Airway Eosinophilic Inflammation and Bronchial Hyperresponsiveness after Allergen Inhalation Challenge in Asthma

Allergen exposure in atopic asthmatic patients is associated with recruitment and activation of eosinophils in the airways. Once activated, eosinophils release toxic products, including the eosinophil cationic protein (ECP), able to damage bronchial structures and to increase bronchial hyperresponsiveness. With this background, the present study was designed to evaluate whether ECP levels in bronchoalveolar lavage (BAL) fluid could reflect, better than BAL eosinophil counts, the cellular activation that follows allergen exposure in atopic asthmatics. Twenty-two atopic patients attended the laboratory on two separate days. On the 1st day, they underwent methacholine (MCh) inhalation challenge to detect the degree of nonspecific bronchial hyperresponsiveness. On the 2nd day, they underwent fiberoptic bronchoscopy and BAL, at baseline or 4–6 h after allergen inhalation challenge. In this latter patient group, MCh challenge was repeated 3–5 h after allergen challenge, 1 h before fiberoptic bronchoscopy. The analysis of the mean baseline FEV₁ values and the degree of bronchial reactivity to MCh (MCh Pd₂₀) on the 1st study day did not demonstrate differences between the two patient groups (p > 0.1, each comparison). In addition, in the allergen-challenged group, MCh Pd₂₀ was decreased significantly after allergen challenge (151.4 μg/ml and 67.6 μg/ml, respectively, before and after challenge; p < 0.05). Evaluation of the different BAL cell types demonstrated that the proportions of eosinophils and epithelial cells were increased significantly in the allergen-challenged group compared with the group evaluated at baseline (p < 0.01 and p < 0.05, respectively). Moreover, ECP levels, corrected by the correspondent albumin levels (ECP/Alb), were higher in the allergen-challenged group compared with the group evaluated at baseline (p < 0.05). In addition, although a positive correlation was demonstrated between BAL eosinophil percentages and ECP/Alb values (r= 0.72, p < 0.05) in the group evaluated at baseline, no links were found between these parameters in the allergen-challenged group (p > 0.1). However, in this latter group, a weak positive correlation was demonstrated between eosinophil percentages and ΔMch, i.e., the increased nonspecific bronchial reactivity, which is observed after allergen challenge (r= 0.55; p < 0.05). Thus, in stable asthmatic patients an ongoing activation of eosinophils parallels their migration, but this eosinophilic inflammation is not strictly related to bronchial reactivity to Mch. By contrast, after allergen inhalation challenge, eosinophil recruitment and activation seem to follow different temporal kinetics, and eosinophilic inflammation may be partially associated with the degree of airway hyperresponsiveness. Accepted for publication: 15 September 1997     

Antioxidant Function of Ambroxol in Mononuclear and Polymorphonuclear Cells in Vitro

This study quantifies the antioxidant function of ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine) in vitro. Polymorphonuclear cells (PMN) and mononuclear cells were isolated from the blood of healthy volunteers (n= 46) to determine reactive oxygen species (ROS) by luminol-enhanced chemiluminescence. Ambroxol or the controls N-acetylcysteine (NAC), nacystelyn (NAL), glutathione (GSH), superoxide dismutase (SOD), catalase, and the combination of SOD/catalase were incubated for 1 or 2 h with zymosan-activated cells in vitro using concentrations ranging from 10⁻⁶ to 10⁻³ mol/liter. Reduction of ROS-mediated luminescence was similar within the cell types. Ambroxol (10⁻⁴ mol/liter) reduced ROS about 75% (1-h incubation) and 98% (2-h incubation), respectively (p < 0.001). SOD and SOD/catalase, but not the H₂O₂-catalyzing substances (NAC, NAL, GSH, and catalase), reduced cellular ROS. This indicates that inflammatory cells predominantly generate O⁻ ₂, which can be scavenged by ambroxol. The antioxidant function of ambroxol with increasing incubation time suggests additional cellular antiinflammatory properties of this substance. Our results indicate that good antioxidant function of ambroxol is related mainly to direct scavenger function of reactive oxygen metabolites such as O⁻ ₂. However, an antioxidative effect of ambroxol may also be associated with the reduction of prooxidative metabolism in inflammatory cells. Concluding from this observation, and because of the well known high affinity of ambroxol for lung tissue, ambroxol may be an alternative in antioxidant augmentation therapy, particularly in pulmonary diseases characterized by an overburden of toxic oxygen metabolites. Accepted for publication: 5 December 1996     

Bronchial and Bronchoalveolar Inflammation in Single Early and Dual Responders after Allergen Inhalation Challenge

To characterize the cellular inflammation at the bronchial and bronchoalveolar levels, we evaluated 43 patients with asthma who were sensitized to house dust mites. On 2 consecutive days patients underwent methacholine challenge and allergen bronchial challenge. In addition, 6, 24, or 72 h after allergen challenge, fiberoptic bronchoscopy with bronchial lavage (BL) and bronchoalveolar lavage (BAL) was performed. Patients belonging to the 6-h, 24-h, or 72-h group were divided further into two subgroups: those with isolated early response to allergen (LAR⁻), and those with dual response to allergen (LAR⁺). The percentage of eosinophils and of epithelial cells in BAL fluid was significantly higher in LAR⁺ than in LAR⁻ patients in the 6-h group (p < 0.05, each comparison), but not 24 or 72 h after (p > 0.05, each comparison). Similarly, the proportion of BL eosinophils was also higher in LAR⁺ than in LAR⁻ patients, both in the 6-h and in the 24-h group (p < 0.05, each comparison). In addition, increased proportions of BL neutrophils were present in the LAR⁺ patients belonging to the 24-h group (p < 0.05). Comparing ``proximal' = BL vs ``distal' = BAL data, we found a significantly higher proportion of epithelial cells in BL compared with BAL, in both LAR⁻ and LAR⁺ subjects, either 6, or 24, or 72 h after challenge (p < 0.01, each comparison) and increased percentages of BL neutrophils and eosinophils in LAR⁺ patients (p < 0.05, each comparison), but not in LAR⁻ patients, in the 24-h group. The percentages of BL or BAL macrophages and lymphocytes did not differ significantly among the different patient groups. These data indicate that the development of LAR after allergen inhalation challenge is associated with an early recruitment of eosinophils and with epithelial desquamation in the airways. In addition, after allergen challenge epithelial desquamation is more pronounced in the proximal than in the distal airways, independently of the type of bronchial response. Accepted for publication: 7 January 1997     

Oxidative Inactivation of Surfactants

Reactive oxygen species (ROS) may play an important role in the chronic pulmonary morbidity of preterm infants. We therefore studied the magnitude and mechanisms of oxidative inactivation of a natural lung surfactant (NLS) and of two surfactants used for treatment of respiratory distress syndrome, beractant and KL₄ surfactant (KL₄). Incubation with Fenton reagents, 2-4 mM peroxynitrite (ONOO⁻) or 0.5 mM hypochlorous acid (OCl⁻), resulted in an increased minimum surface tension (MST) of all surfactants; the order of effect on MST was beractant > KL₄ > NLS. After incubation with Fenton reagents, NLS contained a higher concentration of conjugated dienes (p < 0.01) but lower concentration of malondialdehyde (p < 0.001) than beractant. Protein carbonyl concentrations after treatment with Fenton reagents were higher in NLS and KL₄ than in beractant (p < 0.05). Surface area cycling for 24 h with 2 mM ONOO⁻ or 0.5 mM OCl⁻ caused both beractant and KL₄ to increase the proportion of light subtypes from 8–10% to 26–29%; with Fenton reagents, there was disappearance of the light subtype and formation of ultraheavy subtype 74–91% with poor MST. Natural and therapeutic surfactants differ markedly in their sensitivity to ROS, which may be important for surfactants in therapeutic use because oxidative inactivation may limit their effect. Oxidation of natural surfactant may result in reduced function and contribute to chronic lung disease. Accepted for publication: 8 January 1999     

1,25-Dihydroxycholecalciferol Stimulates ICAM-1 Expression of Human Alveolar Macrophages in Healthy Controls and Patients with Sarcoidosis

Synthesis and release of 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₂) by alveolar macrophages (AM) have been shown to be increased in granulomatous lung disease. ICAM-1 plays a major part in leukocyte homing to sites of chronic inflammation, which is a crucial step during the inflammatory response. Whether 1,25-(OH)₂D₂ alters the ICAM-1 expression of AM in humans has not been studied. Bronchoalveolar lavage (BAL) was performed in 12 healthy volunteers, in 13 patients with sarcoidosis (active disease n= 8, inactive disease n= 5), and in 9 patients with chronic bronchitis. AM were incubated with different concentrations of 1,25-(OH)₂D₂ (10⁻¹¹ to 10⁻⁶ M) with and without priming with interferon-γ (IFN-γ) and with and without preincubation with 10⁻⁸ M dexamethasone. In addition, the metabolites of vitamin D, 24,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol, were used. The AM expression of ICAM-1 (cELISA) and the release of tumor necrosis factor-α (TNF-α) (bioassay) by AM were determined. In healthy volunteers the ICAM-1 expression on AM was significantly and dose-dependently increased by 1,25-(OH)₂D₂, but not by 24,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol. Priming with IFN-γ resulted in an additive effect. Preincubation with dexamethasone inhibited ICAM-1 expression. Addition of 1,25-(OH)₂D₂ after inhibition by dexamethasone increased ICAM-1 expression significantly. TNF-α secretion of AM from healthy volunteers was significantly reduced by 1,25-(OH)₂D₂. In sarcoidosis patients ICAM-1 expression was significantly higher compared with healthy volunteers. Incubation with 1,25-(OH)₂D₂ resulted in a further significant increase of ICAM-1 expression. TNF-α secretion of AM was increased compared with healthy volunteers. 1,25-(OH)₂D₂ reduced TNF-α secretion; however, this difference was not significant. 1,25-(OH)₂D₂ has an immunomodulating effect on human AM both in healthy volunteers and in sarcoidosis patients with enhanced expression of ICAM-1. It may serve as an autocrine mediator in inflammatory lung disease. Accepted for publication: 5 November 1998     

Mechanisms of Ca2+ overload induced by extracellular H2O2 in quiescent isolated rat cardiomyocytes

Rat cardiomyocytes were exposed to H₂O₂ (1–100 μmol/L) for 10 min with washout for 10 min. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using fluo-3. [Ca²⁺]_i increased with 100 μmol/L H₂O₂ and further increased during washout, causing irreversible contracture in one-half of the cells. The increase in [Ca²⁺]_i with 10 μmol/L H₂O₂ was modest with few cells showing irreversible contracture and attenuated by caffeine, and [Ca²⁺]_i gradually decreased during washout and this decrease was accelerated by a calcium-free solution, while 1 μmol/L H₂O₂ did not have any effects on [Ca²⁺]_i or cell viability. Ca²⁺ overload caused during exposure to 100 μmol/L H₂O₂ was attenuated by caffeine with improved cellular viability but not by chelerythrine, KB-R7943 or nifedipine. With 100 μmol/L H₂O₂ calcium-free solution attenuated the increase during exposure and washout while KB-R7943 or chelerythrine partly attenuated further increase during washout but not improved cell viability, but chelerythrine did not have additional effect on calcium-free treatment. Catalase abolished the effects of H₂O₂. We concluded that the increased [Ca²⁺]_i during exposure to 100 μmol/L H₂O₂ was caused both by release of Ca²⁺ from the intracellular store sites including the sarcoplasmic reticulum and by influx through route(s) other than the voltage-dependent Ca²⁺ channels or Na⁺/Ca²⁺ exchanger, although the Na⁺/Ca²⁺ exchanger or protein kinase C-mediated mechanism was partly responsible for a further increase during washout. Received: 1 February 2001, Returned for revision: 19 February 2001, Revision received: 11 April 2001, Accepted: 11 April 2001     

Inhibitory Effects of Oxyradicals on Surfactant Function: Utilizing in Vitro Fenton Reaction

The inhibitory effects of reactive oxygen species (ROS) on the surface tension-lowering abilities of three surfactants were compared: natural lung surfactant (NLS), KL₄ surfactant containing synthetic peptide resembling the hydrophobic/hydrophilic domains of SP-B in an aqueous dispersion of phospholipids, and Survanta? (SUR) containing SP-B and SP-C. The inhibitory concentrations of Fenton reactants (i.e. 0.65 mM FeCl₂, 0.65 mM EDTA, 30 mM H₂O₂), deduced from dose-response plots of FeCl₂ on minimum surface tension (MST) of SUR, were used to assess the Fenton effect on biophysical properties of various surfactants. Neither H₂O₂ (30 mM) nor FeCl₂ with EDTA (both 0.65 mM) alone affected surfactant function, but when mixed together significantly increased (p < 0.01) the MST of SUR compared with KL₄ (p < 0.05) in a FeCl₂ concentration-dependent manner. This effect on NLS was not significant (p= 0.05) at similar phospholipid concentrations. Also, the range of increases in surface adsorption in mN/m at equilibrium surface tension (EST) was 27–40 for SUR, 36–44 for KL₄, and 24–25 for NLS. We speculate that the presence of SP-A and the catalase content in NLS may have protective effects on inactivation of NLS by ROS. We conclude that the in vitro Fenton effect could be a valuable test system for comparing the inactivation range of surfactants by oxyradicals. Accepted for publication: 20 May 1997     

Induction of Manganese Superoxide Dismutase Gene Expression in Bronchoepithelial Cells after Rockwool Exposure

Superoxide dismutases play an important protective role in the lung defense against the pro-oxidative effect of fibrous dusts (e.g. crocidolite fibers). Particularly crocidolite, but also other asbestos fibers, are known to induce cellular antioxidant defense. Although rockwool, a man-made fiber made from rock, is used widely for insulation purposes, its effects on the superoxide dismutases in bronchoepithelial cells have not been investigated. Thus, the purpose of this study was to determine whether human bronchoepithelial cells (BEAS 2B) respond to rockwool fibers (115-4 experimental rockwool fiber) by induction of MnSOD mRNA and an increase of MnSOD activity levels. The results were compared with BEAS 2B cells exposed to silica (α-quartz: DQ12; SiO₂) and UICC (Union Internationale Contre le Cancer) crocidolite (concentrations of all dusts: 0, 2, 5, 10, 25, 50 μg/cm²= 0, 2.4, 6, 12, 30, 60 μg/ml; 24-h exposure) as control fibers. Scanning electron microscopy confirmed close dust cell contact under all experimental settings. Very low MnSOD mRNA baseline levels rose significantly (p < 0.001) in BEAS 2B cells exposed to all three dusts at 2 μg/cm². However, at >25 μg/cm² MnSOD mRNA levels in silica- and crocidolite- but not in rockwool-exposed cells decreased. Slight (no significance) increases of MnSOD activity were observed which decreased at higher dust (>5 μg/cm²) concentrations. These results suggest that: (1) like crocidolite and silica, rockwool accelerates MnSOD gene expression in bronchoepithelial cells; (2) an increase of MnSOD mRNA levels is not accompanied by MnSOD activity elevation; (3) in contrast to rockwool, high concentrations (≥25 μg/cm²) of crocidolite and silica reduced MnSOD activity and MnSOD mRNA levels. Because oxidants (H₂O₂) and crocidolite fibers were shown to reduce SOD activity, lack of active MnSOD protein may be caused by inactivation on a post-translational level. Furthermore, the decline of MnSOD mRNA and MnSOD activity levels coincides with increasing cytotoxicity. In conclusion, rockwool was demonstrated to induce MnSOD gene expression, perhaps because of its pro-oxidative effect in bronchoepithelial cells. In contrast to crocidolite and silica, rockwool fibers are not cytotoxic in this experimental setting. Accepted for publication: 21 August 1997     

Effects of Recycled Paper Dust Extracts on Isolated Guinea Pig Trachea

The effect of paper dust collected at two different locations in a paper recycling plant (PD1 and PD2) on isolated nonsensitized guinea pig tracheal smooth muscle was studied in vitro. Dust extracts were prepared as a 1:10 w/v aqueous solution. Dose-related contractions of guinea pig tracheal rings were elicited with both PD1 and PD2. Pharmacologic studies were performed with atropine (10⁻⁶ M), indometacin (10⁻⁶ M), pyrilamine (10⁻⁶ M), LY171883 (10⁻⁵ M), nordihydroguaiaretic acid (10⁻⁵ M), and TMB8 (10⁻⁵ M). The possible role of endogenous neuropeptides in this constrictor process was studied by depleting neural mediators with capsaicin (5 × 10⁻⁶ M) before challenge with dust extracts. Constrictor effects were partially inhibited by a wide variety of the mediator blocking agents. The effects of both extracts were almost totally inhibited by the anticholinergic agent atropine, suggesting that a principal pathway mediating this response may involve the parasympathetic nervous system. The intracellular calcium-blocking agent TMB8 also induced a reduction of the contractile responses to PD1 and PD2 concsistent with the well established role of intracellular calcium in smooth muscle constriction. Pretreatment with capsaicin significantly increased the contractile activity of paper dust extracts but only at the higher doses of these extracts. This suggests that the effect of paper dust is not initiated by the release of mediators stored in sensory nerves but that the prerelease of these mediators may enhance the constrictor effects of these dusts. We suggest that paper dust extracts cause dose-related airway smooth muscle constriction possibly associated with the release of cholinergic as well as other mediators. The constrictor effect does not require tissue presensitization or the release of neuropeptides from sensory nerves. Accepted for publication: 21 March 1997     

Furosemide-Induced Bronchodilation in the Rat Bronchus: Evidence of a Role for Prostaglandins

Pretreatment with inhaled fuorsemide has been shown to protect against bronchoconstrictive stimuli that indirectly activate airway smooth muscle. However, it is controversial as to whether furosemide acts directly on airway smooth muscle. To investigate this we studied the effect of furosemide on both methacholine (MCh)- and serotonin (5-HT)-induced bronchoconstriction in explanted rat airways. Lungs from 21 Sprague-Dawley rats (269 ± 15 g) were excised, inflated with agarose solution at 37°C (1% w/v, 48 ml/kg), embedded in 4% agarose, and refrigerated to gel the agarose. Lung slices (0.5–1.0 mm thick) were cultured overnight at 37°C. Explants were placed on a dissecting video microscope, and airway area was measured with an image analysis system. MCh or 5-HT was administered directly to explanted airways (final concentrations 3.8 × 10⁻⁶ M and 3.8 × 10⁻⁵ M, respectively). Five min later furosemide (3.7 × 10⁻⁵ M or 3.7 × 10⁻⁴ M) was added and airway area monitored 5, 10, 15, 30, and 60 min later. Results were expressed as a percentage of the maximal response. Significant bronchodilation was seen after 30 min in airways preconstricted with MCh and after 15 min in those preconstricted with 5-HT following 3.7 × 10⁻⁴ M furosemide (p < 0.05). 3.7 × 10⁻⁵ M furosemide caused bronchodilation only at 60 min in airways constricted with 5-HT. The effect was blocked by a 30-min incubation of explants with 10⁻⁶ M indomethacin. The furosemide-induced bronchodilation effect was not observed in airways strongly constricted with 3.8 × 10⁻⁵ M MCh. These findings indicate that in the rat at least, furosemide induces a weak bronchodilator effect present only at high doses, which seems to be dependent on the production of prostaglandins. This effect may be relevant to the observed therapeutic action of furosemide in asthmatics. Accepted for publication: 27 September 1996     

Methacholine Dose-Response Slopes from Maximal Bronchial Challenge Tests in Asthmatic Children: Methodological Aspects

To determine whether the slope of a maximal bronchial challenge test (in which FEV₁ falls by over 50%) could be extrapolated from a standard bronchial challenge test (in which FEV₁ falls up to 20%), 14 asthmatic children performed a single maximal bronchial challenge test with methacholine (dose range: 0.097–30.08 μmol) by the dosimeter method. Maximal dose-response curves were included according to the following criteria: (1) at least one more dose beyond a ΔFEV₁≥ 20%; and (2) a MFEV₁≥ 50%. PD₂₀ FEV₁ was calculated, and the slopes of the early part of the dose-response curve (standard dose-response slopes) and of the entire curve (maximal dose-response slopes) were calculated by two methods: the two-point slope (DRR) and the least squares method (LSS) in % ΔFEV₁×μmol⁻¹. Maximal dose-response slopes were compared with the corresponding standard dose-response slopes by a paired Student's t test after logarithmic transformation of the data; the goodness of fit of the LSS was also determined. Maximal dose-response slopes were significantly different (p < 0.0001) from those calculated on the early part of the curve: DRR_20% (91.2 ± 2.7 ΔFEV₁% ·μmol⁻¹) was 2.88 times higher than DRR_50% (31.6 ± 3.4 ΔFEV₁% ·μmol⁻¹), and the LSS_20% (89.1 ± 2.8% ΔFEV₁·μmol⁻¹) was 3.10 times higher than LSS_50% (28.8 ± 1.5% ΔFEV₁·μmol⁻¹). The goodness of fit of LSS_50% was significant in all cases, whereas LSS_20% failed to be significant in one. These results suggest that maximal dose-response slopes cannot be predicted from the data of standard bronchial challenge tests. Accepted for publication: 12 December 1996     

Byssinosis: Role of Polymer Length on the Effect of Tannin on the Airway β-Adrenergic Receptor

Tannin, isolated from cotton bracts and implicated in the pathogenesis of byssinosis, inhibits isoproterenol and forskolin-stimulated cAMP release from airway cells in part by decreasing cell surface β-adrenergic receptor number and uncoupling the β-adrenergic receptor from its stimulatory G-protein (G_s) and in part by inhibiting adenylyl cyclase activity. We have hypothesized that cotton tannin, because of its long polymer length, interacts with the hydrophobic binding pocket of the β-adrenergic receptor and alters β-adrenergic receptor binding and G_s coupling. In these studies, tannins of three different polymer lengths and molecular masses were isolated from cotton bracts using sequential Amicon ultrafiltration [molecular mass > 10,000 (YM10 retentate), 1,000–10,000 (YM10 filtrate), and 1,000–5,000 Da (YM2 retentate)]. The YM10 retentate (25 μg/ml) decreased chloride secretion (Jnet = 1.11 ± 0.28 (control) to 0.59 ± 0.18 μEq/cm²·h, p < 0.05, n= 6), decreased cell surface β-adrenergic receptor number (18.0 ± 1.8 (control) to 10.6 ± 0.9 fmol/mg protein, p < 0.02, n= 4), and inhibited forskolin-stimulated cAMP release (5,254 ± 1,290 (control) to 2,968 ± 620 pmol/mg protein, p < 0.01, n= 8). In contrast, neither the YM10 filtrate nor the YM2 retentate had any effect on net chloride secretion, β-adrenergic cell surface receptor number, or forskolin-stimulated cAMP release. We conclude that polymer length is essential for the effect of tannin on the β-adrenergic receptor and on adenylyl cyclase. Accepted for publication: 28 June 1998     

Effects of Concanavalin A on Na+-Dependent and Na+-Independent Mechanisms for H+ Extrusion in Alveolar Macrophages

Alveolar macrophages (mφ) possess two parallel mechanisms for plasmalemmal H⁺ extrusion: a V-type H⁺ pump (V-ATPase) and a Na⁺/H⁺ exchanger (NHE). To investigate the coordinated functioning of these H⁺ extruders for mφ intracellular pH (pH_i) regulation, we investigated the effects of the plant lectin concanavalin A (ConA) on resident alveolar mφ from rabbits. ConA (1 μM, 30-min pretreatment) activated the mφ for phagocytosis of opsonized Escherichia coli. ConA activation did not affect the baseline pH_i of mφ or the initial rate of pH_i recovery (dpH_i/dt) from an intracellular acid load (acid-loaded pH_i nadir ≈ 6.9). However, the contributions of Na⁺-independent H⁺ transport (i.e. V-ATPase activity) and Na⁺-dependent H⁺ transport (i.e. NHE activity) to dpH_i/dt were altered significantly. The lectin stimulated Na⁺/H⁺ exchange and inhibited V-ATPase activity. In control mφ, V-ATPase-mediated H⁺ extrusion was responsible for >80% of dpH_i/dt. Conversely, in ConA-treated mφ, Na⁺/H⁺ exchange was responsible for ∼65% of dpH_i/dt, and V-ATPase activity was responsible for only 35% of dpH_i/dt. These results underscore the complex mechanisms and signaling pathways that coordinate the activities of cellular acid-base transporters in mφ pH_i regulation. Accepted for publication: 18 March 1997     

Evidence for pH Sensitivity of Tumor Necrosis Factor-α Release by Alveolar Macrophages

Alveolar macrophages (mφ) participate in inflammatory and immune responses in acidic microenvironments such as the interstitial fluids of tumors and abscesses. Two plasmalemmal H⁺ extruders interact to control the acid-base status of alveolar mφ, namely a V-type H⁺ pump (V-ATPase) and a Na⁺/H⁺ exchanger. The present study examined the effects of extracellular pH (pH_o) and H⁺ transport inhibitors on tumor necrosis factor-α (TNF-α) release induced by endotoxin (lipopolysaccharide) in rabbit alveolar mφ. The amount and activity of TNF-α in mφ-conditioned media were determined by enzyme-linked immunosorbent assay and L929 fibroblast bioassay, respectively. TNF-α release was suppressed progressively at lower pH_o values (≤7.0). Also, bafilomycin A₁ (a specific inhibitor of V-ATPases) significantly reduced the amount and activity of TNF-α in mφ-conditioned media (pH_o 7.4). However, bafilomycin caused a significant increase in the nonspecific cytotoxicity (i.e. bioactivity insensitive to TNF-α antibody) of mφ-conditioned media. The effects of bafilomycin specifically on TNF-α release followed a time course similar to that of acidic pH_o, suggesting that both treatments acted on similar events in the lipopolysaccharide signal transduction pathway. Amiloride (an inhibitor of Na⁺ transporters including the Na⁺/H⁺ exchanger) also suppressed TNF-α release but displayed a time course of action different from the acidic pH_o or bafilomycin. Accepted for publication: 19 June 1997     

The Effect of Hydrogen Peroxide in Human Internal Thoracic Arteries: Role of Potassium Channels,Nitric Oxide and Cyclooxygenase Products

Introduction We investigated both the effect and the role(s) of potassium channels, nitric oxide (NO) and cyclooxygenase (COX) products in the effect of hydrogen peroxide (H₂O₂) in human internal thoracic artery (ITA) rings. Materials and methods Samples of redundant ITA obtained from patients undergoing a coronary artery bypass graft surgery were cut into 3 mm wide rings and suspended in 20 ml organ baths. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. Results H₂O₂ (10⁻⁷–10⁻⁴ M) produced concentration-dependent relaxation responses in human ITA precontracted by phenylephrine. The relaxant responses to H₂O₂ did not differ significantly between endothelium-intact and endothelium-denuded preparations. Incubation of human ITA rings with superoxide dismutase (50 U/ml) did not affect the relaxant responses to H₂O₂, while 1,000 U/ml catalase caused a significant decrease. Incubation of endothelium-intact or endothelium-denuded human ITA rings with voltage-dependent potassium channel blocker 4-aminopyridine (5 mM) significantly inhibited the relaxant responses to H₂O₂. COX inhibitor indomethacin (10⁻⁵ M) also caused a significant inhibition. Incubation with ATP-dependent potassium channel blocker glibenclamide (10⁻⁶ M) or Ca²⁺-activated potassium channel blocker iberiotoxin (10⁻⁷ M) or NO synthase (NOS) blocker -nitro-l-arginine methyl ester (10⁻⁴ M) did not alter relaxant responses of ITA rings to H₂O₂. Conclusion The findings of the present study suggested that H₂O₂-induced relaxation responses in human ITA were neither dependant on the endothelium nor blocked by NOS inhibition but they rather seem to depend on the activation of voltage-dependent potassium channels and COX.     

The Highly Expressed and Inducible Endogenous NAD(P)H:quinone Oxidoreductase 1 in Cardiovascular Cells Acts as a Potential Superoxide Scavenger

It has recently been demonstrated that purified NAD(P)H:quinone oxidoreductase 1 (NQO1) is able to scavenge superoxide (O₂^•−) though the rate of reaction of O₂^•− with NQO1 is much lower than the rate of enzymatic dismutation catalyzed by superoxide dismutase (SOD). This study was undertaken to determine if the endogenously expressed NQO1 in cardiovascular cells could scavenge O₂^•−. We observed that NQO1 was highly expressed in cardiovascular cells, including rat aortic smooth muscle A10 and cardiac H9c2 cells, as well as normal human aortic smooth muscle and endothelial cells. NQO1, but not SOD in the cardiovascular cells was highly inducible by 3H-1,2-dithiole-3-thione (D3T). Cytosols from H9c2 and human aortic smooth muscle cells (HASMCs) were isolated to determine the O₂^•− scavenging ability of the endogenously expressed NQO1 by using pyrogallol autooxidation assay. We showed that cytosols from the above cells inhibited pyrogallol autooxidation in an NADPH or NADH-dependent manner. The NADH/NADPH-dependent inhibition of pyrogallol autooxidation by the cytosols was completely abolished by the NQO1-specific inhibitor, ES936, suggesting that the endogenously expressed NQO1 could scavenge O₂^•−. In the presence of NADH/NADPH, cytosols from D3T-treated cells showed increased ability to scavenge O₂^•− as compared to cytosols from untreated cells. This increased ability to scavenge O₂^•− was also completely reversed by ES936. 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin-trapping experiments using potassium superoxide as a O₂^•− generator further confirmed the ability of NQO1 from HASMCs to scavenge O₂^•−. The spin-trapping experiments also showed that induction of NQO1 by D3T in HASMCs augmented the O₂^•− scavenging ability. Taken together, these results demonstrate that the highly expressed and inducible endogenous NQO1 in cardiovascular cells may act as a potential O₂^•− scavenger.     

Hyperosmotic perfusion of the beating rat heart and the role of the Na+/K+/2CI− co-transporter and the Na+/H+ exchanger

The aim of the present study was to investigate the role of the Na⁺/K⁺/2CI⁻ co-transporter and the Na⁺/H⁺ exchanger on contractile function and electrolyte regulation during hyperosmotic perfusion of the heart. Langendorff perfused rat hearts were subjected to hyperosmolal perfusion in 10-min intervals. Perfusates were made hyperosmotic by adding mannitol to the buffer (370, 450 and 600 mOsmol/kg H₂O). Cardiac contractile function was monitored with a balloon in the left ventricle (LV) coupled to a pressure transducer. Cardiac effluent was sampled repeatedly throughout and after hyperosmotic perfusion and analyzed for content of Na⁺, K⁺ and CI⁻. All three hyperosmotic perfusates initially reduced LV developed pressure (LVDP), but for 370 and 450 mOsmol/kg H₂, LVDP recovered to baseline within 4 min of perfusion. With 600 mOsmol/kg H₂, LVDP recovered slowly and was 50% below baseline after 10 min of hyperosmotic perfusion. Inhibition of the Na⁺/H⁺ exchanger with 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and 3-methyl-sulfonyl-4-piperidinobenzoyl-guanidine methanesulfonate (HOE 694) abolished the recovery of LVDP to the 600 mOsmol/kg H₂ perfusate, whereas inhibition of the Na⁺/K⁺/2CI⁻ co-transporter had no impact on LVDP. Potassium was taken up by the heart during hyperosmotic perfusion and this uptake was significantly reduced with inhibition of the Na⁺/H⁺ exchanger. Intracellular pH was assessed with ³¹P magnetic resonance spectroscopy and hyperosmolality induced a significant alkalosis that was dependent upon the Na⁺/H⁺ exchanger. The rat heart responds to moderate elevations in osmolality with a transient reduction in contractile function, whereas an elevation of 300 mOsmol/kg H₂ persistently reduces contractile function. The Na⁺/H⁺ exchanger, but not the Na⁺/K⁺/2CI⁻ co-transporter, is of importance in contractile recovery and electrolyte regulation during hyperosmotic perfusion in the rat heart. Received: 10 December 1998, Returned for revision: 18 January 1999, Revision received: 1 July 1999, Accepted: 19 July 1999     

The arterial site of action of nitric oxide in the neonatal pig lung determined by microfocal angiography

To determine the site of action of inhaled nitric oxide (iNO) in the newborn pig lung, lungs were isolated and perfused at constant flow for microfocal x-ray angiography. Measurements of pulmonary arterial diameters were made on arteries in the 100–2500 μm diameter range under control conditions, during vasoconstriction caused by hypoxia (decreasing PO₂ from ∼120 to ∼50 Torr), or N^ω-nitro-L-arginine methylester (L-NAME 10⁻⁴ M) administration, with or without vasodilation induced by iNO (40 ppm) or by the NO donor S-nitroso-N-acetylpenicillamine (SNAP 5 × 10⁻⁶ M) given intravascularly. Hypoxia caused constriction only in smaller arteries whereas L-NAME constricted arteries throughout the size range studied. iNO dilated the smaller arteries more than the larger arteries under all study conditions. SNAP was used to provide an intravascular source of NO for comparison to iNO. SNAP also dilated smaller arteries more than larger arteries, but it had a significantly greater effect on the large arteries than did iNO. This suggests that differential accessibility of the vascular smooth muscle to NO between sources, air and blood, is a factor in the diameter dependence of the responses. Accepted for publication: 13 March 2001     

Epigallocatechin-3-Gallate Protects Na<Superscript>+</Superscript> Channels in Rat Ventricular Myocytes Against Sulfite

Sulfite (bisulfite/sulfite) can affect voltage-gated sodium (Na⁺) channels (VGSC) in a concentration-dependent manner in isolated rat ventricular myocytes. In this study, the effect of epigallocatechin-3-gallate (EGCG) on VGSC in isolated ventricular myocytes was studied. Ventricular myocytes were exposed to 10 μM bisulfite/sulfite for 10 min, and EGCG was then administered in different concentrations (10, 30, 50 μg ml⁻¹). Decreased activity of superoxide dismutase, catalase (CAT) and glutathione peroxidase (GPx) was observed after bisulfite/sulfite exposure, with significant increase in Na⁺ currents (I _Na) and alterations in half-activation voltage and half-inactivation voltage. Intracellular reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), hydroxyl (OH^·), and superoxide anion (O₂^·−) were increased. After EGCG treatment, activity of the aforementioned enzymes increased while the ROS level decreased. The effects progressed with increasing amounts of EGCG, up to a level similar to blank control at the dose of 50 μg ml⁻¹ EGCG, EGCG also reduced the I _Na and reversed the alterations in half-activation voltage and half-inactivation voltage. In conclusion, EGCG could protect Na⁺ channels in rat ventricular myocytes against the oxidative damage induced by sulfite as a scavenger of the ROS.