Epithelium-dependent regulation of airways smooth muscle function. A histamine-nitric oxide pathway.

The airway epithelium is responsible for the production of a number of arachidonic acid and non-prostanoid inhibitory factors. Epithelium synthesises nitric oxide (NO) which may be important in regulating the function of airways smooth muscles. We studied in vitro the effect of histamine (100 nM-100 microM) which increases the NO release on rabbit airway smooth muscles induced by 80 mM KC1 in the presence or not of 10(-5) Methylene blue (MB) (inactivator of guanylate cyclase) or N(G)-monomethyl L-arginine (L-NMMA), a NOS inhibitor. All experiments were done in tracheal muscle strips from 28 rabbits with epithelium and after epithelium removal. The additional use of histamine (1 microM) on KC1 contraction induced a relaxation of 10% of the initial contraction. The additional use of L-NMMA decreased the relaxation to 5% of initial contraction. MB rather than L-NMMA increased the contraction significantly (p<0.01). Epithelium removal increased the contraction induced by KC1 (80 mM) and histamine (1 microM) by about 30% (p<0.001). NO release especially from epithelium regulates the airways smooth muscle functions. Damage to the epithelium may contribute to an increase in airways sensitivity, observed in asthma

Epithelium-dependent effect of L-glutamate on airways: involvement of prostaglandins.

We investigated the effect of the excitatory amino acid (EAA) receptor agonists L-glutamate, N-methyl-D-aspartate (NMDA), (RS)-a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainic acid on KCl-induced contractions of rabbit tracheal smooth muscle, as well as the role of epithelium and endogenously produced nitric oxide and prostaglandins on these responses. L-Glutamate decreased KCI-induced contractions up to 30%. This effect was attenuated by epithelium removal, tetrodotoxin, methylene blue and indomethacin but not by NG-nitro-L-arginine methyl ester. While NMDA, AMPA and kainic acid had no effect, the combination of NMDA + kainic acid decreased KCI-induced contractions. These results suggest that, in rabbit trachea, L-glutamate has, at least in part, an epithelium-dependent effect mediated via prostaglandin formation and that the EAA receptors involved are non-classical

Resting Tension Affects eNOS Activity in a Calcium-Dependent Way in Airways

The alteration of resting tension (RT) from 0.5 g to 2.5 g increased significantly airway smooth muscle contractions induced by acetylcholine (ACh) in rabbit trachea. The decrease in extracellular calcium concentration [Ca2+]o from 2 mM to 0.2 mM reduced ACh-induced contractions only at 2.5 g RT with no effect at 0.5 g RT. The nonselective inhibitor of nitric oxide synthase (NOS), NG-nitro-L-arginine methyl ester (L-NAME) increased ACh-induced contractions at 2.5 g RT. The inhibitor of inducible NOS, S-methylsothiourea or neuronal NOS, 7-nitroindazole had no effect. At 2.5 g RT, the reduction of [Ca2+]o from 2 mM to 0.2 mM abolished the effect of L-NAME on ACh-induced contractions. The NO precursor L-arginine or the tyrosine kinase inhibitors erbstatin A and genistein had no effect on ACh-induced contractions obtained at 2.5 g RT. Our results suggest that in airways, RT affects ACh-induced contractions by modulating the activity of epithelial NOS in a calcium-dependent, tyrosine-phosphorylation-independent way

The muscarinic antagonist gallamine induces proliferation of airway smooth muscle cells regardless of the cell phenotype

Background: Muscarinic receptor antagonists are a usual treatment for chronic airway diseases, with increased bronchoconstriction, like asthma and chronic obstructive pulmonary disease. These diseases are usually accompanied by airway remodeling, involving airway smooth muscle cell (ASMC) proliferation. The purpose of this study was to examine the effect of the muscarinic receptor modulator gallamine on rabbit tracheal ASMC proliferation. Methods: ASMCs were incubated with gallamine (1 nM–10 mM), atropine (1 fM–10 mM), and/or acetylcholine (1 nM–1 mM), in the presence or absence of FBS (1% or 10%). Cell proliferation was estimated by incorporation of radioactive thymidine, the Cell Titer AQueous One Solution method and cell number counting after Trypan blue exclusion. The mechanisms mediating cell proliferation were studied using the PI3K and MAPK inhibitors LY294002 (20 μM) and PD98059 (100 μM), respectively. Cell phenotype was studied by indirect immunofluorescence for α-actin, Myosin Heavy Chain and desmin. Results: ASMC incubation with the muscarinic receptor allosteric modulator gallamine or the muscarinic receptor antagonist atropine increased methyl-[3H]thymidine incorporation and cell number in a dose-dependent manner. ASMC proliferation was mediated via PI3K and MAPK activation and was transient. Gallamine antagonized the mitogenic effect of 1% FBS. Furthermore, gallamine had a similar effect on contractile ASMCs, without synergizing with or affecting acetylcholine induced proliferation, or altering the percentage of ASMCs expressing contractile phenotype marker proteins. Conclusions: Gallamine, in the absence of any agonist, has a transient mitogenic effect on ASMCs, regardless of the cell phenotype, mediated by the PI3K and the MAPK signaling pathways. © 2018 Institute of Pharmacology, Polish Academy of Science

Azithromycin has a direct relaxant effect on precontracted airway smooth muscle

Macrolides have been proven to have beneficial bacteriostatic and anti-inflammatory properties, but very little is known about the potential value of their bronchodilatory effect. Therefore, in the present study we investigated the effect of azithromycin on contractile responses of isolated rabbit tracheal strips to carbachol or KCl. Azitbromycin has a relaxant, concentration-dependent effect on tracheal strips precontracted with carbachol (300 nM), significant from the concentration of 1 mu M. The mechanical removal of epithelium did not alter the effect of azithromycin. Azithromycin (100 mu M) also relaxed tracheal strips precontracted with KCl (80 mM) even in the presence of atropine (100 mu M). Moreover, azithromycin (100 mu M) decreased contractions induced by 300 nM and 10 mu M carbachol to 55.4% and 80.5% of initial contraction, respectively. The relaxant effect of azithromycin persisted in both calcium free solution and in the presence of the calcium channel antagonist, verapamil. The relaxant effect of azithromycin was not altered by the pre-treatment of preparations with the inhibitors of Ca2+-ATPase (cyclopiazonic acid), Na+-K+ ATPase (ouabain), Rho-associated kinase [(R)-(+)-17-ans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] (Y-27632) or the non-specific cAMP and cGMP phosphodiesterases inhibitor 3-isobutyl-l-methyl-2,6(1H,3H)-purinedione (IBMX). These results suggest that azithromycin has a concentration-dependent, epithelium-independent, direct relaxant effect on precontracted tracheal strips that is not mediated via inhibition of Ca2+ influx or Ca2+ release from intracellular stores. Also, it is not due to alteration of the function of Na+-K+ ATPase and does not depend on the formation of cAMP/cGMP or the Rho/Rho-activated kinase pathway. (c) 2006 Elsevier B.V. All rights reserved

The mitogenic effect of testosterone and 17 beta-estradiol on airway smooth muscle cells

Airway disease distribution and/or severity exhibit sex differences suggesting that sex hormones are involved in the respiratory system physiology and pathophysiology. The implication of airway smooth muscle cells (ASMCs) in the physiology of the airways and the pathogenetic mechanism of airway remodeling is of great interest. Therefore, we studied the effect of testosterone and 17 beta-estradiol on ASMC proliferation and the mechanisms involved. Cell proliferation was estimated using the methyl-[(3)H]thymidine incorporation and Cell Titer 96 (R) AQueous One Solution Assay methods. ASMC isolated from adult male or female rabbit trachea were incubated with testosterone (1 pM-1 mu M) or 17 beta-estradiol (1 pM-1 mu M), in the presence or absence of the androgen receptor antagonist flutamide (10 nM) or estrogen receptor antagonist ICI182780 (10 nM), as well as of the PI3K inhibitors LY294002 (20 mu M) or wortmannin (1 mu M), or the MAPK inhibitors PD98059 (100 mu M) or U0126 (1 mu M). After 24 h of incubation, testosterone and 17 beta-estradiol increased methyl-[(3)H]thymidine incorporation and cell number, in ASMC isolated from male or female animals. The induction of ASMC proliferation by testosterone or 17 beta-estradiol was inhibited by flutamide or ICI182780 respectively, as well as by LY294002, wortmannin, PD98059 or U0126. In conclusion, testosterone and 17 beta-estradiol have a mitogenic effect on ASMC, which is receptor-mediated and involves the MAPK and PI3K signaling pathways. Moreover, their effect is the same for ASMC from male and female animals. It is possible that gender-related differences in ASMC remodeling, may be influenced by the different patterns of sex steroid hormone secretion in males and females. (C) 2010 Elsevier Inc. All rights reserved

Resting tension effect on airway smooth muscle: the involvement of epithelium

We studied the influence of resting tension (RT) on rabbit tracheal smooth muscle (TSM) contractions induced by acetylcholine or KCl as well as the role of epithelium and the endogenously produced nitric oxide, prostanoids and endothelin on these responses. The alteration of RT from 0.5 to 2.5 g increased the responsiveness of TSM to KCl. The presence of atropine decreased KCl-induced contractions obtained only at 2.5 g RT. The removal of epithelium increased acetylcholine-induced contractions, only at 2.5 g RT. At 0.5 g RT, the presence of L-NAME had no effect on acetylcholine-induced contractions while indomethacin decreased contractions induced by 10(-3) M acetylcholine. At 2.5 g RT, the presence Of L-NAME increased acetylcholine-induced contractions while indomethacin, BQ-123 and BQ-788 had no effect. These results demonstrate that RT affects the responsiveness of TSM differentially, depending on the agonist or integrity of the epithelium. Airway epithelium modulates acetylcholine-induced contractions, only at 2.5 g RT partly via NO release. At 0.5 g RT, the endogenous production of prostanoids by sources other than epithelium modulates the contractility of TSM to acetylcholine. (C) 2004 Elsevier B.V. All rights reserved

Evaluation of airway inflammation in mechanically ventilated patients using cell count and protein concentration

Mechanically ventilated (MV) patients may present airway inflammation and elevated secretion production. However, it is unknown whether cell and/or protein counts in bronchial samples may be useful to evaluate their clinical condition. Our aim was to standardize sampling and propose a new mechanical mucus dissolution in Tracheal-Bronchial secretions. In all patients, bronchial lining fluid aspiration (BLF), Bronchoalveolar lavage (BAL) and Bronchial Washings (BW40, BW5) were performed, while visible bronchial secretions were obtained via bronchoscopy (VBS) and blinded, via a common catheter for tracheobronchial aspiration (AC). Mucus was mechanically or DTT dissolved and cell number was count. Protein, albumin and TNF-α levels were measured, in mucus dissolved samples from control and MV patients. Cell number and protein levels were elevated in mucus dissolved compared to non-dissolved, or DTT dissolved. Cell number and TNF-α levels were elevated in MV patients compared to controls, while protein levels were lower in MV patients. Differences in cell and protein levels were observed in samples acquired using different sampling technics. Therefore, mechanical mucus dissolution provides a proper sample for evaluation, and the sampling technic used can influence the sample’s characteristics. © 2021, The Author(s)