We investigated the role of ABT treatment in

We investigated the role of ABT 702 treatment in MAPKinase activation in mouse microglia in vitro. In Fig. 7D immunocytochemistry results revealed that Iba1 and p-ERK1/2 co-localized in LPS induced mouse microglia. In our previous study we have reported that LPS activates ERK1/2 phosphorylation (Ahmad et al., 2013). Using mouse microglia Nifuroxazide receptor we have shown that LPS treatment induces pERK1/2 and p-P38 MAPKinase activation, which leads increased TNF-α release. Activation of MAPKinase has been demonstrated as a major signaling cascade for TNF-α production in microglia (Ajizian et al., 1999). In this study, data shows that AKI treatment reduced the phosphorylation of ERK and P38 in microglia cells. This result was an agreement with our previous finding where A2AAR agonist attenuated increased TNF-α release in activated microglial cells through MAP Kinase pathway (Ahmad et al., 2013). In conclusion we may demonstrate that inhibition of adenosine kinase attenuates TON induced inflammation and neurotoxicity by stimulating adenosine signaling and inhibiting MAPKinase pathway in activated retinal microglia cells (Fig. 8).
Disclosure
Acknowledgments This work has been supported by the U.S. Department of Defense (Grant number-DM102155) and Vision Discovery Institute (VDI) at GRU, Augusta, GA, USA to GIL.
Introduction Adenosine kinase (AK) catalyzes the phosphorylation of adenosine (Ado) to adenosine monophosphate (AMP) using the γ-phosphate of the co-substrate Mg/ATP−2. AK belongs to the phosphofructokinase B type (PfkB) family of sugar kinases, which includes many proteins such as ribokinase (RK), inosine-guanosine kinase, fructokinase, and 1-phosphofructokinase [1], [2], [3]. Recent studies on AK have focused on designing inhibitors, particularly of the human and parasitic protozoan forms. In humans, it has been proposed that inhibitors of AK could produce various pharmacological effects by increasing intravascular Ado concentrations and act as anti-inflammatory agents [3], [4]. In parasitic protozoa (e.g. plasmodium, toxoplasma, leishmania and trypanosoma), which lack the ability to synthesize purine nucleotides de novo, it has been suggested that AK inhibitors could attenuate their growth by blocking the purine salvage pathway [5], [6]. Until recently, the AK activity and genes were only known to be present in eukaryotic organisms [3]. However, Long et al. [7] have recently reported identification of an enzyme from Mycobacterium tuberculosis (Accession no. P83374) that corresponds to the only known bacterial AK. The identification of AK activity in M. tuberculosis is of much interest because AK can permit the intracellular conversion of nucleoside analogs to toxic anti-metabolites and thus could prove useful in treating multidrug-resistant tuberculosis. The identification of a protein that carried out AK function in M. tuberculosis (referred to here as MTub-AK) was of much importance to us due to our long-standing interest in AK and related enzymes [3]. We have previously carried out extensive studies on the effects of adenosine analogs on mammalian cells, and many different kinds of mutants affected in AK were isolated and characterized [8]. We were also among the first to clone and characterize the AK cDNA as well as its gene structure from mammalian species [2], [9], [10]. Our biochemical studies on AK have led to the discovery of phosphate or pentavalent ion dependency, a property shared by eukaryotic AKs and also other enzymes of the PfkB family [3], [11], [12]. In the present work, we have further characterized MTub-AK and compared its properties with mammalian AKs, both in vitro and in a cellular setting. The results of our studies show that MTub-AK behaves very differently from mammalian AKs in various respects and it is unable to metabolize various adenosine analogs in cellular systems. These results raise important concerns regarding the functioning of this protein as AK in cells.
Materials and methods