Evidence for regulation of a thyrotropin-releasing hormone degradation pathway in GH3 cells

GH3 cells, cloned from a rat anterior pituitary tumor, synthesize and secrete PRL in response to TRH. One of the pathways of TRH degradation is removal of the N-terminal pyroglutamyl residue catalyzed by pyroglutamyl peptide hydrolase (PPH; EC 3.4.11.8). We recently described the synthesis and properties of 5-oxoprolinal, a specific and potent (Ki = 26 nM) inhibitor of PPH. The effect of long term exposure of GH3 cells to 5-oxoprolinal on PPH activity was studied by incubating cells with inhibitor for 3 days, harvesting, washing to remove inhibitor, and assaying for PPH. Unexpectedly, we found a marked (300%) increase in PPH activity. This effect was dependent on the concentration of 5-oxoprolinal (EC50 = 10(-7) M) and was time dependent, with a rapid increase in enzyme activity occurring during the first 24 h. Cycloheximide did not block the increase. The results suggest that the activity of PPH in GH3 cells is subject to complex regulatory mechanisms.