Benjamín Sarriá

Phosphodiesterase 4 inhibition decreases MUC5AC expression induced by epidermal growth factor in human airway epithelial cells

Background: A common pathological feature of chronic inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) is mucus hypersecretion. MUC5AC is the predominant mucin gene expressed in healthy airways and is increased in asthmatic and COPD patients. Recent clinical trials indicate that phosphodiesterase type 4 (PDE4) inhibitors may have therapeutic value for COPD and asthma. However, their direct effects on mucin expression have been scarcely investigated. Methods: MUC5AC mRNA and protein expression were examined in cultured human airway epithelial cells (A549) and in human isolated bronchial tissue stimulated with epidermal growth factor (EGF; 25 ng/ml). MUC5AC mRNA was measured by real time RT-PCR and MUC5AC protein by ELISA (cell lysates and tissue homogenates), Western blotting (tissue homogenates) and immunohistochemistry. Results: EGF increased MUC5AC mRNA and protein expression in A549 cells. PDE4 inhibitors produced a concentration dependent inhibition of the EGF induced MUC5AC mRNA and protein expression with potency values (−log IC50): roflumilast (∼7.5) > rolipram (∼6.5) > cilomilast (∼5.5). Roflumilast also inhibited the EGF induced expression of phosphotyrosine proteins, EGF receptor, and phospho-p38- and p44/42-MAPK measured by Western blot analysis in A549 cells. In human isolated bronchus, EGF induced MUC5AC mRNA and protein expression was inhibited by roflumilast (1 μM) as well as the MUC5AC positive staining shown by immunohistochemistry. Conclusion: Selective PDE4 inhibition is effective in decreasing EGF induced MUC5AC expression in human airway epithelial cells. This effect may contribute to the clinical efficacy of this new drug category in mucus hypersecretory diseases.

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.

Effect of epithelium removal and of enkephalin inhibition on the bronchoconstrictor response to three endothelins of the human isolated bronchus.

The three endothelins ET-1, ET-2 and ET-3 induced a potent contractile response in the human isolated bronchus with an intact epithelium, which proceeded on two different stages. The first stage was observed at low concentrations (high potency) (10(-12) to 10(-9) M) but corresponded to a low intrinsic activity (Emax maximal effect induced by ACh 10(-3) M), the second stage appeared at higher concentrations and corresponded to higher intrinsic activity. The rank order of potency for the two stages of contractile activity was ET-1 greater than ET-2 = ET-3. Removal of the epithelium significantly enhanced the two stages of the contractile responses to the three endothelins and abolished the differences in potency efficacy that were observed between ET-1, ET-2 and ET-3 when the epithelium was present. Phosphoramidon (10(-5) M), an enkephalinase inhibitor, was as potent as epithelium removal in enhancing the contractile responses to these agonists at low concentrations (first stage of contraction, 10(-16) to 10(-9) M). However, with high concentrations of endothelins (greater than 10(-9) M, second stage of contraction), phosphoramidon was less potent than epithelium removal in enhancing the contractile responses. In epithelium-denuded strips, preincubation with phosphoramidon did not further increase the maximal contractions induced by/or the potencies of ET-1, ET-2 or ET-3. After epithelium removal, responses to low doses of endothelins were attenuated by nicardipine (10(-6) M) whereas responses to high doses of the endothelins were not affected, as was also observed when the epithelium was present. (ABSTRACT TRUNCATED AT 250 WORDS)

Antagonism of calcium by zinc in guinea‐pig isolated taenia caeci and trachealis muscle

1 In guinea‐pig isolated taenia caeci and trachealis bathed in a K+‐rich, Ca2+‐free medium, CaCl2 (0.01–10 mm) produced a concentration‐dependent contraction. Zn2+ (0.01–1 mm), Cd2+ (0.01–1 mm), verapamil (0.01–100 μm) and trifluoperazine (1–100 μm) were effective antagonists of CaCl2‐induced responses. 2 Zn2+ and Cd2+ in concentrations from 0.01 to 1 mm were without effect on the tone of taenia and trachea in normal Tris solution. Conversely, Zn2+ and Cd2+, in concentrations of 1 mm, caused contraction of these tissues in a K+‐rich, Ca2+‐free medium. Zn2+ (1 mm)‐induced contractions of taenia and trachea were completely inhibited by verapamil (10 μm). 3 In taenia and trachea skinned of their plasma membranes, tension development induced by Ca2+ (10 μm or 1 μm, respectively) was unaffected by verapamil (100 μm), whereas trifluoperazine (100 μm) depressed the maximal tension produced by Ca2+. Segments of skinned preparations contracted in response to low concentrations of Zn2+ (10 μm) or Cd2+ (10 μm). 4 It is concluded that Zn2+ may suppress Ca2+‐induced spasm by a direct action on the binding sites of the Ca2+ channel.

Non-specific hyperreactivity to pharmacological stimuli in tracheal and lung parenchymal strips of actively sensitized guinea-pigs

The responsiveness of tracheal and lung parenchymal strips isolated from actively sensitized guinea‐pigs to CaCl2 (in K+‐depolarized tissue), KCl, acetylcholine and histamine was compared with that of strips from unsensitized animals. The concentration‐response curves to the mentioned agonists exhibited, in the sensitized group, a left upward displacement (greater maximal effect, lesser effective concentration 50% and a steeper slope) compared with those obtained in the unsensitized group. These results indicate the existence of a non‐specific increase in responsiveness in the airway smooth muscle from sensitized animals.

Functional, biochemical and morphological studies on human bronchi after cryopreservation.

1 . Human isolated bronchi have been investigated as fresh tissue or after storage (7 and 30 days) at — 196°C in foetal calf serum containing 1.8 M dimethyl sulphoxide. 2 . After cryopreservation, the maximal contractile response to acetylcholine (3 μm) was reduced (∼25%) but the difference did not reach significance statistically. Maximal responses to other spasmogens tested (histamine, [Nle10]NKA(4–10), bradykinin, leukotriene D4, U46619, and KC1) did not differ between unfrozen and frozen/thawed tissues. The sensitivity of cyropreserved tissues to the constrictor agents tested was similar to that of fresh tissues. 3 . The accumulation of inositol phosphates produced by acetylcholine in human bronchus in vitro was similar in fresh and cyrostored (30 days) tissues. 4 . Relaxant responses of acetylcholine (0.3 μm)‐precontracted preparations to theophylline, isoprenaline, rolipram and sodium nitroprusside were unchanged after storage with the exception of the sensitivity to rolipram which was diminished in the 30‐days cryostorage group. 5 . Light microscopic examination of sections taken from 30 days cryostored tissues indicates that the epithelium, submucosal tissue and smooth muscle were well preserved. 6 . These experiments suggest that cryopreservation of human bronchi results in maintenance of several morphological, functional (contraction/relaxation) and biochemical properties.

The relaxant effects of cromakalim (BRL 34915) on human isolated airway smooth muscle

SummaryCromakalim (BRL 34915) is a potassium channel opener with therapeutic potential as a bronchodilator in asthma. Cromakalim (0.1–30 μmol/l) inhibited the spontaneous tone of human isolated bronchi n a concentration-related manner being nearly as effective as isoprenaline or theophylline. The order of relaxant potencies (expressed as -log10 IC50 mol/l; mean ±SEM) was isoprenaline (7.29 ± 0.27; n = 8) > cromakalim (5.89 ± 0.12; n = 7) > theophylline (4.07 ±0.13; n = 10). In human bronchi where tone had been raised by addition of histamine (0.1 mmol/l), acetylcholine (0.1 mmol/l) or leukotriene D4 (LTD4, 0.1 μmol/l), the relaxant effect of cromakalim was substantially reduced. Cromakalim suppressed the contraction produced by KCI (25 mmol/l) but not that produced by KCl (120 mmol/l). Tetraethylammonium (8 mmol/l) was without effect against the relaxant action of cromakalimbut procaine (0.5 – 5 mmol/l) and glibenclamide (0.3 μmol/l) antagonised it. Cromakalim (10 μmol/l) produced an upward displacement of concentration-effect curves forKCI (1–100 mmol/l), acetylcholine (1 nmol/l-1 mmol/) and histamine (1 nmol/l-1 mmol/l) but it did not alter the concentration-effect curve for LTD4 (0.1 nmol/l-0.1 μmol/l). When tissues were challenged in the presence of cromakalim (10 μmol/l) with KCI (100 mmol/l), acetylcholine (1 mmol/l) or histamine (1 mmol/l), an enhanced contraction was observed compared to control tissues. This enhancement by cromakalim was absent when tissues were challenged with acetylcholine or histamine in either a Ca2+-free medium (plus EGTA 0.1 mmol/l) or in the presence of verapamil (10 μmol/l). It is concluded that cromakalim is an effective relaxant of human airway smooth muscle in vitro and this activity may depend on the opening of K+ channels in the plasma membrane of smooth muscle cells but other actions cannot be ruled out.

Relaxation of the isolated human internal anal sphincter by sildenafil.

Hypertonicity of the internal anal sphincter (IAS) appears to be involved in the pathogenesis of anal fissure. The relaxant effects of sildenafil, a selective phosphodiesterase 5 (PDE5) inhibitor, on isolated human IAS were investigated.

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

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

Halothane inhibits endothelium-dependent relaxation elicited by acetylcholine in human isolated pulmonary arteries

This study examined whether a clinically relevant concentration of the volatile anaesthetic halothane modifies the endothelium-dependent relaxation produced by acetylcholine (3 nM-10 microM), histamine (1 pM-0.1 microM) and anti-human immunoglobulin E (1:1000) in human isolated pulmonary arteries submaximally precontracted with noradrenaline. An inhibitor of nitric oxide formation, N(G)-nitro-L-arginine (100 microM), attenuated acetylcholine-induced relaxation but failed to inhibit histamine- and anti-human immunoglobulin E-induced relaxation. Indomethacin (2.8 microM, a cyclooxygenase inhibitor) preferentially reduced the relaxation to histamine and anti-human IgE. Halothane (2%) significantly attenuated the relaxation to acetylcholine but had no significant effect on the relaxation elicited by histamine and anti-human IgE. Halothane (2%) enhanced the basal release of prostaglandin I2 by human pulmonary arteries (control 0.31 +/- 0.04 ng mg(-1); treated tissues 0.50 +/- 0.06 ng mg(-1); n = 5; P < 0.05). Halothane (2%) did not alter the responsiveness and sensitivity of preparations to relaxants acting through activation of adenylyl cyclase (forskolin) or guanylyl cyclase (sodium nitroprusside) or by the opening of K(ATP) channels (cromakalim). In conclusion, halothane inhibits the endothelium-dependent relaxation of human pulmonary arteries to acetylcholine by interfering with the nitric oxide pathway at a site before activation of soluble guanylyl cyclase in vascular smooth muscle.