Hammond, James - University of Alberta - Department of Pharmacology

个人简介

BSc (Hons), Physiology & Pharmacology, University of Western Ontario, 1979 PhD, Pharmacology, University of Alberta, 1983

研究领域

The research focus of the Hammond laboratory is on the pharmacological, functional and molecular characterization of the membrane transport systems responsible for the uptake and release of endogenous nucleosides and anticancer/antiviral nucleoside analogues by mammalian cells. Adenosine, the primary physiological substrate for these transporters, is an endogenous bioactive agent with neuromodulator, cardioprotective and vasodilator activities. These actions of adenosine are mediated through extracellular membrane-located adenosine receptors. Uptake into cells via nucleoside transporters is the first step in the metabolism and inactivation of extracellular nucleosides and these transport systems thereby play a significant role in controlling the bioactivity of adenosine. Multiple subtypes of nucleoside transporters have been identified by both functional and molecular approaches. They vary in their sensitivities to inhibition by agents such as nitrobenzylthioinosine (NBMPR) and dipyridamole, and by their dependence on ion (sodium) gradients. Two broad gene families have been identified: 1) Equilibrative nucleoside transporters (SLC29; ENT) that operate by facilitated diffusion and 2) Concentrative sodium/nucleoside co-transporters (SLC28; CNT). Seven of these transporters have been cloned from a number of mammalian (including human) tissues (ENT1, ENT2, ENT3, ENT4, CNT1, CNT2, CNT3). We cloned the canine ENT1 and the mouse ENT1 and ENT2, as well as two novel splice variants of the mouse ENT1 transporter. The physiological and pharmacological significance of the multiple transporter subtypes/variants has yet to be established. It is expected that nucleoside transporter heterogeneity would impact significantly on the actions of many drugs that act through adenosine receptors or by modifying cellular nucleoside metabolism. This has particular relevance to the clinical activities of the cytotoxic nucleoside analogues used in cancer chemotherapy (e.g. cytosine arabinoside, gemcitabine, 2-chlorodeoxyadenosine), as well as drugs that modify vascular function via modulation of the adenosine signalling pathways. Current research in our laboratory: 1) The analysis of the protein structural elements involved in the interaction of drugs and substrates with the multiple subtypes and isoforms of equilibrative nucleoside transporters. Studies involve the generation of point mutants (site-directed mutagenesis) and their expression in transporter-deficient mammalian cell lines. Nucleoside transport activity of the native and recombinant proteins is measured directly by the cellular accumulation of radiolabelled substrates and indirectly using specific probes for the proteins associated with the transporter. 2) The role of protein phosphorylation in the activity and membrane turnover of the ENTs. We have recently cloned two isoforms of the mouse ENT1 that differ in the inclusion or deletion of a potential protein kinase CK2 phosphorylation site. We have also identified the serine residue of ENT1 that is phosphorylated by protein kinase C (PKC). Studies are ongoing to assess the PKC isoforms involved and they association of this regulatory pathway to potential adenosine receptor mediated feedback mechanisms. 3) The role of nucleoside and nucleobase transporters in the regulation of vascular function and the vasodilatory/cardoprotectant role of adenosine. These studies involve the in vitro analysis of vascular function using isolated microvascular endothelial cells (primary and immortalized) and ex vivo preparations of aortic, carotid, and mesenteric vascular rings. The impact of changes in purine metabolism on oxidative stress pathologies is also a focus. 4) Analysis of the phenotypes associated with the genetic knockout of the ENT1 transporter in mice. We have identified a clear vascular phenotype, as well as a soft tissue aberrant mineralization phenotype. Studies are ongoing to understand the mechanisms underlying these biological changes and their relevance to human disease states. The overall goal of these studies is to provide detailed information on the functional and molecular characteristics of the proteins involved in nucleoside translocation. It is expected that these studies will lead ultimately to the development of new sedatives, anti-epileptics, and vasodilators that act through manipulation of adenosinergic systems, as well as novel drugs for use in the on-going battle against cancer and various viral and parasitic diseases.

近期论文

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Ii H, Warraich S, Tenn N, Quinonez D, Holdsworth DW, Hammond JR, Dixon SJ and Séguin CA. (2016) Disruption of biomineralization pathways in spinal tissues of a mouse model of diffuse idiopathic skeletal hyperostosis. Bone 90:37-49. PMID: 27237608. Hughes SJ, Cravetchi X, Vilas G and Hammond JR. (2015) Adenosine A1 receptor activation modulates human equilibrative nucleoside transporter 1 (hENT1) activity via PKC-mediated phosphorylation of serine-281. Cell Signal 27(5):1008-18. PMID: 25725289. Bone DB, Antic M, Vilas G and Hammond JR. (2014) Oxidative stress modulates nucleobase transport in microvascular endothelial cells. Microvasc Res 95C:68-75. PMID: 24976360. Warraich S, Bone DB, Quinonez D, Ii H, Choi DS, Holdsworth DW, Drangova M, Dixon SJ, Seguin CA and Hammond JR. (2013) Loss of equilibrative nucleoside transporter 1 (ENT1) in mice leads to progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. J Bone Miner Res 28(5):1135-49. PMID: 23184610. Park JS and Hammond JR. (2012) Cysteine Residues in the TM9-TM11 Region of the Human Equilibrative Nucleoside Transporter Subtype 1 Play an Important Role in Inhibitor Binding and Translocation Function. Mol Pharmacol 82(5):784-94. PMID: 22837314. Park JS, Hughes SJ, Cunningham FK and Hammond JR. (2011) Identification of Cysteines Involved in the Effects of Methanethiosulfonate Reagents on Human Equilibrative Nucleoside Transporter 1. Mol Pharmacol 80(4):735-46. PMID: 21791574. Bone DB, Choi DS, Coe IR and Hammond JR. (2010) Nucleoside/nucleobase transport and metabolism by microvascular endothelial cells isolated from ENT1 -/- mice. Am J Physiol Heart Circ Physiol 299 (3):H847-H856. PMID: 20543083. Robillard KR, Bone DB, Park JS and Hammond JR. (2008) Characterization of mENT1Delta11, a novel alternative splice variant of the mouse equilibrative nucleoside transporter 1. Mol Pharmacol 74 (1):264-273. PMID: 18413666. Bone DB and Hammond JR. (2007) Nucleoside and nucleobase transporters of primary human cardiac microvascular endothelial cells: Characterization of a novel nucleobase transporter. Am J Physiol Heart Circ Physiol 293:H3325-H3332. PMID: 17921321.