Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Pharmacology and Toxicology

Supervisor

Dr. James Hammond

Abstract

The microvascular endothelium serves as the barrier between the blood and perfused tissues. Proper function of the endothelium is dependent on the ability of the endothelial cells to produce nitric oxide and form tight junctions between themselves. Dysfunction occurs when stresses overwhelm the endothelial cell, with oxidative stress being a leading cause. Intracellular metabolism of purine nucleosides and nucleobases has been implicated in the production of oxidative stress. Nucleosides (e.g. adenosine) and nucleobases (e.g. hypoxanthine) are moved across cell membranes by a specialized family of proteins called equilibrative nucleoside transporters (ENTs).

We characterized primary human cardiac microvascular endothelial cells (MVECs) for expression, function, and regulation of nucleoside and nucleobase transporters. It was discovered that nucleobase transport in these MVECs was mediated by a novel, purine selective transporter that was insensitive to inhibitors of ENTs, which we named equilibrative nucleobase transporter 1 (ENBT1). ENBT1 and the nucleoside selective transporter ENT1 were differentially regulated by a number of intracellular protein kinases, vascular endothelial growth factor, and oxidative stress.

The role of nucleoside and nucleobase transport in the physiology of MVECs was also studied, using primary MVECs isolated from wild-type (WT) and ENT1-/- mice. Altered gene expression of adenosine metabolizing enzymes and adenosine signaling was identified and confirmed at the protein level, however, there was no compensatory changes in other nucleoside or nucleobase transporters. Coinciding with the loss of adenosine transport capabilities, ENT1-/- mice had elevated adenosine plasma concentrations compared to WT and lowered blood pressure.

Unexpectedly, the ENT1-/- began to develop hind limb paralysis at about 12 months old. Post-mortem analysis revealed abnormal mineralization on the spine leading to spinal cord compression. MicroCT analysis was used to determine a time course of development, with mineralization being apparent as early as 2 months old. The pattern of mineralization resembled the human condition known as diffuse idiopathic skeletal hyperostosis (DISH).

Nucleoside and nucleobase transporters are important in the regulation of cardiovascular effects of purines, with special consideration to oxidative stress and blood pressure. We may have also discovered a novel role for ENT1 in the development of DISH.


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