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     

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     

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     

Ramiprilat prevents the development of acute coronary endothelial dysfunction in the dog

We investigated the effect of an infusion of ramiprilat on the development of coronary endothelial dysfunction. In anesthetized dogs, the endothelium-dependent vasodilators acetylcholine (ACh, 5 and 10 μg · min⁻¹ for 1 min) and serotonin (5-HT, 50 and 100 μg · min⁻¹ for 1 min) and the endothelium-independent vasodilator nitroglycerin (NTG, 50 and 100 μg · min⁻¹ for 1 min) were given intracoronarily (i.c.) both prior to and after 60 min of ischemia (I) and 180 min of reperfusion R of a coronary artery. During I/R the dogs received i.c. either saline (N = 22) or ramiprilat (40 ng/kg · min⁻¹, N = 14). At the end of the experiment, a biopsy of the most distal coronary bed was processed for scanning electron microscopy (SEM). Prior to I/R all vasodilators induced a similar dose-related increase in coronary flow in both groups. Following I/R, in controls the responses to ACh and 5-HT were significantly blunted (ACh: −39 % and −34 %; 5-HT: −48 % and −49 %); those to NTG were unchanged. Ramiprilat significantly prevented the blunting of the responses to ACh (−5 %, and −10 %) and 5-HT (−11 %, and −19 %). SEM of control subepicardial arterioles showed adhesion of leukocytes to the endothelium and crater formation. No craters were seen in the ramiprilat-treated dogs. Thus, an acute infusion of ramiprilat significantly prevents the development of coronary endothelial dysfunction. Additionally, the appearance of crater-like changes on the endothelial surface can be taken as a morphological marker of endothelial dysfunction. Received: 19 November 1998, Returned for 1. revision: 17 December 1998, 1. Revision received: 18 January 1999, Returned for 2. revision: 10 February 1999, 2. Revision received: 26 February 1999, Accepted: 1 March 1999     

Interaction between the opposing functional effects of cyclic AMP and cyclic GMP in hypertrophic cardiac myocytes

We tested the hypothesis that in isolated cardiac myocytes, the negative functional effects of cyclic GMP would be blunted when the level of cyclic AMP was increased and that this interaction would be altered in renal hypertensive (One-Kidney-One-Clip, 1K1C) cardiac hypertrophic rabbits. Using isolated control and 1K1C ventricular myocytes, cyclic AMP and cell shortening (%) data were collected: 1) at baseline, 2) after the addition of 8-Br-cGMP 10^{−7, −6, −5} M, and 3) after forskolin (10⁻⁶ M), and adenylate cyclase activator, followed by 8-Br-cGMP 10^{−7, −6,−5} M. Basal levels of cyclic AMP were similar in control vs. 1K1C myocytes (10.2 ± 1.6 vs. 11.3 ± 2.6 pmol/10⁵ myocytes). We found that 8-Br-cGMP decreased the percent shortening in a dose related manner in both control myocytes (5.1 ± 0.6 to 3.2 ± 0.4%) and hypertrophic myocytes (5.2 ± 0.4 to 3.6 ± 0.5). The level of cyclic AMP significantly increased after the addition of 8-Br-cGMP in control myocytes (14.1 ± 2.1), but not in 1K1C myocytes. Forskolin increased the percent shortening in the control myocytes (3.8 ± 0.1 to 4.8 ± 0.4), but no significant increase was noted in the hypertrophic myocytes (3.6 ± 0.3 to 3.7 ± 0.3). The level of cyclic AMP significantly increased after the addition of forskolin in both control (13.9 ± 2.0), and 1K1C cells (14.6 ± 3.8). Forskolin attenuated the negative functional effects of 8-Br-cGMP in the control (4.8 ± 0.4 to 3.2 ± 0.1) and 1K1C myocytes (3.7 ± 0.3 to 2.7 ± 0.3). The adition of 8-Br-cGMP did not affect the level of cyclic AMP after forskolin in either control (13.9 ± 2.0 to 14.8 ± 2.5) or 1K1C myocytes (14.6 ± 3.8 to 13.8 ± 1.9). These data indicated that in hypertrophic cardiac myocytes the negative functional effects of 8-Br-cGMP were similar to control, but the positive functional effects of cyclic AMP were blunted. There was an increase in cyclic AMP levels after addition of 8-Br-cGMP in control but not 1K1C cells. We conclude that in control and hypertrophic myocytes, the effects of cyclic GMP were blunted after forskolin, but this did not seem to be related to cyclic AMP phosphodiesterase activity. Received: 12 October 1999, Returned for 1. revision: 24 November 1999, 1. Revision received: 23 March 2000, Returned for 2. revision: 26 May 2000, 2. received: 16 June 2000, Accepted: 12 July 2000     

Electromechanical effects of leukotriene D4 on ferret tracheal muscle and its muscarinic responsiveness

We investigated the possible electrophysiological processes by which leukotriene D₄ (LTD₄) affects airway smooth muscle and its responsiveness to acetylcholine (ACh). For study in vitro, preparations of ferret tracheal muscle (dissected free of overlying mucosal and submucosal layers) were used. These preparations were arranged so that force transducers and glass intracellular microelectrodes (having tip resistances of 35–60 megohm) could be used to measure isometric force generation and cell membrane potential (Em) simultaneously from muscle stimulated by LTD₄. At rest, the muscle was electrically and mechanically quiescent and had an Em of −59±0.2 mV (mean±SEM). We found that ferret tracheal muscle cells were relatively sensitive to LTD₄, and that both the resulting depolarization (beginning at 10⁻¹⁰ M LTD₄) and force generation (produced by higher concentrations) progressed in a concentration-dependent manner. Depolarization by 10⁻⁹ M LTD₄ elicited electrical oscillations. These oscillations were accompanied by phasic contractile activity at 5 × 10⁻⁹ M LTD₄. Verapamil abolished these oscillations and diminished force substantially. We also found that ACh depolarized and contracted the muscle in a concentration-dependent manner. It caused electrical oscillations at ≥ 10⁻⁶ M. Diltiazem abolished these oscillations and markedly diminished force generation without affecting Em. Preexposure of airway muscle preparations for 20 min to a concentration (10⁻¹⁰ M) of LTD₄ that, by itself, did not produce significant force, substantially augmented the voltage-tension relationship of the muscle upon ACh stimulation. We conclude that there is an electrical basis for the slow, prolonged force generation of airway muscle caused by LTD₄, and that LTD₄ potentiates the electromechanical responsiveness of the airway muscle to muscarinic stimulation.     

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     

Effect of Hyaluronidase on Albumin Diffusion in Lung Interstitium

Albumin diffusion measured in an isolated segment of rabbit lung interstitium with a radioactive tracer (¹²⁵I-albumin) technique was independent of albumin concentration and similar to the free diffusion of albumin in water (Qiu et al, 1998. J Appl Physiol 85: 575–583). We studied the effect of hyaluronidase on the diffusion of albumin. Isolated rabbit lungs were inflated with silicon rubber by way of airways and blood vessels, and two chambers were bonded to the sides of a ∼0.5-cm thick slab enclosing a vessel with an interstitial cuff. One chamber was filled with 2 g/dl albumin solution containing ¹²⁵I-albumin and 0.02 g/dl hyaluronidase. Unbound ¹²⁵I was removed from the tracer by dialysis before use. The other chamber filled with Ringer's solution was placed within a NaI(Tl) scintillation detector. Diffusion of tracer was measured continuously for 120 h. Albumin diffusion coefficient (D) and interstitial area (A) were obtained by fitting the tracer-time curve with the theoretical solution of the equation describing one-dimension diffusion of a solute across a membrane. D averaged 5.2 × 10⁻⁷ cm²/s for albumin diffusion with hyaluronidase, 20% less than that measured previously without hyaluronidase. Hyaluronidase had no effect on A. Results indicated an interaction between albumin and interstitial hyaluronan that was the opposite of the steric effect on albumin excluded volume measured in solution. Accepted for publication: 9 May 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     

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     

Relaxant effects of forskolin on guinea pig tracheal smooth muscle

We investigated the relaxant effects of forskolin, a diterpene derivative isolated from the roots ofColeus forskohlii, on guinea pig airway smooth muscle by measuring the isometric tension of tracheal smooth muscle in vitro and transcutaneous Po₂ during the histamine inhalation test (HIT) in vivo. Forskolin (10⁻⁹–10⁻⁵ M) caused dose-dependent relaxant effects on resting tone and on leukotriene C₄ (10⁻⁷ M)-, leukotriene D₄ (10⁻⁷ M)-, and carbachol (3 × 10⁻⁶ M)-induced contraction of tracheal smooth muscle. Moreover, with propranolol pretreatment the relaxant effect of forskolin on tracheal smooth muscle did not change, whereas with the same pretreatment the relaxant effect of isoproterenol diminished. Forskolin (10⁻⁸–10⁻⁶ M) raised tissue cyclic AMP levels dose-dependently in tracheal smooth muscle (6.7–359.9 pmol/mg protein). Forskolin (1 mg/kg) administered subcutaneously raised the respiratory threshold of (RT-histamine in the HIT. The determination of the RT-histamine by measuring tcPo₂ was possible without anesthesia. These results suggest that forskolin relaxes airway smooth muscle in guinea pigs in vitro and in vivo by raising tissue cyclic AMP levels and that its actions are independent ofβ-adrenoceptors.     

Mechanism of leukotriene-induced contraction of isolated guinea pig tracheal smooth muscle

The mechanical response of guinea pig tracheal smooth muscle to leukotriene (LT) B₄, C₄, D₄ and E₄ was investigated, and the effects of several agents on LT-induced contraction were determined. Agents used in the present study were: verapamil, a Ca-channel blocker; dibutyryl cyclic-AMP (DBcAMP); aminophylline; N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), a calmodulin antagonist; N-(6-aminohexyl)-1-naphthalene-sulfonamide hydrochloride (W-5), resembling W-7 in structure but without calmodulin antagonism. The results were as follows: 1) LTB₄ had a contractile activity, dependent on the [Ca]o concentration; it was suppressed by a [Mg]o increase to 1.2 mM and over. Little effect was observed from other drugs. 2) Contractile activities of LTC₄, D₄ and E₄ were dose-dependent, depending upon [Ca]o as well as LTB₄. 3) Verapamil (5×10⁻⁶−10⁻³ M), DBcAMP (3×10⁻⁶−3×10⁻³ M) and aminophylline (3×10⁻⁸−3×10⁻³ M) suppressed LTs (C₄, D₄, and E₄, 10⁻⁸ M)-induced contraction dose-dependently. However, W-7 and W-5 did not suppress LT-induced contraction. These results suggest that the LTC₄-, D₄-and E₄-induced responses could be explained by the effect of LTC₄, D₄ and E₄ on increasing Ca influx through the plasma membrane.     

A Murine Model of Latex Allergy-Induced Airway Hyperreactivity

Sensitization to latex proteins can cause immediate IgE mast cell-mediated reactions. Health care workers have been found to be particularly at risk because of high exposure. Latex allergy can be produced in mice as demonstrated by IgE and eosinophil responses. Thus the mouse is a potential animal model for studying this disease, but the airway response to latex sensitization in mice has not been evaluated previously. In the present study, we immunized BALB/c mice intranasally with nonammoniated latex proteins. Animals were anesthetized, and lung mechanics were evaluated plethysmographically. Changes in pulmonary conductance (G_L) and compliance (C_dyn) were measured in response to a nonspecific challenge with methacholine or to a direct challenge with intravenous latex antigen. Latex sensitization resulted in elevated levels of IgE and latex-specific IgG₁ as well as interstitial infiltrates consistent with an allergic response. The methacholine dose-response ED₅₀ for G_L was 116.4 μg for the control mice and fell significantly to 20.9 μg for latex-sensitized mice. The ED₅₀ calculated for C_dyn was also significantly lower after latex sensitization. The G_L in latex-sensitized mice challenged with latex antigen fell significantly from a prechallenge value of 1.87 ± 0.41 (S.E.) to 0.198 ± 0.03 ml · s⁻¹· cmH₂O after latex antigen challenge. The results indicate that latex-sensitized mice did exhibit increased airway reactivity in the methacholine challenge test. The latex allergic response in mice is unique in that direct challenge with latex antigen itself also resulted in a significant airway response. Accepted for publication: 10 September 1998     

The Relation of Fat-free Mass to Maximum Exercise Performance in Patients with Chronic Obstructive Pulmonary Disease

To assess the factors determining maximum exercise performance in patients with chronic obstructive pulmonary disease (COPD), we examined nutritional status with special reference to body composition and pulmonary function in 50 stable COPD patients. Nutritional status was evaluated by body weight and body composition, including fat mass (FM) and fat-free mass (FFM) assessed by bioelectrical impedance analysis (BIA). Exercise performance was evaluated by maximum oxygen uptake (Vo _2max) on a cycle ergometer. A total of 50 patients (FEV₁= 0.98 L) was divided randomly into either a study group (group A, n= 25) or validation group (group B, n= 25). Stepwise regression analysis was performed in group A to determine the best predictors of Vo _2max from measurements of pulmonary function and nutritional status. Stepwise regression analysis revealed that Vo _2max was predicted best by the following equation in group A: Vo _2max (mL/min) = 10.223 × FFM (kg) + 4.188 × MVV (L/min) + 9.952 × DL_co (mL/min/mmHg) − 127.9 (r= 0.84, p < 0.001). This equation was then cross-validated in group B: Measured Vo _2max (mL/min) = 1.554 × Predicted Vo _2max (mL/min) − 324.0 (r= 0.87, p < 0.001). We conclude that FFM is an important factor in determining maximum exercise performance, along with pulmonary function parameters, in patients with COPD. Accepted for publication 15 February 2000     

The inotropic response of the isolated,perfused, working rat heart to norepinephrine is attenuated by inhibition of nitric oxide

Experiments on isolated, perfused, working left ventricular (LV) hearts of 66 female Wistar rats were done to examine whether nitric oxide (NO) influences the effects of norepinephrine (NE) on coronary flow as well as on contraction and relaxation. Functional parameters were monitored before and after application of NE at a concentration of 3 × 10⁻⁸ M in the absence and presence of the nitric oxide synthase (NOS) inhibitor L-nitro-arginine (L-NA) at a concentration of 1 × 10⁻⁴ M and of the spontaneous NO donor sodium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolat (DEA/NO) at a concentration of 1 × 10⁻⁷ M. In control experiments, heart rate was varied by electrical stimulation between 200 and 400 beats/min. Within this range of heart rates, coronary flow and cardiac output remained constant, while stroke volume, LV peak pressure and LV dP/dt_max decreased with increasing heart rate. NE increased coronary flow from 7.6 ± 0.4 to 9.8 ± 0.7 ml/min and induced the well-known positive chronotropic and inotropic effects. DEA/NO increased coronary flow; however, the inotropic and lusitropic parameters were not affected. Simultaneous infusion of NE with DEA/NO further increased coronary flow from 9.8 ± 0.7 to 12.1 ± 0.8 ml/min without a significant effect on any other functional parameter. When NOS was inhibited by L-NA, the positive inotropic effect of NE was attenuated. Cardiac output, however, was increased, while coronary flow did not change significantly. Under these conditions, NE increased dP/dt_max by 65.5 ± 5.8% (from 2999 ± 97 to 4929 ± 230 mmHg/s) compared with an increase by 92.8 ± 6.7% (from 3770 ± 82 to 7234 ± 211 mmHg/s) under control conditions. Application of DEA/NO reversed the attenuated inotropic response, but relaxation remained partially impaired. Thus, the presence of NO seems to be necessary for the inotropic effect of NE to become manifest. Received: 9 February 2001, Returned for 1. revision: 22 February 2001, 1. Revision received: 25 May 2001, Returned for 2. revision: 12 June 2001, 2. Revision received: 20 July 2001, Returned for 3. revision: 2 August 2001, Accepted: 20 August 2001     

Bronchodilating effects of the anesthetic ketamine in an in vitro guinea pig preparation

Ketamine, a dissociative anesthetic, is capable of reducing airway resistance and has proved useful in anesthetizing surgical patients with acute or chronic bronchospasm. To determine if ketamine alters smooth muscle tone, the relative responses of large and small airways of the same animal were studied by comparing the pharmacologic reactivity of tracheal smooth muscle strips with that of a specially prepared perfused bronchial tree. Trachealis and bronchial smooth muscle were found to react to methacholine and histamine in a concentration-dependent manner and have similar sensitivities. Ketamine, by itself, in the concentration range 10⁻⁸–10⁻³ M did not alter resting tone as compared to epinephrine, which reduced baseline tone. In tissues precontracted with histamine or methacholine at ED₅₀ doses, ketamine inhibited smooth muscle contraction. In a second series of experiments, the dose-dependent contraction of smooth muscle to histamine and methacholine was reevaluated in the presence of ketamine. Both tissue sensitivity and maximum contractile response to these agonists were reduced by ketamine at 10⁻⁴ M. These data indicate that ketamine alters the in vitro response of guinea pig airways to agonists associated with the asthmatic state. Although ketamine does not reduce airway tone in nonstimulated tissues, its effects on agonist-induced contraction of airway tissues in vitro are consistent with clinical observations that ketamine relieves bronchospasm.     

Atypical Adrenoceptor-mediated Relaxation of Canine Pulmonary Artery Through a Cyclic Adenosine Monophosphate–dependent Pathway

To determine whether functional atypical β-adrenoceptors (β₃-adrenoceptors) are present in pulmonary vascular smooth muscle, we studied isolated canine pulmonary arterial rings under isometric conditions in vitro. Addition of β-adrenoceptor agonists produced a concentration-dependent relaxation of noradrenaline-precontracted tissues, a rank order potency being isoproterenol (1) > salbutamol (0.95) > selective β₃-adrenoceptor agonists, CL 316243 (0.85), and BRL 37344 (0.83). A marked desensitization to salbutamol occurred by pretreatment with salbutamol but not with CL 316243. When β₁-adrenoceptors had been blocked, the relaxant responses to salbutamol were competitively antagonized by the β₂-adrenoceptor antagonist ICI 118551 with a pA₂ value of 7.67 ± 0.21 (mean ± S.E.), but the response to CL 316243 was weekly antagonized by ICI 118551 only at a high concentration of 10⁻⁵ M, where an apparent pA₂ value was 5.24. In contrast, cyanopindolol, a nonselective β-adrenoceptor antagonist, antagonized CL 316243–induced relaxation in a competitive manner with a pA₂ of 6.10 ± 0.11. This pA₂ value was lower than that when salbutamol was used as an agonist (6.69 ± 0.14, p < 0.01). Intracellular 3′,5′-cyclic adenosine monophosphate (cAMP) levels were increased by CL 316243 in a concentration-dependent fashion, an effect that was not altered by ICI 118551. These results suggest that β₃-adrenoceptors may exist in canine pulmonary artery smooth muscle and that stimulation of this atypical receptor causes vasodilation through a cAMP-dependent pathway. Accepted for publication: 17 June 1999     

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     

Effects of carbamylcholine chloride on human antral gastrin mRNA levels

The effects of the muscarinic receptor agonist, carbamylcholine chloride (carbachol), on gastrin release and gastrin mRNA levels in human antral mucosa (n=15) were determined. During a-2-h incubation period, carbachol (10⁻⁶−10⁻⁴M) decreased gastrin mRNA levels to 71±8% (10⁻⁶M), 40±8% (10⁻⁵M), and 33±5% (10⁻⁴M) of control levels. Carbachol (10⁻⁵M) decreased intracellular gastrin (from 1634±103 to 1272±126 pg/mg tissue protein), while it increased gastrin release into the medium (from 609±48 to 918±68 pg/ml per mg tissue protein). After 6-and 9-h culture, carbachol gradually increased gastrin mRNA levels, by 96±12% and 126±23%, respectively. Atropine sulfate (10⁻⁵ M) completely inhibited the carbachol-induced changes. Cycloheximide markedly decreased tissue gastrin concentration, but increased gastrin mRNA levels, whereas it had no effects on gastrin release. These findings suggested that carbachol may have a time-related biphasic action on human antral gastrin biosynthesis.     

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