Chromaffin cells of the adrenal medulla are a primary neuroendocrine output of the sympathetic nervous system. When stimulated, they secrete a host of transmitter molecules, including catecholamines and neuropeptides, through the fusion of dense core secretory granules with the cell surface. At basal firing rates, set by the sympathetic tone, chromaffin cells selectively release catecholamines at a modest rate. Stress-mediated sympathetic activation leads to elevated catecholamine secretion and also evokes neuropeptide release. Catecholamines and neuropeptides are copackaged in the same granules; thus, it is unclear how this activity-dependent differential transmitter release is achieved. In this report, we use electrophysiological, electrochemical, fluorescence, and immunocytochemical approaches to quantify transmitter release under physiological electrical stimulation at the single cell level. We provide data to show that chromaffin cells selectively release catecholamine under basal firing conditions but release both neuropeptides and catecholamines under conditions that match acute stress. We further show that this differential transmitter release is achieved through a regulated activity-dependent dilation of the granule fusion pore. Thus, chromaffin cells may regulate release of different transmitters through a simple size-exclusion mechanism.

Chromaffin cells of the adrenal medulla are innervated by the sympathetic nervous system. Stimulation causes chromaffin cells to fire action potentials, leading to the exocytosis of various classes of transmitters into the circulation. Low-frequency electrical stimulation (action potentials delivered at 0.5 Hz) causes adrenal chromaffin cells to selectively release catecholamines through a kiss-and-run fusion event. Elevated electrical stimulation (action potentials at 15 Hz) evokes fusion pore dilation, full granule collapse, and additional release of the neuropeptide-containing proteinaceous granule core. Here we apply single-cell electrophysiological, electrochemical, and fluorescence measurements to investigate the cellular mechanism for this shift in exocytic behavior. We show that at low-frequency stimulation, a filamentous-actin cell cortex plays a key role in stabilizing the kiss-and-run fusion event. Increased stimulation disrupts the actin cortex, driving full granule collapse. We show that pharmacological perturbation of the actin cortex supersedes stimulus frequency in controlling exocytic mode. Finally, we show that nonmuscle myosin II activation contributes to the cytoskeleton-dependent control of the fusion event. Inhibition of myosin II or myosin light chain kinase under elevated stimulation frequencies inhibits fusion pore dilation and maintains the granule in a kiss-and-run mode of exocytosis. These results demonstrate an essential role for activity-evoked cytoskeletal rearrangement and the action of myosin II in the regulation of catecholamine and neuropeptide exocytosis and represent an essential element of the sympathetic stress response.

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.

Under low stimulation, adrenal chromaffin cells release freely-soluble catecholamines through a restricted granule fusion pore while retaining the large neuropeptide-containing proteinacious granule core. Elevated activity causes dilation of the pore and release of all granule contents. Thus, physiological differential transmitter release is achieved through regulation of fusion pore dilation. We examined the mechanism for pore dilation utilizing a combined approach of peptide transfection, electrophysiology, electrochemistry and quantitative imaging techniques. We report that disruption of dynamin I function alters both fusion modes. Under low stimulation, interference with dynamin I does not affect granule fusion but blocks its re-internalization. In full collapse mode, disruption of dynamin I limits fusion pore dilation, but does not block membrane re-internalization. These data suggest that dynamin I is involved in both modes of exocytosis by regulating contraction or dilation of the fusion pore and thus contributes to activity-dependent differential transmitter release from the adrenal medulla.

Adrenal medullary chromaffin cells are innervated by the sympathetic splanchnic nerve and translate graded sympathetic firing into a differential hormonal exocytosis. Basal sympathetic firing elicits a transient kiss-and-run mode of exocytosis and modest catecholamine release, whereas elevated firing under the sympathetic stress response results in full granule collapse to release catecholamine and peptide transmitters into the circulation. Previous studies have shown that rearrangement of the cell actin cortex regulates the mode of exocytosis. An intact cortex favors kiss-and-run exocytosis, whereas disrupting the cortex favors the full granule collapse mode. Here, we investigate the specific roles of two actin-associated proteins, myosin II and myristoylated alanine-rich C-kinase substrate (MARCKS) in this process. Our data demonstrate that MARCKS phosphorylation under elevated cell firing is required for cortical actin disruption but is not sufficient to elicit peptide transmitter exocytosis. Our data also demonstrate that myosin II is phospho-activated under high stimulation conditions. Inhibiting myosin II activity prevented disruption of the actin cortex, full granule collapse, and peptide transmitter release. These results suggest that phosphorylation of both MARCKS and myosin II lead to disruption of the actin cortex. However, myosin II, but not MARCKS, is required for the activity-dependent exocytosis of the peptide transmitters.

Adrenal chromaffin cells release multiple transmitters in response to sympathetic stimulation. Modest cell firing, matching sympathetic tone, releases small freely soluble catecholamines. Elevated electrical firing rates matching input under sympathetic stress results in release of catecholamines as well as semi-soluble vaso- and neuro-active peptides packaged within the dense core of the secretory granule. This activity-dependent differential transmitter release has been shown to rely on a mechanistic shift in the mode of exocytosis through the regulated dilation of the secretory fusion pore between granule and cell surface membranes. However, biochemical description of the mechanism regulating fusion pore dilation remains elusive. In the experimental setting, electrical stimulation designed to mimic sympathetic input, is achieved through single-cell voltage-clamp. While precise, this approach is incompatible with biochemical and proteomic analysis, both of which require large sample sizes. We address this limitation in the current study. We describe a bulk chemical stimulation paradigm calibrated to match defined electrical activity. We utilize calcium and single-cell amperometric measurements to match extracellular potassium concentrations to physiological electrical stimulation under sympathetic tone as well as acute stress conditions. This approach provides larger samples of uniformly stimulated cells for determining molecular players in activity-dependent differential transmitter release from adrenal chromaffin cells.

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Adrenal chromaffin cells release catecholamines and neuropeptides upon stimulation. Both transmitter classes are packaged in the same secretory organelle. At basal firing rates, highly soluble catecholamines are released from chromaffin cells by ‘kiss and run’ fusion events. Under acute stress, elevated firing leads to full granule collapse releasing both catecholamine as well as a neuropeptide‐containing proteinacious granule core. Thus, activity dependent transmitter release is regulated by a transition in secretion mode. Here we employ electrochemical, electrophysiological and fluorescence based approaches to investigate the role of actin and myosin in regulating this transition. We show that under basal firing rates, F‐actin networks are maintained stabilizing ‘kiss and run’ fusion events. Under the same stimulation conditions, disruption of F‐actin resulted in conversion to full‐collapse exocytosis. Also, under increased cell stimulation, activation of myosin II and disruption of F‐actin is necessary to drive the full collapse of the granule. Under the same conditions stabilization of F‐actin and inhibition of myosin II resulted in a conversion to ‘kiss and run’ exocytosis. Our results demonstrate that the transition from ‘kiss and run’ to ‘full collapse’ exocytosis depends upon both F‐actin and myosin II, helping to provide essential regulation of the fusion mode and a proper stress response.This work was supported by a grant from the National Institutes of Health (NIH) 5/T/32/HL07887