title: NAADP-sensitive two-pore channels are present and functional in gastric smooth muscle cells.
abstract: Nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as an important modulator of Ca(2+) release from the endo-lysosomal system in a variety of cells by a new and ubiquitous class of endo-lysosomal ion channels known as the two-pore channels (TPCs). However, the role of TPCs in NAADP action in smooth muscle is not known. In the present work, we investigated the effects of NAADP in gastric smooth muscle cells and its ability to release Ca(2+) by TPCs. We show that Ca(2+) signals mediated by NAADP were inhibited by disrupting Ca(2+) handling by either acidic organelles (using bafilomycin A1) or the Endoplasmic Reticulum (using thapsigargin, ryanodine or 2-APB). Transcripts for endogenous TPC1 and TPC2 were readily detected and recombinant TPCs localized to the endosomes and/or lysosomes. Overexpression of wild-type TPCs but not pore mutants enhanced NAADP-mediated cytosolic Ca(2+) signals. Desensitizing the NAADP pathway inhibited Ca(2+)-responses to extracellular stimulation with carbachol but not ATP. Taken together, these results indicate that NAADP likely induces Ca(2+) release from the endolysosomal system through TPCs which is subsequently amplified via the ER in an agonist-specific manner. Thus, we suggest a second messenger role for NAADP in smooth muscle cells.
authors.affiliation: Department of Pharmacology, Federal University of São Paulo (UNIFESP), São Paulo, Brazil.
authors.forename: Gustavo J S
authors.initials: GJ
authors.lastname: Pereira
authors.affiliation: Department of Pharmacology, Federal University of São Paulo (UNIFESP), São Paulo, Brazil.
authors.forename: Hanako
authors.initials: H
authors.lastname: Hirata
authors.affiliation: Department of Pharmacology, Federal University of São Paulo (UNIFESP), São Paulo, Brazil.
authors.forename: Lúcia G
authors.initials: LG
authors.lastname: do Carmo
authors.affiliation: Interdisciplinary Center for Gene Therapy, Federal University of São Paulo, São Paulo, Brazil.
authors.forename: Roberta S
authors.initials: RS
authors.lastname: Stilhano
authors.affiliation: Department of Pharmacology, Federal University of São Paulo (UNIFESP), São Paulo, Brazil.
authors.forename: Rodrigo P
authors.initials: RP
authors.lastname: Ureshino
authors.affiliation: Department of Pharmacology, Federal University of São Paulo (UNIFESP), São Paulo, Brazil.
authors.forename: Natalia C
authors.initials: NC
authors.lastname: Medaglia
authors.affiliation: Interdisciplinary Center for Gene Therapy, Federal University of São Paulo, São Paulo, Brazil.
authors.forename: Sang W
authors.initials: SW
authors.lastname: Han
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford, UK.
authors.forename: Grant
authors.initials: G
authors.lastname: Churchill
authors.affiliation: Department of Pharmacology, Federal University of São Paulo (UNIFESP), São Paulo, Brazil.
authors.forename: Claudia
authors.initials: C
authors.lastname: Bincoletto
authors.affiliation: Department of Cell and Developmental Biology, University College London, London, UK.
authors.forename: Sandip
authors.initials: S
authors.lastname: Patel
authors.affiliation: Department of Pharmacology, Federal University of São Paulo (UNIFESP), São Paulo, Brazil. Electronic address: ssmaili@unifesp.br.
authors.forename: Soraya S
authors.initials: SS
authors.lastname: Smaili
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism; D009249:NADP / Q000031:analogs & derivatives* / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013270:Stomach / Q000166:cytology
chemical_list: D015220:Calcium Channels; D011994:Recombinant Proteins; D009249:NADP; C024376:NAADP; D002118:Calcium
keywords: Ca(2+); Gastric smooth muscle cells; Lysosomes; NAADP; TPCs