1. The Mechanisms Of Priming Of Peritoneal Macrophages By RU486 Injection In Rat 2. The Mechanisms Of Increased Expression Of Protein Kinase Cα By Glucocorticoids

Our laboratory has long been interested in glucocorticoid receptor (GR) function in severe stress and shock. Xu et al have reported that the binding capacity of GR was decreased after stress, systemetic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). In addition, GR blockade rats (injected with GR antagonist RU486) were more sensitive to endotoxemia or hemorrhage and the pathological changes were exacerbated. Based on these observations, Fan and Xu proposed that the decrease of GR may be involved in the pathogenesis of shock, SIRS and MODS. In present work, we hypothesized that leukocytes may be primed by injection of RU486. In order to validate this working hypothesis, we firstly studied the changes of basal and LPS induced nitric oxide (NO) in the peritoneal macrophages from RU486 injected rats. The results showed that both basal and LPS induced NO production were significantly higher in peritoneal macrophages from RU486 injected rats than control. It was verified that RU486 injection resulted in macrophage priming.As the mechanisms of priming are so complicated that many possibilities including the changes of G protein coupled receptor, phospholipidase D, protein kianase C and intracellular Ca2+ have been considered. Our previous work hasindicated that PKCa and [Ca2+]i play important roles in priming of leukocytes, so in the present work we investigated the changes of [Ca2+]i and PKCa in macrophages from RU486 injected rats. The results showed that both [Ca2+]i and PKCa were significantly higher in peritoneal macrophages from RU486 injected rats as compared with control rats suggesting the involvement of [Ca2+]i and PKCa associated signal pathways in RU486 induced priming of macrophages.Since plasma corticosterone is also elevated except the blockade of GR by RU486 injection, the role of each in macrophage priming should be defined. In order to prevent the elevation of plasma corticosterone, unilateral adrenalectomy (Adxl, left adrenals were removed) was carried out. Rats were divided into three groups: Control, RU486 injection(RU), RU486 injected to Adxl (RU+Adxl) rats. The results showed that NO production and [Ca2+]i decreased significantly in RU+Adx group as compared with RU group but were still much high than control rats. In contrast, the level of PKCa. was decreased to the level of the control rats, i.e. the increase of PKCa. in RU group may be reversed completely by Adxl. Taken together, these results indicated that the elevation of GC plays an important role in RU486 injection induced macrophage priming. Furthermore, these indexes still increased by GC in spite of GR was blocked about 90% (as demonstrated in our experiments) by injection of RU486, which suggested that GC might function via GR independent pathway.Considering that adrenal glands secret many hormones other than GC, adrenalectomy also affected the serum concentration of these hormones, so we investigated the effects of GR blockade and GC on NO production, [Ca2+]i and PKCa in vitro in murine macrophage cell line RAW264.7. The following results were obtained: 瓽C inhibited NO production in RAW264.7 cells which was reversed completely by RU486 and not affected by the concentrations of GC at the same concentration of RU486. This results indicated unequivocally that the inhibitory action of NO production by GC is GR-mediated exclusively. These results also suggested that in the physiological state, the NO production in macrophages is inhibited by the physiological concentration of plasma GC while GR blockade inhibited this effect of GC and resulted in the increase of NO production andmacrophage priming in vivo. Based on these in vitro results, the decrease of NO production in RU+Adxl rats as compared with the RU rats may be due to the changes of some factors other than GC in Adxl rats. 〥examethasone (Dex) increased [Ca2+]i significantly in RAW264.7 cells while RU486 only partly reversed the effect. (DDex and corticosterone (B) could increase PKCa in RAW264.7 cells, which was not blocked by R...