FIGURE 4.

Secretagogue-stimulated exocytosis of SgII in COS-7 and A35C cells. A and B, regulated secretion of EAP fusion proteins. COS-7 cells (A) and A35C cells (B) expressing SIG-EAP or SgII-EAP were exposed for 30 min to DMEM alone (−) or DMEM supplemented with the indicated concentration of A23187 (+). EAP type secretion was assayed as described under “Experimental Procedures.” Release of EAP is expressed either as % of EAP activity secretion (A, left) or relative to enzymatic activity released in the absence of secretagogue (A, right, and B). Values are given as the mean ± S.E. of triplicate determinations. Results from one of at least three independent experiments are shown. †, p > 0.05; ***, p < 0.001, as compared with control, analysis of variance with Dunnett's post-hoc test. C, stimulated release of SgII-HA from A35C-S7 cells. A35C-S7 cells were sequentially exposed for 30 min to DMEM (−) and then DMEM containing the indicated secretagogues (+). The resulting supernatants were subjected to immunoblotting. The amount of secreted SgII-HA was quantified by densitometry in three independent secretion experiments. The means ± S.E. of fold increases in stimulated (+) over basal (−) conditions were as follows: A23187, 5.9 ± 1.2; KCl, 4.8 ± 0.8; nicotine, 4.9 ± 1.7. D, TIRFM image of a characteristic A35C cell expressing SgII-GFP. Only the pool of vesicles within ∼140 nm from the plasma membrane in the z axis (corresponding to the estimated penetration depth of the evanescent wave used to excite GFP) is seen. Scale bar, 5 μm. E, secretagogue-evoked release of SgII-GFP observed by TIRFM. The arrow in the overall view of a transfected A35C cell prior (−1 s) to stimulation with 1 μm A23187 (left) indicates the exocytotic event depicted in the adjacent sequential images. Shown on the right is the quantification of the fluorescence intensity changes measured from the sequential images.