Stress triggered changes in gene expression in adrenal medulla: transcriptional responses to acute and chronic stress

Abstract

1. Stress elicits activation of several transcription factors involved in the regulation of catecholamine biosynthetic enzyme gene expression depending on its duration or repetition. However, the dynamic of the conversion of transient transcriptional activation with acute stress to sustained changes in transcription in response to repeated exposure to stress in adrenomedullary catecholaminergic systems is not clear. 2. Here, we analyzed changes in levels of phospho-CREB (P-CREB), phospho-ERK1/2 (P-ERK1/2) and Fra-2 by Western Blot analysis in adrenal medulla of Sprague Dawley male rats exposed to single or repeated immobilization stress (IMO). For single stress, rats were immobilized for 5 min, 30 min, or 2 h and sacrificed immediately afterwards. In the repeated stress conditions, animals were immobilized for 2 h daily on each consecutive day prior to the final day (day 2 for 2x IMO, day 6 for 6x IMO) in which the rats were immobilized for a session lasting 5 min, 30 min or 2 h. There were two control groups, an absolute control (AC) not exposed to stress, and a handled control (HC) gently handled daily for 6 days. 3. Phosphorylation of CREB was rapid and elevated at the earliest time examined, even with single stress. However, with a second daily episode of stress the increase in P-CREB was observed for at least the entire duration of the stress. In contrast, phosphorylation of ERK1/2 was only significant after brief exposure to a single IMO. The elevation of Fra-2 protein level was slower, but was significant after 2 h of a single IMO. With repeated IMO, there were marked elevations of Fra-2 throughout the 2 h IMO, which were especially pronounced at the end of the immobilization. 4. The transient nature of the phosphorylation of CREB may be responsible for the short-lived induction of transcription of catecholamine biosynthetic enzymes after brief exposure to a single immobilization stress. The sustained phosphorylation of CREB throughout the repeated stress coupled with induction of Fra-2 may mediate the longer lasting responses to repeated stress.

Fig. 1.

Changes in phosphorylation of CREB in the adrenal medulla with different durations and repetition of immobilization stress. Representative immunoblots of IMO-induced changes in levels of P-CREB protein are shown on the upper panel. Blots were stripped and re-probed for β-actin, which was used as a loading control. Summary data of the experiments are shown on the lower panel. ^* p < 0.05 vs. absolute control (AC), ^§ p < 0.05 vs. 1× IMO, ^# p < 0.05 vs. handled control (HC).

Fig. 2.

Changes in phosphorylation of ERK1/2 in the adrenal medulla with different durations and repetition of immobilization stress. Representative immunoblots of IMO-induced changes in levels of P-ERK1/2 protein are shown on the upper panel. Summary data of the experiments are shown on the lower panel. ^* p < 0.05 vs. absolute control (AC).

Fig. 3.

Changes in phosphorylation of Fra-2 in the adrenal medulla with different durations and repetition of immobilization stress. Representative immunoblots of IMO-induced changes in levels of Fra-2 protein are shown on the upper panel. The two bands were previously shown by phosphatase treatment to be specific for Fra-2 and differ only in phosphorylation (Liu et al., 2005) so both were combined for the summary data of the experiments shown on the lower panel ^* p < 0.05 vs. absolute control (AC), ^§ p < 0.05 vs. 2 h 6× IMO.