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. 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 Vargatef receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation. (21), and decay constant of the evanescent field (1/e depth) was determined to be 160 Vargatef 28 nm. Different combinations of fluorescent dyes were used to label the cells. In experiments aimed at simultaneously determining vesicle fusion and the intracellular calcium concentration ([Ca2+](22) and z distances according to Johns (23) were performed using homemade macros for IgorPro (WaveMetrics Inc., Lake Oswego, OR). [Ca2+]for 5 min at 4 C, and 10-l aliquots of supernatant were transferred to wells of a 96-well plate placed on ice. The 96-well plate was set in a FLUOstar OPTIMA Microplate Luminometer (BMG LABTECH GmbH, Offenburg, Germany), and 100 l of rLuciferase/Luciferin reagent was automatically injected into each well Vargatef at room temperature (25 C). Immediately before the start of the experiments, N2a cells were bathed in Mg2+-free Locke’s buffer for 1 h at 37 C. Then cells were exposed for 5 min to either plain extracellular Locke’s buffer or Locke’s buffer supplemented with 100 m ARL 67156, a competitive inhibitor of ecto-ATPases (24), with 500 m NEM or with the two compounds, and their medium was collected to measure basal ATP concentration. Thereafter, cells were stimulated by adding ionomycin (10 m, final concentration in extracellular medium) prepared in either plain Locke’s buffer or Locke’s buffer containing the above-mentioned supplements. Five minutes later, extracellular medium was again collected to measure evoked ATP concentration. In another set of experiments, cells were grown for 72 h in the absence or the presence of BoNT/A (30 nm). Both controls and toxin-treated cells were incubated with ARL 67156 (100 m; 5 min) and subsequently challenged with ionomycin (10 m; 5 min) to elicit ATP release. ATP concentration was determined by comparison with a calibration curve generated with ATP standards diluted in the same buffer as the samples. Data Analysis Pooled data are shown as the means S.E.; denotes the number of individual cells, vesicles, exocytotic events, or STICs considered in each particular analysis. Statistical differences were determined by the Student’s test for unpaired samples. A value equal or smaller than 0.05 was taken as the limit of significance. RESULTS P2X7 Receptors Trigger Exocytosis as Assayed AIGF by Membrane Capacitance Measurements It has been reported that N2a cells transiently transfected with pro-opiomelanocortin undergo calcium-regulated release of -endorphin located to dense-core granules (25, 26). On the other hand, N2a cells express ionotropic purinergic P2X7 receptors whose activation promotes Ca2+ entry into the cell and the ensued increase in [Ca2+](12). Thus, the question arises as to whether P2X7 receptors might be coupled to exocytosis in this neuroblastoma cell line. We first addressed this issue by determining the potential change in membrane capacitance elicited by P2X7 receptor stimulation. Biological membranes behave as electrical capacitors whose capacitance is in direct relation to their surface. Because exocytosis involves the fusion of the vesicle membrane with the plasma membrane, it implies an increase in cellular surface, which can be detected as a Vargatef change in the Vargatef capacitance of the cell by using the whole-cell configuration of the patch clamp technique (20, 27). In these experiments we employed N2a cells seeded at a low density.