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     

Effects of Lidocaine on Cytosolic pH Regulation and Stimulus-Induced Effector Functions in Alveolar Macrophages

Local anesthetics influence a variety of stimulus-induced effector functions in leukocytes. The present study determined the effects of lidocaine on intracellular pH (pH_i) regulation, superoxide production, and tumor necrosis factor-α (TNF-α) release in alveolar macrophages (mφ). Resident mφ were obtained by bronchoalveolar lavage of rabbits. The cells were subjected to an intracellular acid load, and subsequent pH_i recovery was followed in the presence or absence of bafilomycin A₁, a specific inhibitor of V-type H⁺-translocating ATPase (V-ATPase) or amiloride, an inhibitor of Na⁺/H⁺ exchange. Lidocaine slowed pH_i recovery in a dose-dependent manner. Pretreatment (1 h) with 2.5 mM lidocaine abolished Na⁺/H⁺ exchange and reduced the V-ATPase-mediated component of pH_i recovery. Lidocaine also significantly depressed the superoxide production induced by phorbol ester. TNF-α release induced by endotoxin was not affected significantly by the local anesthetic. Macrophage viability (trypan blue exclusion) and cellular ATP concentration were unaffected. These results indicate that lidocaine inhibits pH_i regulatory mechanisms in alveolar mφ. This disruption of pH_i regulation could contribute to inhibitory actions of lidocaine on mφ effector functions. Accepted for publication: 28 January 1997     

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     

Effects of Plasmalemmal V-ATPase Activity on Plasma Membrane Potential of Resident Alveolar Macrophages

The acid-base status and functional responses of alveolar macrophages (m) are influenced by the activity of plasmalemmal V-type H⁺-pump (V-ATPase), an electrogenic H⁺ extruder that provides a possible link between intracellular pH (pH_i) and plasma membrane potential (E_m). This study examined the relationships among E_m, pH_i, and plasmalemmal V-ATPase activity in resident alveolar m from rabbits. E_m and pH_i were measured using fluorescent probes. E_m was –46 mV and pH_i was 7.14 at an extracellular pH (pH_o) of 7.4. The pH_i declined progressively at lower pH_o values. Decrements in pH_o also caused depolarization of the plasma membrane, independent of V-ATPase activity. The pH effects on E_m were sensitive to external K⁺, and hence, probably involved pH-sensitive K⁺ conductance. H⁺ were not distributed at equilibrium across the plasma membrane. V-ATPase activity was a major determinant of the transmembrane H⁺ disequilibrium. Pump inhibition with bafilomycin A₁ caused cytosolic acidification, due most likely to the retention of metabolically generated H⁺. V-ATPase inhibition also caused depolarization of the plasma membrane, but the effects were mediated indirectly via the accompanying pH_i changes. V-ATPase activity was sensitive to E_m. E_m hyperpolarization (valinomycin-clamp) reduced V-ATPase activity, causing an acidic shift in baseline pH_i under steady-state conditions and slowing pH_i recovery from NH₄Cl prepulse acid-loads. The findings indicate that a complex relationship exists among E_m, pH_i, and pH_o that was partially mediated by plasmalemmal V-ATPase activity. This relationship could have important consequences for the expression of pH- and/or voltage-sensitive functions in alveolar m.     

Vacuolar H+-ATPase in ocular ciliary epithelium

The mechanisms controlling the production of aqueous humor and the regulation of intraocular pressure are poorly understood. Here, we provide evidence that a vacuolar H⁺-ATPase (V-ATPase) in the ocular ciliary epithelium is a key component of this process. In intracellular pH (pH_i) measurements of isolated ciliary epithelium performed with 2′,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF), the selective V-ATPase inhibitor bafilomycin A₁ slowed the recovery of pH_i in response to acute intracellular acidification, demonstrating the presence of V-ATPase in the plasma membrane. In isolated rabbit ciliary body preparations examined under voltage-clamped conditions, bafilomycin A₁ produced a concentration-dependent decrease in short-circuit current, and topical application of bafilomycin A₁ reduced intraocular pressure in rabbits, indicating an essential role of the V-ATPase in ciliary epithelial ion transport. Immunocytochemistry utilizing antibodies specific for the B1 isoform of the V-ATPase 56-kDa subunit revealed localization of V-ATPase in both the plasma membrane and cytoplasm of the native ciliary epithelium in both rabbit and rat eye. The regional and subcellular distribution of V-ATPase in specific regions of the ciliary process was altered profoundly by isoproterenol and phorbol esters, suggesting that change in the intracellular distribution of the enzyme is a mechanism by which drugs, hormones, and neurotransmitters modify aqueous humor production.     

Cotton Smoke Inhalation Primes Alveolar Macrophages for Tumor Necrosis Factor-α Production and Suppresses Macrophage Antimicrobial Activities

The present study determined the effects of cotton smoke inhalation on the functioning of alveolar macrophages (mφ). Smoke inhalation led to dose-dependent impairment of respiratory gas exchange by 48 h postexposure and pulmonary edema by 96 h. Maximal effects were observed in animals ventilated with 54 breaths of cotton smoke (3-min exposure, 18 breaths/min). Macrophages were obtained at 48 h postexposure by bronchoalveolar lavage of rabbits subjected to 54 breaths of smoke or room air (control). Phagocytosis of opsonized bacteria and adherence to solid substratum were reduced in smoke-exposed mφ. Smoke inhalation primed mφ for release of tumor necrosis factor-α (TNF-α) induced by lipopolysaccharide (LPS). Smoke-exposed mφ were also primed for TNF-α release induced by phorbol myristate acetate, which suggests that the priming event occurred downstream of protein kinase C activation in the signal transduction pathway. Further, smoke exposure attenuated the inhibitory effects of phosphodiesterase inhibitors on LPS-induced TNF-α release. Thus, the priming event may be mediated through cAMP and/or protein kinase A. The data indicate that cotton smoke inhalation suppresses the antimicrobial activities of alveolar mφ and can lead to excessive mφ production of TNF-α. These mφ effects would be expected to contribute to the pathophysiological abnormalities associated with smoke inhalation injury. Accepted for publication: 19 February 1998     

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     

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     

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     

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     

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     

Budesonide Down-Regulates Eosinophil Locomotion But Has No Effects on ECP Release or on H2O2 Production

Treatment of allergic asthma with inhaled corticosteroids results in local down-regulation of proinflammatory cytokine synthesis and in marked decrease in tissue eosinophilia. Blood concentrations of inhaled corticosteroids, although significantly lower than those measured in the lung, may still have antiinflammatory effects on circulating eosinophils, reducing their ability to migrate. The aim of our study was to evaluate in vitro the activity of budesonide on blood eosinophils by measuring their chemotactic response, eosinophil cationic protein (ECP) release, and hydrogen peroxide (H₂O₂) production in the presence of different drug concentrations similar to those obtained at airway level (10⁻⁸ and 10⁻⁷ M) and at blood level (10⁻¹⁰ and 10⁻⁹ M). Partially purified blood eosinophils, isolated from 23 asthmatic subjects, were used to evaluate the activity of budesonide on: (1) chemotaxis toward the activated fifth component of complement (C5a, 0.1 μg/ml) or recombinant human (rh) interleukin (IL)-5 (200 pg/ml), (2) ECP release by cells stimulated with tetradecanoylphorbol acetate (TPA) and (3) H₂O₂ production by TPA-activated cells. The chemotactic response to C5a was down-regulated significantly by budesonide only by the highest concentrations tested (10⁻⁸ and 10⁻⁷ M); differently, budesonide was effective in inhibiting eosinophil migration toward rhIL-5, at all concentrations tested (p < 0.01, each comparison). By contrast, no drug-induced modifications were observed in ECP release or in H₂O₂ production (p > 0.05, each comparison). We conclude that concentrations of budesonide similar to those obtained in vivo are effective in inhibiting eosinophil locomotion but not in down-regulating the release of reactive oxygen species and granule-associated proteins. Accepted for publication: 11 February 1999     

Effect of Terbutaline on Exercise Capacity and Pulmonary Function in Patients with Chronic Obstructive Pulmonary Disease

The objective of this study was to investigate the effects of a single dose of a β₂-agonist, terbutaline (Bricanyl Turbuhaler?), on resting lung function and exercise capacity in patients with chronic obstructive lung disease. Using a double-blind, placebo-controlled, randomized crossover study and outpatients from a department of pulmonary medicine at a major inner-city hospital, we examined 26 individuals with chronic obstructive lung disease who met the criteria of 40% ≤ FEV₁≤ 70% of predicted, FEV₁/FVC ≤ 70%, and ΔFEV₁≤ 200 ml 20 min after inhalation of 1 mg of terbutaline. The patients inhaled 2.5 mg of terbutaline and matched placebo. At rest, terbutaline caused significant increases in D_LCO, MVV_12sec, and all spirometric indices derived from the flow-volume loop, the increases also being significantly larger than those after inhalation of 1 mg of terbutaline for FEV₁, FVC, and PEF. The peak work rate was unchanged after terbutaline. Oxygen uptake, ventilation, and tidal volume at peak work rate increased significantly, whereas carbon dioxide elimination increased insignificantly. Cumulative oxygen uptake and carbon dioxide elimination during progressive exercise to exhaustion and 10 min of recovery were significantly higher after terbutaline. We concluded that despite significant improvements in resting lung function, inhalation of 2.5 mg of terbutaline did not increase exercise capacity, but it increased cumulative oxygen uptake and carbon dioxide elimination during exercise and recovery, presumably because of a thermogenic effect of terbutaline. Accepted for publication: 29 April 1999     

Suppressive Effect of Prostaglandin E1 on Pulmonary Hypertension Induced by Monocrotaline in Rats

The effect of administering prostaglandin E₁ (PGE₁) on the extent of monocrotaline (MCT)-induced pulmonary hypertension and cytokine production [interleukins (IL) 1 and 6 and tumor necrosis factor (TNF)] by macrophages during MCT induction of pulmonary hypertension was studied. Right ventricle/left ventricle plus septum weight ratios (RV/LV + S) were used as an index of the development of pulmonary hypertension. Administering PGE₁ at a dose of 0.2 mg/kg/day for 4 weeks reduced significantly the RV/LV + S ratio from 0.428 ± 0.070 to 0.243 ± 0.059 (p < 0.01) and decreased the production of these cytokines: IL-1, from 4.675 ± 3.558 to 1.800 ± 0.722 units; IL-6, from 0.322 ± 0.121 to 0.060 ± 0.039 units; and TNF, from 0.578 ± 0.369 to 0.004 ± 0.004 units. In another series of experiments, a significant reduction of the RV/LV + S ratio was noted for only 1 week when we administered PGE₁ immediately after the injection of MCT. We confirmed that histopathologic improvements of lungs were noted by administering 0.2 mg/kg PGE₁ for 4 weeks. In another experiment, PGE₁ at a concentration of 2 μg/ml suppressed a rise in the cytosolic Ca²⁺ concentration of lipopolysaccharide-stimulated peritoneal macrophages of rats in vitro, suggesting that PGE₁ suppressed cytokine production by macrophages through the suppression of the Ca²⁺ influx. These results suggest that administering PGE₁ may be effective in the treatment of some forms of pulmonary hypertension in humans. Accepted for publication: 20 August 1998     

Cytosolic pH regulation in density-defined subpopulations of bronchoalveolar macrophages

Bronchoalveolar macrophages (m) represent a heterogeneous population of morphologically and functionally distinct cells. In mixed populations of bronchoalveolar m, cytosolic pH (pH _i ) regulation has been shown to involve both Na⁺-dependent and -independent mechanisms for H⁺ extrusion, i.e., passive H⁺ extrusion in exchange for extracellular Na⁺ (Na⁺-H⁺ exchange or NHE) and active H⁺ extrusion by plasmalemmal vacuolar-type H⁺-ATPase (V-ATPase), respectively. The present studies explored the possibility that individual subpopulations of bronchoalveolar m possess distinct ensembles of H⁺ extrusion mechanisms. Rabbit bronchoalveolar m were separated into five density-defined subpopulations using a discontinuous density gradient. Scanning and transmission electron microscopy revealed morphological differences between the subpopulations. The number of plasmalemmal projections and electron-dense inclusions increased with increments in cell density. The subpopulations were also functionally distinct. Fe receptor-mediated phagocytosis increased in the increasing density subpopulations. Despite these differences, all subpopulations displayed Na⁺-dependent and -independent mechanisms for pH _i recovery from intracellular acid loads (ammonia prepulse technique). We conclude that NHE and V-ATPase activities were present in each subpopulation. These findings support the use of mixed populations to study pH _i homeostasis in bronchoalveolar m. Offprint requests to: Akhil Bidani     

Involvement of Tachykinins in Endotoxin-Induced Airway Hyperresponsiveness

Inhaled endotoxin, lipopolysaccharide (LPS), has been shown to result in bronchial hyperresponsiveness (BHR) to endogenous bronchoconstrictive mediators such as histamine. To determine the role of sensory neuropeptides released from bronchopulmonary C-fibers in LPS-induced BHR, 24 guinea pigs were allocated randomly to the following four groups. Animals in Groups I and IV were challenged with intratracheal instillation of 100 μl of saline vehicle, and those in Groups II and III with 1 mg of LPS (Escherichia coli, 0111:B4) in 100 μl of saline. Groups III and IV also received a high dose capsaicin (HDC) treatment to deplete tachykinins from C-fibers 1–2 weeks prior to the experiment. Animals were anesthetized and paralyzed, and total lung resistance (R_L) and compliance (C_dyn) were measured continuously during the experiment. Dose responses of R_L and C_dyn to histamine (0–8 μg/kg, intravenously) and capsaicin (0–1.6 μg/kg, intravenously), a specific C-fiber stimulant, were obtained prior to and at 1, 2, and 3 h following LPS/saline vehicle challenge. At 2 h after LPS, ΔR_L caused by histamine (8 μg/kg) was significantly higher in Group II (1.145%) than that in Group I (280%; p < 0.05); similarly, ΔR_L caused by capsaicin (1.6 μg/kg) was also increased after LPS (Group I, 107%; Group II, 267%; p < 0.05). Although HDC treatment completely abolished the bronchomotor response to capsaicin in both Groups III and IV, it enhanced the LPS-induced BHR to histamine (8 μg/kg; Group III, 1.834%; p < 0.05). In conclusion, these results suggest that the role of tachykinins in LPS-induced BHR may be dependent upon the type and the route of administration of the bronchoactive substance studied. Accepted for publication: 13 December 1996     

Oxidant-induced inhibition of myocardial calcium-independent phospholipase A2

We discovered the acute inhibition of myocardial phospholipase A₂ activity by micromolar concentrations of tert-butyl hydroperoxide and hydrogen peroxide. Specifically, freshly isolated adult rat cardiomyocytes were treated with the oxidants for 30 min, and phospholipase A₂ activity was assessed in cell subcellular fractions using (16∶0, [³H]18∶1) plasmenylcholine and phosphatidylcholine substrates in the absence or presence of calcium. Calcium-independent phospholipase A₂ activity was inhibited by approx 50% in both the cytosolic and membrane fractions by the oxidants. The inhibition of the phospholipase A₂ activity was concentration dependent and preceded detectable changes in cell viability as assessed by lactate dehydrogenase release and rod-shaped morphology. Taking into account that oxidized sn-2 fatty acyl groups must first be hydrolyzed by phospholipase A₂ to be repaired by glutathione peroxidase, we suggest that the observed inhibition of phospholipase A₂ activity by oxidants compromises the myocyte's ability to deal with lipid peroxidation. This conclusion was further validated by the experiments in which pretreatment with the calcium-independent phospholipase A₂ inhibitor bromoenol lactone exacerbated cardiotoxicity of tert-butyl hydroperoxide in myocyte cultures.     

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     

Pleural Tissue Hyaluronan Produced by Postmortem Ventilation in Rabbits

We developed a method that used Alcian blue bound to hyaluronan to measure pleural hyaluronan in rabbits postmortem. Rabbits were killed, then ventilated with 21% O₂–5% CO₂–74% N₂ for 3 h. The pleural liquid was removed by suction and 5 ml Alcian blue stock solution (0.33 mg/ml, 3.3 pH) was injected into each chest cavity. After 10 min, the Alcian blue solution was removed and the unbound Alcian blue solution (supernatant) separated by centrifugation and filtration. The supernatant transmissibility (T) was measured spectrophotometrically at 613 nm. Supernatant Alcian blue concentration (Cab) was obtained from a calibration curve of T versus dilutions of stock solution Cab. Alcian blue bound to pleural tissue hyaluronan was obtained by subtracting supernatant Cab from stock solution Cab. Pleural tissue hyaluronan was obtained from a calibration curve of hyaluronan versus Alcian blue bound to hyaluronan. Compared with control rabbits, pleural tissue hyaluronan (0.21 ± 0.04 mg/kg) increased twofold, whereas pleural liquid volume decreased by 30% after 3 h of ventilation. Pleural effusions present 3 h postmortem without ventilation did not change pleural tissue hyaluronan from control values. Thus ventilation-induced pleural liquid shear stress, not increased filtration, was the stimulus for the increased hyaluronan produced from pleural mesothelial cells. Accepted for publication: 16 September 1999     

Effects of BIIB513 on ischemia-induced arrhythmias and myocardial infarction in anesthetized rats

Na⁺/H⁺ exchange (NHE) plays an important role in the regulation of the intracellular pH (pH_i) and in cardiac cell injury induced by ischemia and reperfusion. In the present study, we investigated the effects of BIIB513, a selective NHE-1 inhibitor on myocardial ischemia induced arrhythmias and myocardial infarction, provoked by 30 minutes of left main coronary artery occlusion followed by 2 hours of reperfusion in an anesthetized rat model. Intravenous administration of BIIB513 (0.01–3.0 mg/kg) did not induce changes in blood pressure or heart rate. BIIB513 (0.01, 0.1, 0.3, 1.0, 3.0 mg/kg) given prior to the coronary artery occlusion dose-dependently reduced ventricular premature beats, ventricular tachycardia, and a complete suppression of ventricular fibrillation down to the dose of 0.1 mg/kg. BIIB513 (0.01, 0.1, 0.3, 1.0, 3.0 mg/kg) given prior to the coronary artery occlusion dose-dependently reduced the infarct size with an ED50 value of 0.16 mg/kg. BIIB513 (1.0 mg/kg) given prior to reperfusion also reduced infarct size by 47.3 ± 13.1%. The reduction in infarct size was accompanied by a decrease in circulating levels of creatine phosphokinase (CPK). In conclusion, the present study demonstrates the cardioprotective ability of NHE-1 inhibition during myocardial ischemia and reperfusion by reducing serious ventricular arrhythmias and myocardial infarct size in anesthetized rats. Received: 18 November 1999, Returned for 1.revision: 9 December 1999, 1.Revision received: 2 May 2000, Returned for 2.revision: 24 May 2000, 2.Revision received: 5 June 2000, Accepted: 7 June 2000