apyrase (a soluble NTPDase) in the absence of IP attenuated hepatic injury after IRI. become implicated in CD39 transcriptional rules. In fact, Sp1 siRNA treatment was associated with attenuated CD39 induction, and improved hepatic injury protection of the liver (4C8) While the underlying mechanisms of hepatic IP remain unclear, it would be highly desirable to use pharmacological means to recapitulate IP-dependent liver safety (2). Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) hydrolyzes both extracellular ATP and ADP to AMP. AMP is definitely rapidly degraded to adenosine via the ubiquitously indicated 5-ecto-nucleotidase (CD73) (9C13). Earlier studies suggest that extracellular adenosine is an important pathway for liver safety from ischemia and swelling (14C18). For example, we previously shown that extracellular adenosine production by CD73 mediates safety during murine hepatic IP (17). Additional studies recently shown the catalysis of extracellular nucleotides by CD39 is required for liver Borneol regeneration following partial hepatectomy (19). Based on the fact that extracellular AMP primarily stems from CD39-dependent ATP/ADP-phosphohydrolysis, we hypothesized a central part of CD39 in IP-mediated liver protection. To test this hypothesis, we combined pharmacological and genetic studies to address the part of CD39 with this aspect of hepatic IRI. Materials and Methods Mice All animal experiments were in accordance with German recommendations and authorized by the University or college of Tbingen, Germany. Mice deficient in CD39 (CD39?/?)(20) were compared to littermate settings matched in age, gender and excess weight (CD39+/+; WT). In some experiments, mice were treated with sodium polyoxotungstate (POM-1, Na6[H2W12O40], 3 mg/kg/h, i.a., 30 min prior to IP or IR) (21, 22), apyrase from potatoes (Sigma, 5U apyrase i.p., 30 min prior to IP or IR), AMP (100 l/h of 4 mg/kg, i.a.) (21, 22), Sp1 small interfering RNA (Sp1 siRNA, Dharmacon RNA Systems, Lafayette, CO, 2 mg/kg in transfection reagent, siPORT Amine; Ambion, Austin, TX, i.v., 24 hours prior to IP or IR),(23) or nonsense siRNA (NS siRNA, Silencer Bad Control #1 siRNA, Ambion, 2 mg/kg in transfection reagent, i.v., 24 hours prior Borneol to IP or IR). Technique of portal triad occlusion Partial hepatic ischemia was performed via portal triad occlusion with the use of a hanging-weight system as explained previously (24). Mice underwent 30 min ischemia, followed by 3 h reperfusion or IP (3 cycles of 5 min ischemia/5 min reperfusion) prior to IR (24). Sham mice underwent exposure of the portal triad without IR or IP. Real-time RT-PCR and Western blot To measure Sp1 and CD39 transcript levels, the median lobe was excised, followed by isolation of RNA and quantification Borneol of mRNA by real-time RT-PCR relative to -actin (21, 23). For western blot of Sp1, the median lobe was excised and proteins were resolved by SDS-PAGE, transferred to nitrocellulose and probed with anti-Sp1 antibody (Abcam, Cambridge, USA). Serum markers of liver injury Lactate dehydrogenase (LDH, Borneol Randox, Crumlin, UK), aspartate (AST) and alanine (ALT) aminotransferases (Teco Diagnostics, Anaheim, CA, USA) were measured using commercially available kits. Histological sections The median and remaining liver lobes were placed in OCT Tissue-Tek, frozen, sectioned and stained with H&E. Exam/rating was carried out by a pathologist blinded to the experimental group using a semi-quantitative grading level of 0C4 for histopathological assessment of liver necrosis (25): 0=no liver Rabbit Polyclonal to iNOS (phospho-Tyr151) necrosis, 1=solitary cell necrosis, 2=up to 30% lobular necrosis, 3=up to 60% lobular necrosis, and 4=more than 60% lobular necrosis. Immunohistochemical staining was performed having a polyclonal goat anti-mouse IgG antibody against CD39 (sc-33558 rabbit polyclonal IgG, Santa Cruz, Heidelberg, Germany) or using a bad control rabbit immunoglobulin portion (DakoCytomation, Glostrup, Denmark). Adenosine measurements The remaining and median liver lobes were eliminated and immediately snap freezing with clamps pre-cooled to the temp of liquid Borneol nitrogen within a time lag of 3C5 mere seconds. The frozen cells was pulverized under liquid nitrogen,.