However, the expression levels of ENT1 and ENT2 were consistent with studies of murine Ent1 and Ent2 expression in a model of partial hepatic ischemia and reperfusion (Fig. 2A). Indeed, murine
Ent1 and Ent2 transcript and protein levels were repressed following 45 minutes of liver ischemia and 2 hours reperfusion (Fig. 2B,C). Together, these studies demonstrate that hepatic ENT1 and ENT2 transcript levels are repressed during conditions of limited oxygen availability, indicating the likelihood of a transcriptional regulated endogenous protective pathway directed towards enhancing extracellular adenosine levels and signaling during liver ischemia and reperfusion injury. After having shown that ENT1 and ENT2 are transcriptionally regulated during
liver ischemia and reperfusion, as occurs during human liver transplantation, we next pursued GDC-0068 solubility dmso studies to address check details their functional contributions to the regulation of extracellular adenosine levels and outcomes of hepatic ischemia and reperfusion injury. For this purpose, we exposed mice to 45 minutes of partial hepatic ischemia and 2 hours of reperfusion. In order to address the functional role of ENTs, we pretreated the experimental animals with intravenous dipyridamole (0.5 mg/25g mouse intravenously) 15 minutes prior to the onset of liver ischemia (Fig. 3A). Indeed, studies using high-performance liquid chromatography (HPLC) to measure hepatic adenosine levels in ischemic livers that were shock-frozen immediately following 45 minutes of hepatic triad occlusion revealed elevations of adenosine
levels following ischemia. Importantly, these elevations of adenosine were further enhanced in mice pretreated with dipyridamole (Fig. 3B). Subsequent functional studies of the clinical Pregnenolone outcome of hepatic ischemia and reperfusion injury revealed that mice pretreated with dipyridamole experienced attenuated plasma levels of AST and ALT and a less severe degree of hepatic tissue injury 2 hours (Fig. 3C,D) and 24 hours (Fig. 3E,F) following hepatic ischemia, indicating a protective role of dipyridamole in liver ischemia and reperfusion. Together, these studies demonstrate that ENT inhibition with dipyridamole is associated with liver protection from ischemia and reperfusion injury by way of enhancing hepatic adenosine levels and signaling events. After having shown that nonspecific inhibition of ENTs with dipyridamole is associated with elevated hepatic adenosine levels and concomitant liver protection from ischemia, we next pursued studies to address the functional contributions of Ent1 versus Ent2. For this purpose we exposed previously described mice for Ent1 or Ent2 to liver ischemia,[13, 19] and measured hepatic adenosine levels and assessed liver injury.