In this study we provide what we believe to be the first evidence that a specific protease system present in the extracellular environment of the chromaffin cell can cleave CgA into bioactive fragments that inhibit secretagogue-stimulated catecholamine release. Plasmin digestion of CgA produced peptides that inhibited nicotine-stimulated catecholamine release from both PC12 cells and primary bovine adrenal chromaffin cells. This suggests that the digest contains peptides that may participate in an autocrine negative-feedback mechanism controlling catecholamine release from chromaffin cells that may have important implications in cardiovascular regulation (11
). In the presence of α2
-antiplasmin, the major physiologic inhibitor of plasmin, CgA cleavage into both large and small Mr
products was inhibited, indicating that the cleavage was due to plasmin activity.
As a mechanism for concentrating the activity of plasminogen in the environment into which CgA is secreted, we found that PC12 cells bound plasminogen in a specific, saturable, and reversible manner. Both a high-affinity (Kd
= 77 nM) and a low-affinity (Kd
= 8.87 μM) interaction of plasminogen with these cells was detected. The identification of a high-affinity plasminogen-binding site on these cells is particularly noteworthy because the Kd
values measured for plasminogen binding to eukaryotic cells are typically in the 1 μM range (24
). This high-affinity plasminogen-binding site is predicted to be more than 90% occupied at plasma and extracellular fluid concentrations of plasminogen (2 μM) (19
). Rapid processing of CgA occurred at plasmin concentrations corresponding to activation of 1% and 10% of circulating plasminogen. Because of the presence of plasminogen-binding sites, the plasminogen concentration in the environment of the chromaffin cell is predicted to be markedly increased, compared with the circulating plasminogen concentration, an effect that would further amplify the cleavage of CgA. Accordingly, we found that in the presence of cells, processing of CgA was enhanced, compared with processing in the presence of buffer. Cell-enhanced cleavage was inhibited in the presence of EACA, a lysine analogue that interferes with plasminogen binding to the cells, implicating plasminogen-binding sites in promoting the enhanced cleavage. We performed these experiments at the physiologic circulating concentration of CgA, 1.5 nM. The concentration of CgA within the chromaffin vesicle is extremely high, approximately 4 mM, so that following exocytosis, the concentration of CgA in the local extracellular space may be as high as 0.4 mM (41
). Thus, this increase in substrate concentration also would favor even more rapid processing of CgA in the environment of the cell after the release reaction. Thus, catecholaminergic cells have the ability to concentrate and spatially organize plasmin activity in the local environment into which CgA is secreted, providing a logical microanatomic and physiologic rationale for a mechanism of local proteolytic processing of CgA.
As a mechanism for further amplification of plasmin formation, binding sites for the plasminogen activators, t-PA (27
) and u-PA (27
), are present on PC12 cells, also. We confirmed the published report that t-PA can also interact with PC12 cells (27
). The t-PA binding was saturable because the binding was 78% inhibitable by unlabeled t-PA. The cells had a high capacity for t-PA. Arginine and EACA also inhibited t-PA binding. Thus, the recognition specificity appears to be similar to t-PA–binding sites described on other cell types (32
). t-PA is synthesized by these cells (21
) and is coreleased with catecholamines and CgA upon chromaffin cell stimulation (21
). The released t-PA potentially can bind to its receptors for further amplification of this system.
The importance of the local cellular fibrinolytic system in catecholamine release was tested in experiments using t-PA overexpressing PC12 cells. Overexpression of t-PA resulted in markedly diminished secretagogue-stimulated catecholamine release (Figure ). Conversely, when plasmin activity was inhibited using an anticatalytic mAb, nicotine-stimulated catecholamine secretion was substantially increased in these cells (Figure ). Taken together, these results suggest a major role for fibrinolytic molecules in the regulation of catecholamine secretion. In addition, these results are consistent with the interpretation that overexpression of t-PA in these cells results in excessive plasminogen activation at the chromaffin cell surface, with a resultant increase in CgA processing to bioactive peptides that inhibit catecholamine release, an effect that can be reversed by specifically inhibiting plasmin.
Thus, the results of this study demonstrate that (a) the fibrinolytic enzyme plasmin proteolytically cleaves CgA, the major secretory protein of chromaffin cells; (b) proteolysis of CgA by plasmin is sufficient to liberate bioactive fragments that inhibit secretagogue-stimulated catecholamine release from these cells; (c) augmented activity of the plasminogen/t-PA system by overexpression of t-PA leads to decreased catecholamine secretion, and conversely, inhibition of plasmin activity enhances catecholamine release in PC12 cells; (d) plasminogen binds to both a high-affinity (Kd
= 77 nM) and to a lower-affinity (Kd
= 8.87 μM) binding site on PC12 pheochromocytoma cells; (e) binding of plasminogen to its cellular receptors on these cells promotes the processing of CgA by plasmin. Expression of plasminogen and t-PA binding sites, taken together with recent results demonstrating expression of t-PA (21
) and trafficking of expressed t-PA to catecholamine storage vesicles (21
), suggest the presence of a local catecholaminergic cell plasminogen/t-PA system that participates in cell-associated neuroendocrine prohormone processing. We present a working model for this system in Figure . These interactions between CgA and plasmin(ogen) represent a heretofore unrecognized relationship between catecholaminergic and fibrinolytic pathways and a novel autocrine/paracrine system that may have a dramatic impact upon catecholamine secretion. In addition, CgA and t-PA are expressed together in a variety of neuronal and neuroendocrine tissues (21
) so that these interactions between CgA and components of the fibrinolytic system may have broad implications for the processing of CgA and, perhaps, other prohormones throughout the neuroendocrine system. Also, these results demonstrating an interaction between CgA and plasminogen/t-PA may suggest a new paradigm for prohormone processing in the neuroendocrine system, one consisting of coordinated coexpression, cotrafficking (to the secretory vesicle), and cosecretion of a neuroendocrine prohormone substrate (CgA), along with the activator (t-PA) of the protease system (that is locally concentrated by specific cell-surface binding sites) required for processing in the extracellular space after secretagogue-mediated exocytotic secretion.
Figure 10 Proposed working model for a local (autocrine/paracrine) chromaffin cell plasminogen/plasmin system and its interactions with CgA. Upon stimulation of the chromaffin cell by a secretagogue, CgA and catecholamines (Cats) are coreleased by exocytosis. Plasminogen (more ...)