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Rat submandibular glands secrete nanovesicles with NTPDase and 5'-nucleotidase activities.

ABSTRACT: Extracellular nucleotides modulate a wide number of biological processes such as neurotransmission, platelet aggregation, muscle contraction, and epithelial secretion acting by the purinergic pathway. Nucleotidases as NTPDases and ecto-5'-nucleotidase are membrane-anchored proteins that regulate extracellular nucleotide concentrations. In a previous work, we have partially characterized an NTPDase-like activity expressed by rat submandibular gland microsomes, giving rise to the hypothesis that membrane NTPDases could be released into salivary ducts to regulate luminal nucleotide concentrations as was previously proposed for ovarian, prostatic, and pancreatic secretions. Present results show that rat submandibular glands incubated in vitro release membrane-associated NTPDase and ecto-5'-nucleotidase activities. Electron microscopy images show that released membranes presenting nucleotidase activity correspond to exosome-like vesicles which are also present at microsomal fraction. Both exosome release and nucleotidase activities are raised by adrenergic stimulation. Nucleotidase activities present the same kinetic characteristics than microsomal nucleotidase activity, corresponding mainly to the action of NTPDase2 and NTPDase3 isoforms as well as 5'-nucleotidase. This is consistent with Western blot analysis revealing the presence of these enzymes in the microsomal fraction.

SUBMITTER: Gonzalez DA

PROVIDER: S-EPMC4336312 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Rat submandibular glands secrete nanovesicles with NTPDase and 5'-nucleotidase activities.

González Débora A DA Egido Patricia P Balcarcel Noelia B NB Hattab Claude C van Haaster Martín M Barbieri MM Pelletier Julie J Sévigny Jean J Ostuni Mariano A MA

Purinergic signalling 20141219 1

Extracellular nucleotides modulate a wide number of biological processes such as neurotransmission, platelet aggregation, muscle contraction, and epithelial secretion acting by the purinergic pathway. Nucleotidases as NTPDases and ecto-5'-nucleotidase are membrane-anchored proteins that regulate extracellular nucleotide concentrations. In a previous work, we have partially characterized an NTPDase-like activity expressed by rat submandibular gland microsomes, giving rise to the hypothesis that m ...[more]

PMID: 25523180

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Project description:Macrophages are key elements in the inflammatory process, whereas depending on the micro-environmental stimulation they exhibit a pro-inflammatory (classical/M1) or an anti-inflammatory/reparatory (alternative/M2) phenotype. Extracellular ATP can act as a danger signal whereas adenosine generally serves as a negative feedback mechanism to limit inflammation. The local increase in nucleotides communication is controlled by ectonucleotidases, such as members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family and ecto-5'-nucleotidase/CD73 (ecto-5'-NT). In the present work we evaluated the presence of these enzymes in resident mice M1 (macrophages stimulated with LPS), and M2 (macrophages stimulated with IL-4) macrophages. Macrophages were collected by a lavage of the mice (6-8 weeks) peritoneal cavity and treated for 24 h with IL-4 (10 ng/mL) or LPS (10 ng/mL). Nitrite concentrations were measured using the Greiss reaction. Supernatants were harvested to determine cytokines and the ATPase, ADPase and AMPase activities were determined by the malachite green method and HPLC analysis. The expression of selected surface proteins was evaluated by flow cytometry. The results reveal that M1 macrophages presented a decreased ATP and AMP hydrolysis in agreement with a decrease in NTPDase1, -3 and ecto-5'-nucleotidase expression compared to M2. In contrast, M2 macrophages showed a higher ATP and AMP hydrolysis and increased NTPDase1, -3 and ecto-5'-nucleotidase expression compared to M1 macrophages. Therefore, macrophages of the M1 phenotype lead to an accumulation of ATP while macrophages of the M2 phenotype may rapidly convert ATP to adenosine. The results also showed that P1 and P2 purinoreceptors present the same mRNA profile in both phenotypes. In addition, M2 macrophages, which have a higher ATPase activity, were less sensitive to cell death. In conclusion, these changes in ectoenzyme activities might allow macrophages to adjust the outcome of the extracellular purinergic cascade in order to fine-tune their functions during the inflammatory set.

Dataset Information

Rat submandibular glands secrete nanovesicles with NTPDase and 5'-nucleotidase activities.

Publications

Rat submandibular glands secrete nanovesicles with NTPDase and 5'-nucleotidase activities.

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