Figure 5. Uncaging of PI(4,5)P₂, but not DAG augments exocytosis.

(a) Left panel: mean whole-cell capacitance responses during the test pulse of chromaffin cells loaded with cg-PI(4,5)P₂ (data from compounds 1a,b and 2a,b pooled, uncaging group: blue, control group: grey). Different secretion phases are indicated. Right panel: cell-wise quantification. IRP = Immediately Releasable Pool; RRP = Readily Releasable Pool, slope = slope determined by linear fit of sliding capacitance. (b) Structure of caged DAG (cg-DAG). (c) Titration to determine the intracellular cg-PI(4,5)P₂ concentration by comparison of coumarin fluorescence in cells loaded with known cg-DAG concentrations via the patch pipette, yielding [cg-PI(4,5)P₂]=29 µM. (d) Left panel: depolarization-induced capacitance (average trace) elicited by the test pulse (same stimulation as in Figure 4a) in cells exposed to DAG uncaging (green), or not (grey, control). No augmentation was seen. Middle and right panel: quantification of IRP, RRP, total secretion and slope revealed no significant changes. Scale bars 20 fF/1 s. Statistical testing by unpaired Student’s t-test; **p<0.01; ***p<0.001. Number of cells (n): panel a: n = 50 (wild type control), n = 49 (wild type PI(4,5)P₂ uncaging); panel c: n = 16 (cg-DAG 5 µM), n = 3 (cg-DAG 15 µM), n = 6 (cg-DAG 30 µM), n = 4 (cg-DAG 45 µM), n = 5 (cg-DAG 100 µM), n = 14 (cg-PI(4,5)P₂), panel d: we used two different cg-DAG concentrations (cg-DAG, 45 µM = 6 cells and cg-DAG, 30 µM = 15 cells); pooled results are shown; n = 20 (wild type control), n = 21 (wild type DAG-uncaging).

Figure 5—figure supplement 1. Blocking PI(4,5)P₂-degradation to DAG augments recovery of the RRP.

These experiments measure exocytosis (capacitance changes) induced by sudden intracellular Ca²⁺ elevations. (a) Ca²⁺ uncaging (at arrow) stimulates fast and slow components of exocytosis. The response to a second Ca²⁺ uncaging (Stimulation 2, 100 s after Stimulation 1) is smaller. We reasoned that Ca²⁺ not only triggers the release of secretory vesicles from chromaffin cells, but also activates PLC, leading to PI(4,5)P₂ hydrolysis. To test whether the lack of full recovery after the first stimulation might be due to an induction of PLC activity, we blocked PLC pharmacologically. Top panel: intracellular [Ca²⁺] (mean) following uncaging (at 0.5 s, see arrow). Bottom panel: average capacitance traces. Black traces are the first stimulation, red traces a second stimulation delivered 100 s later. Shown are mean traces from all measured cells. The inactive analog U73343 of the PLC inhibitor was present in the pipette (concentration 10 μM). (b) Similar experiment, but including the active PLC inhibitor (U73122, 10 μM) in the patch-clamp pipette. (c) The preflash (before uncaging) [Ca²⁺] (mean ±SEM) was unchanged between experiments performed with the active and the inactive compound (blue and green bars, respectively, a two-tailed Student’s t-test was used to test for differences between means, Stimulation 1: p=0.733; Stimulation 2: p=0.936). (d) Kinetic analysis of capacitance traces was used to identify the Readily-Releasable Pool (RRP) and the Slowly-Releasable Pool (SRP). The fractional recovery of the RRP (mean ±SEM) was significantly augmented by the active PLC-inhibitor (tested by a Student’s t-test p=0.0379 for RRP and p=0.323 for SRP). Number of cells, n = 36 (U73343), n = 36 (U73122). *p<0.05.