NAADP-sensitive two-pore channels are present and functional in gastric smooth muscle cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as an important modulator of Ca2+ 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 Ca2+ by TPCs. We show that Ca2+ signals mediated by NAADP were inhibited by disrupting Ca2+ 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 Ca2+ signals. Desensitizing the NAADP pathway inhibited Ca2+-responses to extracellular stimulation with carbachol but not ATP. Taken together, these results indicate that NAADP likely induces Ca2+ 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. (C) 2014 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Biotechnology and Biological Sciences Research CouncilFed Univ São Paulo UNIFESP, Dept Pharmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Interdisciplinary Ctr Gene Therapy, BR-04044020 São Paulo, BrazilUniv Oxford, Dept Pharmacol, Oxford OX1 3QT, EnglandUCL, Dept Cell & Dev Biol, London, EnglandFed Univ São Paulo UNIFESP, Dept Pharmacol, São Paulo, BrazilUniversidade Federal de São Paulo, Interdisciplinary Ctr Gene Therapy, BR-04044020 São Paulo, BrazilFAPESP: 2008/11.515-3CNPq: 482030/2010-0CAPES: BB/G013721/1Web of Scienc