br Materials and methods br
Materials and methods
Discussion PKA and Epac are two principal effector proteins involved in cAMP signaling; they are expressed in a wide range of tissues and control diverse biological functions . The existence of two cAMP effectors provides more precise regulation of cAMP signaling pathways in different spatiotemporal manners. Recently, Epac and PKA were suggested to play independent, synergistic or opposite roles in a specific cellular function. For example, hyperoxia-induced BDNF (brain-derived neurotrophic factor) secretion in developing airway smooth muscle was found to be Epac2-dependent, whereas PKA inhibition does not influence this process . PKA exclusively mediates the inhibitory effects of prostaglandin E2 (PGE2) on collagen I expression; however, Epac1 exclusively inhibits fibroblast proliferation . Moreover, endothelial nitric oxide (NO) release mediates vascular relaxation through the synergistic activation of PKA and Epac . As shown in our study, LPS induces lower Epac1 and Epac2 expression but has no impact on PKA. Cigarette smoke extract (CSE) decreases Epac1 expression but does not affect Epac2 and PKA expression , . Findings from these previous studies were consistent with the results from our study and suggest that reduced Epac1 expression might be the common phenomenon in inflammation. Epac and PKA also exert different effects on LPS-induced inflammation. LPS-stimulated NO production in murine BV2 microglial Evodiamine mg has been shown to primarily depend on PKA because Epac1-mediated Rap1 activation is not required for the induction of NO production . Although 8CPT increases NF-κB activity in LPS-treated RAW 264.7 murine macrophages, NF-κB is also activated by cAMP production through Epac1-mediated Rap1 activation . Activation of Epac with 8CPT didn’t inhibit LPS-induced increases in TNF-α mRNA levels in J774 and RAW 264.7 macrophages, whereas the activation of PKAI with 6BnZ inhibited by 50% . However, conflicting evidence has also been reported. In CSE-induced inflammation in human airway smooth muscle (ASM) cells, 8CPT prevents CSE-induced p65 nuclear translocation and subsequently decreases CSE-induced IL-8 release . Our study provides the first indication that 8CPT inhibits TNF-α release from MH-S cell (Fig. 1A) and the expression of cytokine mRNAs in LPS-induced lungs (Fig. 3). The results obtained with the co-culture system further confirm the effects of 8CPT on epithelial cells and macrophages. Based on the animal experiments, 8CPT exerts an anti-inflammatory effect on LPS-induced lung injury, significantly decreases the number of white blood cells (WBC) in the BALF (Fig. 2C), decreases MPO activity (Fig. 2D), up-regulates the LPS-induced down-regulation of Epac1 expression in whole lung (Fig. 5), and inhibits activation of small GTPases, such as Rac (Fig. 6), but does not affect the MAPK pathway (Fig. 5). Although we did not determine the precise mechanism governing the effects of 8CPT on LPS-induced ALI, our data indicate that PKA activation does not decrease the levels of pro-inflammatory mediators after 6 h of LPS stimulation (Fig. 1A) but did have an effect on endothelial permeability (Fig. 1B). Moreover, in the present study, we did not assess the in vivo effect of the Epac inhibitor ESI-09. However, according to a recent report, ESI-09 inhibits complete Freund’s adjuvant-induced mechanical hyperalgesia , further suggesting that an Epac inhibitor might be a potential therapeutic target for other diseases. All these conclusions depend on pharmacological activators and inhibitors of Epac and PKA. Nonetheless, the drugs used in this study are not perfect. Some questions have been raised regarding these activators/inhibitors. 8CPT is a strong Epac activator and is even stronger than cAMP, but it exhibits weak activation and lower affinity for PKA. Simultaneously, 8CPT is also a good substrate for both PDE5 and PDE10 and can act as an inhibitor of PDE1, 2 and 6 , . The inhibitor of Epac ESI-09 has been shown to act as a chemical with general protein-denaturing properties and does not selectively inhibit Epac . H-89 might inhibit other kinases in addition to PKA ; 6BnZ is a preferential PKA activator and is the most PKA-selective and PDE-resistant cAMP analogue . We have shown a lack of an anti-inflammatory effect on the PKA-dependent pathway, but this compound is likely to have effects on other pathway.