Tissue Plasminogen Activator (t-PA) Is Targeted to the Regulated Secretory Pathway

Tissue-type plasminogen activator (t-PA) is a serine protease that plays a central role in the regulation of intravascular thrombolysis. The acute release of t-PA in vivo is induced by a variety of stimuli including exercise, trauma, and neural stimulation. These types of stimuli also result in sympathoadrenal activation and exocytotic release of amines and proteins from catecholamine storage vesicles of the adrenal medulla and sympathetic neurons. Therefore, we tested the hypothesis that t-PA is packaged in and released directly from catecholamine storage vesicles, using several chromaffin cell sources including the rat pheochromocytoma PC-12 chromaffin cell line, primary cultures of bovine adrenal chromaffin cells, and human pheochromocytoma. t-PA was expressed in chromaffin cells as detected by Northern blotting, immunoprecipitation of 35SMet-labeled t-PA, and specific t-PA enzyme-linked immunosorbent assay of cell homogenates. In addition, chromaffin cell t-PA was enzymatically active by fibrin zymography. To explore the subcellular localization of the expressed t-PA, PC-12 cells were labeled with 3Hnorepinephrine, homogenized, and subjected to sucrose density fractionation. 3HNorepinephrine and t-PA antigen were co-localized to the same subcellular fraction with a major peak at 1.4 M sucrose, consistent with the buoyant density of catecholamine storage vesicles. In addition, catecholamine storage vesicle lysates isolated from human pheochromocytoma tumors were enriched approximately 30-fold in t-PA antigen, compared with tumor homogenate. Furthermore, exposure of PC-12 cells or primary bovine adrenal chromaffin cells to chromaffin cell secretagogues (60 microM nicotine, 55 mM KCl, or 2 mM BaCl2) resulted in co-release of t-PA in parallel with catecholamines. These data demonstrate that t-PA is expressed in chromaffin cells, is sorted into the regulated pathway of secretion, and is co-released with catecholamines by chromaffin cell stimulation. Catecholamine storage vesicles may be an important reservoir and sympathoadrenal activation an important physiologic mechanism for the rapid release of t-PA. In addition, expression of t-PA by chromaffin cells suggests a role for this protease in the proteolytic processing of chromaffin cell proteins.