Abstract
Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca2+/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 Ca2+-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 Ca2+ 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 Ca2+ 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.
Highlights
Purinergic signaling is already present at early stages of embryogenesis wherein it is involved in proliferation, migration, and differentiation of cells from multiple structures (6 –9)
P2X7 Receptor-mediated ATP Exocytosis in Neuroblastoma Cells present study we have addressed this issue by identifying in N2a cells several essential proteins of the exocytotic apparatus of sympathetic neurons, including the vesicular catecholamine and nucleotide transporters, and by measuring exocytosis with both electrophysiological and imaging techniques in the presence of selective P2X7 receptor agents
Our results indicate that P2X7 receptors are coupled to exocytosis and to the regulation of secretory vesicle dynamics in N2a cells, and they suggest that the exocytotic release of ATP induced by P2X7 receptor stimulation could mediate the tonic inhibition of neurite formation exerted by P2X7 receptors in N2a cells
Summary
N2a, Neuro-2a; BoNT/A, botulinum neurotoxin A; BBG, Brilliant Blue G; BzATP, 2Ј,3Ј-O-(4-benzoyl)-benzoyl ATP; NEM, N-ethylmaleimide; STIC, small transient inward current; TIRFM, total internal reflection fluorescence microscopy; SNARE, soluble N-ethylmaleimide factor attachment protein (NSF) receptor; fF, femtofaraday; LDCV, large dense-core vesicle. Biochemical measurements based on the luciferin-luciferase reaction indicated that, in effect, N2a cells release ATP. In clusters of N2a cells we report the detection of small transient inward currents (STICs), which are likely generated by the quantal release of ATP. Our results indicate that P2X7 receptors are coupled to exocytosis and to the regulation of secretory vesicle dynamics in N2a cells, and they suggest that the exocytotic release of ATP induced by P2X7 receptor stimulation could mediate the tonic inhibition of neurite formation exerted by P2X7 receptors in N2a cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
