Population of DCVs blocked by ryanodine. (A) Frequency distribution of individual amperometric events according to charge (pC) in control (white; 918 events) and ryanodine-treated cells (stripes; 2,536 events). The difference between the two distributions is shown on an expanded x axis in the inset. (B) Examples of amperometric events recorded from mouse chromaffin cells. The first two traces show a spike, the second one with a pre-spike foot. The third one represents an SAF. (A includes all events, both SAFs and spikes. The criteria for classifying an event as an SAF are given in Materials and methods.) (C; left) The frequency of the spikes and the SAFs in control solution (0.08 ± 0.01 s⁻¹ for both; n = 12). Spike frequency increases in the presence of ryanodine (0.67 ± 0.14 s⁻¹; P < 0.0008; n = 13). (Right) Ryanodine increases the mean charge of spikes (0.14 ± 0.03 to 0.31 ± 0.03 pC; P < 0.0003; n = 12 for control and n = 13 for treated cells) and SAFs (0.02 ± 0.00 to 0.04 ± 0.01 pC; P < 0.01; n = 12 for control and n = 13 for treated cells).

Effect of 2 µM Tg plus 20 mM caffeine and buffering free [Ca²⁺]_i at 500 nM with EGTA and CaCl₂. (A) Diagram representing the protocol to decrease the stores with Tg and caffeine. (B) Tg and caffeine decrease syntilla frequency to 0.09 ± 0.04 s⁻¹ (n = 8) compared with Tg alone (0.33 ± 0.09 s⁻¹; n = 15; P < 0.04), caffeine alone (0.57 ± 0.14 s⁻¹; n = 12; P < 0.01), and with the free [Ca²⁺]_i buffered at 500 nM (0.49 ± 0.08 s⁻¹; n = 23; P < 0.001). (C) Tg and caffeine increase the frequency of all amperometric events to 0.44 ± 0.09 s⁻¹ (n = 8), SAFs to 0.15 ± 0.02 s⁻¹ (n = 8), and spikes to 0.33 ± 0.09 s⁻¹ (n = 8) when compared with Tg alone (0.13 ± 0.05 s⁻¹; n = 9; P < 0.01) for all events: 0.08 ± 0.02 s⁻¹ (n = 7; P < 0.02) for SAFs, 0.07 ± 0.03 s⁻¹ (n = 9; P < 0.02) for spikes, and caffeine alone (0.10 ± 0.04 s⁻¹; n = 10; P < 0.01) for all events; 0.04 ± 0.02 s⁻¹ (n = 10; P < 0.001) for SAFs, 0.07 ± 0.03 s⁻¹ (n = 10; P < 0.02) for spikes, and when the free [Ca²⁺]_i is buffered at 500 nM (0.10 ± 0.03 s⁻¹; n = 10; P < 0.01) for all events: 0.04 ± 0.01 s⁻¹ (n = 9; P < 0.001) for SAFs and 0.07 ± 0.02 s⁻¹ (n = 10; P < 0.02) for spikes. (D) Tg and caffeine do not alter the quantal charge of amperometric events, compared with Tg alone, caffeine alone, or when the free [Ca²⁺]_i is buffered at 500 nM. (E) Tg and caffeine increase global [Ca²⁺]_i to 505.3 ± 172 nM (n = 5) compared with typical basal global [Ca²⁺]_i in Tg alone (130.0 ± 23 nM; n = 7; P < 0.03) and caffeine alone (71.4 ± 16 nM; n = 7; P < 0.02). When free [Ca²⁺]_i is buffered at 500 nM, the mean basal global [Ca²⁺]_i (397.8 ± 69 nM; n = 6) is not significantly different compared with Tg and caffeine (P = 0.59). Error bars, ±SEM.

Relationship between Ca²⁺ released into the syntilla microdomain and exocytotic events. (A; top) Spatiotemporal profiles of the Ca²⁺ syntilla arising from RYR2s near the plasma membrane for each experimental condition. Concentric curves represent different [Ca²⁺]'s within the syntilla microdomain. Note that the ordinate presents one of the spatial axes; the other two are the same as the one shown and together constitute a hemisphere (see Materials and methods). (Bottom) Syntilla frequency for each of the four experimental conditions listed above the top panel. (B) Relationship of the SI for the entire cell versus frequency of exocytotic events from the entire cell surface. The plot shows that the frequency of amperometric events declines to a baseline level as the SI increases. The relationship drawn here is based on a 10-µM syntilla microdomain (A, green line). The equation of the curve to which the data are fit:(4)is based on a two-state model as described in Materials and methods, where DCVs are either in an inhibited or releasable state. A least-squares fit of Eq. 4 to the data points in B was performed, resulting in F₀ = 7.75 and k = 104.10. The correlation coefficient (R²) of the fit is 0.993.

Effect of 100 µM ryanodine. (A) Changes in cytosolic [Ca²⁺] measured with the Ca²⁺-indicator dye, fluo-3, and expressed on a pseudo-color scale as the change in fluorescence over the baseline fluorescence (ΔF/F₀). Image threshold is 15% of ΔF/F₀. Syntillas are larger and more frequent under normal conditions (left), and they are smaller and less frequent or completely blocked in the presence of ryanodine (right). (B) Representative amperometric trace from a cell in the absence (left) and presence (right) of ryanodine. In the presence of syntillas (left; control), amperometric events are smaller and less frequent. When syntillas are blocked or decreased (right; 100 µM ryanodine), amperometric events are larger and more frequent. (C; left) 100 µM ryanodine decreases syntilla frequency (left bar graph) from 0.81 ± 0.12 (n = 4) to 0.28 ± 0.09 s⁻¹ (n = 18; P < 0.02). Conversely, ryanodine increases exocytotic frequency (right bar graph) from 0.16 ± 0.03 (n = 12) to 0.73 ± 0.15 s⁻¹ (n = 13; P < 0.002). Error bars ± SEM. (Right) 100 µM ryanodine also decreases the mean signal mass of the individual syntilla from 28.5 ± 3.9 (n = 13) to 6.2 ± 0.9 × 10⁻²⁰ moles (n = 18; P < 0.00001) and increases the mean charge per amperometric event from 0.08 ± 0.01 (n = 12) to 0.28 ± 0.03 pC (n = 13; P < 0.0003).

Effect of 100 µM ryanodine in unpatched cells. In the results shown here, the same protocol was used as in Figs. 1 and 2, except that the cells were not patched and hence the cytosol was left undisturbed. (A) All amperometric events. (Left) The frequency of all events in control (0.09 ± 0.02 s⁻¹; n = 28) versus ryanodine (0.49 ± 0.14 s⁻¹; n = 9; P < 0.0002). (Right) The magnitude of all events for control (0.18 ± 0.2 pC; n = 28) versus ryanodine (0.26 ± 0.03 pC; n = 9; P < 0.02). (B) Amperometric events separated into spikes and SAFs. (Left) Control frequency of spikes (0.08 ± 0.02 s⁻¹; n = 28) and SAFs (0.01 ± 0.00 s⁻¹; n = 28) versus ryanodine for spikes (0.42 ± 0.13 s⁻¹; n = 9; P < 0.03) and for SAFs (0.05 ± 0.02 s⁻¹; n = 9; P < 0.02). (Right) The magnitude of spikes (0.20 ± 0.03 pC; n = 28) and SAFs (0.07 ± 0.01 pC; n = 21) in control versus ryanodine for spikes (0.30 ± 0.04 pC; n = 9; P < 0.02) and for SAFs (0.06 ± 0.01 pC; n = 9; P = 0.6).

Effect of 2 µM Tg with caffeine. Chromaffin cells were treated with 2 µM of the SERCA inhibitor Tg for 20 min or longer and briefly exposed (1 min) to 20 mM caffeine by picospritzer to reduce internal Ca²⁺ stores. (A; left) When Ca²⁺ stores are reduced and cytosolic Ca²⁺ is buffered with EGTA as described in Materials and methods, the mean frequency of syntillas is reduced from 0.78 ± 0.15 (n = 15) to 0.42 ± 0.01 s⁻¹ (n = 23; P < 0.03), and the amperometric event frequency is increased from 0.17 ± 0.02 (n = 20) to 0.39 ± 0.06 s⁻¹ (n = 10; P < 0.0003). (Right) When the stores are reduced, there is no detectable change in the mean syntilla signal mass (25.9 ± 2.3 [n = 47] and 22.1 ± 3.4 [n = 36], control and treated, respectively), but the mean charge of amperometric events increases (0.083 ± 0.014 [n = 20] and 0.143 ± 0.023 pC [n = 10; P < 0.026]). A and B include both spikes and SAFs. (B) Frequency distribution of individual amperometric events according to charge (pC) in control (white; 731 events) and when stores are reduced by Tg and caffeine treatment (stripes; 908 events). The difference between the two distributions is shown on an expanded x axis in the inset in blue. (C; left) The frequency in control solution with buffer (0.09 ± 0.01 s⁻¹ for spikes and 0.08 ± 0.01 s⁻¹ for SAFs) is the same as in control solution without buffer as given in Fig. 2 C. After treatment with Tg and caffeine, the spike frequency increases to 0.30 ± 0.05 s⁻¹ (P < 0.00002; n = 10). (Right) The mean charge of spikes (0.13 ± 0.02 vs. 0.17 ± 0.02 pC) or SAFs (0.02 ± 0.00 pC for control and treatment) in control and after Tg plus caffeine treatment.