Antioxidation theory of non-steroidal anti-inflammatory drugs based upon the inhibition of luminol-enhanced chemiluminescence from the myeloperoxidase reaction

The action of non-steroidal anti-inflammatory drugs (NSAIDS) has been ascribed to their ability to block the reaction of arachidonate with cyclooxygenase/peroxidase, thus inhibiting the cellular production of inflammation mediators such as prostaglandins and leukotrienes. However, this and other polymorphonuclear leukocyte (PMN) peroxidases such as myeloperoxidase (MPO) would still be capable of producing destructive oxidants which contribute to inflammation. Sulindac sulfide (Clinoril^® sulfide) has recently been shown to scavenge oxidant products of prostaglandin cyclooxygenase/peroxidase and MPO. The MPO–H₂O₂–Cl^– reaction is a potent antimicrobial/cytotoxic system which produces HOCl, a strong oxidant. MPO itself has the ability to oxidize drugs and cellular components, and may be the main oxidant in PMN defenses. An antioxidant/free radical scavenger action of NSAIDs against the MPO system could be a primary mechanism of their anti-inflammatory effects. Other antioxidant/free radical scavengers have anti-inflammatory effects.MPO activity has previously been quantified using chemiluminescence (CL). In this study, NSAIDs from various classes were tested for their ability to inhibit luminol-enhanced CL from MPO. The most potent NSAIDs against MPO-CL were BW755C, phenylbutazone, indomethacin and sulindac sulfide. Salicylates and arylacetic acid derivatives, such as naproxen, also decreased MPO-CL. These drugs are also effective against CL from PMNs, of which MPO may be a main source. This effect of NSAIDs on MPO suggests that NSAIDs may impair the killing mechanism of the PMN, preventing cell destruction and release of inflammation mediators. PMN MPO appears to be a target for the antioxidant/free radical scavenging effects of NSAIDs.     

Altered function of synovial fluid granulocytes in patients with acute inflammatory arthritis

In rheumatoid arthritis (RA) a chronic inflammatory state exists in which the synovial fluid is periodically filled with large numbers of polymorphonuclear leukocytes (PMNs). Oxygen radicals produced by these cells have been implicated as mediators of tissue damage and may be directly involved in the pathogenesis of RA. We examined the production of oxygen radicals by synovial fluid PMNs (SFPMNs) and peripheral blood PMNs (PB-PMNs) by measuring chemiluminescence (CL) as well as Superoxide anion (O ₂ ^– ) release. Increased spontaneous CL in the presence of luminol and increased CL in response to phorbol myristate acetate (PMA) was observed in SF-PMNs when compared to PB-PMNs. When zymosan was used as the stimulus in the absence of luminol, a slightly lower CL response was observed in SF-PMNs as compared to PB-PMNs. No significant differences were observed in the generation of O ₂ ^– generation with any stimulus. Preincubation of normal PBPMNs in 10% synovial fluid enhanced the luminol-dependent spontaneous and PMA-stimulated CL as well as zymosan-stimulated CL. When O ₂ ^– release from normal PB-PMNs pretreated with 10% synovial fluid was compared to untreated controls, enhancement of spontaneous O ₂ ^– release was observed. PMA- and zymosan-stimulated responses did not differ significantly from controls. Increased spontaneous and PMA-stimulated release of myeloperoxidase (MPO) was also observed in normal PB-PMNs pretreated with synovial fluid. These findings may explain the increased luminol-dependent CL since this type of CL requires the presence of MPO. Our findings suggest that the enhanced chemiluminescence observed in normal PMNs treated with synovial fluids may be related to increases in spontaneous O ₂ ^– generation and myeloperoxidase release. Increased MPO release may account for enhanced CL observed in SF-PMNs.     

Luminol-independent chemiluminescence by phagocytes is markedly enhanced by dexamethasone,not by other glucocorticosteroids

The effect of several glucocorticosteroids on the generation of reactive oxygen species (ROS) was examined. The ROS assessed were O ₂ ^– , H₂O₂, OH·, and chemiluminescence (CL) (determined in the presence or absence of luminol), generated by both opsonized zymosan-stimulated neutrophils or monocytes and by the xanthine-xanthine oxidase system. Except for luminol-independent CL, only high concentrations (10^–4 M) of steroids could decrease each ROS. In contrast, luminol-independent CL generation in the phagocyte system was increased in a dose-dependent manner by the addition of dexamethasone, but not by any other steroid. Further, in lymphocyte cultures stimulated with Con A for four days, luminol-independent CL generation was demonstrated and enhanced by the addition of dexamethasone, although CL generation was not detected in the absence of dexamethasone. These findings provide evidence that CL does not always represent light specific to ROS, and they suggest the possibility that dexamethasone induces emission of light at sites of inflammation.     

Effects of HC 20-511 (ketotifen) on chemiluminescence of human neutrophils

The effects of ketotifen on the chemiluminescence (CL) and chemotaxis of human neutrophils were studied in vitro. Stimulations of neutrophils by concanavalin A (Con A), calcium ionophore A23187 (CI) and formyl-methionyl-leucyl-phenylalanine (FMLP) were strongly suppressed in contrast with that by zymosan, which was only slightly inhibited in CL assay; the inhibitory effect of ketotifen was dose dependent. The addition of ketotifen even after stimulations of CI and FMLP also inhibited CL response. The neutrophils which were activated by incubation with FMLP (10^–7 M) at 37°C for 60 min emitted much greater light in CI-induced CL assay than those without the pretreatment with FMLP. Such enhanced CL of activated cells was also markedly suppressed by ketotifen. On the other hand, ketotifen did not show any inhibitory effect on the direct movement of neutrophils by FMLP (10^–7 M) at the concentrations which inhibited CL responses. These unique pharmacological activities of ketotifen are encouraging for its potential clinical usage as an antiinflammatory agent in some disorders associated with neutrophils as well as for its experimental usefulness for the analysis of various functions of neutrophils.     

Sulindac sulfide selectively increases sensitivity of ABCC1 expressing tumor cells to doxorubicin and glutathione depletion

ATP-binding cassette (ABC) transpo rters ABCC1 (MRP1), ABCB1 (P-gp), and ABCG2 (BCRP) contribute to chemotherapy failure. The primary goals of this study were to characterize the efficacy and mechanism of the non­steroidal anti-inflammatory drug (NSAID), sulindac sulfide, to reverse ABCC1 mediated resistance to chemother­apeutic drugs and to determine if sulindac sulfide can influence sensitivity to chemotherapeutic drugs independently of drug efflux. Cytotoxicity assays were performed to measure resistance of ABC-expressing cell lines to doxoru­bicin and other chemotherapeutic drugs. NSAIDs were tested for the ability to restore sensitivity to resistance selected tumor cell lines, as well as a large panel of standard tumor cell lines. Other experiments characterized the mechanism by which sulindac sulfide inhibits ABCC1 substrate and co-substrate (GSH) transport in isolated membrane vesicles and intact cells. Selective reversal of multi-drug resistance (MDR), decreased efflux of doxor­ubicin, and fluorescent substrates were demonstrated by sulindac sulfide and a related NSAID, indomethacin, in resistance selected and engineered cell lines expressing ABCC1, but not ABCB1 or ABCG2. Sulindac sulfide also inhibited transport of leukotriene C₄ into membrane vesicles. Sulindac sulfide enhanced the sensitivity to doxoru­bicin in 24 of 47 tumor cell lines, including all melanoma lines tested (7-7). Sulindac sulfide also decreased intra­cellular GSH in ABCC1 expressing cells, while the glutathione synthesis inhibitor, BSO, selectively increased sensitivity to sulindac sulfide induced cytotoxicity. Sulindac sulfide potently and selectively reverses ABCC1-mediated MDR at clinically achievable concentrations. ABCC1 expressing tumors may be highly sensitive to the direct cytotoxicity of sulindac sulfide, and in combination with chemotherapeutic drugs that induce oxidative stress.     

Polymorphonuclear leukocyte response to stimulation in vitro during pregnancy

Oxidative metabolism of polymorphonuclear leukocytes (PMNLs) isolated from pregnant women in the third trimester and from controls were studied using zymosan-induced chemiluminescence (CL) and f-Met-Leu-Phe-stimulated superoxide (O ₂ ^– ) generation. CL was significantly increased during pregnancy, but a decrease was noted in cytochromec reduction. Total cellular levels of -glucuronidase and lysozyme were diminished in PMNLs from pregnant subjects, with unaltered concentrations of cytosol lactate dehydrogenase. The capacity of PMNLs from pregnant women to degranulate did not differ from controls. It is suggested that during pregnancy, in vivo stimulation of PMNLs may occur to account for these changes.     

Time course of superoxide generation by leukocytes—The MCLA chemiluminescence system

This study was performed to examine the pattern of Superoxide (O ₂ ^– ·) generation from leukocytes using the O ₂ ^– · specific chemiluminescence (CL) method.Cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (MCLA) was used as a CL probe. The appropriate conditions of the MCLA method was first determined for the evaluation of the time course of O ₂ ^– · generation by leukocytes. The time course of O ₂ ^– · generation obtained by the MCLA-CL system was compared with that by the luminol-dependent CL, electron spin resonance (ESR)/spin trapping, and cytochromec systems. Following stimulation by three different stimulants (PMA, OZ, FMLP), leukocytes continuously generated O ₂ ^– · for up to 5 h in the MCLA-CL system, irrespective of the kind of stimulation. The curves obtained by generation ceased more rapidly in the luminol-CL, ESR/spin trapping, and cytochromec systems. A 50% activity of the initial value was observed at 70 min in the MCLA-CL system, but 30, 10 and 35 min in the other systems, respectively. The CL or O ₂ ^– · generation value decreased to less than 1% (possible termination) at 300, 90, 120 and 180 min, respectively. With the exception of ESR studies with OZ, the cell viability was not significantly affected in any of the trials. These results indicate that leukocytes can generate O ₂ ^– · much longer than previously estimated and that the MCLA-CL-system is the most suitable system for the measurement of the O ₂ ^– · generation by leukocytes.     

Comparison of the effects of antioxidant non-steroidal anti-inflammatory drugs against myeloperoxidase and hypochlorous acid luminol-enhanced chemiluminescence

The interaction of myeloperoxidase (MPO) with H₂O₂ and Cl^– provides a potent antimicrobial/cytotoxic system for polymorphonuclear leukocytes (PMNs). MPO-related cytotoxicity may be associated with the formation of toxic oxidant MPO intermediates, HOCl, or both. MPO itself is able to oxidize drugs and cellular components. Non-steroidal anti-inflammatory drugs (NSAIDs) able to act as antioxidant free radical scavengers have recently been shown to inhibit luminol-enhanced chemiluminescence (CL) which results from the MPO–H₂O₂–Cl^– reaction. CL is a measure of the activity of this reaction. At that time it was not clear whether the source of CL which these NSAIDs affected was HOCl or components of the initial MPO–H₂O₂–Cl^– reaction. A NSAID antioxidant mechanism could affect MPO oxidant intermediates and HOCl.This study compares the effects of antioxidant NSAIDs, methylprednisone and free radical scavengers against MPO-based and NaOCl-based luminol-enhanced CL. Most NSAIDs which affected both MPO and NaOCl-CL appeared to share similar mechanisms, suggesting that MPO oxidant internediates and HOCl are susceptible to NSAID effects. However, most NSAIDs were more effective against MPO-CL. The effect of these NSAIDs against MPO-CL followed the profile of NSAIDs effective in previous studies against PMN-CL. One exception to this was methylprednisone, which has no effect on PMN or MPO-CL, yet inhibited NaOCl-CL. This and other data suggest that MPO and not HOCl-related reactions are a major source of PMN-CL. Less effective NSAIDs affected NaOCl-CL better than MPO-CL. While both HOCl and MPO oxidant intermediates may be affected by NSAIDs, it appears that MPO oxidant intermediates or MPO itself are the primary target for NSAID antioxidant free radical scavenging mechanisms. These antioxidant effects impair the major killing system of the PMN and may be NSAIDs' primary anti-inflammatory mechanism. Although our data suggests the production of superoxide anion and hydroxyl radical from the MPO–H₂O₂–Cl^– reaction, the actual presence or involvement of these free radical species is not confirmed herein.     

Benoxaprofen: A pro-oxidant anti-inflammatory drug?

Benoxaprofen inhibited the random motility and migration to the leucoattractants endotoxin-activated serum (EAS) and f-met-leu-phe of human polymorphonuclear leucocytes (PMNL)in vitro. Inhibition of random and leucoattractant-induced migration was observed at drug concentrations of >1×110^–6 M and 1×10^–5 M respectively. Benoxaprofenper se was not leucotactic but was pro-oxidative in that it stimulated PMNL hexose-monophosphate shunt activity, chemiluminescence, myeloperoxidase-mediated iodination reactions and degranulation. The drug also mediated auto-oxidation of PMNL as measured by cellular auto-lodination. The relationship between benoxaprofen-mediated inhibition of PMNL migration and activation of oxidative metabolism was investigated using the anti-oxidants ascorbate and levamisole at concentrations of 10^–2 M and 10^–3 M respectively. These agents prevented the decreased motility and auto-oxidation of PMNL induced by 10^–4 M benoxaprofen. Benoxaprofen (10^–4 M) did not inhibit the migration of PMNL from 3 children with chronic granulomatous disease thus showing that intact PMNL oxidative metabolism is required for the induction of drug-mediated inhibition of cell motility. Ingestion of therapeutic doses of benoxaprofen for 7 days by normal adults gave serum drug concentrations greater than those required for detectable effects on PMNL functionsin vitro (mean serum value 126 g/ml). Co-incubation of normal PMNL with serum from individuals who had ingested the drug caused decreased cell migration and increased chemiluminescence. These results show that benoxaprofen inhibits PMNL migration as a consequence of pro-oxidant properties and despite its withdrawal may be the prototype of the pro-oxidative anti-inflammatory drug.     

Ascorbate and cysteine-mediated selective neutralisation of extracellular oxidants during N-formyl peptide activation of human phagocytes

The effects of sodium ascorbate and cysteine (2.5×10^–5M–2.5×10^–4 M) on the intensity and profile of luminol-enhanced chemiluminescence, superoxide generation, extracellular myeloperoxidase (MPO) activity and auto-iodination were measured in purified human polymorphonuclear leukocytes activated by the leukoattractant FMLPin vitro. Chemiluminescence studies were also performed using a whole-blood method. Cysteine (10^–4 M–2.5×10^–4M) and ascorbate (2.5×10^–5M–2.5×10^–4M) caused significant inhibition of the early extracellular peak of FMLP-activated chemiluminescence and increased the intensity of the later occurring intracellular peak in both PMNL and blood. At the same concentrations both agents scavenged superoxide released by FMLP-activated PMNL, inhibited oxidant generation by extracellular MPO and decreased FMLP-induced auto-oxidation of PMNL. Administration of a single 1 gram oral dose of ascorbate to adult human volunteers was associated with significant reduction and enhancement respectively of the extracellular and intracellular luminolenhanced chemiluminescence responses of FMLP-activated blood. These results show that the water soluble anti-oxidants cysteine and especially ascorbate selectively neutralise the reactivity of harmful reactive oxidants released by phagocytes, whilst the intracellular generation of antimicrobial oxidants remains intact.     

Effects on extra- and intracellularly localized,chemoattractant-induced,oxygen radical production in neutrophils following modulation of conditions for ligand-receptor interaction

Results obtained with the luminol-dependent chemiluminescence (CL) technique show that with this technique, generation of oxygen radicals from an extra-as well as from an intracellular source is quantified. This investigation was performed in order to study the relationship between intra and extracellularly generated radicals in human granulocytes stimulated with the chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP). A difference in time course between extra and intracellular CL was observed. The extracellular response reached a maximum value after 1–2 min, whereas the intracellular response reached a maximum value after 5–7 min. The ED₅₀ values for the two responses were the same, whereas the onset time was a little longer for the intracellular response. Both high and low concentrations of FMLP gave rise to CL. However, the ratio between the extra and the intracellular response differed depending on the concentration of FMLP; the ratio was decreased at low concentrations of FMLP. The same type of change was obtained when the ligand-receptor ratio was decreased through modulation of the number of exposed receptors. The ratio between extra and intracellular activities was also changed by cytochalasin B, removal of Ca²⁺, or removal of Na⁺. The role of the extra and intracellular oxygen radical production as well as possible regulatory mechanisms are discussed.     

Effects of histamine agonists and antagonists on luminol-dependent chemiluminescence of granulocytes

Histamine inhibited luminol-dependent chemiluminescence (CL) of granulocytes in a dose-dependent manner, with an ID₅₀ of about 3×10^–5 M. Dimaprit, a selective H₂-agonist, produced a histamine-like effect. Furthermore, cimetidine, ranitidine, and TZU 0460, which are selective H₂-antagonists, but not mepyramine, a selective H₁-antagonist, blocked the inhibitory effect of histamine on CL. Thus it may be concluded that the inhibitory effect of histamine is mediated via histamine H₂-receptors. H₁- and H₂-antagonistsper se, except at extremely high concentrations, had no effect on CL of granulocytes.     

Effect of corticosteroids on the production of superoxide and hydrogen peroxide and the appearance of chemiluminescence by phagocytosing polymorphonuclear leukocytes

Human polymorphonuclear leukocytes were incubated with either methylprednisolone sodium succinate, hydrocortisone sodium succinate, or distilled water, and then latex spherules were added as target particles for phagocytosis. At low concentrations of these corticosteroids (0.04–0.22 mM), no effect was observed on O₂ ^–· production, H₂O₂ production, or chemiluminescence. At high concentrations of these steroids (2.7 mM), a significant inhibition was observed in both O₂ ^–· production and H₂O₂ production. At 2.7 mM, methylprednisolone sodium succinate significantly decreased chemiluminescence, whereas hydrocortisone sodium succinate was without effect on chemiluminescence.     

Dihydropyridine receptors in transverse tubules from normal and dystrophic chicken skeletal muscle

Summary Calcium overload is a fundamental pathogenic event associated with chronic muscle degeneration in muscular dystrophies. The possibility that l-type voltage-dependent calcium channels were involved in the etiology of chicken muscular dystrophy was investigated by studying the dihydropyridine receptors in transverse tubule membranes isolated from skeletal muscle of normal (line 412) and dystrophic (line 413) chickens. The yield of T-tubular protein from dystrophic muscle was considerably increased compared with that from normal muscle (2.51±0.18 vs 1.04±0.31 mg protein × 100 g muscle^–1). The binding of the calcium channel antagonist (+) [³H]PN200-110 to the dihydropyridine receptor in transverse tubule preparations was relatively slow, markedly affected by temperature and required divalent cations. (+) [³H]PN200-110 equilibrium binding assays revealed a single class of high-affinity sites and showed that maximum binding capacity (B_max) (3.17±0.47 for normal and 3.51±0.52 pmol × mg protein^–1 for dystrophic transverse tubules) and dissociation constant (K_d) (0.32±0.07 and 0.26±0.09 nm, respectively) were not significantly different in normal and dystrophic membranes. Kinetic studies indicated that normal and dystrophic transverse tubules did not differ significantly in association (2.54×10⁶ and 2.27×10⁶ m ^–1 s^–1, respectively) and dissociation (8.5×10^–4 and 9.3×10^–4 s^–1, respectively) rate constants. Since dissociation kinetics for both preparations were monoexponential under all the experimental conditions employed, no low-affinity binding sites for (+) [³H]PN200-110 could be detected in chicken transverse tubules membranes. However, immunoblot assay, using a monoclonal antibody, revealed that dystrophic transverse tubules as compared with normal membranes were enriched twofold with the ₁-subunit of the dihydropyridine receptor. Therefore, although dihydropyridine-binding sites were not altered in transverse tubule membranes from dystrophic chicken skeletal muscle, both the increased yield in T-tubule vesicles and the enhanced immunodetection of the ₁-subunit of the dihydropyridine receptor, suggest that total content in dihydropyridine receptor is higher in dystrophic than in normal muscle.     

CL 316,243, a selective β3-adrenergic agonist, inhibits protein breakdown in rat skeletal muscle

The in vitro effect of CL 316,243 (CL), a selective ₃-adrenoceptor agonist in the rate of overall proteolysis, the activity of proteolytic systems (lysosomal, Ca²⁺-dependent, ATP-dependent, and ATP-independent) and in the process of protein synthesis was investigated in rat skeletal muscles. The rate of overall proteolysis in soleus muscle from rats incubated with CL (10^–4 and 10^–5 M) or epinephrine (10^–5 M) was significantly decreased. In vitro rates of maximal activity of Ca²⁺-dependent proteolysis in soleus muscles were decreased by about 41% in the presence of 10^–5 M CL. No change was observed in the activities of the lysosomal, ATP-dependent or ATP-independent proteolytic systems. The anti-proteolytic effect of CL or epinephrine was partially prevented by 10^–5 M SR 59230A, a selective ₃-adrenoceptor antagonist. The increase of proteolysis induced by food deprivation in soleus was abolished by in vitro addition of 10^–5 M CL. No change in proteolysis was observed in extensor digitorum longus (EDL) muscles incubated with any concentration of the ₃-adrenoceptor agonist tested. Rates of protein synthesis were not affected by 10^–4 M CL neither in soleus nor EDL. The data suggest that a ₃-adrenoceptor-mediated inhibition of Ca²⁺-dependent proteolysis participates of the antiproteolytic effect of catecholamines in oxidative muscles.     

Stimulation of neutrophil oxidative metabolism by indomethacin

Human neutrophils exposed to indomethacin demonstrate an enhanced capacity for superoxide ion (O ₂ ^– ) generation when stimulated with opsonized zymosan. Enhancement is not seen with indomethacin-treated cells exposed to solube oxidative stimuli. To further investigate this phenomenon, O ₂ ^– generation, chemiluminescence, and phagocytosis were assessed in human neutrophils preincubated with indomethacin. Zymosan-stimulated O ₂ ^– release was increased from 150 to 300% of controls in neutrophils exposed to 400 g/ml. indomethacin. Enhancement was not reversed by removal of indomethacin from the medium prior to addition of the stimulus and was dose-dependent at drug concentrations of 5 to 400 /ml. Neutrophils exposed to methacin alone also generated more O ₂ ^– than control cells, although this increment was not sufficient to account for the degree of enhancement seen when indomethacintreated cells were exposed to zymosan. Neutrophil cehmiluminescence induced by zymosan was also increased by exposure to indomethacin, and at a drug concentration of 400 g/ml (1.1 mM), enhancement randed from 253 to 333% of controls. As was observed with O ₂ ^– generation, chemiluminescence of neutrophils was increased in the presence of indomethacin alone, although, to a degree far less than was seen when drug-treated cells were stimulated with zymosan. Phagocytosis of radiolabeledS. aureus by neutrophils incubated with indomethacin was increased 13±5% over controls (P<0.01,n=5), but was unaltered by incubation of cells with the buffer used to solubilize the drug. The modest degree of enhancement of phagocytosis suggests that increased particle uptake is not the sole mechanism of oxidative enhancement. The data are in keeping with the hypothesis that indomethacin has a direct effect on the neutrophil plasma membrane and/or the O ₂ ^– -forming oxidase.     

Enhancement by cimetidine of chemotactic peptide-stimulated ATP release and chemiluminescence in human neutrophils

As determined by luciferase-luciferin, we recently found that the H₂-blocker CIM considerably increased the ATP release from fMLP-stimulated PMN. This observation correlates well with our previous report [1] regarding the enhancement of superoxide output (chemiluminescence) in human neutrophils by CIM plus fMLP.In order to compare the ATP release from PMN of different donors, a standard procedure has been developed consisting of the determination of the ATP present initially in the cell suspension (without stimulation), ATP release after stimulation with fMLP, and ATP release in the presence of CIM plus fMLP. The whole ATP content per neutrophil was determined after ultrasonication of the cells as well. The mean value of the initially present ATP was 0.45×10^–17 mol/cell in the suspension.Stimulation with fMLP plus CIM yielded within 5–10 minutes considerably higher ATP amounts than fMLP alone. The corresponding and statistically significantly different mean values were 2.46×10^–17 mol/PMN (s.d.=1.047) and 1.38×10^–17 mol/PMN (s.d.=0.55), respectively.The whole ATP per neutrophil was found to be 1.22×10^–15 mol (mean; s.d.=0.60) and thus, the stimulation with CIM plus fMLP released about 2.0 per cent, with fMLP alone about 1.0 per cent of the whole ATP.CIM without fMLP did not enhanced the ATP release during the reaction time applied. On the other hand, fMLP-stimulated, lucigenin-amplified chemiluminescence determinations were carried out in the presence of CIM as well; contrarily to our previous method, CIM was dissolved in PBS without DMSO, because DMSO inhibited the chemiluminescence slightly. Our previous results showing an enhancement of the chemiluminescence by CIM could be justified.     

Enhancement of normal neutrophil chemiluminescence by chronic granulomatous disease neutrophils

Neutrophils and other phagocytic cells from patients with chronic granulomatous disease (CGD) lack the ability to generate reactive oxygen intermediates (ROI), although other phagocytic functions appear to be intact. The effects of CGD neutrophils on the ability of normal neutrophils to produce ROI as measured by luminol-enhanced chemiluminescence (CL) were examined. Normal neutrophils (2×10⁵) had a peak CL response to phorbol myristate acetate (PMA; 20 ng/ml) of 6.5±0.9 mV, while the CL response of CGD neutrophils was completely absent. However, the addition of CGD neutrophils (8×10⁵) to normal neutrophils (2×10⁵) markedly increased the peak CL response to PMA to 11.0+-1.1 mV (P < 0.001). The peak response of normal neutrophils (2×10⁵) alone to the peptideN-formyl-methionyl-leucyl-phenylalanine (FMLP; 10^–6 M) was 9.0±1.1mV, and this increased to 22.2±3.5 mV (P < 0.001) when 8×10⁵ CGD neutrophils were added and to 18.9±3.6 mV (P<0.005) when 4×10⁵ CGD neutrophils were added. Thus, CGD neutrophils increase the release of ROI from normal cells, suggesting nonoxidative regulatory factors in ROI production.This work was supported in part by grants GI517-02 and 0008-08 from the Cystic Fibrosis Foundation and HD-09800 and AI-15332 from the National Institutes of Health.     

Nicotinic receptor distribution in the human thalamus: autoradiographical localization of [H]nicotine and [I]α-bungarotoxin binding

The thalamus plays a major role in relaying and transforming information that is relayed to the cortex and in turn modulates cortical outputs. The reticular nucleus projects to the other thalamic nuclei, modulating and integrating their activity. The distribution of high affinity nicotine and α-bungarotoxin (αBTX) receptors in the human thalamus has been investigated by radioligand autoradiography in post mortem human tissue. [³H]nicotine binding in the human thalamus was high in most thalamic nuclei, especially in the lateral dorsal, the medial geniculate, lateral geniculate and anterior nuclei. The distribution of [¹²⁵I]αBTX binding was quite distinct from [³H]nicotine binding. [¹²⁵I]αBTX binding was generally lower (<0.26–11.62 fmol/mg protein compared with 6.68–36.17 fmol/mg protein for nicotine binding) and concentrated in the reticular nucleus, with discrete groups of cells displaying higher binding in the latter. These results indicate differences between the distribution of nicotinic receptors in humans and those previously reported in mice and monkeys. Changes in high affinity nicotine and αBTX receptors in the thalamus may contribute to symptoms observed in neuropathological conditions associated with disorders of perception and movement such as Dementia with Lewy Bodies, Alzheimer's Disease and Schizophrenia.     

Indomethacin is a competitive inhibitor of the binding of the chemotactic peptide formyl-Met-Leu-Phe to human polymorphonuclear leukocytes

Indomethacin, a non-steroidal anti-inflammatory drug, competes with the tritiated chemotactic peptide formyl-Met-Leu-Phe (FMLP) for binding to human neutrophils (PMN). This competition which occurs in the concentration range 10^–6–10^–3 M, with an IC₅₀ of 7×10^–5 M, is inversely proportional to the concentration of albumin present in the incubation medium. These data explain why indomethacin is able to inhibit, in the same concentration range and with the same IC₅₀, many of the physiological responses of PMN elicited by the chemotactic peptide formyl-Met-Leu-Phe.