Mast cell mediators in citric acid-induced airway constriction of guinea pigs

We demonstrated previously that mast cells play an important role in citric acid (CA)-induced airway constriction. In this study, we further investigated the underlying mediator(s) for this type of airway constriction. At first, to examine effects caused by blocking agents, 67 young Hartley guinea pigs were divided into 7 groups: saline + CA; methysergide (serotonin receptor antagonist) + CA; MK-886 (leukotriene synthesis inhibitor) + CA; mepyramine (histamine H1 receptor antagonist) + CA; indomethacin (cyclooxygenase inhibitor) + CA; cromolyn sodium (mast cell stabilizer) + CA; and compound 48/80 (mast cell degranulating agent) + CA. Then, we tested whether leukotriene C4 (LTC4) or histamine enhances CA-induced airway constriction in compound 48/80-pretreated guinea pigs. We measured dynamic respiratory compliance (Crs) and forced expiratory volume in 0.1 s (FEV0.1) during either baseline or recovery period. In addition, we detected histamine level, an index of pulmonary mast cell degranulation, in bronchoalveolar lavage (BAL) samples. Citric acid aerosol inhalation caused decreases in Crs and FEV0.1, indicating airway constriction in the control group. This airway constriction was significantly attenuated by MK-886, mepyramine, cromolyn sodium, and compound 48/80, but not by either methysergide or indomethacin. Both LTC4 and histamine infusion significantly increased the magnitude of CA-induced airway constriction in compound 48/80-pretreated guinea pigs. Citric acid inhalation caused significant increase in histamine level in the BAL sample, which was significantly suppressed by compound 48/80. These results suggest that leukotrienes and histamine originating from mast cells play an important role in CA inhalation-induced noncholinergic airway constriction.     

Reactive oxygen species in sustained airway constriction induced by citric acid aerosol inhalation

We tested if there is a direct relationship between reactive oxygen species and citric acid-induced airway constriction. Guinea pigs were divided into two groups: control and dimethylthiourea (a hydroxyl radical scavenger). The animals in each group were further separated into four subgroups: baseline, recovery 2-3 min, recovery 10 min, and recovery 20 min. Each animal was anesthetized, cannulated, paralyzed, and artificially ventilated. Citric acid aerosol inhalation caused the following significant changes in the control group during the recovery period: airway constriction for at least 20 min, increases in luminol-amplified t-butyl hydroperoxide-initiated chemiluminescence counts in the bronchoalveolar lavage samples at 2-3 and 20 min, an increase in bronchoalveolar lavage fluid substance P level at 2-3 min, and elevations in the bronchoalveolar lavage fluid total cell and neutrophil numbers at 20 min. All citric acid-induced alterations were prevented by dimethylthiourea pretreatment. These results suggest that citric acid inhalation induces the initial release of reactive oxygen species and tachykinins, which causes further cellular infiltration and sustained airway constriction.     

Effects of actinomycin D on airway constriction induced by tachykinins and capsaicin in guinea-pigs.

The effects of actinomycin D on airway constriction induced by tachykinins was studied in guinea-pigs in vitro and in vivo. Actinomycin D significantly inhibited the constriction of isolated guinea-pig trachea induced by neurokinin A (NKA) and eledoisin. Conversely, substance P (SP)- and physalaemine-induced constrictions were not affected by actinomycin D. The same selectivity in the inhibitory action of actinomycin D against tachykinins was also observed in in vivo. Actinomycin D given i.v. specifically inhibited the increase in airway resistance induced by NKA. I.v. injection of NKA caused not only airway constriction but also transient systemic hypotension. Interestingly, actinomycin D injected i.v. inhibited only airway constriction and the systemic hypotension induced by NKA was not affected. These results clearly suggest that actinomycin D specifically inhibits NKA-induced airway constriction in guinea-pigs. Actinomycin D also had an inhibitory action on the airway constriction induced by capsaicin. In the case of capsaicin-induced constriction, actinomycin D was more effective on the later phase of constriction than on the acute phase. The airway constriction induced by capsaicin is thought to be mediated by the release of SP and NKA from sensory nerve endings, and the persistent increase in airway resistance induced by capsaicin is thought to be due mainly to NKA.     

Involvement of hydroxyl radicals in neurogenic airway plasma exudation and bronchoconstriction in guinea-pigs in vivo.

1. Cigarette smoke induces plasma exudation in the airways of rodents by activation of capsaicin-sensitive 'sensory-efferent' nerves. The response is mediated predominantly by substance P (SP) and the magnitude of exudation is regulated by neutral endopeptidase (NEP). The component(s) of the smoke responsible for the activation of the nerves may be reactive oxygen radicals. We investigated the effect of the hydroxyl radical scavenger dimethylthiourea (DMTU), a regulator of superoxide anion, superoxide dismutase (SOD), and a regulator of hydrogen peroxide, catalase, on plasma exudation (measured using Evans blue dye) induced by cigarette smoke in guinea-pig main bronchi in vivo. The effect of DMTU on plasma exudation and non-cholinergic bronchoconstriction (measured as pulmonary insufflation pressure, PIP) induced by electrical stimulation of the vagus nerves was also assessed. Interaction between hydroxyl radicals and NEP was assessed with the NEP inhibitor phosphoramidon. 2. In each of the experiments, cigarette smoke increased plasma exudation by approximately 200% above air-exposed controls. Acute administration of DMTU (1.5 g kg-1, i.v. for 20 min) significantly reduced cigarette smoke-induced plasma exudation by 69%. In contrast, neither SOD (240,000 u kg-1, i.v.) nor catalase (400,000 u kg-1, i.v.) significantly affected the exudative response. 3. Chronic pretreatment with DMTU (1.25 g kg-1 over 4 days) significantly reduced bronchial plasma exudation induced by cigarette smoke by 72%. Phosphoramidon (1.5 mg kg-1, i.v.) completely reversed the inhibition by DMTU of cigarette smoke-induced plasma exudation. 4. Vagal stimulation increased plasma exudation by approximately 200% and PIP by approximately 250%. Acute treatment with DMTU had no significant inhibitory effect on these responses, whereas chronic pretreatment inhibited them by approximately 80%. Phosphoramidon reversed the inhibition by chronic DMTU. 5. SP (1 nmol kg-1) increased plasma exudation by approximately 250%, a response which was not inhibited by either acute or chronic DMTU. 6. We conclude that hydroxyl radicals, rather than superoxide anion or hydrogen peroxide, are involved in the induction of neurogenic plasma exudation and bronchoconstriction induced by cigarette smoke or by electrical stimulation of the vagus nerves. These radicals also affect the activity of NEP. Acute DMTU may affect directly the neural actions of hydroxyl radicals contained in the cigarette smoke. Chronic pretreatment with DMTU may inhibit the neurogenic airway responses by effects on tachykinin biosynthesis and/or axonal transport.     

Effects of the tripeptide substance P antagonist, FR113680, on airway constriction and airway edema induced by neurokinins in guinea-pigs.

FR113680 is a newly developed tripeptide substance P (SP) receptor antagonist. The effects of FR113680 on airway constriction and airway edema induced by neurokinins were investigated in guinea-pigs. In in vitro experiments, FR113680 inhibited the contraction of isolated guinea-pig trachea induced by SP and neurokinin A (NKA) in a dose-dependent manner with IC50 values of 2.3 x 10(-6) and 1.5 x 10(-5) M, respectively. The tracheal contraction induced by histamine and acetylcholine was not affected by FR113680. FR113680 (5 x 10(-5) M) also significantly inhibited the atropine-resistant contraction of isolated guinea-pig bronchi induced by electrical field stimulation. In in vivo experiments, FR113680 given i.v. inhibited SP-induced airway constriction in guinea-pigs at doses of 1 and 10 mg kg-1. However, FR113680 only inhibited NKA- and capsaicin-induced airway constriction by 40-50% even at a dose of 10 mg kg-1. FR113680 also inhibited SP-induced airway edema in guinea-pigs with the same potency as it inhibited SP-induced airway constriction. Histamine-induced airway constriction and airway edema were not affected at a dose of 10 mg kg-1. These results suggest that FR113680 preferentially inhibits responses induced by NK1 receptor activation (SP-induced airway constriction and airway edema), but is less effective on a NK2 receptor-induced response (airway constriction by NKA and neurogenic stimulation).     

Propranolol-induced airway hyperreactivity in guinea-pigs.

In anaesthetized, spontaneously breathing guinea-pigs, enhanced bronchoconstrictor responses (increases RL) to histamine were measured following intravenous injection of practolol, (+/-)-propranolol, (+)- and (-)-propranolol. Propranolol enhanced not only histamine- but 5-hydroxytryptamine (5-HT)-induced bronchoconstrictions and its effects lasted up to 2 h. This increased airway sensitivity was not due to beta-adrenoceptor blockade because: (a) similar effects were produced by racemic propranolol and its two isomers (+)- and (-)-propranolol and (b) whilst equal doses of (+/-)- and (+)-propranolol produced the same potentiation of histamine bronchoconstriction, only (+/-)-propranolol also caused a measurable beta-adrenoceptor blockade in the airways. The enhanced histamine- and 5-HT-induced bronchoconstrictions were antagonized by the leukotriene antagonist FPL 55712 and by the lipoxygenase/cyclo-oxygenase inhibitor BW755c. The results demonstrate that endogenously released leukotrienes can produce not only a direct bronchospasm but may enhance the effects of other bronchoconstrictor agents. The relevance of this leukotriene-mediated hyperreactivity to the non-specific airway hyperreactivity seen in asthmatics is discussed.     

Roles of reactive oxygen species,NF-kappaB,and peroxiredoxins in glycochenodeoxycholic acid-induced rat hepatocytes death

The aim of this study was to determine the roles of reactive oxygen species (ROS), NF-kappaB and antioxidants in glycochenodeoxycholic acid (GCDC, 0-400 micromol/l, 0.5- 3 h)-induced hepatocytes death. The differential uptake of ethidium bromide and acridine orange revealed that apoptotic death occurred dose-dependently in GCDC-treated hepatocytes whereas necrotic death was prominent especially at higher GCDC concentrations (> or =200 micromol/l). ROS generation measured fluorometrically either by a confocal laser microscope or by a microplate fluorescence reader was increased dose-dependently. The dose-dependent NF-kappaB activation with the significant IkappaB-alpha decrease preceded both hepatocyte cell death and the alteration of antioxidant enzymes. The Cu/Zn-SOD level among several antioxidants, we checked, remained unchanged. In contrast, the catalase level and its enzymatic activity were markedly decreased only at 400 micromol/l. The Prx I and Prx II, newly defined antioxidant enzymes reducing H(2)O(2) levels were decreased at the 200 and 400 micromol/l. These observations point to ROS generation in the GCDC-treated hepatocyte as the proximate event that triggers NF-kappaB activation, IkappaB-alpha proteolysis, Prx depletion, and finally cell death. And oxidative stress may be more related to necrotic cell death in GCDC-treated hepatocytes.     

Mast cells in citric acid-induced cough of guinea pigs

It was demonstrated previously that mast cells play an important role in citric acid (CA)-induced airway constriction. To investigate the role of mast cells in CA-induced cough, three experiments were carried out in this study. In the first experiment, 59 guinea pigs were employed and we used compound 48/80 to deplete mast cells, cromolyn sodium to stabilize mast cells, MK-886 to inhibit leukotriene synthesis, pyrilamine to antagonize histamine H(1) receptor, methysergide to antagonize serotonin receptor, and indomethacin to inhibit cyclooxygenase. In the second experiment, 56 compound 48/80-pretreated animals were divided into two parts; the first one was used to test the role of exogenous leukotriene (LT) C(4), while the second one to test the role of exogenous histamine in CA-induced cough. Each animal with one of the above pretreatments was exposed sequentially to saline (baseline) and CA (0.6 M) aerosol, each for 3 min. Then, cough was recorded for 12 min using a barometric body plethysmograph. In the third experiment, the activation of mast cells upon CA inhalation was investigated by determining arterial plasma histamine concentration in 17 animals. Exposure to CA induced a marked increase in cough number. Compound 48/80, cromolyn sodium, MK-886 and pyrilamine, but not indomethacin or methysergide, significantly attenuated CA-induced cough. Injection of LTC(4) or histamine caused a significant increase in CA-induced cough in compound 48/80-pretreated animals. In addition, CA inhalation caused significant increase in plasma histamine concentration, which was blocked by compound 48/80 pretreatment. These results suggest that mast cells play an important role in CA aerosol inhalation-induced cough via perhaps mediators LTs and histamine.     

Catecholamines in hyperpnoea-induced airway constriction of guinea pigs

1. We found previously that propranolol augments hyperpnoea-induced bronchoconstriction (HIB). This study was performed to investigate the underlying mechanism of this aug- menting action of propranolol. 2. In the first series, 45 young Hartley guinea-pigs were divided into five groups: control; propranolol; adrenalectomy; metoprolol and reserpine. Each animal underwent three periods: baseline, hyperpnoea, and recovery. For each animal 1 ml of arterial blood was sampled during the baseline and recovery periods. 3. Treatments of propranolol, metoprolol, and reserpine caused significant decreases in both dynamic respiratory compliance (Crs) and forced expiratory volume in 0.1 s (FEV0.1) during the baseline period. Hyperpnoea caused slight but not significant decreases in Crs, FEV0.1, and maximal expiratory flow at 50% total lung capacity (TLC) (V(max50)) during the recovery period in the control group. Propranolol, but not other treatments, significantly augmented these decreases (indicating HIB). Plasma noradrenaline and adrenaline levels in the reserpine group were not detectable. The above treatments or hyperpnoea did not induce any significant effect on the plasma noradrenaline level. Plasma adrenaline level of the control group was higher than that of either adrenalectomy or reserpine group during the baseline and the recovery periods. 4. In the second series, we avoided repeated blood samplings. Forty-eight animals were evenly divided into two groups: control and propranolol. Each group was again evenly divided into three subgroups: baseline; hyperpnoea, and recovery. Five minutes into the recovery period, we demonstrated HIB in the control group. In terms of V(max50), this HIB was significantly augmented by propranolol. Plasma noradrenaline and adrenaline levels, however, were not significantly altered by either hyperpnoea or propranolol. 5. Taken together, these data suggest that propranolol-augmented HIB has no direct relationship with decreased catecholamine activity.     

Role of leukotrienes in airway hyperresponsiveness in guinea-pigs.

1. Repeated aerosolization of leukotriene C4 (LTC4) to guinea-pigs produced leftward shift in their pulmonary resistance (RL) dose-response curves to inhaled acetylcholine (ACh) without increasing the maximum responses. 2. Repeated LTC4 aerosolization did not increase airway eosinophils. 3. The 5-lipoxygenase-activating protein (FLAP) inhibitor, MK-886, prevented the leftward shift in RL dose-response curves to ACh following repeated antigen challenge in guinea-pigs. 4. MK-886 did not inhibit the increased maximal RL produced by repeated antigen challenge, nor inhibit the airway eosinophilia induced by repeated antigen challenge. 5. Our findings suggest that leukotrienes may account for the leftward shift in pulmonary resistance responses caused by antigen but do not cause the airway eosinophilia nor enhanced maximum broncho-constrictor response to antigen.     

Role of substance P and tachykinin receptor antagonists in citric acid-induced cough in pigs

The purpose of this work was to investigate the role of tachykinins in cough induced by citric acid (0.8 M) in pigs. With this object, we have studied the effect of citric acid on substance P content in the tracheo-bronchial tree and the effects of substance P and of tachykinin receptor antagonists on citric acid-induced cough. Citric acid exposure significantly increased substance P concentration in both broncho-alveolar and tracheal lavage fluids, while it decreased significantly the substance P content in tracheal mucosa. Substance P did not elicit cough, but significantly potentiated the citric acid-induced cough frequency. Tachykinin NK(1), NK(2) or NK(3) receptor antagonists, SR 140333 (nolpitantium), SR 48968 (saredutant) and SR 142801 (osanetant), respectively, significantly inhibited citric acid-induced cough. The same inhibitory effect of tachykinin receptor antagonists was observed, when substance P was nebulised before citric acid challenge. We conclude that citric acid induces in pigs a release of substance P in the tracheo-bronchial tree, which plays a sensitising role on the cough reflex. The involvement of tachykinin NK(1), NK(2), NK(3) receptors are also demonstrated in this reflex.     

Inflammatory cells and oxygen radicals

At sites of inflammation, multiple inflammatory cells including eosinophils, neutrophils, and macrophages are capable of generating reactive oxygen species (ROS), which can contribute to development of various diseases. In case of allergic inflammation, for example, the lung cells obtained by bronchoalveolar lavage (BAL) following antigen challenge generates superoxide anion at nanomolar concentrations. Eosinophils obtained from BAL following a segmental allergen challenge generate more superoxide anion than eosinophils obtained from the peripheral circulation. Such ROS may contribute not only to tissue injury but also to inflammatory reactions. For example, hydrogen peroxide can stimulate both neutrophil and eosinophil adhesion as an autocrine or paracrine mediator via the upregulation of beta2 integrin. Furthermore, ROS may alter morphological or functional properties of endothelial cells, including permeability and adhesion molecule expression. Thus, ROS can promote adhesive interaction between inflammatory and endothelial cells, which could culminate in manifestations of inflammatory diseases such as bronchial asthma.     

Mechanism of protection against "reperfusion injury" by aprotinin. Roles of polymorphonuclear leucocytes and oxygen radicals

We have proposed that reperfusion injury results from the overproduction of reactive oxygen metabolites by PMN as a result of increased sensitivity to oxygen during the low oxygen phase. Aprotinin was demonstrated to inhibit this overproduction and also radical production evoked by chemotactic peptide, 50% inhibitions occurring in the range 5-20 microM. Over this range, no effect on cell viability was observed. This inhibitory effect may provide a scientific basis for the protective effect of aprotinin in reperfusion induced injury.     

Role of endogenous nitric oxide in allergen-induced airway responses in guinea-pigs

1. Endogenous nitric oxide (NO) can be detected in exhaled air and accumulates in inflamed airways. However its physiological role has not been fully elucidated. In this study, we investigated a role for endogenous NO in allergen-induced airway responses. Sensitised guinea-pigs were treated with N^G-nitro-L-arginine methyl ester L-NAME (2.0 mM) or aminoguanidine (AG) (2.0 mM) 30 min before the allergen challenge, and 3 and 4 h after the challenge. Alternatively, L-arginine (2.4 mM) treatment was performed 30 min before, and 2 and 3 h after the challenge. In all groups, ovalbumin (OVA) challenge (2 mg ml⁻¹ for 2 min) was performed, and airway responses, NO production, infiltration of inflammatory cells, plasma exudation and histological details were examined.
2. Allergen-challenged animals showed an immediate airway response (IAR) and a late airway response (LAR), which synchronised with an increase in exhaled NO. Treatment with L-NAME and AG did not affect IAR while they significantly blocked LAR (72% and 80% inhibition compared to vehicle) and production of NO (35% and 40% inhibition). On the other hand, treatment with L-arginine did not affect IAR but potentiated LAR (74% augmentation).
3. In bronchoalveolar lavage (BAL) fluid, allergen-induced increases in eosinophils were reduced by 48% for L-NAME treatment compared to vehicle, and increased by 56% for L-arginine treatment.
4. Treatment with L-NAME significantly decreased airway microvascular permeability to both Monastral blue (MB) and Evans blue (EB) dye (50.6% and 44% inhibition).
5. We conclude that allergen-induced LAR is closely associated with NO production, and that NO plays a critical role in inflammatory cell infiltration and plasma exudation in the allergic condition.

     

Modification by indomethacin of airway contractile responses in normal and sensitized guinea-pigs

Active sensitization of guinea-pigs resulted in an increase in responsiveness and sensitivity of tracheal and lung parenchymal strips to CaCl2 (in K+-depolarised tissue), KCl, acetylcholine and histamine. Indomethacin (5 microM) preferentially enhanced the response of tracheal strips from normal animals to histamine and to a lesser extent acetylcholine but not to CaCl2 or KCl. A similar trend was observed in sensitized tissues. Indomethacin pretreatment did not cause changes in responsiveness or sensitivity of lung parenchymal strips from normal or sensitized guinea-pigs to the agonists tested. It is concluded that immunological sensitization produced a non-specific hyperresponsiveness in trachea and lung parenchymal strips. Conversely, cyclooxygenase inhibition by indomethacin elicited a selective increase in the responsiveness to certain agonists in central but not in the peripheral airways.     

Possible role of leukotrienes in propranolol-induced airway hyperreactivity in sensitized guinea-pigs

In anesthetized guinea-pigs previously sensitized to ovalbumin by intratracheal immunization, the intravenous administration of propranolol (3.1 mg/kg) produced an increase in the respiratory insufflation pressure induced by ovalbumin. Atropine (2 mg/kg), zolertine (1 mg/kg) and indomethacin (3.1 mg/kg) did not antagonize the propranolol-induced airway hyperreactivity. Indomethacin at higher dose (10 mg/kg) and BW755c (10 mg/kg) decreased the potentiation of the respiratory insufflation pressure to ovalbumin. Our results suggest that besides the beta-blockade, propranolol-induced bronchial hyperreactivity to ovalbumin in sensitized guinea-pigs could be mediated by leukotrienes.     

Immediate anaphylactic bronchoconstriction induces airway hyperreactivity in anaesthetized guinea-pigs.

1. The possible acute occurrence of airway hyperreactivity after immediate-type bronchial anaphylaxis has been investigated in anaesthetized guinea-pigs actively sensitized to ovalbumin (OA). 2. Aerosol challenge (OA 10 mg ml-1, 5 s) provoked immediate bronchoconstriction which was substantially, although incompletely, reversed by isoprenaline (Iso) infusion (1 microgram kg-1 min-1) for 10 min). 3. Bronchoconstrictor responses to 5-hydroxytryptamine (5-HT) were enhanced in challenged animals when compared to those in non-challenged animals that had also received Iso. This was seen as a leftward shift in the location of the dose-response curve for the bronchoconstrictor effect of 5-HT (dose-ratio 2.45, 95% confidence limits 1.77-3.38; P less than 0.01). This phenomenon was associated with pulmonary infiltration of polymorphonuclear leukocytes, which was not modified by Iso treatment. 4. Iso infusion alone caused a slight enhancement of airway reactivity seen as a small leftward shift of the dose-response curve for the bronchoconstrictor effect of 5-HT (dose-ratio 1.51, 95% confidence limits 1.07-2.13; P less than 0.05). 5. These results support a causal relationship between acute pulmonary inflammation and airway hyperreactivity in an animal model of human allergic asthma.     

Prejunctional GABA-B inhibition of cholinergic,neurally-mediated airway contractions in guinea-pigs

GABA is a known inhibitory neurotransmitter in the CNS. Recent studies have also demonstrated the presence of GABA in peripheral tissue, including lung. To delineate a role for GABA in lung, the effect of GABA and selective GABA agonists and antagonists on neuronally-induced airway contractions in guinea pigs were studied. In vitro, tracheal contractions induced by electrical field stimulation (EFS) were inhibited by tetrodotoxin and atropine indicating that the contractions were mediated by neuronal release of acetylcholine. The contractions caused by EFS, but not those by exogenous acetylcholine, were inhibited by GABA (EC₅₀ = 4.5 μM) and the selective GABA-B agonist baclofen (EC₅₀ = 9 μM), but not by the GABA-A agonist, muscimol. The inhibitory effect of baclofen was not affected by the GABA-A antagonist, bicuculline, but was significantly reversed with the GABA-B antagonists, 3-aminopropylphosphonic acid (3-APPA) (pA₂ = 4.5) and 2-hydroxysaclofen (pA₂ = 4.1).In vivo, vagal nerve stimulation (5 V, 20 Hz, 0.5 ms, 5 s) in anesthetized, mechanically ventilated guinea-pigs caused cholinergic-dependent bronchospasms that were inhibited by intravenous GABA (3 and 10 mg/kg) and baclofen (1–10 mg/kg), but not by muscimol. The inhibitory effects of GABA and baclofen against vagal bronchospasm were blocked by 3-APPA (5 mg/kg, iv), but not by bicuculline. Responses to the GABA-B agonists were unaltered after the treatment of animals with phentolamine or propranolol to block α-adrenergic and β-adrenergic receptors, respectively. Bronchospasm due to intravenous methacholine was also unchanged by GABA and baclofen. These results demonstrate that GABA inhibits cholinergic bronchoconstriction in guinea-pigs and does so by a mechanism involving inhibitory prejunctional GABA-B receptors.     

帕金森病与氧自由基

该文对近年来在帕金森病与氧自由基关系方面的研究工作做了回顾 ,许多事实表明 ,氧自由基在帕金森病的发病过程中起着重要作用 ,帕金森病患者机体处于氧应激状态 ,帕金森病病变部位的选择性决定了其氧自由基产生的特点 ,其它有关帕金森病的致病学说均与氧自由基有着密切关系。