Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Pharmacology and Toxicology

Supervisor

Dr. James Hammond

Abstract

Adenosine is important in the regulation of vascular tone and the Equilibrative Nucleoside Transporter 1 (ENT1) is a regulator of adenosine concentrations. Thus, changes in ENT1 expression and activity may alter vascular tone regulation. We therefore examined the cardiovascular phenotype of ENT1-null (ENT1-/-) as compared to wild type mice (ENT1+/+). Heart rates (HRs) and blood pressures (BPs) of ENT1-/- and ENT1+/+ littermate male mice were obtained while conscious and anaesthetized, and metabolic data were collected over a 24 hr period. Vascular reactivity was assessed in aortic ring segments obtained from 3 month old ENT1-/- and ENT1+/+ mice during both normoxic and hypoxic conditions. Gene expression was obtained using qPCR. KCl administration mediated significantly greater constriction (~32% increase independent of tension applied or age) in ENT1-/- rings compared to ENT1+/+. However, no significant differences between the two genotypes were observed for the different receptor agonists. KCl works independent of receptors and carries out direct vasoconstriction via depolarization whereas receptor agonists work indirectly through cellular signaling mechanisms. In hypoxic conditions, ENT1-/- mice exhibited diminished response to phenylephrine following pretreatment with increasing concentration of adenosine as well as in the presence of ENT1 inhibitor NBMPR suggesting an ENT1 independent effect of NBMPR. Length tension curves on the aortas were carried out and resulted in ­­ an increase in ENT1-/-curve when the same tension was applied. HR and BP analysis showed elevated blood pressure while the mice were conscious. Morphological assessment of vascularized tissues was performed using H&E and Alizarin Red Staining. Visualization of H&E stain yielded significant differences in luminal area however no significant differences were seen in arterial wall thickness between ENT1+/+ and ENT1-/- mice at the age of 3 months. Alizarin Red staining yielded no evidence of calcium deposits in the arterial walls. The enhanced effect of KCl in ENT1-/- aortic rings suggests a physiological difference in the regulation of the vasoconstriction response. The increased blood pressure coupled with the stiffening of the aortic wall suggests an increase on the mechanical load of the heart, possibly the early stages of hypertension. The studies suggest a vascular phenotype associated with a loss of ENT1. This model will continue to aid in unraveling the role of adenosine in vascular regulation.

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