Biomedical subjects

H Kankaanranta

Publications and source records attributed to H Kankaanranta.

At least 19 recordsLinked to original sources

PubMed2002

Increased alveolar nitric oxide concentration in asthmatic patients with nocturnal symptoms.

Nocturnal asthma symptoms and impaired lung function at night are related to inflammatory activity in the peripheral lung compartment. Exhaled nitric oxide (NO) measurement at multiple exhalation flow rates can be used to separately assess alveolar and bronchial NO production and inflammation. The authors hypothesised that asthmatic patients with nocturnal symptoms have a higher alveolar NO concentration than those with only daytime symptoms. The authors asked 40 patients with newly-diagnosed steroid-naïve asthma about their nocturnal asthma symptoms through the use of a written questionnaire. Alveolar NO concentration and bronchial NO flux were assessed in the 40 asthmatics and 40 healthy controls. Nineteen of the 40 patients reported nocturnal symptoms. Patients with nocturnal symptoms had a higher alveolar NO concentration (1.7+/-0.3 (mean+/-SEM) parts per billion (ppb)) than patients without nocturnal symptoms (0.8+/-0.3 ppb, p=0.012) or healthy controls (1.0+/-0.1 ppb, p=0.032). Bronchial NO flux was higher both in patients with (2.4+/-0.4 nL x s(-1), p<0.001) and without (2.6+/-0.4 nL x s(-1), p<0.001) nocturnal symptoms, compared to controls (0.7+/-0.1 nL x s(-1)). Nocturnal symptoms in asthmatic patients are related to a higher alveolar nitric oxide concentration. The results suggest that assessment of alveolar nitric oxide concentration can be used to detect the parenchymal inflammation in asthmatic patients with nocturnal symptoms.

Adult↗

PubMed2001

Beclomethasone, budesonide and fluticasone propionate inhibit human neutrophil apoptosis.

Inhaled glucocorticoids are widely used to treat chronic obstructive pulmonary disease without much evidence of efficiency in this disease where neutrophils may contribute to the pathophysiology. This prompted us to test the effects of several currently used inhaled and systemic glucocorticoids on human neutrophil apoptosis. Beclomethasone, budesonide, dexamethasone, fluticasone propionate, hydrocortisone and prednisolone inhibited apoptosis in a concentration-dependent manner as assessed by flow cytometric analysis, annexin-V binding and morphological analysis. The maximal inhibition of apoptosis was 50-60%. The order of potency was fluticasone propionate (EC(50) 0.6+/-0.2 nM) approximately equal to budesonide (EC(50) 0.8+/-0.2 nM)> dexamethasone approximately equal to prednisolone approximately equal to beclomethasone approximately equal to hydrocortisone. The inhibitory effects of glucocorticoids were reversed by mifepristone. Moreover, glucocorticoids slightly enhanced the inhibitory effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on neutrophil apoptosis. The present data suggests that budesonide and fluticasone propionate prolong human neutrophil survival by inhibiting apoptosis at clinically relevant drug concentrations via an effect on glucocorticoid receptor.

Androstadienes↗

PubMed2001

Bi-directional effects of the elevation of intracellular calcium on the expression of inducible nitric oxide synthase in J774 macrophages exposed to low and to high concentrations of endotoxin.

Nitric oxide produced through the action of inducible nitric oxide synthase (iNOS) is an important mediator in immune responses of the host. Various extracellular factors, including inflammatory stimuli, affect intracellular free Ca2+ levels ([Ca2+](i)), modulating cellular signalling and gene expression. In the present study we investigated the effects of increased ([Ca2+](i)) on NO production through the iNOS pathway in J774 macrophages. Thapsigargin (TG), a Ca2+-ATPase inhibitor, and the Ca2+ ionophore A23187 were used as tools to induce an increase in ([Ca2+](i)) in the cytosol. This increase was confirmed by the fura 2 method. The production of NO was measured as accumulated nitrite in the cell culture medium; iNOS protein and iNOS mRNA were detected by Western blotting and reverse-transcriptase-mediated PCR respectively. The activation of nuclear factor kappaB (NF-kappaB) was investigated by electrophoretic mobility-shift assay. TG (100 nM) induced a marked synthesis of iNOS mRNA, iNOS protein and NO in cells primed with a low concentration of endotoxin [lipopolysaccharide (LPS) 1 ng/ml], which on its own induced barely detectable NO synthesis. Stimulation by a high concentration of LPS (100 ng/ml) induced a marked expression of iNOS and NO production. Under these conditions, treatment with TG hindered the synthesis of iNOS protein and NO production by accelerating the degradation of iNOS mRNA. Treatment with TG (100 nM) did not affect the NF-kappaB activity induced by low (1 ng/ml) or high (100 ng/ml) concentrations of LPS. Viability of the cells was confirmed by the 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyaniline ("XTT") method; apoptosis was ruled out by propidium iodide staining and flow cytometry. A23187 (1 microM) also transiently increased ([Ca2+](i)) and had opposite effects on NO production depending on the LPS concentration. Our results show that increased ([Ca2+](i)) induced the stimulation or suppression of NO production through iNOS in macrophages depending on the state of cell activation. These findings suggest that the receptor-mediated increase in ([Ca2+](i)) might be an important factor in the control of the balance between the up-regulation and down-regulation of inflammatory genes, including that encoding iNOS, depending on the phase of the inflammatory response.

Animals↗

PubMed2001

Suppression of T-cell activation by Lactobacillus rhamnosus GG-degraded bovine casein.

Earlier data indicate that Lactobacillus rhomnosus GG ATCC 53103 (L. GG), a commensal intestinal bacterial strain, promotes the degradation of proteins in the gut in vivo, and bovine casein hydrolysed with L. GG-derived proteases suppresses lymphocyte proliferation in vitro. The present study aimed to evaluate the effect of L. GG-degraded bovine casein on T-cell activation, i.e. IL-2 mRNA expression and protein kinase C (PKC) translocation. To this end, Northern blot analyses for IL-2 mRNA expression and PKC assays with and without L. GG-degraded casein were carried out on T cells isolated from 11 healthy adults. Cell cultures in 8-11 experiments contained 1 mg ml(-1) bovine casein in degraded or undegraded form in the presence of a mitogen, i.e. phorbol 12,13-dibutyrate plus calcium ionophore (PBDu + A23187) or anti-CD3. Also IL-2, IL-4 and IFN-gamma syntheses were determined in 24-h culture supernatants. IL-2 mRNA expression was reduced in experiments with L. GG-degraded casein. In parallel, the IL-2 concentration in PBDu + A23187-stimulated culture supernatants, expressed as geometric means (95% confidence interval), decreased from 15,892 (7174-35,203) pg ml(-1) to 4744 (2095-10,742) pg ml(-1) when containing L. GG-degraded casein. L. GG-degraded casein inhibited PKC translocation, the action resembling that of PKC inhibitor, RO31-8220. These results extend previous data on L. GG-degraded casein, showing in vitro the suppression of T-cell activation.

Animals↗

PubMed2001

Induction of nitric oxide synthesis by probiotic Lactobacillus rhamnosus GG in J774 macrophages and human T84 intestinal epithelial cells.

BACKGROUNDS AND AIMS: Probiotic Lactobacillus rhamnosus GG (LGG) has proved to be beneficial in the treatment of viral- and antibiotic-associated diarrhea but the mechanisms of action remain unknown. Nitric oxide (NO) is involved in the protective mechanisms in the gastrointestinal tract and may contribute to some of the beneficial effects of probiotics. The aim of the present study was to investigate if induction of NO synthesis is involved in the cellular actions of LGG. METHODS: NO synthesis and its regulation were measured in cultures of J774 murine macrophages and human T84 colon epithelial cells. NO production was measured as its metabolite nitrite accumulated into the culture medium. Inducible nitric oxide synthase (iNOS) protein and iNOS mRNA were detected by Western blot and RT-PCR, respectively. RESULTS: In J774 macrophages, LGG induced a low level production of NO in the presence of gamma interferon (IFNgamma) and it was inhibited by NOS inhibitors, cycloheximide and by a NF-kappa B inhibitor pyrrolidinedithiocarbamate. Accordingly, LGG and IFNgamma-stimulation increased iNOS mRNA and protein levels. T84 cells produced NO in response to LGG when first primed with a combination of IL-1beta, TNFalpha and IFNgamma. Lipoteichoic acid (LA), an antigenic structure in gram-positive bacteria, also induced NO formation in J774 cells in the presence of IFNgamma suggesting that LA may be the active component in LGG. CONCLUSIONS: LGG induces NO production in J774 macrophages and in human T84 colon epithelial cells through induction of iNOS by a mechanism involving activation of transcription factor NF-kappaB. Induction of iNOS and low-level synthesis of NO may be involved in the protective actions of LGG in the gastrointestinal tract.

Cell Line↗

PubMed2001

Extended exhaled NO measurement differentiates between alveolar and bronchial inflammation.

Lower respiratory tract inflammation can be detected by measuring exhaled nitric oxide (NO) concentration at a single exhalation flow rate, but this does not differentiate between alveolar and bronchial NO production. We assessed alveolar NO concentration and bronchial NO flux with an extended method of measuring exhaled NO at several exhalation flow rates in 40 patients with asthma, 17 patients with alveolitis, and 57 healthy control subjects. Bronchial NO flux was higher in asthma (2.5 +/- 0.3 nl/s, p < 0.001) than in alveolitis (0.7 +/- 0.1 nl/s) and healthy control subjects (0.7 +/- 0.1 nl/s). Alveolar NO concentration was higher in alveolitis (4.1 +/- 0.3 ppb, p < 0.001) than in asthma (1.1 +/- 0.2 ppb) and healthy control subjects (1.1 +/- 0.1 ppb). In asthma, bronchial NO flux correlated with serum level of eosinophil protein X (EPX) (r = 0.60, p < 0.001) and bronchial hyperresponsiveness (r = 0.55, p < 0.001). In alveolitis, alveolar NO concentration correlated inversely with pulmonary diffusing capacity (r = -0.55, p = 0.022) and pulmonary restriction. Glucocorticoid treatment or allergen avoidance normalized bronchial NO flux in asthma and decreased alveolar NO concentration toward normal in alveolitis. In conclusion, extended exhaled NO measurement can be used to separately assess alveolar and bronchial inflammation and to assess disease activity/severity in asthma and alveolitis.

Adult↗

PubMed2001

Inhaled fluticasone decreases bronchial but not alveolar nitric oxide output in asthma.

Exhaled nitric oxide (NO) concentration is a noninvasive measure of airway inflammation and is increased in asthma. Inhaled glucocorticoids decrease exhaled NO concentration, but the relative contributions of alveolar and bronchial levels to the decrease in exhaled NO concentration are unknown. Alveolar NO concentration and bronchial NO flux can be separately approximated by measuring exhaled NO at several exhalation flow rates. The effect of steroid treatment on alveolar and bronchial NO output in asthma was studied. Alveolar NO concentration and bronchial NO flux were assessed in 16 patients with asthma before and during treatment with inhaled fluticasone for 8 weeks and in 16 healthy controls. Before the treatment, asthmatics had increased bronchial NO flux (mean+/-SEM: 3.6+/-0.4 versus 0.7+/-0.1 nL x s(-1), p<0.001) but normal alveolar NO concentration (1.2+/-0.5 versus 1.0+/-0.2 parts per billion (ppb), p>0.05) compared with controls. Inhaled fluticasone decreased bronchial NO flux from 3.6+/-0.4 to 0.7+/-0.1 nL x s(-1) (p<0.01) but had no effect on alveolar NO concentration (before: 1.2+/-0.5; after: 1.2+/-0.1 ppb, p>0.05). The forced expiratory volume in one second improved, whereas asthma symptom score and serum levels of eosinophil cationic protein and eosinophil protein X decreased during the treatment. In conclusion, inhaled fluticasone decreases bronchial but not alveolar nitric oxide output simultaneously with clinical improvement in patients with asthma.

Administration, Inhalation↗

PubMed2000

Enhancement of human eosinophil apoptosis by fluticasone propionate, budesonide, and beclomethasone.

Beclomethasone, budesonide, dexamethasone, and fluticasone propionate enhanced human eosinophil apoptosis in a concentration-dependent manner in vitro as assessed by flow cytometric analysis and morphological analysis. The order of potency was fluticasone propionate (EC(50) 3.7+/-1.8 nM) approximately budesonide (EC(50) 5.0+/-1.7 nM)>beclomethasone (EC(50) 51+/-19 nM)>dexamethasone (EC(50) 303+/-40 nM). Hydrocortisone, prednisolone, and prednisone (up to 1 microM) did not induce any significant increase in eosinophil apoptosis. The apoptosis promoting effects of glucocorticoids on eosinophils were reversed by an antagonist of glucocorticoid receptor mifepristone. The survival-prolonging effect of tumor necrosis factor (TNF)-alpha was reversed by dexamethasone and fluticasone (1 microM). In contrast, fluticasone, and dexamethasone (1 microM) did not reverse the survival-prolonging effects of interleukins-3 and -5 or granulocyte-macrophage colony-stimulating factor (GM-CSF). The results suggest that fluticasone and budesonide induce eosinophil apoptosis at clinically achievable drug concentrations via an effect on glucocorticoid receptor.

Androstadienes↗

PubMed2000

Inhibition by nitric oxide-releasing compounds of E-selectin expression in and neutrophil adhesion to human endothelial cells.

The effects of two chemically unrelated nitric oxide (NO)-releasing compounds were studied on adhesion molecule expression in and neutrophil adhesion to human umbilical vein endothelial cells. Incubation of confluent monolayers of endothelial cells with increasing concentrations of lipopolysaccharide stimulated the adhesion of polymorphonuclear leukocytes to endothelial cells. Flow cytometric analysis showed that lipopolysaccharide treatment upregulated the expression of adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells. A novel NO-releasing compound GEA 3175 (1,2,3, 4-oxatriazolium, -3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl)sulfonyl]amino]-, hydroxide inner salt) inhibited lipopolysaccharide-induced adhesion being more potent than the earlier known NO donor S-nitroso-N-acetylpenicillamine. The increased E-selectin expression induced by lipopolysaccharide was significantly attenuated by the two NO donors tested whereas ICAM-1 expression remained unaltered. The present data show that NO donors inhibit E-selectin expression in and neutrophil adhesion to lipopolysaccharide-stimulated vascular endothelial cells. Thus, by inhibiting leukocyte adhesion NO donors may reduce leukocyte infiltration and leukocyte-mediated tissue injury in inflammation and ischemia-reperfusion injury.

Cell Adhesion↗

PubMed2000

Delayed eosinophil apoptosis in asthma.

BACKGROUND: Eosinophilic inflammation of the airways is a key characteristic of asthma. A defect in apoptosis might contribute to the chronic tissue eosinophilia associated with asthma. OBJECTIVE: Our purpose was to examine whether the rate of apoptosis differs between peripheral blood eosinophils from asthmatic patients and healthy volunteers. METHODS: Peripheral blood was obtained from volunteers with asthma and from control volunteers. Eosinophils were isolated by CD16-negative selection to >99% purity and were cultured for 48 hours. The number of apoptotic eosinophils in the culture was assessed by flow cytometric analysis of relative DNA content in propidium iodide-stained cells. Eosinophil apoptosis is expressed as apoptosis index (number of apoptotic cells/total number of cells). RESULTS: Eosinophils from asthmatic patients not taking steroid medication survived longer (apoptosis index 0.25) than those of healthy control subjects (apoptosis index 0.40, P <.05). In contrast, the rate of apoptosis in eosinophils from patients concurrently taking steroids (apoptosis index 0.46) is higher than that of those not using steroids (P <.01) and not different from that of healthy subjects. To assess whether endogenous IL-3, IL-5, and GM-CSF production contributes to the delayed eosinophil apoptosis, the effects of the corresponding neutralizing antibodies were studied on eosinophil longevity. Neutralization of GM-CSF, but not of IL-3 or IL-5, increased slightly but significantly (P <.01) the rate of apoptosis in eosinophils obtained from patients with asthma. To assess whether beta(2)-agonist medication could contribute to the observed differences, we determined the in vitro effects of albuterol, fenoterol, and salmeterol on eosinophil apoptosis. All beta(2)-agonists inhibited eosinophil apoptosis by 12% to 19%. A possibility existed that a prior in vivo exposure to IL-5, GM-CSF, or beta(2)-agonists would explain the observed difference. To study this, eosinophils were incubated with GM-CSF, IL-5, and albuterol for 2 to 3 hours, followed by washout of the added compounds, and were subsequently cultured for 48 hours. However, an exposure to GM-CSF (7 pmol/L) or IL-5 (10 pmol/L) for 15 to 180 minutes was not a sufficient signal to prevent eosinophil apoptosis. In contrast, exposure to albuterol (100 nmol/L) for 120 minutes was sufficient to induce a significant (P <.05) decrease in eosinophil apoptosis. CONCLUSIONS: The results suggest that eosinophil apoptosis is delayed in asthma. This delay may be partly explained by production of GM-CSF. The in vitro effects of beta(2)-agonists suggest that beta(2)-agonist use might contribute to the prolonged eosinophil survival through inhibition of apoptosis and thus may worsen eosinophilia in asthmatic patients. Use of inhaled glucocorticoids seems to totally reverse the delayed eosinophil apoptosis in asthma.

Adrenal Cortex Hormones↗

PubMed2000

Increased bronchial nitric oxide production in patients with asthma measured with a novel method of different exhalation flow rates.

The concentration of nitric oxide (NO) in exhaled air is increased in patients with asthma, suggesting that measuring fractional exhaled NO concentration (FE(NO)) may be used to monitor asthmatic airway inflammation. However, increased FE(NO) is not specific for asthma, as other inflammatory lung diseases may also increase FE(NO). To augment the specificity of FE(NO) measurement, we tested a novel theoretical modelling of pulmonary NO dynamics that allows the approximation of alveolar NO concentration and bronchial NO flux separately by measuring FE(NO) at several exhalation flow rates. We measured FE(NO) at four exhalation flow rates in 10 steroid-naive asthmatics, 5 patients with extrinsic allergic alveolitis, and in 10 healthy controls. Both the asthmatics and the patients with alveolitis had significantly higher FE(NO) than the healthy controls. The increased NO concentration originated from the bronchial level in the asthmatics and from the alveolar level in the patients with alveolitis. In the second part of the study we assessed the repeatability of FE(NO) test, within-day and day-to-day (during two weeks) variation in FE(NO), and the effects of mouth pressure and cigarette smoking on FE(NO) in healthy volunteers. Repeatability of 10 subsequent measurements was high (coefficient of variation (CV) 4.6% +/- 0.4%), and no diurnal variation was found. The day-to-day variation during a 2-week period gave a CV of 10.6% +/- 1.0%. The magnitude of mouth pressure (5-20 cmH2O in adults, 5-40 cmH2O in children) during measurement had no effect on FE(NO). Smoking a cigarette caused a small and transient but statistically significant increase in FE(NO) at 1 and 5 min after smoking. In conclusion, FE(NO) measurement is highly repeatable with low day-to-day variation among healthy subjects. Our results also suggest that the present novel method of measuring FE(NO) at several exhalation flow rates can be used to approximate alveolar and bronchial contributions to FE(NO) separately and thus increase the clinical value of this test.

Adult↗

PubMed2000

Inhibition of extracellular signal-regulated kinase suppresses endotoxin-induced nitric oxide synthesis in mouse macrophages and in human colon epithelial cells.

Macrophages produce large amounts of nitric oxide (NO) in response to proinflammatory cytokines and lipopolysaccharide (LPS) by expressing inducible isoform of NO synthase (iNOS). We examined the role of extracellular signal-regulated kinase p42/44(MAPK) (Erk1/2) in signal transduction pathways leading to induction of NO synthesis in response to LPS in J774 mouse macrophages and T-84 human colon epithelial cells. LPS activated Erk1/2 and induced iNOS and subsequent NO production. Erk1/2 activation was inhibited by PD 98059, a specific inhibitor of mitogen-activated protein kinase kinase (Mek) that is an upstream activator of Erk1/2. At corresponding concentrations PD 98059 reduced LPS-induced NO formation by 40 to 50% by inhibiting iNOS expression in J774 and T-84 cells. Inhibition of iNOS expression was not mediated by nuclear factor-kappaB because PD 98059 had no effect on nuclear factor-kappaB activity in J774 macrophages. In addition, PD 98059 reduced LPS-induced L-arginine transport into the cells as measured in J774 macrophages, whereas the availability of tetrahydrobiopterin was not a limiting factor in NO production after PD 98059. Our results indicate that Erk1/2 activation mediates up-regulation but is not essential for LPS-induced iNOS expression.

Animals↗

PubMed1999

Effects of K(+) channel inhibitors on relaxation induced by flufenamic and tolfenamic acids in guinea-pig trachea.

The effects of different K(+) channel inhibitors on flufenamic- and tolfenamic-acids-induced relaxation were studied in prostaglandin F(2alpha) (1 microM) precontracted guinea-pig trachea. Flufenamic and tolfenamic acids (each 0.1-33 microM) and lemakalim (0.01-33 microM), but not indomethacin (0.1-33 microM), caused relaxation. Iberiotoxin (33 and 100 nM) inhibited flufenamic- and tolfenamic-acids-, but not lemakalim-, induced relaxation. Iberiotoxin (100 nM) inhibited nifedipine (10 nM-10 microM)-induced relaxation. Nifedipine (0.1 microM) inhibited the blockade of fenamate-induced relaxation by iberiotoxin. Apamin (0.1 and 1 microM) did not affect flufenamic- and tolfenamic-acids- and lemakalim-induced relaxation. Glibenclamide (10 and 33 microM) inhibited lemakalim-, but not flufenamic- and tolfenamic-acids-, induced relaxation. 4-Aminopyridine (0.5 and 2 mM) inhibited flufenamic- and tolfenamic- acids- and lemakalim-induced relaxation. Flufenamic- and tolfenamic-acids-induced relaxation is likely to be activation of Ca(2+)-activated K(+) channels and differs from that of lemakalim.

Animals↗

PubMed1999

Nitric oxide-releasing compounds inhibit neutrophil adhesion to endothelial cells.

In the present work, we demonstrated that chemically different nitric oxide (NO)-releasing compounds inhibit tumor necrosis factor alpha (TNF-alpha)-induced polymorphonuclear leukocyte adhesion to endothelial cells in vitro. Two mesoionic oxatriazole derivatives GEA 3162 (1,2,3,4-oxatriazolium,5-amino-3(3, 4-dichlorophenyl)-chloride) and GEA 3175 (1,2,3,4-oxatriazolium, -3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl)sulfonyl]amino]-, hydroxide inner salt) were compared to the earlier-known NO donor SIN-1 (3-morpholino-sydnonimine). GEA 3162 (3-10 microM) and GEA 3175 (10-30 microM) inhibited human polymorphonuclear leukocyte adhesion to B(4) endothelial cells in a dose-dependent manner being more potent than SIN-1. In the present model, leukocytes rather than endothelial cells seemed to be the target of the effect of NO. Flow cytometric analysis showed that NO-releasing compounds did not alter TNF-alpha induced CD11/CD18 surface expression in polymorphonuclear leukocytes. The inhibitory action of NO-releasing compounds on adhesion paralleled with the increased synthesis of cGMP in polymorphonuclear leukocytes. Analogues of cGMP inhibited polymorphonuclear leukocyte adhesion indicating a role for cGMP in the action of NO donors. The results suggest that exogenous NO in the form of NO-releasing compounds inhibits polymorphonuclear leukocyte adhesion to endothelial cells, which may be implicated in the regulation of leukocyte migration and leukocyte-mediated tissue injury.

Animals↗

PubMed1999

Effects of nitric oxide donors GEA 3162 and SIN-1 on ethanol-induced gastric ulceration in rats.

Low doses of the intragastrically (i.g.) administered nitric oxide (NO) donors, 1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162; 0.3 mg/kg) and 3-morpholino-sydnonimine (SIN-1; 1 mg/kg), inhibited gastric ulceration induced by ethanol (94%) in anesthetized rats. In contrast, higher doses of these NO donors administered i.g. exacerbated the damage. When administered intravenously, the NO donors had no effect on ethanol-induced gastric lesions although a clear blood pressure-lowering effect was seen. Neither the inhibition nor the exacerbation of ulceration was correlated with changes in blood pressure or prostaglandin E2 release from the mucosal tissue. The relatively small difference between the gastroprotective and damaging doses suggests that orally administered NO donors, especially in the case of GEA 3162, may have a narrow gastric safety margin.

Animals↗

PubMed1999

No detectable NO synthesis from L-arginine or N(G)-hydroxy-L-arginine in fMLP-stimulated human blood neutrophils despite production of nitrite, nitrate, and citrulline from N(G)-hydroxy-L-arginine.

Nitric oxide (NO) is a well-documented effector molecule in rodent phagocytes but its synthesis in human neutrophils has been controversial. In this study, NO production in human neutrophils activated by chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) was measured in the presence of L-arginine (L-Arg) and N(G)-hydroxy-L-arginine (OH-L-Arg), the precursor and intermediate amino acids in NO synthesis, respectively. Incubation of fMLP-activated neutrophils with OH-L-Arg resulted in a production of nitrite, nitrate, and citrulline that was greater than with unstimulated neutrophils but was not inhibited by the NOS inhibitors L-NMMA and L-NIO or the cytochrome P450 inhibitor troleandomycin and was not seen when OH-L-Arg was replaced with L-Arg. This nitrite, nitrate, and citrulline production was not associated with any detectable NO synthesis because no increases in cyclic GMP were observed in the presence of phosphodiesterase inhibitors and in the presence or absence of superoxide dismutase. Moreover, no increases in the formation of the reaction product of NO with superoxide, peroxynitrite, were observed on addition of either OH-L-Arg or L-Arg to activated neutrophils, as assessed either by dihydrorhodamine oxidation or protein nitration. This suggests that, in spite of the production of nitrite, nitrate, and citrulline, commonly used indicators of NO formation, normal human blood neutrophils, are not producing detectable amounts of either NO or peroxynitrite when stimulated with fMLP in the presence of OH-L-Arg.

Arginine↗

PubMed1999

Expression and activation of protein kinase C-zeta in eosinophils after allergen challenge.

Protein kinase (PK) C is an increasingly diverse family of enzymes that has been implicated in a range of cellular functions within the eosinophil. Using isoform-specific polyclonal antibodies, we have explored the expression of PKC isoforms in circulating eosinophils. Initial studies demonstrated the presence of the alpha, betaI, betaII, and zeta and the low-level expression of the delta, epsilon, iota, and micro isoforms but no detectable expression of the gamma, eta, and theta isoforms in both normal and asthmatic subjects. There was no difference in the total protein expression between these two groups. Subsequent studies examined the expression and activation of PKC isoforms in circulating eosinophils from asthmatic patients before and 24 h after a late asthmatic response to an inhaled allergen. Cellular fractionation showed PKC-alpha and PKC-betaII to be mainly located in the cytosol, whereas PKC-betaI was constitutively more expressed in the membrane. No changes in expression or subcellular localization of these isoforms were seen after allergen challenge. In contrast, PKC-zeta expression was increased after allergen challenge, and we demonstrated a significant PKC-zeta translocation to the membrane, in keeping with activation of the enzyme. Our results suggest that 24 h after allergen exposure of asthmatic patients, there is increased expression and activation of eosinophil PKC-zeta that correlates with late asthmatic responses recorded between 4 and 10 h postallergen challenge.

Adult↗

PubMed1999

SB 203580, an inhibitor of p38 mitogen-activated protein kinase, enhances constitutive apoptosis of cytokine-deprived human eosinophils.

The role of p38 mitogen-activated protein (MAP) kinase, and extracellular-regulated protein kinase -1 and -2 in regulating constitutive apoptosis and interleukin (IL)-5-induced survival of human eosinophils have been investigated. Two populations of donors were identified whose eosinophils, in the absence of exogenous cytokines, underwent apoptosis at different rates. Eosinophils were thus arbitrarily classified as either "fast"- or "slow"-dying cells, where greater or less than 15% of the cells were apoptotic at 2 days, respectively. The selective p38 MAP kinase inhibitor, SB 203580, increased constitutive eosinophil apoptosis in both populations (EC(50) approximately 2 microM) as evinced from morphological analysis, flow cytometry, and DNA laddering. The ability of SB 203580 to kill eosinophils was not due to nonspecific toxicity or through the inhibition of prostanoid or leukotriene production. Exposure of eosinophils to IL-5, at a concentration (10 pM) that enhanced survival maximally, abolished SB 203580-induced apoptosis. In contrast PD 098059, which selectively blocks MAP kinase kinase (MEK) 1, did not affect apoptosis of fast- or slow-dying eosinophils, or the enhanced survival of cells effected by IL-5. Collectively, these results suggest that: 1) the basal activity of p38 MAP kinase may regulate the survival of cytokine-deprived eosinophils through inhibition of apoptosis, 2) the enhancement of eosinophil survival effected by IL-5 is mediated by a mechanism(s) divorced from the activation of p38 MAP kinase, and 3) neither spontaneous eosinophil apoptosis nor their enhanced survival by IL-5 involves the activation of MEK-1.

Apoptosis↗