Figure 5.

Detection and real-time monitoring of ER stress during endotoxemia using ES-TRAP. (A) Effect of systemic ER stress on the level of serum SEAP in ex vivo gene transferred mice. Hepa1/SEAP cells (2.5 × 10⁵ cells) were implanted into the peritoneal cavity of C57BL/6 mice. After 24–48 h, thapsigargin (1 mg/kg body weight) was administered into eight mice. Serum was sampled at 2 and 24 h, and activity of SEAP was evaluated. (B) Activity of SEAP in organs of transgenic ES-TRAP mice. Tissue extracts were prepared from perfused organs of wild-type mice (WT) and ES-TRAP mice and subjected to chemiluminescent assay, as described in Materials and Methods. Activity of SEAP per 1 µg total protein was evaluated and compared among different organs. (C) Effect of systemic ER stress on the level of serum SEAP in ES-TRAP mice. Thapsigargin (1 mg/kg body weight) was administered into four mice (body weight 25 g) intraperitoneally, and serum was sampled at 8 and 24 h. (D) Induction of ER stress by LPS in macrophages. NR8383 alveolar macrophages were exposed to LPS (1 µg/ml) for 3–6 h, and expression of grp78 was examined by northern blot analysis. (E–G) Effects of endotoxemia in ES-TRAP mice on induction of ER stress in various organs (E), serum SEAP activity (F) and tissue SEAP activity in the spleen (G). ES-TRAP mice (body weight 20 g, n = 8) were administered without (−) or with (+) LPS (200 µg/mouse) intraperitoneally, and organs (8 h) and serum (2–48 h) were collected for northern blot analysis of grp78 (E) and SEAP assay (F and G). In (G), activity of SEAP in the spleen before [LPS(−)] and 8 h after the administration with LPS [LPS(+)] is shown. (H and I) Lack of in vivo response of SEAP to hyperthermia. ES-TRAP mice (body weight 22 g, n = 4) were heated at 42°C for 15 min, and brains, lungs and livers were collected before or 2 and 6 h after the whole body hyperthermia. Expression of hsp70 and grp78 was examined by northern blot analysis (H). Serum was also sampled every 2 h until 6 h and subjected to chemiluminescent assay to evaluate activity of SEAP (I). Data are expressed as means ± SE. Asterisks indicate statistically significant differences (P < 0.05). N.S., not significant.