Jgp_201711894 893..896

Chromaffin cells are neuroendocrine cells that reside within the medulla of the adrenal glands. They release several hormones in amounts and proportions that vary greatly in response to homeostatic needs and to a wide range of challenges and stresses. In addition to catecholamines, the contents of chromaffin cell secretory granules include the prohormone chromogranin A, the hormone processing enzyme tissue plasminogen activator (tPA), and the tPA inhibitor plasminogen activator inhibitor (PAI). Products released from chromaffin cells have distal targets, including the vasculature, heart, and pancreas. In addition, local actions of chromaffin cell secretions provide feedback loops for local paracrine/ autocrine actions. Different biological triggers elicit varied responses in chromaffin cells to regulate processes as diverse as blood pressure, blood clotting, angiogenesis, and metabolism in a coordinated manner. Elucidating the intricate control mechanisms that tune and orchestrate these diverse responses represents a major challenge to current research in physiology and endocrinology. In this issue of JGP, Bohannon et al. introduce a new concept to this field by suggesting that the pH rise within chromaffin cell granules that occurs when the fusion pore opens initiates a chemical reaction between tPA and PAI before these large proteins are released. The autocrine control mechanisms of the adrenal medulla exploit proximity as part of their physiological strategy. For example, tPA released from chromaffin cells binds to the chromaffin cell surface, where its target, plasminogen, also binds (Bai et al., 2012). The close proximity of tPA and plasminogen enhances processing of plasminogen to plasmin by tPA (Fig. 1). Tethering recently generated plasmin at the surface of a chromaffin cell means that it is ready and waiting to process chromogranin A to several biologically active products as soon as it is released from chromaffin cells. Thus, two processing steps are enhanced by proximity. tPA can act locally within the adrenal medulla as illustrated in Fig. 1, or enter the circulation to regulate processes such as blood clotting. The limited space around chromaffin cells makes it easier for the processing enzyme to find its prohormone substrate. The article by Bohannon et al. (2017) takes the localization of endocrine factors to a new level by suggesting that containment within the same vesicle enhances the inactivation of tPA by its inhibitor PAI. Because PAI is a suicide substrate of tPA, the inhibition is irreversible. Bohannon et al. (2017) show that individual vesicles contain both molecules but that their chemical reaction is prevented during storage by the low pH of the vesicle lumen. The authors show that raising the pH within vesicles leads to inactivation of tPA by PAI, even when there is no exocytosis. During exocytosis, the luminal pH of the vesicle rises as soon as the fusion pore opens. Protons have an exceptionally high mobility and can flow very easily, even through a narrow pore that completely blocks the passage of larger molecules. As a result, the pH of the vesicle lumen rises well before the efflux of any of the signaling molecules. Most signaling molecules escape through fusion pores in times of under 100 ms for small molecules or hundreds of milliseconds for larger molecules. However, tPA is different and remains confined within vesicles for several seconds. Bohannon et al. (2017) confirm this finding and further show that PAI lingers in vesicles much longer when the vesicles also contain tPA. They also use the polarization of membrane dye fluorescence to show that the membrane at the release site maintains the high curvature characteristic of omega-shaped narrow pores during tPA release. Thus, when a tPA-containing vesicle fuses with the plasma membrane, the fusion pore remains narrow for an unusually long time, keeping larger proteins within the vesicle. Based on these observations, Bohannon et al. (2017) put forward the intriguing idea that the unusually long residence and close proximity of tPA and PAI within vesicles after the pH goes up creates a nanoscale chamber where the two molecules can chemically react. As a result, instead of tPA and PAI being released as distinct species, they emerge as a covalent complex lacking prohormone processing activity. It is a very reasonable idea; there should be enough time according to the kinetics of the reaction in vitro. The next step, which will probably be difficult, is showing that a fraction of secreted tPA is indeed inactivated by PAI. Until then, Bohannon et al. (2017) have planted the seed of a new and important