Endocrine Abstracts (2006) 11 P632

Processing and sorting of pro-opiomelanocortin is an important checkpoint in regulating release of ACTH from secretory vesicles in pituitary cells

A Warhurst1, RL Oliver1, RA Davies2, LE Pritchard2 & A White1

1University of Manchester, Manchester, United Kingdom; 2AstraZeneca, Alderley Park, United Kingdom.

Pro-opiomelanocortin (POMC) is endoproteolysed by prohormone convertase-1 (PC1) to ACTH within the secretory pathway in pituitary cells, where the regulation of ACTH release is essential for mediating the stress response. However POMC is present in the human circulation, indicating that not all POMC is processed. This suggests that regulation of trafficking and processing of POMC are important in determining ACTH release. To investigate this, we analysed steady-state release of POMC and ACTH from AtT20 pituitary adenoma cells utilising specific immunometric assays.

In cell lysates, we detected high levels of POMC (110 pmol/well) relative to ACTH (13 pmol/well), which remained constant over 48 h. In medium, we observed greater accumulation of POMC (24–1180 pmol/well) relative to ACTH (2–74 pmol/well) from 1–48 h, suggesting that excess POMC is constitutively secreted.

Stimulation of cells with BaCl2 (1 mM) caused an acute 5 fold increase in ACTH secretion within 15 min while POMC levels did not change. Repeated stimulation with BaCl2 at 15 min intervals for 60 min exhausted the secretion of ACTH, but release of POMC was not altered. Similarly, acute stimulation of cells with CRH (3 nM) increased ACTH, but not POMC release. Hydrocortisone (10–1000 ) inhibited POMC and ACTH secretion but only after 24 h. These results show that the POMC sorted to secretory granules, is processed for dynamic release of ACTH, but that most POMC secretion occurs constitutively.

Stable over-expression of PC1 in cells produced a marked increase in ACTH secretion after BaCl2 stimulation, suggesting that PC1 upregulation enhances post-Golgi sorting of POMC. Conversely, RNAi knockdown of PC1 resulted in a decrease in ACTH with a concomitant increase in POMC, proving that regulation of processing enzymes can impact on the secretory process.

In summary, PC1 may play a regulatory role in the sorting process and the mechanisms for sorting POMC into secretory pathways appear to provide key checkpoints to regulate secretion of ACTH.

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