J.L. Ortiz

Leukotriene receptors on human pulmonary vascular endothelium.

1 Cysteinyl‐leukotrienes cause contractions and/or relaxations of human isolated pulmonary vascular preparations. Although, the localization and nature of the receptors through which these effects are mediated have not been fully characterized, some effects are indirect and not mediated via the well‐ described LT1 receptor. 2 In human pulmonary veins (HPV) with an intact endothelium, leukotriene D4 (LTD4) induced contraction above basal tone. This response was observed at lower concentrations of LTD4 in the presence of nitric oxide synthase inhibitor Nω‐nitro‐L‐arginine (L‐NOARG). Contractions (in the absence and presence of L‐NOARG) were partially blocked by the LT1 antagonists (MK 571 and ICI 198615). 3 LTD4 relaxed HPV previously contracted with noradrenaline. This relaxation was potentiated by LT1 antagonists, but was abolished by removal of the endothelium. LTD4 also relaxed human pulmonary arteries (HPA) precontracted with noradrenaline but this effect was not modified by LT1 antagonists. 4 The results suggest that contraction of endothelium‐intact HPV by LTD4 is partially mediated via LT1 receptors. Further, in endothelium‐intact HPV, this contraction was opposed by a relaxation induced by LTD4, dependent on the release of nitric oxide, which was mediated, at least in part, via a non‐LTi receptor. In addition, LTD4 relaxation on contracted HPA was not mediated by LT1 receptors. 5 The mechanical effects of LTD4 on human pulmonary vasculature are complex and involve both direct and indirect mechanisms mediated via at least two types of cysteinyl‐leukotriene receptors.

Inhibitory Effects of N-acetylcysteine on Superoxide Anion Generation in Human Polymorphonuclear Leukocytes

It has been suggested that reactive oxygen species released by activated polymorphonuclear leukocytes (PMN) in man is one mechanism of tissue injury. Therapeutic action aimed at increasing antioxidant defence mechanisms is still a clinical challenge. This study examines the activity of N‐acetylcysteine, a known antioxidant, in the protection of PMN exposed in‐vitro to the chemoattractant peptide fMet‐Leu‐Phe (FMLP), the protein kinase C activator phorbol myristate acetate or the lipid peroxidation promoter t‐butyl hydroperoxide.

Effects of selective phosphodiesterase inhibitors on platelet-activating factor- and antigen-induced airway hyperreactivity, eosinophil accumulation, and microvascular leakage in guinea pigs

There is currently interest in the potential use of selective inhibitors of cyclic nucleotide phosphodiesterases (PDE) in the treatment of asthma. In this study we examined the effects of three selective PDE inhibitors, milrinone (PDE III), rolipram (PDE IV) and zaprinast (PDE V), on the broncoconstriction produced by antigen and histamine, the airway hyperreactivity and microvascular leakage after aerosol exposure to platelet-activating factor (PAF) and antigen, and the antigen-induced eosinophil infiltration in guinea-pig lung. Inhaled rolipram (0.01–10 mg ml−1) inhibited dose dependently the bronchospasm produced by aerosol antigen (5 mg ml−1) an anaesthetised, ventilated guinea-pigs. Rolipram (10 mg ml−1) produced maximal inhibition of antigen-induced bronchoconstriction but only partial inhibition of the response to aerosol histamine (1 mg ml−1). Milrinone and zaprinast (each 10 mg ml−1) showed weak, or no, inhibitory effects against bronchoconstriction produced by aerosol antigen or histamine. Pretreatment with rolipram (10 mg kg−1, i.p.) prevented airway hyperreactivity to histamine which develops 24 h after exposure of conscious guinea-pigs to aerosol PAF (500 μg ml−1) or antigen (5 mg ml−1). The pulmonary eosinophil infiltration obtained with 24 h of antigen-exposure was inhibited by rolipram. In contrast, milrinone and zaprinast (each 10 mg kg−1, i.p.) failed to reduce either the airway hyperreactivity of the eosinophil accumulation in these animals. Rolipram (1–10 mg ml−1) reduced the extravasation of Evans blue after aerosol PAF (500 μg ml−1) at all airway levels while a lower dose (0.1 mg ml−1) was only effective at intrapulmonary airways. Rolipram (0.01–1 mg ml−1) markedly reduced airway extravasation produced by inhaled antigen (5 mg ml−1). Zaprinast (1–10 mg ml−1) was also effective against airway microvascular leakage produced by aerosol PAF or antigen while milrinone (10 mg ml−1) had no antiexudative effect. These data support previous suggestions that pharmacological inhibition of PDE IV results in anti-spasmogenic and anti-inflammatory effects in the airways and may be useful in the treatment of asthma.

Cigarette smoke-induced pulmonary endothelial dysfunction is partially suppressed by sildenafil.

Cigarette smoke mediated oxidative stress and endothelial dysfunction are important processes in the pathogenesis of several lung disorders. In this study we evaluated the effect of PDE5 inhibition on pulmonary artery endothelial dysfunction induced by cigarette smoke in vitro. Human pulmonary artery endothelial cells (HPAEC) were incubated in the absence or presence of PDE5 inhibitor sildenafil (10 nM-1 microM), PKG agonist 8-Br-cGMP (1mM), or the antioxidants dyphenyleneiodonium (DPI 1 microM) and N-acetylcysteine (NAC 1mM) for 30 min. Then, cigarette smoke extract (CSE) was added for 24h. CSE (2.5-10%)-induced ROS generation was suppressed by DPI, and partially reversed by sildenafil and 8-Br-cGMP. Decreases in intracellular levels of cGMP and extracellular NO induced by CSE were reversed by sildenafil and DPI. Furthermore, CSE-induced pg91(phox) and PDE5 mRNA overexpression were suppressed by both sildenafil and DPI. CSE (2.5-10%) induced upregulation of IL-6, IL-8 and Ang-2, and decreased Ang-1 expression in parallel to apoptosis which were partially suppressed by sildenafil, 8-Br-cGMP, DPI and NAC. This study demonstrates that PDE5 inhibition attenuates the oxidant burden and the inflammatory and remodeling effects of CSE in human HPAEC which may contribute to the therapeutic value of PDE5 inhibitors for pulmonary disorders coursing with endothelial dysfunction.

Cigarette smoke exposure up‐regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences

BACKGROUND AND PURPOSE Pulmonary arteries from smokers and chronic obstructive pulmonary disease patients show abnormal endothelium‐dependent vascular reactivity. We studied the effect of cigarette smoke extract (CSE) on endothelin receptor B (ETB) expression in human pulmonary artery endothelial cells (HPAECs) and its role in endothelial dysfunction.

Cooling-induced contraction of trachea isolated from normal and sensitized guinea-pigs

SummaryFast (−7°C/min) cooling of guinea-pig isolated trachea produced a rapidly developing, transient contraction followed by relaxation. Cooling-induced contraction was dependent on temperature (30, 20 or 10°C) and responses in trachea obtained from actively sensitized guinea pigs were significantly greater (20 and 10°-C) than those observed in normal trachea. Cooling to 20°C was selected for subsequent experiments. Pre-treatment with sufficient concentrations of atropine, clemastine, cromoglycate, indomethacin, or nordihydroguaiaretic acid did not depress contraction to cooling in either normal or sensitized trachea. This indicates a direct effect of cooling. The contraction. produced by cooling was resistant to verapamil (1 μmol/l) or dantrolene (0.3 mmol/l). Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium; all of them at 10–100 μmol/l) inhibited contraction in sensitized and normal trachea. Activators of protein kinase C (phorbol 12,13-diacetate, 1 μmol/l) enhanced while inhibitors (H-7, 20 μmol/l; staurosporine, 10 μmol/l) depressed cooling-induced contraction in both normal and sensitized tissues. Incubation (20 min) in a Ca2+ -free solution inhibited cooling-induced contraction in normal but not in sensitized trachea. Exposure to a low Na+ (25 mmol/l) or a K+-free medium abolished contraction to cooling in normal and sensitized trachea. Ouabain (0.1–10 μmol/l) and vanadate (0.01–5 mmol/l) inhibited cooling-induced-contraction to a greater extent in normal than in sensitized trachea. Polymyxin B (0.5 mmol/l) selectively depressed responses to cooling in sensitized trachea. In a separate series of experiments, it was shown that sensitized trachea was hyperresponsive to ouabain and vanadate. Previous cooling to 20°C abolished responses to ouabain but only attenuated those to vanadate. These results are compatible with an enhancement of Na+,K+-ATPase and Ca2+-ATPase activities in sensitized trachea and further support the notion that intracellularly stored Ca2+ plays a decisive role in the activation of sensitized tracheal muscle.

Rolipram inhibits airway microvascular leakage induced by platelet-activating factor, histamine and bradykinin in guinea-pigs

Abstract— Rolipram (0·1–1000 μg kg−1, i.v.) reduced the increase in microvascular permeability induced by platelet‐activating factor (PAF; 50 ng kg−1, i.v.) at different sites of the guinea‐pig airways. Rolipram (1–100μg kg−1, i.v.) inhibited histamine (30μg kg−1, i.v.)‐and bradykinin (0·3 μg kg, i.v.)‐induced airway microvascular leakage. These effects of rolipram were obtained at doses which inhibit histamine (7–20 μg kg−1 min−1)‐induced bronchoconstriction (IC50 = 3 ± 1 μg kg, i.v.) without depressing arterial blood pressure in the guinea‐pig. Aminophylline (50 mg kg−1) did not change the effect of PAF. The anti‐exudative effect of rolipram is of potential therapeutic value in asthma.

Direct effect of cigarette smoke on human pulmonary artery tension

The effect of chronic cigarette smoke on pulmonary artery (PA) tension has been studied extensively; nevertheless, the direct effect of cigarette smoke is poorly understood. We investigated the direct effect of cigarette smoke extract (CSE) on PA tension in non-smokers, smokers, and COPD patients in vitro. PA samples from 35 patients who underwent lung resection were examined by measuring isometric tension in response to increasing serotonin concentrations. CSE dose dependently inhibited the response to serotonin in smokers and COPD patients, and to a lesser extent in non-smokers. CSE-induced relaxation was similarly inhibited by the nonspecific nitric oxide synthase (NOS) inhibitor l-NOARG and the specific inducible NOS (iNOS) inhibitor l-NIL, mainly in non-smokers and smokers, and to a lesser extent in COPD patients. Immunostaining of iNOS in PA samples was greater for smokers and COPD patients compared with non-smokers, which explains the lesser effect of CSE on PA tension in non-smokers. Moreover, CSE induced the release of nitrite via iNOS in human PA smooth muscle cells. In conclusion, CSE inhibition of serotonin-induced PA contraction was mediated mainly by iNOS in non-smokers, smokers, and COPD patients, but in different ways, which may be explained by differential iNOS expression in the PA of these patients.

Phosphodiesterase-4 inhibition improves corticosteroid insensitivity in pulmonary endothelial cells under oxidative stress

Several clinical studies have shown that smoking in asthmatics and chronic obstructive pulmonary disease patients is closely associated with corticosteroid refractoriness. In this work, we have analyzed glucocorticoid insensitivity in human pulmonary artery endothelial cells (HPAECs) under cigarette smoke extract (CSE) exposure as well as the possible additive effects of the combination therapy with a phosphodiesterase (PDE)‐4 inhibitor.

Bosentan inhibits cigarette smoke-induced endothelin receptor expression in pulmonary arteries

The endothelin (ET) system contributes to lung vascular tension and remodelling in smokers and chronic obstructive pulmonary disease (COPD) patients. This study examined the effect of cigarette smoke (CS) on ET receptor A (ETA) and B (ETB) expression in human pulmonary artery smooth muscle cells (HPASMCs) and human small intrapulmonary arteries, as well as their functional consequences. CS extract (CSE) increased ETA and ETB expression in HPASMCs and small intrapulmonary arteries, which was attenuated by bosentan, the ETA antagonist BQ123 and the ETB antagonist BQ788, and by blocking ET-1 with a monoclonal antibody against ET-1, suggesting a feed-forward mechanism mediated by ET-1 release. ET receptor (ETR) antagonism attenuated the CSE-induced HPASMC proliferation. Furthermore, CSE exposure increased the acute ET-1-induced small intrapulmonary artery contraction, which was attenuated by bosentan, BQ123 and BQ788. Pulmonary arteries from smokers and COPD patients showed a higher expression of ETA and ETB than those of nonsmoker patients. These results show a novel mechanism by which ETR blockade attenuates CS-induced ETR overexpression and, subsequently, small intrapulmonary artery tension. These data may be of potential value to explain therapeutic effects of bosentan in some forms of disproportionate pulmonary hypertension in COPD patients.