Targeting of tissue plasminogen activator into the regulated secretory pathway of neuroendocrine cells

Plasma levels of tissue plasminogen activator (tPA) increase rapidly in res ponse to specific vasoactive agents, trauma, and neural stimulation. This r esponse has been attributed to acute release of tPA from stored pools withi n the vascular endothelium and from catecholamine storage vesicles of chrom affin cells. We have tested directly whether tPA can be sorted into the reg ulated secretory pathway using the murine pituitary-derived neuroendocrine cell line AtT-20 transfected with tPA cDNA. Clones of AtT-20 cells expressi ng tPA were isolated, and targeting of tPA into the regulated secretory pat hway was demonstrated by (1) stimulation of tPA secretion with 8-bromo-cAMP , the secretagogue which promotes the release of dense granule contents; (2 ) colocalization with ACTH, an endogenous protein that is stored in dense c ore granules; and (3) retention of newly synthesized tPA in the cell for pr olonged periods of time. Laser scanning confocal microscopy analysis of cel ls immunostained with antibodies to tPA and ACTH showed colocalization at t he tips of the neuritic processes under the cytoplasmic membrane, a region where dense granules are known to migrate after maturation. Treatment of th e cells with 5 mM 8-bromo-cAMP for 30 min resulted in a 2.41 +/- 0.36-fold increase in tPA secretion. Both the magnitude of the stimulatory effect and the fraction of the intracellular tPA released were the same regardless of the tPA expression level in the various clones. Pulse-chase experiments sh owed that a portion of newly synthesized tPA is retained in the cell for at least 4 h and is released into the culture medium in response to 8-bromo-c AMP. These studies indicate that tPA, under the appropriate conditions, can be targeted into the regulated secretory pathway and can be stored for lat er release by cellular stimuli. (C) 1999 Elsevier Science B.V. All rights r eserved.