Physiological stimulation regulates the exocytic mode through calcium activation of protein kinase C in mouse chromaffin cells

Abstract

Adrenal medullary chromaffin cells release catecholamines and neuropeptides in an activity-dependent manner controlled by the sympathetic nervous system. Under basal sympathetic tone, catecholamines are preferentially secreted. During acute stress, increased sympathetic firing evokes release of both catecholamines as well as neuropeptides. Both signalling molecules are co-packaged in the same large dense core granules, thus release of neuropeptide transmitters must be regulated after granule fusion with the cell surface. Previous work has indicated this may be achieved through a size-exclusion mechanism whereby, under basal sympathetic firing, the catecholamines are selectively released through a restricted fusion pore, while less-soluble neuropeptides are left behind in the dense core. Only under the elevated firing experienced during the sympathetic stress response do the granules fully collapse to expel catecholamines and neuropeptides. However, mechanistic description and physiological regulation of this process remain to be determined. We employ electrochemical amperometry, fluid-phase dye uptake and electrophysiological capacitance noise analysis to probe the fusion intermediate in mouse chromaffin cells under physiological electrical stimulation. We show that basal firing rates result in the selective release of catecholamines through an Omega-form 'kiss and run' fusion event characterized by a narrow fusion pore. Increased firing raises calcium levels and activates protein kinase C, which then promotes fusion pore dilation until full granule collapse occurs. Our results demonstrate that the transition between 'kiss and run' and 'full collapse' exocytosis serves a vital physiological regulation in neuroendocrine chromaffin cells and help effect a proper acute stress response.

Figure 1. Amperometric spike amplitude and charge increase in elevated external calcium

(a) Chromaffin cells were pre-loaded with fura, bathed in Ringer solutions with low (0.5 mM), normal (2.8 mM) and high (10 mM) Ca²⁺ content and stimulated with trains of action potentials at 0.5 Hz or at 15 Hz in normal Ca²⁺. Relative cytosolic Ca²⁺ levels were determined as the average value over the final 50% of the stimulus train (after they reached a steady value) and are reported as the ratio of light emitted under 360 and 390 nm excitation. (b) The mean rate of catecholamine release (slope of the spike rising phase; nA/s, black bars) as well as mean spike charge (pC, white bars) for each condition are shown in the category plot. Example single amperometric spikes for each Ca²⁺ condition are shown above each category. [Ca²⁺]_o values are indicated below each category. Sample numbers from left to right (numbers of spikes from cells/number of cells): 99/4, 1301/15, 1143/7, 2819/14. These data show that mean spike size and charge grow with calcium influx.

Figure 2. Endocytic uptake of 40 kDa dextran is blocked under low Ca²⁺

(a) A cartoon representation of the protocol is provided. A fluorescence-based size-exclusion assay was used to probe the fusion pore diameter by determining fluid-phase uptake of fluorescently labelled 40 kDa dextran. The predicted molecular diameter of this marker is approx. 7 nm. (i) Under conditions of ‘kiss and run’ exocytosis, the dextran molecules are too large to fit through the fusion pore, (ii) but are readily incorporated into endosomes formed under ‘full collapse’. (b) Cells were bathed in a Ringer solution containing 1 μM Texas Red-labelled 40 kDa dextran and either 0.5, 2.8 or 10 mM Ca²⁺ and stimulated. Following stimulation the cells were imaged and internalized fluorescence was measured as the mean image brightness within the cell cytosol (broken white line). (c) Mean dye uptake was measured during 0.5 Hz stimulation under various [Ca²⁺]_o values and at 15 Hz normal Ca²⁺. Sample numbers were from left to right (number of cells/number of spikes): 7/99; 18/1301; 7/1143; 13/2819. The fluorescence signal is expressed as a function of amperometric spike number (i.e. dye uptake per fusion event). These data indicate that dye is excluded from endosomal uptake under low Ca²⁺ influx, but is readily internalized under higher Ca²⁺ influx.

Figure 3. Cell-capacitance noise analysis predicts Ω-figures under 0.5 Hz stimulation

(a) The equivalent electrical circuit of a patch-clamped cell and the equation predicting the capacitance variance (σ²_cell; see the Experimental section for a description of the terms). ‘Kiss and run’ exocytosis results in accumulation of Ω-figures and additional electrical elements: granule capacitance (C_g), resistance (R_g) and fusion-pore conductance (G_fp; shaded box on circuit). These elements contribute to the variance equation (σ²_Ω, shaded box in lower equation) to increase capacitance noise by a factor scaled by the number of Ω-figures present (‘n’). (b) Capacitance variance predicted for the equivalent circuit is plotted against command sine-wave frequency under full granule collapse (σ²_cell) and ‘kiss and run’ fusion (σ²_cell+Ω). The balance between exocytosis and endocytosis is predicted to result in the equilibrium accumulation of 2.88 granules in the cell membrane during stimulation (as described in the text). Therefore the σ²_cell+Ω calculations include the accumulation of 2.88 Ω-figures. The contribution of the Ω-figures (σ²_Ω) is the difference between the contiuous and dotted lines. (c) The variance of the capacitance signals calculated at the 650 Hz are plotted for σ²_cell and σ²_cell+Ω against fusion-pore conductance. Cartoons represent the status of granule fusion at various pore conductances.

Figure 4. Capacitance variance predicts a Ca²⁺-dependent dilation of the fusion pore

(a) Cells held in the perforated-patch configuration were stimulated with action-potential waveforms and evoked cell capacitance is plotted. Mean cell capacitance (continuous lines), and the standard deviation (σ, broken lines) before and after the pulse are shown. (b) Stimulus-dependent development of the cell variance signal is shown for 0.5 and 15 Hz stimulation. For clarity, variance data are plotted against stimulus number. (c) Capacitance variance (σ²) measured under control, 0.5 and 15 Hz stimulation (n=7, 19 and 13 cells respectively) is plotted along with variance fitted by the model, incorporating accumulation of 2.88 ‘kiss and run’ Ω-figures under 0.5 Hz, but no Ω-figures under 15 Hz stimulation. Broken lines represent variance from unstimulated (σ²_cell) and Ω-figure-decorated (σ²_cell+Ω) cells. Cartoons below each category show the hypothesized fusion mode. (d) Cell capacitance variance was measured at 0.5 Hz stimulation under various value of [Ca²⁺]_o and at 15 Hz normal Ca²⁺. As in (c), dotted lines indicate smooth (σ²_cell) versus Ω-figure-decorated cell membrane (σ²_cell+Ω). Cartoon representations of the hypothesized fusion mode are shown below each category. Sample sizes from left to right (n=cells): 13, 19, 17, 13.

Figure 5. Activation or inhibition of conventional PKC shifts the mode of granule fusion

Cells were stimulated at 0.5 Hz or 15 Hz in 2.8 mM Ca²⁺ under conditions in which PKC activity was pharmacologically blocked or activated. Top panel: analysis of evoked rates of catecholamine release (initial spike slope) are provided for all conditions. Pretreatment of cells with PMA increased the rate of catecholamine release measured under 0.5 Hz stimulation to that measured under 15 Hz stimulation, while inhibition of PKC with Gö 6983 and Ro-31-8220 (pooled together as ‘PKC Inh.’) acted to decrease the 15 Hz-evoked rate of catecholamine release to that measured under 0.5 Hz. Sample numbers from left to right (number of spikes/number of cells): 1301/15, 821/13, 209/4, 2819/14, 3940/20. Middle panel: endocytic uptake of fluorescent 40 kDa dextran was measured and normalized to the number of amperometric spikes for each condition. Dye uptake was blocked by inhibition of PKC. In all other conditions, dye was internalized equally efficiently on an ‘all-or-none’ basis when normalized to the number of release events. Sample numbers from left to right (n=cells) 18, 10, 9, 13, 18. Bottom panel: capacitance variance measured under control, PKC-blocked and PKC-activated conditions. As in Figure 3, dotted lines represent variance values from smooth (σ²_cell) and Ω-figure-decorated (σ²_cell+Ω) membranes. Cell variance was higher under 0.5 Hz than 15 Hz stimulation. A block of PKC under 0.5 Hz stimulation had no effect on variance. A block of PKC under 15 Hz stimulation raised cell variance to the level of 0.5 Hz control conditions. Pre-treatment of cells with PMA and stimulation at 0.5 Hz resulted in decreased variance matching the 15 Hz control condition. Sample numbers for these data were from left to right (n=cells) 19, 36, 15, 13, 58. Control data are re-plotted from previously shown Figures. Summary cartoon: taken together, all three techniques predict that granule fusion occurs through a ‘kiss and run’ mechanism under modest Ca²⁺ influx, or when PKC is inactive.