Figure 4

Calcium responses to UTP in immunolabeled chromaffin cells. A-C. Calcium images showing a group of chromaffin cells before (A, control), during stimulation with 100 μM UTP in presence of extracellular calcium (B) and during stimulation in the virtual absence of extracellular calcium (C, "UTP+EGTA"). At the end of each experiment cells were stimulated with 10 μM nicotine. Cells were allowed to rest for 10 min between consecutive stimulations. The fura-2 fluorescence ratio F₃₄₀/F₃₈₀ was determined for each cell in a field on a pixel-by-pixel basis. Images were coded in pseudocolor to show differences in the F₃₄₀/F₃₈₀ ratio. The images corresponding to UTP stimulation (B and C) were captured ~5 s after the UTP challenges (i.e. at the response peak in presence of extracellular calcium); D. Pseudocolor image of PNMT/TH immunofluorescence ratio (labeling index) and corresponding scale. Intensely colored red and green cells are adrenergic and noradrenergic cells, respectively. Scale bar = 50 μm; E. Time courses of changes in F₃₄₀/F₃₈₀ fluorescence ratio for two AD-cells (cells 1 and 2, also depicted in A-D) and two NA-cells (cells 3 and 4). The lines denote superfusions with UTP or nicotine in the presence or virtual absence (middle traces) of extracellular calcium.