Hammond, James R.

Abstract Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory spondyloarthropathy, characterized by ectopic calcification of spinal tissues. Symptoms include spine pain and stiffness, and in severe cases dysphagia and spinal cord compression. The etiology of DISH is unknown and there are no specific treatments. Recent studies have suggested a role for purine metabolism in the regulation of biomineralization. Equilibrative nucleoside transporter 1 (ENT1) transfers hydrophilic nucleosides, such as adenosine, across the plasma membrane. In mice lacking ENT1, we observed the development of calcified lesions resembling DISH. By 12 months of age, ENT1–/– mice exhibited signs of spine stiffness, hind limb dysfunction, and paralysis. Micro–computed tomography (µCT) revealed ectopic mineralization of paraspinal tissues in the cervical-thoracic region at 2 months of age, which extended to the lumbar and caudal regions with advancing age. Energy-dispersive X-ray microanalysis of lesions revealed a high content of calcium and phosphorus with a ratio similar to that of cortical bone. At 12 months of age, histological examination of ENT1–/– mice revealed large, irregular accumulations of eosinophilic material in paraspinal ligaments and entheses, intervertebral discs, and sternocostal articulations. There was no evidence of mineralization in appendicular joints or blood vessels, indicating specificity for the axial skeleton. Plasma adenosine levels were significantly greater in ENT1–/– mice than in wild-type, consistent with loss of ENT1—a primary adenosine uptake pathway. There was a significant reduction in the expression of Enpp1, Ank, and Alpl in intervertebral discs from ENT1–/– mice compared to wild-type mice. Elevated plasma levels of inorganic pyrophosphate in ENT1–/– mice indicated generalized disruption of pyrophosphate homeostasis. This is the first report of a role for ENT1 in regulating the calcification of soft tissues. Moreover, ENT1–/– mice may be a useful model for investigating pathogenesis and evaluating therapeutics for the prevention of mineralization in DISH and related disorders. © 2013 American Society for Bone and Mineral Research.

Nucleoside and nucleobase uptake is integral to mammalian cell function, and its disruption has significant effects on the cardiovasculature. The predominant transporters in this regard are the equilibrative nucleoside transporter subtypes 1 (ENT1) and 2 (ENT2). To examine the role of ENT1 in more detail, we have assessed the mechanisms by which microvascular endothelial cells (MVECs) from ENT1−/−mice transport and metabolize nucleosides and nucleobases. Wild-type murine MVECs express mainly the ENT1 subtype with only trace levels of ENT2. These cells also have a Na+-independent equilibrative nucleobase transport mechanism for hypoxanthine (ENBT1). In the ENT1−/−cells, there is no change in ENT2 or ENBT1, resulting in a very low level of nucleoside uptake in these cells, but a high capacity for nucleobase accumulation. Whereas there were no significant changes in nucleoside transporter subtype expression, there was a dramatic increase in adenosine deaminase and adenosine A2areceptors (both transcript and protein) in the ENT1−/−tissues compared with WT. These changes in adenosine deaminase and A2areceptors likely reflect adaptive cellular mechanisms in response to reduced adenosine flux across the membranes of ENT1−/−cells. Our study also revealed that mouse MVECs have a nucleoside/nucleobase transport profile that is more similar to human MVECs than to rat MVECs. Thus mouse MVECs from transgenic animals may prove to be a useful preclinical model for studies of the effects of purine metabolite modifiers on vascular function.

To better understand the role of equilibrative nucleoside transporters (ENT) in purine nucleoside-dependent physiology of the cardiovascular system, we investigated whether the ENT1-null mouse heart was cardioprotected in response to ischemia (coronary occlusion for 30 min followed by reperfusion for 2 h). We observed that ENT1-null mouse hearts showed significantly less myocardial infarction compared with wild-type littermates. We confirmed that isolated wild-type adult mouse cardiomyocytes express predominantly ENT1, which is primarily responsible for purine nucleoside uptake in these cells. However, ENT1-null cardiomyocytes exhibit severely impaired nucleoside transport and lack ENT1 transcript and protein expression. Adenosine receptor expression profiles and expression levels of ENT2, ENT3, and ENT4 were similar in cardiomyocytes isolated from ENT1-null adult mice compared with cardiomyocytes isolated from wild-type littermates. Moreover, small interfering RNA knockdown of ENT1 in the cardiomyocyte cell line, HL-1, mimics findings in ENT1-null cardiomyocytes. Taken together, our data demonstrate that ENT1 plays an essential role in cardioprotection, most likely due to its effects in modulating purine nucleoside-dependent signaling and that the ENT1-null mouse is a powerful model system for the study of the role of ENTs in the physiology of the cardiomyocyte.