title: P2X7 receptors trigger ATP exocytosis and modify secretory vesicle dynamics in neuroblastoma cells.
abstract: Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca(2+)/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca(2+)-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca(2+) concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca(2+) and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation.
authors.affiliation: Departamento de Toxicología y Farmacología, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Gutiérrez-Martín
authors.forename: Diego
authors.initials: D
authors.lastname: Bustillo
authors.forename: Rosa
authors.initials: R
authors.lastname: Gómez-Villafuertes
authors.forename: Jesús
authors.initials: J
authors.lastname: Sánchez-Nogueiro
authors.forename: Cristina
authors.initials: C
authors.lastname: Torregrosa-Hetland
authors.forename: Thomas
authors.initials: T
authors.lastname: Binz
authors.forename: Luis Miguel
authors.initials: LM
authors.lastname: Gutiérrez
authors.forename: María Teresa
authors.initials: MT
authors.lastname: Miras-Portugal
authors.forename: Antonio R
authors.initials: AR
authors.lastname: Artalejo
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D019898:Autocrine Communication / Q000187:drug effects / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D005089:Exocytosis; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D019899:Paracrine Communication / Q000187:drug effects / Q000235:genetics; D058486:Receptors, Purinergic P2X7 / Q000235:genetics / Q000378:metabolism*; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D050493:Vesicular Monoamine Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D007476:Ionophores; D009363:Neoplasm Proteins; D058486:Receptors, Purinergic P2X7; D050600:SNARE Proteins; C493450:Slc18a1 protein, mouse; C493453:Slc18a2 protein, mouse; D050493:Vesicular Monoamine Transport Proteins; D015759:Ionomycin; D000255:Adenosine Triphosphate; D002118:Calcium