPS-induced nitrite production in the murine macrophage cell line J774. Since the only known mechanism of action of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we conclude that expression of iNOS can be critically regulated by endogenous polyamines.

Inhibition of inducible nitric oxide synthase expression by novel nonsteroidal anti-inflammatory derivatives with gastrointestinal-sparing properties.

1. The effects of novel nitric oxide-releasing nonsteroidal anti-inflammatory compounds (NO-NSAIDs) on induction of nitric oxide (NO) synthase by bacterial lipopolysaccharide (LPS) were examined in a murine cultured macrophage cell line, J774. 2. LPS-induced nitrite production was markedly attenuated by the nitroxybutylester derivatives of flurbiprofen (FNBE), aspirin, ketoprofen, naproxen, diclofenac and ketorolac, with each compound reducing accumulated nitrite levels by > 40% at the maximum concentrations (100 micrograms ml-1) used. 3. Further examination revealed that nitrite production was inhibited in a concentration-dependent (1-100 micrograms ml-1) manner by FNBE which at 100 micrograms ml-1 decreased LPS-stimulated levels by 63.3 +/- 8.6% (n = 7). The parent compound flurbiprofen was relatively ineffective over the same concentration-range, inhibiting nitrite accumulation by 24 +/- 0.9% (n = 3) at the maximum concentration used (100 micrograms ml-1). 4. FNBE reduced LPS-induced nitrite production when added to cells up to 4 h after LPS. Thereafter, FNBE caused very little or no reduction in nitrite levels. Furthermore NO-NSAIDs (100 micrograms ml-1) did not inhibit the metabolism of L-[3H]-arginine to citrulline by NO synthase isolated from LPS-activated macrophages. 5. Western blot analysis demonstrated that NO synthase expression was markedly attenuated following co-incubation of J774 cell with LPS (1 microgram ml-1; 24 h) and FNBE (100 micrograms ml-1; 24 h). Thus taken together, these findings indicate that NO-NSAIDs inhibit induction of NO synthase without directly affecting enzyme activity. 6. In conclusion our results indicate that NO-NSAIDs can inhibit the inducible L-arginine-NO pathway, and are capable of suppressing NO synthesis by inhibiting expression of NO synthase. The clinical implications of these findings remain to be established.

Regulation of L-arginine transport and nitric oxide release in superfused porcine aortic endothelial cells.

1. We have investigated whether changes in extracellular ion composition and substrate deprivation modulate basal and/or bradykinin-stimulated L-arginine transport and release of nitric oxide (NO) and prostacyclin (PGI2) in porcine aortic endothelial cells cultured and superfused on microcarriers. 2. Saturable L-arginine transport (Km = 0.14 +/- 0.03 mM; Vmax = 2.08 +/- 0.54 nmol min-1 (5 x 10(6) cells)-1) was pH insensitive and unaffected following removal of extracellular Na+ or Ca2+. 3. Cationic arginine analogues, including L-lysine and L-ornithine, inhibited L-arginine transport, whilst 2-methylaminoisobutyric acid, beta-2-amino-bicyclo[2,2.1]-heptane-2-carboxylic acid, L-phenylalanine, 6-diazo-5-oxo-norleucine, L-glutamine, L-cysteine and L-glutamate were poor inhibitors. 4. Deprivation of L-arginine (30 min to 24 h) reduced intracellular free L-arginine levels from 0.87 +/- 0.07 to 0.40 +/- 0.05 mM (P < 0.05) and resulted in a 40% stimulation of L-arginine, L-lysine and L-ornithine transport. 5. L-arginine and NG-monomethyl-L-arginine (L-NMMA), but not N omega-nitro-L-arginine methyl ester (L-NAME), trans-stimulated efflux of L-[3H]arginine. 6. Depolarization of endothelial cells with 70 mM K+ reduced L-arginine influx and prevented the stimulation of transport by 100 nM bradykinin, but agonist-induced release of NO and PGI2 was still detectable. 7. Basal rates of L-arginine transport and NO release were unaffected during superfusion of cells with a nominally Ca(2+)-free solution. Bradykinin-stimulated L-arginine transport was insensitive to removal of Ca2+, whereas agonist-induced NO release was abolished. 8. Although bradykinin-stimulated NO release does not appear to be coupled directly to the transient increase in L-arginine transport, elevated rates of L-arginine influx via system y+ in response to agonist-induced membrane hyperpolarization or substrate deprivation provide a mechanism for enhanced L-arginine supply to sustain NO generation.

Anti-thrombotic effects of a nitric oxide-releasing, gastric-sparing aspirin derivative.

Effects of a nitroxybutylester derivative of aspirin (NCX 4215) on platelet aggregation and prostanoid synthesis were compared to the effects of aspirin. NCX 4215 was approximately seven times more potent than aspirin as an inhibitor of thrombin-induced human platelet aggregation in vitro, but did not inhibit platelet thromboxane synthesis or gastric prostaglandin synthesis. NCX 4215 released nitric oxide when incubated in the presence of platelets and increased platelet levels of cGMP within 10 min of exposure, while aspirin did not. The anti-aggregatory effects of NCX 4215 in vitro were significantly attenuated by 10 microM hemoglobin. In ex vivo studies of ADP- or collagen- or thrombin-induced rat platelet aggregation, aspirin and NCX 4215 had comparable inhibitory effects 3 h after administration. Aspirin (10-120 mg/kg) caused extensive hemorrhagic erosion formation in the stomach of the rat within 3 h of oral administration, while NCX 4215 did not produce significant damage at doses of up to 300 mg/kg, nor when given daily for two weeks at 166 mg/kg. NCX 4215 did not alter systemic arterial blood pressure when administered intravenously to the rat. These studies demonstrate that NCX 4215 has comparable or enhanced anti-thrombotic activity to that of aspirin, but does not cause gastric damage or alter systemic blood pressure. The anti-thrombotic actions of NCX 4215 are, at least in part, due to generation of nitric oxide.

Induction of L-arginine transport and nitric oxide synthase in vascular smooth muscle cells: synergistic actions of pro-inflammatory cytokines and bacterial lipopolysaccharide.

1. The interactions between pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) on L-arginine transporter and inducible nitric oxide synthase (iNOS) activities were examined in rat cultured aortic smooth muscle cells. 2. LPS induced a concentration (0.01-100 micrograms ml-1) and time (8-24 h)-dependent stimulation of nitrite production which was accompanied by a parallel increase in L-arginine transport. 3. Unlike LPS, activation of smooth muscle cells with either interferon-gamma (IFN-gamma, 100 u ml-1), tumour necrosis factor-alpha (TNF-alpha, 300 u ml-1) or interleukin-1 alpha (IL-1 alpha, 100 u ml-1) failed to stimulate L-arginine transport or increase nitrite accumulation. 4. When applied in combination with LPS (100 micrograms ml-1) both IFN-gamma and TNF-alpha, but not IL-1 alpha, enhanced the effects observed with LPS alone. Furthermore, activation of cells with LPS and IFN-gamma had no effect on uptake of the neutral amino acid L-citrulline but selectively increased the Vmax for L-arginine transport 2.8 fold and nitrite levels from 24 +/- 7 to 188 +/- 14 pmol micrograms-1 protein 24 h-1. 5. The substrate specificity, Na- and pH-independence of saturable L-arginine transport in both unactivated (K(m) = 44 microM, Vmax = 3 pmol micrograms-1 protein min-1) and activated (K(m) = 75 microM, Vmax = 8.3 pmol micrograms-1 protein min-1) smooth muscle cells were characteristic of the cationic amino acid transport system y+. 6. Cycloheximide (1 microM) abolished induction of L-arginine transport and nitrite accumulation in response to LPS and IFN-gamma. In contrast, the glucocorticoid dexamethasone (10 microM, 24 h) selectively inhibited nitrite production. 7. Our results demonstrate that pro-inflammatory mediators selectively enhance transport of L-arginine under conditions of sustained NO synthesis by vascular smooth muscle cells. In addition, the differential inhibition of iNOS and L-arginine transporter activity by dexamethasone suggests that distinct signalling pathways mediate induction of the cationic transport protein and iNOS. The close coupling between substrate supply and NO production may have important implications in the pathogenesis of several disease states including endotoxin shock.

Flurbinitroxybutylester: a novel anti-inflammatory drug has enhanced antithrombotic activity.

We have recently shown that the introduction of a nitroxybutylester moiety into flurbiprofen, to form Flurbi-NO, results in a compound with markedly reduced undesired effects in the gastrointestinal tract. This effect has been shown to be linked to nitric oxide release from the Flurbi-NO. Here we have investigated whether this is associated with a reduction in platelet aggregability in vivo, as assessed in a mouse model of thromboembolism and a rat model of platelet aggregation, and found in both models that Flurbi-NO is more potent than flurbiprofen at inhibiting collagen-induced platelet aggregation. Further in vitro studies using human washed platelets and cells in culture suggest that this is due to the release of NO from Flurbi-NO following the action of (possibly plasma) esterases. Together with our earlier data, these results strongly suggest that Flurbi-NO and other members of this class of drugs, have particular potential as anti-thrombotic agents devoid of gastrointestinal side effects.

Discrimination between citrulline and arginine transport in activated murine macrophages: inefficient synthesis of NO from recycling of citrulline to arginine.

1. The kinetics, specificity, pH- and Na(+)-dependency of L-citrulline transport were examined in unstimulated and lipopolysaccharide (LPS)-activated murine macrophage J774 cells. The dependency of nitric oxide production on extracellular arginine or citrulline was investigated in cells activated with LPS (1 microgram ml-1) for 24 h. 2. In unstimulated J774 cells, transport of citrulline was saturable (Kt = 0.16 mM and Vmax = 32 pmol micrograms-1 protein min-1), pH-insensitive and partially Na(+)-dependent. In contrast to arginine, transport of citrulline was unchanged in LPS-activated (1 microgram ml-1, 24 h) cells. 3. Kinetic inhibition experiments revealed that arginine was a relatively poor inhibitor of citrulline transport, whilst citrulline was a more potent inhibitor (Ki = 3.4 mM) of arginine transport but only in the presence of extracellular Na+. Neutral amino acids inhibited citrulline transport (Ki = 0.2-0.3 mM), but were poor inhibitors of arginine transport. 4. Activated J774 cells did not release nitrite in the absence of exogenous arginine. Addition of citrulline (0.01-10 mM), in the absence of exogenous arginine, could only partially restore the ability of cells to synthesize nitrite, which was abolished by 100 microM NG-nitro-L-arginine methyl ester or NG-iminoethyl-L-ornithine. 5. Intracellular metabolism of L-[14C]-citrulline to L-[14C]-arginine was detected in unstimulated J774 cells and was increased further in cells activated with LPS and interferon-gamma. 6. We conclude that J774 macrophage cells transport citrulline via a saturable but nonselective neutral carrier which is insensitive to induction by LPS. In contrast, transport of arginine via the cationic amino acid system y+ is induced in J774 cells activated with LPS.7. Our findings also confirm that citrulline can be recycled to arginine in activated J774 macrophage cells. Although this pathway provides a mechanism for enhanced arginine generation required for NO production under conditions of limited arginine availability, it cannot sustain maximal rates of NO synthesis.

Selective targeting of nitric oxide synthase inhibitors to system y+ in activated macrophages.

Amino acid transport systems mediating uptake of nitric oxide (NO) synthase inhibitors were characterized in the murine macrophage cell line J774. Treatment of J774 cells with bacterial endotoxin (LPS, 1 microgram ml-1, 24 h) selectively increased the transport capacity for NG-monomethyl-L-[14C]arginine (L-NMMA), whereas transport of NG-nitro-L-[3H]arginine (L-NNA) was unaffected. Inhibition studies established that the cationic transport system y+ mediates uptake of L-arginine, L-NMMA and NG-iminoethyl-L-ornithine (L-NIO). A neutral transporter, with low substrate specificity and insensitive to LPS, mediates uptake of L-citrulline, L-NNA and its methyl ester L-NAME. We conclude that enhanced expression of the y+ transporter in LPS-stimulated macrophages may facilitate the targeting of selective inhibitors of inducible NO synthase to activated cells generating NO in endotoxin shock.

Selective inhibition by dexamethasone of induction of NO synthase, but not of induction of L-arginine transport, in activated murine macrophage J774 cells.

1. Effects of dexamethasone on induction of nitric oxide (NO) synthase and L-arginine transport by lipopolysaccharide (LPS) were examined in a murine cultured macrophage cell line J774. Metabolism of L-arginine to L-citrulline and subsequent changes in intracellular amino acids pools were correlated with changes in nitrite production. 2. Despite a high intracellular concentration of arginine in activated J774 cells, LPS (1 microgram ml-1, 8 h) induced a 2.4 fold increase in arginine transport. Treatment of cells with cycloheximide (1 microgram ml-1) inhibited the time-dependent (1-8 h) induction of NO synthase and arginine transport mediated by LPS. 3. Induction of NO synthase by LPS (1 microgram ml-1, 24 h) alone was accompanied by a marked increase in arginine utilisation leading to decreased intracellular arginine levels and elevated intracellular and extracellular L-citrulline levels. These changes were further enhanced in the presence of interferon-gamma (IFN-gamma, 100 units ml-1, 24 h). 4. Dexamethasone (1 microM) abolished the increases in both nitrite and citrulline production induced by LPS alone but only partially reversed the combined effects of LPS and IFN-gamma. In contrast, treatment of cells with dexamethasone (10 microM) had no effect on the LPS-mediated induction of arginine transport or the decrease in intracellular arginine concentration. 5. We conclude that induction of arginine transporter activity in LPS-stimulated J774 cells involves de novo synthesis of carrier proteins, which increases transport of exogenous arginine during enhanced NO production. Moreover, the intracellular signalling pathways mediating induction of arginine transport and of NO synthase by LPS in activated macrophages diverge, since only the latter is sensitive to dexamethasone.

Bacterial endotoxin rapidly stimulates prolonged endothelium-dependent vasodilatation in the rat isolated perfused heart.

1. The effects of bacterial lipopolysaccharide (Escherichia coli 0111-B4; LPS) on coronary vascular tone were examined in the isolated perfused heart of the rat. The role of nitric oxide and/or prostaglandin products of the cyclo-oxygenase pathway in mediating the actions of LPS were also investigated. 2. Coronary vascular tone was raised and maintained by a continuous perfusion of the thromboxane-mimetic U46619 (5 nM). LPS perfusion (0.1-100 micrograms ml-1) caused a concentration-dependent fall in coronary tone without any significant change in the force of cardiac contractility. 3. At 5 micrograms ml-1, LPS reduced perfusion pressure by 38 +/- 9 mmHg. This effect was rapid in onset, maximal within the first 5 min and sustained for 90 +/- 10 min (n = 6). 4. The vasodilatation induced by LPS was dependent on the presence of an intact endothelium and abolished following endothelial damage caused by air embolism. 5. NG-nitro-L-arginine methylester (L-NAME; 50 microM) or NG-nitro-L-arginine (L-NOARG; 50 microM) blocked the vasodilatation induced by LPS (5 micrograms ml-1). The inhibition caused by these arginine analogues was partially reversed by 1 mM L- but not D-arginine. 6. The vasodilator action of LPS was also completely blocked by the glucocorticoid, dexamethasone (10 microM) but unaffected by indomethacin (10 microM). 7. These results suggest that LPS evokes rapid release of nitric oxide (NO) in the microvasculature of the rat isolated heart presumably via activation of the constitutive L-arginine-NO pathway in the endothelium. Furthermore, the lack of effect of indomethacin suggests that prostaglandins released via the cyclo-oxygenase pathway are not involved in mediating this action of LPS.

Arginine uptake and metabolism in cultured murine macrophages.

Transport of L-arginine and nitrite production were examined in the murine macrophage cell line J774. Lipopolysaccharide (LPS) induced a dose- and time-dependent stimulation of nitrite production which was further increased in the presence of interferon-gamma (IFN-gamma). Nitrite synthesis was dependent on extracellular arginine and inhibited in the presence of L-lysine. Treatment of cells with LPS and IFN-gamma caused a reduction in intracellular L-arginine concentration which was accompanied by increases in the levels of L-citrulline in both the cells and culture medium. These findings indicate that activation of J774 cells with LPS produces an increase in both L-arginine transport and nitrite synthesis. The elevated rate of L-arginine transport in activated J774 cells may provide a mechanism for sustained substrate supply during enhanced utilization of L-arginine for the generation of nitric oxide.