Equilibrative nucleoside transporter (ENT)-1-dependent elevation of extracellular adenosine protects the liver during ischemia and reperfusion.

UNLABELLED: Ischemia and reperfusion-elicited tissue injury contributes to morbidity and mortality of hepatic surgery and during liver transplantation. Previous studies implicated extracellular adenosine signaling in liver protection. Based on the notion that extracellular adenosine signaling is terminated by uptake from the extracellular towards the intracellular compartment by way of equilibrative nucleoside transporters (ENTs), we hypothesized a functional role of ENTs in liver protection from ischemia. During orthotopic liver transplantation in humans, we observed higher expressional levels of ENT1 than ENT2, in conjunction with repression of ENT1 and ENT2 transcript and protein levels following warm ischemia and reperfusion. Treatment with the pharmacologic ENT inhibitor dipyridamole revealed elevations of hepatic adenosine levels and robust liver protection in a murine model of liver ischemia and reperfusion. Studies in gene-targeted mice for Ent1 or Ent2 demonstrated selective protection from liver injury in Ent1(-/-) mice. Treatment with selective adenosine receptor antagonists indicated a contribution of Adora2b receptor signaling in ENT-dependent liver protection. CONCLUSION: These findings implicate ENT1 in liver protection from ischemia and reperfusion injury and suggest ENT inhibitors may be of benefit in the prevention or treatment of ischemic liver injury.

Human equilibrative nucleoside transporters 1 and 2 may be differentially modulated by A2B adenosine receptors in placenta microvascular endothelial cells from pre-eclampsia.

Pre-eclampsia is associated with elevated maternal blood pressure and proteinuria, altered fetal growth, and increased plasma adenosine concentration in the mother and the fetus. Human equilibrative nucleoside transporters 1 (hENT1) and hENT2 are crucial to maintain physiological plasma levels of adenosine, thus modulating its several biological effects through adenosine receptor activation. However, it is unknown whether hENTs and adenosine receptors are expressed in human placental microvascular endothelium (hPMEC). To assay whether the increased fetal plasma adenosine concentration in pre-eclampsia results from altered hENT-mediated transport, and the potential involvement of adenosine receptors in this phenomenon, we investigated hENTs and A2A and A2B adenosine receptors expression and function in hPMEC. Cells were isolated and cultured from normal pregnancies (n=17) or pre-eclampsia with adequate-for-gestational age fetuses (n=7). hENT1, hENT2, A2A and A2B adenosine receptors were expressed and functional in hPMEC. Extracellular adenosine concentration was higher (4-fold) in pre-eclampsia versus normal pregnancies. hPMEC from pre-eclampsia exhibit increased total transport (hENT1+hENT2), and maximal velocity (Vmax) for hENT2- (2-fold), but reduced Vmax for hENT1-mediated adenosine transport (75%), with no changes in apparent Km. hENT2 expression was increased (4.5-fold), but hENT1 protein abundance was reduced (80%) in pre-eclampsia. Equally, A2A expression was reduced (50-80%) in pre-eclampsia. CGS-21680 (A2A agonist) did not alter hENTs expression or activity, but ZM-241385 (A2A antagonist) blocked pre-eclampsia effects and increased hENT1-mediated transport in normal pregnancies. Thus, A2B adenosine receptors may differentially modulate hENTs in hPMEC, which could be a mechanism attempting to re-establish physiological extracellular adenosine levels in pre-eclampsia.

Determination of adenosine effects and adenosine receptors in murine corpus cavernosum.

This study tested the hypothesis that adenosine, in murine corpora cavernosa, produces direct relaxation of smooth muscle cells and inhibition of contractile responses mediated by sympathetic nerve stimulation. Penes were excised from anesthetized male C57BL/6 mice, dissected, and cavernosal strips were mounted to record isometric force. Adenosine, 2-chloroadenosine (stable analog of adenosine), and 2-phenylaminoadenosine (CV1808) (A2(A)/A2(B) agonist) produced concentration-dependent relaxations of phenylephrine-contracted tissues. Relaxation to 2-chloroadenosine was inhibited, in a concentration-dependent manner, by 2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH58261; A2(A) antagonist; 10(-9)-10(-6) M) and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamida (MRS1706; A2(B) antagonist; 10(-8)-10(-6) M). The combination of both antagonists abrogated 2-chloroadenosine-induced relaxation. Electrical field stimulation (EFS; 1-32 Hz) of adrenergic nerves produced frequency-dependent contractions that were inhibited by compounds that increase adenosine levels, such as 5'-iodotubercidin (adenosine kinase inhibitor), erythro-9-(2-hydroxy-3-nonyl)adenine (adenosine deaminase inhibitor), and dipyridamole (inhibitor of adenosine transport). The adenosine A1 receptor agonist N(6)-cyclopentyladenosine (C8031) right-shifted contractile responses to EFS, with a significant inhibitory effect at 10(-6) M. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine (C101) (10(-7) M) enhanced contractile responses to EFS and eliminated the inhibitory effects of 5'-iodotubercidin. Dipyridamole and 5'-iodotubercidin had no effect on adenosine-mediated relaxation. In summary, adenosine directly relaxes cavernosal smooth muscle cells, by the activation of A2(A)/A2(B) receptor subtypes. In addition, adenosine negatively modulates sympathetic neurotransmission, by A1 receptor subtype activation, in murine corpora cavernosa. Adenosine may subserve dual roles in modulating the physiological mechanisms of erection in mice.