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Allergic sensitization increases the amount of extracellular ATP hydrolyzed by guinea pig leukocytes.

ABSTRACT: Increased levels of ATP have been found in the bronchoalveolar lavage of patients with asthma, and subjects with this disease, but not healthy subjects, develop bronchospasm after nebulization with ATP. Because the main mechanism for controlling the noxious effects of extracellular ATP is its enzymatic hydrolysis, we hypothesized that allergic sensitization is accompanied by a decreased functioning of such hydrolysis. In the present study, peripheral blood leukocytes from sensitized and non-sensitized guinea pigs were used for determining the extracellular metabolism (as assessed by inorganic phosphate production) of ATP, ADP, AMP, or adenosine, and for detecting possible changes in the expression (qPCR and Western blot) of major ectonucleotidases (NTPDase1, NTPDase3, and NPP1) and purinoceptors (P2X1, P2X7, P2Y4, and P2Y6). Contrary to our hypothesis, we found that leukocytes from allergic animals produced higher amounts of inorganic phosphate after stimulation with ATP and ADP, as compared with leukocytes from non-sensitized animals. Although at first glance, this result suggested that sensitization caused higher efficiency of ectonucleotidases, their mRNA and protein expressions were unaffected. On the other hand, after sensitization, we found a significant increase in the protein expression of P2X7 and P2Y4, two purinoceptors known to be responsible for ATP release after activation. We concluded that allergic sensitization increased the amount of ATP hydrolyzed by ectonucleotidases, the latter probably not due to the enhanced efficiency of its enzymatic breakdown, but rather due to an increased release of endogenous ATP or other nucleotides, partly mediated by enhanced expression or P2X7 and P2Y4 receptors.

SUBMITTER: Chavez J

PROVIDER: S-EPMC6439100 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Allergic sensitization increases the amount of extracellular ATP hydrolyzed by guinea pig leukocytes.

Chávez Jaime J Vargas Mario H MH Martínez-Zúñiga Jesús J Falfán-Valencia Ramcés R Ambrocio-Ortiz Enrique E Carbajal Verónica V Sandoval-Roldán Rosa R

Purinergic signalling 20190113 1

Increased levels of ATP have been found in the bronchoalveolar lavage of patients with asthma, and subjects with this disease, but not healthy subjects, develop bronchospasm after nebulization with ATP. Because the main mechanism for controlling the noxious effects of extracellular ATP is its enzymatic hydrolysis, we hypothesized that allergic sensitization is accompanied by a decreased functioning of such hydrolysis. In the present study, peripheral blood leukocytes from sensitized and non-sens ...[more]

PMID: 30637575

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Project description:Asthma is a heterogeneous disease of the airways characterized by chronic inflammation associated with bronchial and smooth muscle hyperresponsiveness. Currently, different murine models for the study of asthma show poor bronchial hyperresponsiveness due to a scarcity of smooth muscle and large airways, resulting in a failure to reproduce smooth muscle hyperreactivity. Thus, we aimed to standardize a guinea pig model of chronic allergic lung inflammation mimicking airway smooth muscle hyperreactivity observed in asthmatics (Asth). Animals were randomly divided into a control group (Ctrl), which received saline (0.9% NaCl), and the Asth group, subjected to in vivo sensitization with ovalbumin (OVA) nebulization. Morphological analysis was performed by hematoxylin-eosin staining. Bronchial hyperresponsiveness was evaluated by nebulization time in the fifth, sixth, and seventh inhalations (NT5-7) and tracheal isometric contractions were assessed by force transducer. Total antioxidant capacity was measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and protein expression by Western blot. Histologically, the Asth group developed peribronchial cellular infiltrate, epithelial hyperplasia and smooth muscle thickening. After the fourth nebulization, the Asth group developed bronchial hyperreactivity. The trachea from the Asth group contracted after in vitro stimulation with OVA, differing from the Ctrl group, which showed no response. Additionally, airway smooth muscle hyperreactivity to carbachol and histamine was observed in the Asth group only in intact epithelium preparations, but not to KCl, and this effect was associated with an augmented production of reactive oxygen species. Moreover, lung inflammation impaired the relaxant potency of isoproterenol only in intact epithelium preparations, without interfering with nifedipine, and it was found to be produced by transforming growth factor-β negative modulation of β adrenergic receptors and, furthermore, big-conductance Ca2+-sensitive K+ channels. These effects were also associated with increased levels of phosphatidylinositol 3-kinases but not extracellular signal-regulated kinases 1/2 or phosphorylation, and augmented α-actin content as well, explaining the increased smooth muscle mass. Furthermore, pulmonary antioxidant capacity was impaired in the Asth group. Therefore, we developed a standardized and easy-to-use, reproducible guinea pig model of lung inflammation that mimics airway smooth muscle hypercontractility, facilitating the investigation of the mechanisms of bronchial hyperresponsiveness in asthma and new therapeutic alternatives.

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Allergic sensitization increases the amount of extracellular ATP hydrolyzed by guinea pig leukocytes.

Publications

Allergic sensitization increases the amount of extracellular ATP hydrolyzed by guinea pig leukocytes.

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