Neuroendocrine chromaffin cells selectively secrete a variety of transmitter molecules into the circulation as a function of sympathetic activation. Activity-dependent release of transmitter species is controlled through regulation of the secretory fusion pore. Under sympathetic tone, basal synaptic excitation drives chromaffin cells to selectively secrete modest levels of catecholamine through a restricted secretory fusion pore. In contrast, elevated sympathetic activity, experienced under stress, results in fusion pore expansion to evoke maximal catecholamine release and to facilitate release of co-packaged peptide transmitters. Therefore, fusion pore expansion is a key control point for the activation of the sympatho-adrenal stress response. Despite the physiological importance of this process, the molecular mechanism by which it is regulated remains unclear. Here we employ fluorescence imaging with electrophysiological, and electrochemical-based approaches to investigate the role of dynamin I in the regulation of activity-mediated fusion pore expansion in mouse adrenal chromaffin cells. We show that under elevated stimulation, dynamin I is dephosphorylated at Ser-774 by calcineurin. We also demonstrate that disruption of dynamin I-syndapin binding, an association regulated by calcineurin-dependent dynamin dephosphorylation, limits fusion pore expansion. Lastly, we show that perturbation of N-WASP function (a syndapin substrate) limits activity-mediated fusion pore expansion. Our results suggest that fusion pore expansion is regulated by a calcineurin-dependent dephosphorylation of dynamin I. Dephosphorylated dynamin I acts via a syndapin/N-WASP signaling cascade to mediate pore expansion.

Hill J, Lee SK, Samasilp P, Smith C. Pituitary adenylate cyclaseactivating peptide enhances electrical coupling in the mouse adrenal medulla. Am J Physiol Cell Physiol 303: C257-C266, 2012. First published May 16, 2012; doi:10.1152/ajpcell.00119.2012.-Neuroendocrine adrenal medullary chromaffin cells receive synaptic excitation through the sympathetic splanchnic nerve to elicit catecholamine release into the circulation. Under basal sympathetic tone, splanchnicreleased acetylcholine evokes chromaffin cells to fire action potentials, leading to synchronous phasic catecholamine release. Under elevated splanchnic firing, experienced under the sympathoadrenal stress response, chromaffin cells undergo desensitization to cholinergic excitation. Yet, stress evokes a persistent and elevated adrenal catecholamine release. This sustained stress-evoked release has been shown to depend on splanchnic release of a peptide transmitter, pituitary adenylate cyclase-activating peptide (PACAP). PACAP stimulates catecholamine release through a PKC-dependent pathway that is mechanistically independent of cholinergic excitation. Moreover, it has also been reported that shorter term phospho-regulation of existing gap junction channels acts to increase junctional conductance. In this study, we test if PACAP-mediated excitation upregulates cell-cell electrical coupling to enhance chromaffin cell excitability. We utilize electrophysiological recordings conducted in adrenal tissue slices to measure the effects of PACAP stimulation on cell coupling. We report that PACAP excitation increases electrical coupling and the spread of electrical excitation between adrenal chromaffin cells. Thus PACAP acts not only as a secretagogue but also evokes an electrical remodeling of the medulla, presumably to adapt to the organism's needs during acute sympathetic stress. acute stress; catecholamine; connexin-43; connexin-36; gap junction ADRENAL MEDULLARY CHROMAFFIN cells are neural crest-derived neurosecretory cells that release catecholamine into the bloodstream (2). Chromaffin cells are innervated by the bifurcating sympathetic splanchnic nerve. Under sympathetic tone, corresponding to the "rest and digest" metabolic state, the splanchnic nerve fires at a modest rate to maintain catecholamine homeostasis. Splanchnic-released acetylcholine binds to nicotinic ionotropic receptors on the chromaffin cell membrane, leading to action potential firing and controlled, phasic catecholamine release (1,7,13,18). Under the acute sympathetic stress response, the splanchnic nerve fires at a heightened rate, leading to rapid and robust cholinergic-evoked catecholamine secretion (25). Yet, the cholinergic pathway rapidly desensitizes (4, 5, 29) while elevated catecholamine secretion persists (44). Pituitary adenylate cyclase-activating peptide (PACAP) is a noncholinergic splanchnic-derived peptide transmitter released selectively during elevated splanchnic firing. PACAP has been identified as the peptide transmitter driving tonic catecholamine secretion after desensi...

Samasilp P, Lopin K, Chan SA, Ramachandran R, Smith C. Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells.

SNAP25 is a t-SNARE protein mediating exocytosis in neuronal cell types. Previous results with a construct lacking the nine C-terminal residues (SNAP25D9) showed changed fusion pore properties, suggesting a model for fusion pore mechanics that couple C-terminal zipping of the SNARE complex to the opening of a proteolipid fusion pore (Fang 2008 PNAS,105:15388). The deleted fragment contains the positively charged residues R198andK201, which are located in layers 7-8 of the SNARE complex at its C-terminal end. To study the role of these two residues in the structure and dynamics of the fusion pore, carbon fiber amperometry and cell-attached capacitance measurement of individual fusion and release events were performed using bovine chromaffin cells overexpressing wild type SNAP-25 or the point mutation constructs R198Q/E, K201Q/E to substitute the positively charged amino acid with a neutral or positively charged one. The R198E/K201E mutants increased fusion pore duration similar to SNAP25D9. Furthermore, the R198E/R198Q also showed a smaller fusion pore conductance. These results support the conclusion that fusion pore properties depend on the precise arrangements at the C-terminal end of the SNARE complex. Supported by NIH grant GM085808. 2207-Pos Board B193Activity-Dependent Fusion Pore Dilation Mediated by a Dynamin I-Syndapin Pathway Prattana Samasilp, Bryan Doreian, Shyue-An Chan, Corey Smith. Chormaffin cells of adrenal medulla serve a primary role in setting a proper homeostatic status under basal sympathetic tone as well as a physiological response to sympathetic stress. In order to function under these diverse conditions, chromaffin cells exhibit a differential release of catecholamine and co-packaged peptide transmitter molecules. Under basal sympathetic firing, catecholamine is selectively secreted through a restricted fusion pore of ''Kiss and Run'' exocytic mode. On the other hand, under acute stress, elevated sympathetic activity increases cytosolic calcium, leading to dilation of fusion pore and finally full granule collapse into plasma membrane. Both catecholamine and peptide transmitters are expelled in this condition. Thus, activity-dependent differential transmitter release is regulated by fusion pore dilation. Previous studies have shown that dynamin I plays a critical role in controlling fusion pore dilation in chromaffin cells. Here, we employ electrochemical, electrophysiological and fluorescence based approaches to investigate the molecular mechanism responsible for dynamin I-dependent fusion pore dilation. We show that syndapins (synaptic dynamin-associated proteins) are a primary molecular component of dynamin I-dependent fusion pore regulation. Disruption of dynamin I-syndapin interaction decreases normal activity-mediated catecholamine release. Our results suggest that fusion pore dilation is regulated by a dynamin I-syndapin-dependent signaling mechanism. 2208-Pos Board B194Oxidation and Palmitoylation of SNAP-25B In Vitro Nozomi Ogawa, Alex DaBell, Dixon J. Woodbury. SNAP-25 plays a cr...

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