After activation AKT phosphorylates target
After activation, AKT phosphorylates target proteins involved in cell growth, metabolism and survival (Manning and Cantley, 2007). For example, AKT phosphorylates the pro-apoptotic protein BAD, preventing it from binding to and inactivating Bcl-xL in mitochondria (Datta et al., 1997). In turn, Bcl-xL exerts its anti-apoptotic effect and contributes to cell survival. In cerebral ischemia, BAD has been shown to be a key molecule regulating the balance between cell survival and death signals (Kamada et al., 2007).
Materials and methods
Discussion Another finding of the present study is that EP1 receptor inhibition increases AKT phosphorylation even in normal conditions (Fig. 2A, lane 2). One possible explanation for this finding is that there is a constitutive interaction between EP1 receptors and the PTEN/AKT pathway and that EP1 receptors negatively regulate AKT phosphorylation. Inhibition of EP1 receptors interferes with this negative interaction, leading to an increase in AKT phosphorylation. Thus, EP1 inhibition could potentiate the AKT pathway and prepare the caudatin to better withstand stressful events. Once activated, AKT phosphorylates several downstream proteins involved in cell metabolism and cell fate (Manning and Cantley, 2007). One of the well known targets is the Bcl-2 family member BAD. BAD is a BH3 domain protein and it exerts its pro-apoptotic activity by binding and neutralizing the function of anti-apoptotic BCl-xL (Yang et al., 1995). In normal conditions, BAD is phosphorylated at serine-136 by p-AKT (Datta et al., 1997). Serine-136 phosphorylation makes it possible for BAD to bind to 14-3-3 proteins and to be sequestered in the cytosol (Datta et al., 2000). Following cell stress, the loss of AKT activity leads to BAD dephosphorylation and translocation to mitochondria, where it binds to BCl-xL. The reduced availability of BCL-xL leads to the formation of BAX homodimers and to activation of the mitochondrial cell death pathway (Zha et al., 1996). Our data demonstrate that OGD causes BAD protein translocation to mitochondria, an effect prevented in part by inhibition of EP1 receptors leading to improved cell survival. Thus, the neuroprotection mediated by EP1 receptor inhibition in hippocampal slices involves the activation of the AKT pathway, which in turn prevents the translocation of BAD into mitochondria. We observed that the PGE2 analog 17-pt-PGE2 is not neurotoxic. This phenomenon was also observed in our earlier study in which 17-pt-PGE2 did not cause brain injury in vivo (Manabe et al., 2004). Similarly, in neuronal cell cultures, 17-pt-PGE2 did not induce cell death unless NMDA was added to the culture (Carlson, 2003). Therefore, PGE2 is toxic only in the setting of NMDA receptor activation. One possible explanation for this finding is that, because PGE2 alters Ca2+ homeostasis via effects of EP1 receptors on the Na+/Ca2+ exchanger (Kawano et al., 2006), the deleterious actions of this prostaglandin are not expressed unless the Na+/Ca2+ exchanger is activated during excitotoxicity. Based on the observations that EP1 receptors are predominantly present in neurons (Kawano et al., 2006) and that OGD-induced cell death in CA1 region involves neurons, we would anticipate that the effect of SC51089 is mainly neuronal. However, interactions with other cells cannot be ruled out at this time. Further studies are needed to identify the cell type involved in the effect of SC51089 in brain slices.
PGE is a major contributor to exaggerated pain sensitivity during inflammation. Its production requires the activity of at least one of the two cyclooxygenase (COX) isoforms, constitutively expressed COX-1 or inducible COX-2, with COX-2 being particularly relevant for inflammation-induced PGE formation , . Most classical non-steroidal anti-inflammatory drugs (NSAIDs) block COX-1 and COX-2 to similar degrees, while the more recently developed “coxibs” are COX-2-selective. Although these drugs often provide excellent relief from inflammatory pain, in particular their long-term use is frequently associated with side effects. Traditional NSAIDs cause upper gastrointestinal tract ulcerations , and the use of COX-2-selective inhibitors is associated with an increased risk of cardiovascular events .