Contraction Of Human Airways Research Articles

Attenuation of murine and human airway contraction by a peptide fragment of the cytoskeleton regulatory protein gelsolin.

We have previously reported that mice genetically deficient in the actin binding protein gelsolin exhibit impaired airway smooth muscle (ASM) relaxation. Primary cultured ASM cells from these mice demonstrate enhanced inositol triphosphate (IP3) synthesis and increased intracellular calcium in response to Gq-coupled agonists. We hypothesized that this was due to increased intracellular availability of unbound phosphatidylinositol 4,5-bisphosphate (PIP2), based on the fact that gelsolin contains a short peptide region that binds PIP2, presumably making it a less available substrate. We now questioned whether a peptide that corresponds to the PIP2 binding region of gelsolin could modulate ASM signaling and contraction. The 10 amino acid sequence of the gelsolin peptide within the PIP2-binding region was incubated with primary cultures of human ASM cells, and IP3 synthesis was measured in response to a Gq-coupled agonist. Gelsolin peptide-treated cells generated less IP3 under basal and bradykinin or acetylcholine (Gq-coupled) conditions. Acetylcholine-induced contractile force measured in isolated tracheal rings from mice and human tracheal muscle strips in organ baths was attenuated in the presence of the gelsolin peptide. The gelsolin peptide also attenuated methacholine-induced airway constriction in murine precision-cut lung slices. Furthermore, this peptide fragment delivered to the respiratory system of mice via nebulization attenuated subsequent methacholine-induced increases in airway resistance in vivo. The current study demonstrates that introduction of this small gelsolin peptide into the airway may be a novel therapeutic option in bronchoconstrictive diseases.

Cannabinoids inhibit cholinergic contraction in human airways through prejunctional CB1 receptors

Marijuana smoking is widespread in many countries, and the use of smoked synthetic cannabinoids is increasing. Smoking a marijuana joint leads to bronchodilation in both healthy subjects and asthmatics. The effects of Δ(9) -tetrahydrocannabinol and synthetic cannabinoids on human bronchus reactivity have not previously been investigated. Here, we sought to assess the effects of natural and synthetic cannabinoids on cholinergic bronchial contraction. Human bronchi isolated from 88 patients were suspended in an organ bath and contracted by electrical field stimulation (EFS) in the presence of the phytocannabinoid Δ(9) -tetrahydrocannabinol, the endogenous 2-arachidonoylglycerol, the synthetic dual CB1 and CB2 receptor agonists WIN55,212-2 and CP55,940, the synthetic, CB2 -receptor-selective agonist JWH-133 or the selective GPR55 agonist O-1602. The receptors involved in the response were characterized by using selective CB1 and CB2 receptor antagonists (SR141716 and SR144528 respectively). Δ(9) -tetrahydrocannabinol, WIN55,212-2 and CP55,940 induced concentration-dependent inhibition of cholinergic contractions, with maximum inhibitions of 39, 76 and 77% respectively. JWH-133 only had an effect at high concentrations. 2-Arachidonoylglycerol and O-1602 were devoid of any effect. Only CB1 receptors were involved in the response because the effects of cannabinoids were antagonized by SR141716, but not by SR144528. The cannabinoids did not alter basal tone or contractions induced by exogenous Ach. Activation of prejunctional CB1 receptors mediates the inhibition of EFS-evoked cholinergic contraction in human bronchus. This mechanism may explain the acute bronchodilation produced by marijuana smoking.

C-027 Inhibits IgE-mediated passive sensitization bronchoconstriction and acts as a histamine and serotonin antagonist in human airways

Atopic asthma is poorly controlled by current therapies. Newer therapies and novel antihistamines are, therefore, required to treat patients whose atopic asthma is not controlled. For the first time, C-027 is shown to antagonize histamine, IgE-mediated and serotonin-induced contraction in human airways and vessels. Human precision-cut lung slices (PCLS, 250 μm thick), containing an airway or blood vessel, were pretreated with either C-027 (2 hours) or with vehicle alone and were contracted with histamine or serotonin. Known antihistamine was used as a comparator in antihistamine studies. Also, human airways were contracted via IgE passive sensitization in the presence or absence of C-027 or fexofenadine. Affinity of C-027 toward human G-protein coupled receptors was also determined, as well as the drug's biodistribution in murine model. C-027 was shown to have the highest affinity toward human histamine and serotonin receptors. Subsequently, C-027 was shown to antagonize histamine- and serotonin-induced airway and vascular smooth muscle contraction, respectively, and histamine-released bronchocontraction mediated by IgE passive sensitization in human small airways. C-027 also inhibited histamine-mediated single-cell calcium ion release. Low levels of C-027 were found in murine brain tissue. Collectively, these data suggest that C-027 markedly inhibits IgE-induced bronchoconstriction and antagonizes histamine and serotonin-contraction with little biodistribution in the brain. The compound may offer a future therapy for allergen-induced airway hyperresponsiveness in patients with asthma.

Human Airway Contraction and Formoterol-Induced Relaxation Is Determined by Ca2+ Oscillations and Ca2+ Sensitivity

The etiology of airway hyperresponsiveness associated with asthma requires an understanding of the regulatory mechanisms mediating human airway smooth muscle cell (SMC) contraction. The objective of this study was to determine how human airway SMC contraction (induced by histamine) and relaxation (induced by formoterol) are regulated by Ca(2+) oscillations and Ca(2+) sensitivity. The responses of human small airways and their associated SMCs were studied in human lung slices cut from agarose-inflated lungs. Airway contraction was measured with phase-contrast video microscopy. Ca(2+) signaling and Ca(2+) sensitivity of airway SMCs were measured with two-photon fluorescence microscopy and Ca(2+)-permeabilized lung slices. The agonist histamine induced contraction of human small airways by stimulating both an increase in intracellular Ca(2+) concentration in the SMCs in the form of oscillatory Ca(2+) waves and an increase in Ca(2+) sensitivity. The frequency of the Ca(2+) oscillations increased with histamine concentration, and correlated with increased contraction. Formoterol induced airway relaxation at low concentrations by initially decreasing SMC Ca(2+) sensitivity. At higher concentrations, formoterol additionally slowed or inhibited the Ca(2+) oscillations of the SMCs to relax the airways. The action of formoterol was only slowly reversed. Human lung slices provide a powerful experimental assay for the investigation of small airway physiology and pharmacology. Histamine induces contraction by simultaneously increasing SMC Ca(2+) signaling and Ca(2+) sensitivity. Formoterol induces long-lasting relaxation by initially reducing the Ca(2+) sensitivity and, subsequently, the frequency of the Ca(2+) oscillations of the airway SMCs.

A model of allergen-driven human airway contraction: β 2 pathway dysfunction without cytokine involvement

In our previous in vitro model, allergen incubation of passively sensitized human airways reduced the response to salbutamol. However, whether cytokines play a role in this model is still unknown. To investigate interleukin 1beta and tumor necrosis factor a expression in allergen-challenged human airways. Nonasthmatic airways (n = 13) were passively sensitized by overnight atopic serum incubation and then challenged with allergen for 1 hour (n = 9). After repeated washouts, airways were immersed in physiologic salt solution for 6 hours and finally in formaldehyde for immunohistochemical studies. The effect of co-incubation in anti-interleukin 1beta and anti-tumor necrosis factor a specific neutralizing antibodies on salbutamol response was also studied (n = 4). No differences were found among control, sensitized, and challenged rings in the number of inflammatory cells. The percentage of basement membrane covered by epithelium was similar in the different conditions. There was a higher percentage of degranulating to total mast cells in allergen-challenged rings than in sensitized rings (P < .001). A significant correlation was observed between allergen-induced contraction and mast cell degranulation (r = 0.88; P < .001). The sensitization procedure was validated by paired allergen-induced contractions. No expression of the 2 cytokines was detectable up to 6 hours after allergen challenge, and specific neutralizing antibodies did not attenuate the impaired response to salbutamol in allergen-challenged rings. These data suggest that in our in vitro model of allergic inflammation, beta2 pathway dysfunction can occur without cytokine involvement, thus supporting previous results that suggest a role for leukotrienes.

Epinastine (WAL 801CL) inhibits the electrical field stimulation-induced cholinergic contraction in guinea pig and human airways in vitro.

Epinastine is an antihistamine drug with binding affinities at 5-hydroxytryptamine (5-HT) receptors. The current study was performed to investigate whether epinastine could modulate the cholinergic contraction in guinea pig and human airways in vitro. Isolated guinea pig and human airway preparations were suspended in organ baths containing modified Krebs-Henseleit solution. Electrical field stimulation was applied to elicit cholinergic contractions. Epinastine produced a concentration-dependent inhibition of the cholinergic contraction in guinea pig airways and pretreatment with methysergide (5-HT1/2/7 antagonist) significantly attenuated these inhibitory effects of epinastine. Pretreatment with tropisetron (5-HT3/4 antagonist), ketanserin (5-HT2 antagonist), SDZ216-525 (5-HT1A antagonist) or phentolamine (alpha-adrenergic antagonist) had no effect. Epinastine did not displace the concentration-response curve to acetylcholine. These results suggest that epinastine inhibits the cholinergic contraction in guinea pig airways through stimulation of prejunctional 5-hydroxytryptamine receptors, located to postganglionic cholinergic nerves. Inhibitory effects of epinastine on the cholinergic contraction in human airways in vitro were also demonstrated, which suggests that a similar mechanism might be present in human airways. The pharmacological profile of epinastine, which shows binding affinity at the 5-hydroxytryptamine7 receptor but not at the 5-hydroxytryptamine1 receptor subtypes corroborates the hypothesis that the inhibitory prejunctional 5-hydroxytryptamine receptor on cholinergic nerves is of the 5-hydroxytryptamine7 subtype.

Contraction of human airways by oxidative stress: Protection by n-acetylcysteine

We examined the in vitro effects of tert-butylhydroperoxide (tBu-OOH) in human bronchial muscle. tert-Butylhydroperoxide produced concentration-dependent contractions of bronchial rings (maximum effect was 56.5 ± 9.6% of contraction by 1 mM acetylcholine; effective concentration 50% was ∼100 μM). tert-Butylhydroperoxide (0.5 mM)-induced contraction was enhanced by epithelial removal but abolished by indomethacin (cyclooxygenase inhibitor) and zileuton (lipoxygenase inhibitor). tert-Butylhydroperoxide produced a transient rise in intracellular calcium in human cultured airway smooth muscle cells (HCASMC). The bronchial reactivity to acetylcholine and histamine was not altered by tBu-OOH. In HCASMC, tBu-OOH (0.5 mM, 30 min) increased malondialdehyde levels (MDA; from 7.80 ± 0.83 to 26.82 ± 1.49 nmol mg −1 protein), accompanied by a decrease of reduced glutathione (GSH; from 16.7 ± 2.6 to 6.9 ± 1.9 nmol mg −1 protein) and an increase of oxidized glutathione (from 0.09 ± 0.03 to 0.18 ± 0.03 nmol mg −1 protein). N-acetylcysteine (0.3 mM) inhibited by approximately 60% the bronchial contraction resulting from tBu-OOH (0.5 mM) and protected cultured cells exposed to tBu-OOH (MDA was lowered to 19.51 ± 1.19 nmol mg −1 protein, and GSH content was replenished). In summary, tBu-OOH caused contraction of human bronchial muscle mediated by release of cyclo-oxygenase and lipoxygenase products without producing airways hyperreactivity. N-acetylcysteine decreases tBu-OOH-induced contraction and protects human cultured airway smooth muscle cells exposed to tBu-OOH.

Effects of β2‐adrenoceptor agonists on anti‐IgE‐induced contraction and smooth muscle reactivity in human airways

1. The beta 2-adrenoceptor agonists, salbutamol, salmeterol and RP 58802 relaxed basal tone of human isolated bronchial smooth muscle. Salmeterol- and RP 58802-induced relaxations persisted for more than 4 h when the medium was constantly renewed after treatment. 2. Salbutamol, salmeterol and RP 58802 reversed histamine-induced contractions in human airways (pD2 values: 6.15 +/- 0.21, 6.00 +/- 0.19 and 6.56 +/- 0.12, respectively). 3. Anti-IgE-induced contractions were significantly inhibited immediately after pretreatment of preparations with beta 2-adrenoceptor agonists (10 microM). However, when tissues were treated with beta 2-agonists and then washed for a period of 4 h, salmeterol was the only agonist which significantly inhibited the anti-IgE response. 4. Histamine response curves were shifted to the right immediately after pretreatment of tissues with the beta 2-adrenoceptor agonists (10 microM; 20 min), but maximal contractions were not affected. After a 4 h washing period, the histamine curves were not significantly different from controls. Concentration-effect curves to acetylcholine (ACh) or leukotriene C4 (LTC4) were not significantly modified after beta 2-agonist pretreatment. 5. These results suggest that beta 2-adrenoceptor agonists may prevent anti-IgE-induced contraction by inhibition of mediator release rather than alterations of those mechanisms involved in airway smooth muscle contraction.

Extracellular calcium and human isolated airway muscle: ionophore A23187 induced contraction

In order to investigate the role played by extracellular calcium mobilization in activating human airway contraction, we studied the effects of A23187, a calcium ionophore, in human isolated bronchial spiral strips. In this preparation, ionophore induced a concentration dependent contraction from 10 −7 M to 10 −5 M which resulted from a direct effect on smooth muscle cells and was not a consequence of mediator release. Ionophore-induced contraction was dependent upon an entry of extracellular calcium which did not occur through the verapamil sensitive voltage dependent channel. Maximal ionophore contraction was 97 ± 11% (n = 5) of the maximal histamine contraction but only 46 ± 11% (n = 5) of the maximal carbachol contraction. However, when extracellular calcium concentration was doubled to 5 mM before addition of ionophore, the significant difference in amplitue between carbachol and ionophore maximal contraction was abolished. At physiological calcium concentrations addition of carbachol or histamine to the plateau of the ionophore maximal contraction produced a significant increase in the tension. Verapamil blocked the increase in ionophore tension produced only by histamine. These results suggest that (i) calcium mobilization from the extracellular source alone can produce contraction comparable in magnitude to that induced by histamine or carbachol. (ii) Extracellular calcium mobilization through different pathways has a cumulative effect on human airway contraction.

The calcium dependence of histamine, carbachol and potassium chloride-induced contraction in human airways in vitro

The effect of depletion of calcium from the Krebs Henseleit (KH) solution and the addition of EDTA (1 mM) on contraction of human isolated bronchus by histamine, carbachol and potassium chloride (KCl) was examined. Contractions to histamine and carbachol were almost totally abolished in calcium free KH in the presence of 1 mM EDTA which only reduced KCl responses to 76% of control values. Verapamil (10 −6, 10 −5, 10 −4 M) decreased histamine and KCl contractile responses in a dose-related manner while having no significant effect on carbachol-induced tension. The intracellular calcium antagonist TMB8 10 −4 M caused a slight but significant decrease in histamine contraction but was without effect on the maximal response of the other two agonists. TMB8 decreased the potency of both histamine and carbachol. These results suggest that histamine, carbachol and KCl utilise different pools of calcium for contraction, that only part of the calcium entry for all agonists occurs via voltage-dependent calcium channels and that intracellular calcium stores may play a small role in contraction to these agonists.

Formyl peptide-induced contraction of human airways in vitro.

Formylmethionylleucylphenylalanine (FMLP) is a synthetic analogue of bacterial chemotactic factors. We studied the contraction of human airway tissue in vitro by FMLP. FMLP induced a concentration-dependent contraction of all bronchial spiral strips studied (n = 45). The maximum tension generated in response to FMLP was 86.6 +/- 7.0% (SE) of the maximum response to histamine. The contraction was not reduced by the histamine H1-receptor antagonist pyrilamine, the cyclooxygenase and lipoxygenase inhibitors indomethacin and BW755C, the muscarinic antagonist atropine, or capsaicin which depletes stores of substance P. The concentration-response curve was shifted to the right by the polypeptide antagonist N-t-BOC-phenylalanylleucylphenylalanylleucylphenylalanine and the leukotriene antagonist FPL 55712. When 2 successive FMLP concentration-response curves were performed the maximum response was significantly reduced from 114.8 +/- 9.1% of the histamine maximum to 39.3 +/- 6.1%. The contraction of human airways in vitro by an agent that is structurally and functionally similar to chemotactic peptides released from bacteria may have important implications in airway disease.