Morphine peripheral analgesia depends on activation of the PI3Kgamma/AKT/nNOS/NO/KATP signaling pathway.

Morphine is one of the most prescribed and effective drugs used for the treatment of acute and chronic pain conditions. In addition to its central effects, morphine can also produce peripheral analgesia. However, the mechanisms underlying this peripheral action of morphine have not yet been fully elucidated. Here, we show that the peripheral antinociceptive effect of morphine is lost in neuronal nitric-oxide synthase null mice and that morphine induces the production of nitric oxide in primary nociceptive neurons. The activation of the nitric-oxide pathway by morphine was dependent on an initial stimulation of PI3Kgamma/AKT protein kinase B (AKT) and culminated in increased activation of K(ATP) channels. In the latter, this intracellular signaling pathway might cause a hyperpolarization of nociceptive neurons, and it is fundamental for the direct blockade of inflammatory pain by morphine. This understanding offers new targets for analgesic drug development.

Hydrogen sulfide improves neutrophil migration and survival in sepsis via K+ATP channel activation.

RATIONALE: Recovering the neutrophil migration to the infectious focus improves survival in severe sepsis. Recently, we demonstrated that the cystathionine gamma-lyase (CSE)/hydrogen sulfide (H(2)S) pathway increased neutrophil recruitment to inflammatory focus during sterile inflammation. OBJECTIVES: To evaluate if H(2)S administration increases neutrophil migration to infectious focus and survival of mice. METHODS: Sepsis was induced by cecal ligation and puncture (CLP). MEASUREMENTS AND MAIN RESULTS: The pretreatments of mice with H(2)S donors (NaHS or Lawesson's reagent) improved leukocyte rolling/adhesion in the mesenteric microcirculation as well as neutrophil migration. Consequently, bacteremia levels were reduced, hypotension and lung lesions were prevented, and the survival rate increased from approximately 13% to approximately 80%. Even when treatment was delayed (6 h after CLP), a highly significant reduction in mortality compared with untreated mice was observed. Moreover, H(2)S pretreatment prevented the down-regulation of CXCR2 and l-selectin and the up-regulation of CD11b and G protein-coupled receptor kinase 2 in neutrophils during sepsis. H(2)S also prevented the reduction of intercellular adhesion molecule-1 expression in the endothelium of the mesenteric microcirculation in severe sepsis. Confirming the critical role of H(2)S on sepsis outcome, pretreatment with dl-propargylglycine (a CSE inhibitor) inhibited neutrophil migration to the infectious focus, enhanced lung lesions, and induced high mortality in mice subjected to nonsevere sepsis (from 0 to approximately 80%). The beneficial effects of H(2)S were blocked by glibenclamide (a ATP-dependent K(+) channel blocker). CONCLUSIONS: These results showed that H(2)S restores neutrophil migration to the infectious focus and improves survival outcome in severe sepsis by an ATP-dependent K(+) channel-dependent mechanism.

Hydrogen sulfide augments neutrophil migration through enhancement of adhesion molecule expression and prevention of CXCR2 internalization: role of ATP-sensitive potassium channels.

In this study, we have addressed the role of H(2)S in modulating neutrophil migration in either innate (LPS-challenged naive mice) or adaptive (methylated BSA (mBSA)-challenged immunized mice) immune responses. Treatment of mice with H(2)S synthesis inhibitors, dl-propargylglycine (PAG) or beta-cyanoalanine, reduced neutrophil migration induced by LPS or methylated BSA (mBSA) into the peritoneal cavity and by mBSA into the femur/tibial joint of immunized mice. This effect was associated with decreased leukocyte rolling, adhesion, and P-selectin and ICAM-1 expression on endothelium. Predictably, treatment of animals with the H(2)S donors, NaHS or Lawesson's reagent, enhanced these parameters. Moreover, the NaHS enhancement of neutrophil migration was not observed in ICAM-1-deficient mice. Neither PAG nor NaHS treatment changed LPS-induced CD18 expression on neutrophils, nor did the LPS- and mBSA-induced release of neutrophil chemoattractant mediators TNF-alpha, keratinocyte-derived chemokine, and LTB(4). Furthermore, in vitro MIP-2-induced neutrophil chemotaxis was inhibited by PAG and enhanced by NaHS treatments. Accordingly, MIP-2-induced CXCR2 internalization was enhanced by PAG and inhibited by NaHS treatments. Moreover, NaHS prevented MIP-2-induced CXCR2 desensitization. The PAG and NaHS effects correlated, respectively, with the enhancement and inhibition of MIP-2-induced G protein-coupled receptor kinase 2 expression. The effects of NaHS on neutrophil migration both in vivo and in vitro, together with CXCR2 internalization and G protein-coupled receptor kinase 2 expression were prevented by the ATP-sensitive potassium (K(ATP)(+)) channel blocker, glybenclamide. Conversely, diazoxide, a K(ATP)(+) channel opener, increased neutrophil migration in vivo. Together, our data suggest that during the inflammatory response, H(2)S augments neutrophil adhesion and locomotion, by a mechanism dependent on K(ATP)(+) channels.

The multifaceted photocytotoxic profile of hypericin.

Photodynamic therapy (PDT) is an established anticancer treatment employing a phototoxin (photosensitizer), visible light and oxygen. The latter is photochemically converted into reactive oxygen species, which are highly toxic to the cells. Hypericin, a natural pigment of hypericum plants, is prominent among photosensitizers. The unique perylenequinone structure of hypericin is responsible for its intriguing multifaceted photochemical cytotoxicity. The diverse photodynamic action of hypericin targets a range of subcellular organelles most importantly the mitochondria and the endoplasmic reticulum (ER)-Golgi complex. Hypericin exerts its phototoxicity through intricate mechanisms, implicating key proteins, vital enzymes, organelle membranes and changes in cellular homeostasis. This, depending on drug and light administration conditions, leads to cell death, which occurs mainly by the induction of apoptosis and/or necrosis. Cell photosensitization with hypericin is also associated with the stimulation of macroautophagy, which may promote cell demise when the apoptotic machinery is defective. Herein, we aim to integrate the most important findings with regard to hypericin photocytotoxicity, into a unified scenario, detailing its potential in cancer photomedicine.

The diverse roles of glutathione-associated cell resistance against hypericin photodynamic therapy.

The diverse responses of different cancers to treatments such as photodynamic therapy of cancer (PDT) have fueled a growing need for reliable predictive markers for treatment outcome. In the present work we have studied the differential response of two phenotypically and genotypically different breast adenocarcinoma cell lines, MCF7 and MDA-MB-231, to hypericin PDT (HYP-PDT). MDA-MB-231 cells were 70% more sensitive to HYP PDT than MCF7 cells at LD50. MCF7 were found to express a substantially higher level of glutathione peroxidase (GPX4) than MDA-MB-231, while MDA-MB-231 differentially expressed glutathione-S-transferase (GSTP1), mainly used for xenobiotic detoxification. Eighty % reduction of intracellular glutathione (GSH) by buthionine sulfoximine (BSO), largely enhanced the sensitivity of the GSTP1 expressing MDA-MB-231 cells to HYP-PDT, but not in MCF7 cells. Further inhibition of the GSH reduction however by carmustine (BCNU) resulted in an enhanced sensitivity of MCF7 to HYP-PDT. HYP loading studies suggested that HYP can be a substrate of GSTP for GSH conjugation as BSO enhanced the cellular HYP accumulation by 20% in MDA-MB-231 cells, but not in MCF7 cells. Studies in solutions showed that L-cysteine can bind the GSTP substrate CDNB in the absence of GSTP. This means that the GSTP-lacking MCF7 may use L-cysteine for xenobiotic detoxification, especially during GSH synthesis inhibition, which leads to L-cysteine build-up. This was confirmed by the lowered accumulation of HYP in both cell lines in the presence of BSO and the L-cysteine source NAC. NAC reduced the sensitivity of MCF7, but not MDA-MB-231, cells to HYP PDT which is in accordance with the antioxidant effects of L-cysteine and its potential as a GSTP substrate. As a conclusion we have herein shown that the different GSH based cell defense mechanisms can be utilized as predictive markers for the outcome of PDT and as a guide for selecting optimal combination strategies.

Mitochondria are a primary target of hypericin phototoxicity: synergy of intracellular calcium mobilisation in cell killing.

Hypericin, a naturally occurring anthraquinone synthesised by hypericum, upon light activation exhibits photodynamic cytotoxicity attributed mainly to the production of reactive oxygen species. This study aimed to elucidate the primary subcellular targets and mechanistic aspects of hypericin photosensitization in human prostate carcinoma cells. Depletion of intracellular glutathione (>85%) via inhibition of gamma-glutamyl-cysteine synthase had no effect on hypericin (5 microM) phototoxicity, thus precluding any direct oxidative involvement of H2O2. There was no change in intracellular SOD activity immediately after hypericin irradiation (1.5-5 J cm(-2)). Evaluation of the lysosomal enzyme hexosaminidase activity showed: (a) 60% cell loss 22 h following irradiation (1.5 J cm(-2)) and (b) a steady rate of lysosomal leakage to the cytosol (25%), at the same time and irradiation. However, lysosomal damage appears to be a slower process compared to the rapid loss of mitochondrial function, as reflected from parallel tetrazolium to formazan assays. The activity of cytosolic and mitochondrial aconitase, an enzyme exquisitely sensitive to oxidation, revealed a dose correlated loss of activity in the mitochondria immediately following hypericin photoactivation. The use of ionomycin, which modulates both internal Ca2+ stores and external Ca2+ transport during hypericin photosensitization, profoundly enhanced photocytotoxicity. Our data supports a direct mitochondrial hypericin phototoxicity that does not involve glutathione/H2O2 homeostasis. Further a potential synergistic treatment combining mitochondrial targeting of photosensitisers and Ca2+ mobilisation was identified.

Firefly luciferin-activated rose bengal: in vitro photodynamic therapy by intracellular chemiluminescence in transgenic NIH 3T3 cells.

Photodynamic therapy (PDT) of cancer (1, 2) is a well-established treatment modality that uses light excitation of a photosensitive substance to produce oxygen-related cytotoxic intermediates, such as singlet oxygen or free radicals (3, 4). Although PDT is advantageous over other forms of cancer treatments because of its limited side effects, its main disadvantage is the poor accessibility of light to more deeply lying malignancies. External light sources such as lasers or lamps can be applied either noninvasively to reach tumors that lie well within the penetration depth of the light or in a minimally invasive fashion (interstitial treatments) in which optical fibers are placed intratumorally through needles. Even with the second approach, light distribution over the tumor is not homogeneous and nonidentified metastatic disease is left untreated. CL, the chemical production of light, is exemplified by firefly light emission mediated by the enzymatic (luciferase + ATP) oxidation of D-luciferin to oxyluciferin (5). This mobile light source is a targetable alternative to external sources of illumination. Here we show the in vitro photodynamic effect of rose bengal activated by intracellular generation of light, in luciferase-transfected NIH 3T3 murine fibroblasts.

Crystal and microparticle effects on MDCK cell superoxide production: oxalate-specific mitochondrial membrane potential changes.

We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O2-) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O2- was measured in digitonin-permeabilized MDCK cells by lucigenin (10 microM) chemiluminescence. [(14)C]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O2- in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O2- or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 microg/cm(2). Free oxalate (750 microM), at the level released from COM with EDTA (1 mM), increased O2- (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O2-, implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (deltapsi(m)) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca(2+) transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca(2+) from internal stores. Thus, COM-induced mitochondrial O2- requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O2- production, which is probably regulated by deltapsi(m).

Evidence for intracellular aggregation of hypericin and the impact on its photocytotoxicity in PAM 212 murine keratinocytes.

We have assessed photoinduced toxicity of hypericin in PAM 212 murine keratinocytes and the relationship between concentration, incubation time and light fluence to evaluate the effect of intracellular aggregation at high concentrations. Confocal microscopy was used to establish the subcellular localization of hypericin at 5 and 50 microM and incubation times of 1 and 3 h. From fluorescence uptake time course studies, intracellular hypericin was demonstrated to exist predominantly in the monomeric form for up to 26 h incubation at 5 microM. However, there was a pronounced aggregation effect at 50 microM, with intracellular hypericin fluorescence levels initially showing an increase followed by a decrease with incubation time. This effect was subsequently shown to exert an effect on the phototoxicity of hypericin. On irradiation, the photocytotoxicity for 1 and 7 h incubation with 50 microM hypericin was comparable, whereas using 5 microM the photocytotoxicity showed good correlation with the intracellular fluorescence measurements at 1 and 7 h incubation.

Metabolic fate of the increased yeast amino Acid uptake subsequent to catabolite derepression.

Catabolite repression (CCR) regulates amino acid permeases in Saccharomyces cerevisiae via a TOR-kinase mediated mechanism. When glucose, the preferred fuel in S. cerevisiae, is substituted by galactose, amino acid uptake is increased. Here we have assessed the contribution and metabolic significance of this surfeit of amino acid in yeast undergoing catabolite derepression (CDR). L-[U-(14)C]leucine oxidation was increased 15 ± 1 fold in wild type (WT) strain grown in galactose compared to glucose. Under CDR, leucine oxidation was (i) proportional to uptake, as demonstrated by decreased uptake and oxidation of leucine in strains deleted of major leucine permeases and (ii) entirely dependent upon the TCA cycle, as cytochrome c1 (Cyt1) deleted strains could not grow in galactose. A regulator of amino acid carbon entry into the TCA cycle, branched chain ketoacid dehydrogenase, was also increased 29 ± 3 fold under CCR in WT strain. Protein expression of key TCA cycle enzymes, citrate synthase (Cs), and Cyt1 was increased during CDR. In summary, CDR upregulation of amino acid uptake is accompanied by increased utilization of amino acids for yeast growth. The mechanism for this is likely to be an increase in protein expression of key regulators of the TCA cycle.

Hypericin hydroquinone: potential as a red-far red photosensitizer?

Hypericin hydroquinone is the product of two-electron reduction of hypericin (quinone), a potent phenanthroperylenequinone photosensitizer. In contrast to the quinone, the hydroquinone exhibits strong absorbance in the far-red spectral region. Herein we provide initial evidence on the potential of hypericin hydroquinone as a far-red photosensitizer.

Necrostatin: a potentially novel cardioprotective agent?

BACKGROUND: Necrostatin-1 (Nec-1), a small tryptophan-based molecule, was recently reported to protect the cerebral cortex against ischemia-reperfusion (I/R) injury. We investigated the actions of Nec-1 and its so-called inactive analog, Nec-1i, in the setting of myocardial I/R injury. MATERIALS AND METHODS: The actions of Nec-1 and Nec-1i were examined in cultured C2C12 and H9c2 myocytes, cardiomyocytes isolated from male Sprague-Dawley rats, Langendorff isolated perfused C57Bl/6J mouse hearts and an in vivo open-chest C57Bl/6J mouse heart model. RESULTS: Nec-1 at 30 microM and 100 microM (but not 100 microM Nec-1i) reduced peroxide-induced cell death in C2C12 cells from 51.2 +/- 1.1% (control) to 26.3 +/- 2.9% (p < 0.01 vs control) and 17.8 +/- 0.9% (p < 0.001), respectively. With H9c2 cells cell death was also reduced from 73.0 +/- 0.4% (control) to 56.7 +/- 0% (30 microM Nec-1, p < 0.05) and 45.4 +/- 3.3% (100 microM Nec-1, p < 0.01). In the isolated perfused heart Nec-1 (30 microM) reduced infarct size (calculated as a percentage of the risk area) from 48.0 +/- 2.0% (control) to 32.1 +/- 5.4% (p < 0.05). Nec-1i (30 microM) also reduced infarct size (32.9 +/- 5.1%, p < 0.05). In anesthetized C57Bl/6J mice Nec-1 (1.65 mg/kg), given intraperitoneally to coincide with reperfusion following left anterior descending artery ligation (30 min), also reduced infarct size from 45.3 +/- 5.1% (control) to 26.6 +/- 4.0% (p < 0.05), whilst Nec-1i (1.74 mg/kg) was ineffective (37.8 +/- 6.0%). Stimulus-induced opening of the mitochondrial permeability transition pore (MPTP) in rat cardiomyocytes, as reflected by the time until mitochondrial depolarisation, was unaffected by Nec-1 or Nec-1i at 30 muM but increased at 100 muM i.e. 91% (p < 0.05 vs control) and 152% (p < 0.001) for Nec-1 and Nec-1i, respectively. CONCLUSION: This is the first study to demonstrate that necrostatins inhibit myocardial cell death and reduce infarct size, possibly via a mechanism independent of the MPTP.

Renal oxidative vulnerability due to changes in mitochondrial-glutathione and energy homeostasis in a rat model of calcium oxalate urolithiasis.

Calcium oxalate monohydrate (COM) crystals are the commonest component of kidney stones. Oxalate and COM crystals in renal cells are thought to contribute to pathology via prooxidant events. Using an in vivo rat model of crystalluria induced by hyperoxaluria plus hypercalciuria [ethylene glycol (EG) plus 1,25-dihydroxycholecalciferol (DHC)], we measured glutathione and energy homeostasis of kidney mitochondria. Hyperoxaluria or hypercalciuria without crystalluria was also investigated. After 1-3 wk of treatment, kidney cryosections were analyzed by light microscopy. In kidney subcellular fractions, glutathione and antioxidant enzymes were measured. In mitochondria, oxygen consumption and superoxide formation as well as cytochrome c content were measured. EG plus DHC treatment increased formation of renal birefringent crystal. Histology revealed increased renal tubular pathology characterized by obstruction, distension, and interstitial inflammation. Crystalluria at all time points led to oxidative stress manifest as decreased cytosolic and mitochondrial glutathione and increased activity of the antioxidant enzymes glutathione reductase and -peroxidase (mitochondria) and glucose-6-phosphate dehydrogenase (cytosol). These changes were followed by a significant decrease in mitochondrial cytochrome c content at 2-3 wk, suggesting the involvement of apoptosis in the renal pathology. Mitochondrial oxygen consumption was severely impaired in the crystalluria group without increased mitochondrial superoxide formation. Some of these changes were also evident in hyperoxaluria at week 1 but were absent at later times and in all calciuric groups. Our data indicate that impaired electron flow did not cause superoxide formation; however, mitochondrial dysfunction contributes to pathological events when tubular crystal-cell interactions are uncontrolled, as in kidney stones disease.

Heat-shock protein 25 ameliorates calcium oxalate crystal-mediated oxidative stress in renal epithelial cells.

OBJECTIVE: To investigate whether the antioxidant protection attributed to small heat-shock proteins (sHSPs) affects calcium oxalate stone formation, a pro-oxidant disease. MATERIALS AND METHODS: Canine distal tubular epithelial cells (Madin-Darby canine kidney, MDCK cells) were grown as confluent monolayers. Treatment regimens included control and HS-treated cells (37 degrees C and 42 degrees C for 1 h) with or without calcium oxalate monohydrate (COM) or free oxalate treatment (28 microg/cm2) 16 h later. In digitonin-permeabilized cells, O2- was measured by lucigenin-enhanced chemiluminescence over a 5-min period, to measure mitochondrial O2- production. Protein expression was assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis Western blot analysis using specific antibodies. RESULTS: COM significantly increased O2- production in MDCK cells. HS treatment, which up-regulated HSP25 expression, significantly decreased this O2- production (P < 0.05) but had no effect in control cells. In COM-treated cells (20 h) there was a marked and significant down-regulation of both HSP 25, HSP 70 and heme oxygenase-1 expression compared to cells treated with HS alone (P < 0.05). Free oxalate had no effect on HSP 25 expression. CONCLUSIONS: The results suggest that the COM-induced increase in mitochondrial O2- production in MDCK cells is ameliorated by HSP 25 up-regulation via HS. Specific COM inhibition of HSP 25, HSP 70 and heme oxygenase-1 up-regulation suggests that COM-induced reactive oxygen species damage is unable to benefit from HSP-associated physiological resistance.

Cardioprotection initiated by reactive oxygen species is dependent on activation of PKCepsilon.

To examine whether cardioprotection initiated by reactive oxygen species (ROS) is dependent on protein kinase Cepsilon (PKCepsilon), isolated buffer-perfused mouse hearts were randomized to four groups: 1) antimycin A (AA) (0.1 microg/ml) for 3 min followed by 10 min washout and then 30 min global ischemia (I) and 2 h reperfusion (R); 2) controls of I/R alone; 3) AA bracketed with 13 min of N-2-mercaptopropionyl- glycine (MPG) followed by I/R; and 4) MPG (200 microM) alone, followed by I/R. Isolated adult rat ventricular myocytes (ARVM) were exposed to AA (0.1 microg/ml), and lucigenin was used to measure ROS production. Murine hearts and ARVM were exposed to AA (0.1 microg/ml) with or without MPG, and PKCepsilon translocation was measured by cell fractionation and subsequent Western blot analysis. Finally, the dependence of AA protection on PKCepsilon was determined by the use of knockout mice (-/-) lacking PKCepsilon. AA exposure caused ROS production, which was abolished by the mitochondrial uncoupler mesoxalonitrile 4-trifluoromethoxyphenylhydrazone. In addition, AA significantly reduced the percent infarction-left ventricular volume compared with control I/R (26 +/- 4 vs. 43 +/- 2%; P < 0.05). Bracketing AA with MPG caused a loss of protection (52 +/- 7 vs. 26 +/- 4%; P < 0.05). AA caused PKCepsilon translocation only in the absence of MPG, and protection was lost on the pkcepsilon(-/-) background (38 +/- 3 vs. 15 +/- 4%; P < 0.001). AA causes ROS production, on which protection and PKCepsilon translocation depend. In addition, protection is absent in PKCepsilon null hearts. Our results imply that, in common with ischemic preconditioning, PKCepsilon is crucial to ROS-mediated protection.

Elevated immunoglobulin E against recombinant Brugia malayi gamma-glutamyl transpeptidase in patients with bancroftian filariasis: association with tropical pulmonary eosinophilia or putative immunity.

A major allergen of the lymphatic filarial nematode Brugia malayi, a homologue of gamma-glutamyl transpeptidase (gamma-GT), is involved in the pathology of tropical pulmonary eosinophilia (TPE) through its potent allergenicity and the induction of antibodies against the host pulmonary epithelium. To investigate the immunoglobulin G (IgG) subclass and IgE responses to recombinant B. malayi gamma-GT, we analyzed the results obtained from 51 patients with differing clinical manifestations of bancroftian filariasis. gamma-GT-specific IgG1, rather than IgG4, was the predominant IgG subclass, particularly in patients with TPE (geomean, 6,321 ng/ml; range, 78 to 354,867 ng/ml) and was 75 times higher than in patients with elephantiasis (CP) (P < 0.003) and 185 times higher than in endemic normal individuals (ENL) (P < 0.010). IgG2 responses were low and IgG3 was almost absent, with no significant differences among the groups. gamma-GT-specific IgG4 responses were significantly elevated in those with subclinical microfilaremia (MF) compared to the CP and ENL groups and correlated with the presence of circulating filarial antigen (CAg). More significantly, gamma-GT-specific IgE antibody levels were strikingly elevated in patients with TPE (geomean, 681 ng/ml; range, 61 to 23,841 ng/ml) and in the ENL group (geomean, 106 ng/ml; range, 13 to 1,405 ng/ml) whereas the gamma-GT-specific IgE level was 44 and 61 times lower in those with MF and CP, respectively (P < 0.001). Elevated gamma-GT-specific IgE/IgG4 ratios were demonstrated in patients with TPE (ratio, 45) and ENL (ratio, 107). Because expression of gamma-GT in Brugia infective third-stage larvae (L3) was demonstrated by immunoblot analysis, the elevated gamma-GT-specific IgE antibodies appear to be associated not only with pulmonary pathology but also with possible resistance to infection in lymphatic filariasis.

Inhibition of neutrophil superoxide production by uremic concentrations of guanidino compounds.

In uremia, diminished reactive oxygen intermediate production is an important consequence of impaired neutrophil function. The effects of guanidino compounds, which are known uremic toxins, on neutrophil reactive oxygen intermediate production in vitro were studied. Neutrophils from healthy volunteers were exposed for 3 h to individual guanidino compounds or mixed guanidino compounds (GCmix), at concentrations observed in uremic plasma. After removal of the guanidino compounds, the neutrophils were activated by adhesion, N-formylmethionylleucylphenylalanine, phorbol myristate acetate, or opsonized zymosan, and superoxide production was measured by monitoring lucigenin chemiluminescence. The direct effects of guanidino compounds on superoxide production in activated neutrophils were also measured. The energy status (ATP and creatine phosphate), antioxidant status (total glutathione), and glycolytic flux (lactate production) were measured. GCmix pretreatment decreased superoxide production in activated neutrophils (activated by N-formylmethionylleucylphenylalanine or zymosan) by 50% (P < 0.01), decreased ATP concentrations by 60% (P < 0.05), and inhibited glycolytic flux (lactate production) by 45% (P < 0.01) but did not alter glutathione concentrations. Simultaneous GCmix exposure and activation did not inhibit NADPH oxidase activity in cell lysates but inhibited superoxide formation in zymosan-activated intact neutrophils; this inhibition was reversed after removal of the guanidino compounds. Guanidinosuccinic acid, guanidinopropionic acid, and guanidinobutyric acid, when tested individually, were each as potent as GCmix. The inhibition of neutrophil superoxide generation by guanidino compounds results from decreased energy status. Micromolar concentrations of guanidino compounds significantly inhibit neutrophil metabolism, with serious implications for the functions of neutrophils in host defenses.