Figure 2
From: Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium
ATP is released both constitutively and by purinergic receptor activation. (A) left: Representative confocal image of quinacrine labeling of endogenous ATP stores located ~100 μm into the slice. Dashed line, basement membrane. White box indicates region that is expanded to right. Scale bar, 100 μm. right: Expanded view of OE showing punctate fluorescent ATP stores. White ○’s indicate typical puncta selected for further analysis. (B-C) Representative traces of normalized changes in fluorescence over time from individual puncta measured from time-series recordings. (B) Shown are background fluorescence measured in tissue devoid of punctate fluorescence (dark gray), and data that display a linear or exponential decrease in fluorescence recorded in control physiological Ringer’s solution (black) or 50 μM exogenous ATP (light gray). (C) Representative trace of a punctum displaying dynamic fluctuations in fluorescence recorded following ATP application. Legend pertains to both B and C. (D) Half-time decrease in ATP release (mean + SEM) measured from time-series recordings 400 s after application of Control Ringer’s solution (white bar) or 50 μM ATP (grey bar). *, p < 0.0001 v. control (unpaired Student’s t-test). (E) Mean (+ SEM) % of released ATP measured from time-series recordings after application of Control Ringer’s solution or 50 μM ATP. (F) Mean (+ SEM) % of released ATP measured from Z-stack recordings after application of Control Ringer’s solution, 50 μM ATP, UTP, αβmeATP, or Bz-ATP, or PPADS (25 μM) or apyrase (3 units/ml, 1 hr pre-incubation) ± 50 μM ATP. *, p < 0.01 v. Control (one-way ANOVA with Bonferroni’s planned comparison test). (G) Luciferin-luciferase assays were used to quantify the amount of constitutively released ATP or evoked ATP release following stimulation with 50 μM P2X1,7 receptor agonist Bz-ATP in the absence or presence of neonatal OE tissue slices. *, p < 0.05 v. Bz-ATP only (one-way ANOVA with Bonferroni’s planned comparison test). (H) Peak calcium responses (mean + SEM) elicited from fluo-4 AM loaded P2X2-transfected HEK-293 cells following stimulation with ATP in the absence of OE tissue (5 μM, 1 μM, 500 nM or 50 nM) or with 50 μM Bz-ATP in the presence of an OE slice (Tissue + Bz-ATP). Bz-ATP-evoked calcium transients were not observed in transfected HEK-293 cells in the absence of an OE slice (data not shown).