title: ATP release and Ca2+ signalling by human bronchial epithelial cells following Alternaria aeroallergen exposure.
abstract: Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) that was dependent on the coordinated activation of specific purinergic receptor (P2Y2 and P2X7) subtypes. In the present study, pretreatment with a cell-permeable Ca2+-chelating compound (BAPTA-AM) significantly inhibited ATP release, indicating dependency on [Ca2+]i. Alternaria-evoked ATP release exhibited a greater peak response and a slightly lower EC50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca2+]i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA-AM significantly blocked Alternaria-stimulated incorporation of fluorescent lipid (FM1-43)-labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA-AM. These results indicate that an important mechanism for Alternaria-induced ATP release is Ca2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria-induced ATP release; however, the sustained increase in [Ca2+]i typically observed following Alternaria exposure appeared to be independent of protease-activated receptor (PAR2) stimulation.
authors.affiliation: S. M. O'Grady: Department of Animal Science, University of Minnesota, 480 Haecker Hall, 1364 Eckles Avenue, St Paul, MN 55108, USA. ograd001@umn.edu.
authors.forename: Scott M
authors.initials: SM
authors.lastname: O'Grady
authors.forename: Nandadavi
authors.initials: N
authors.lastname: Patil
authors.forename: Tamene
authors.initials: T
authors.lastname: Melkamu
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Maniak
authors.forename: Cheryl
authors.initials: C
authors.lastname: Lancto
authors.forename: Hirohito
authors.initials: H
authors.lastname: Kita
mesh_terms: D000255:Adenosine Triphosphate / Q000276:immunology / Q000378:metabolism*; D000485:Allergens / Q000494:pharmacology*; D000528:Alternaria / Q000276:immunology*; D000946:Antigens, Fungal / Q000494:pharmacology*; D001980:Bronchi / Q000166:cytology / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D020013:Calcium Signaling; D002460:Cell Line; D005089:Exocytosis; D006801:Humans; D011480:Protease Inhibitors / Q000494:pharmacology; D020545:Respiratory Mucosa / Q000166:cytology / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism
chemical_list: D000485:Allergens; D000946:Antigens, Fungal; D011480:Protease Inhibitors; D000255:Adenosine Triphosphate