Extracellular adenosine (Ado) has been implicated as central signaling molecule during conditions of limited oxygen availability (hypoxia), regulating physiologic outcomes as diverse as vascular leak, leukocyte activation, and accumulation. Presently, the molecular mechanisms that elevate extracellular Ado during hypoxia are unclear. In the present study, we pursued the hypothesis that diminished uptake of Ado effectively enhances extracellular Ado signaling. Initial studies indicated that the half-life of Ado was increased by as much as fivefold after exposure of endothelia to hypoxia. Examination of expressional levels of the equilibrative nucleoside transporter (ENT)1 and ENT2 revealed a transcriptionally dependent decrease in mRNA, protein, and function in endothelia and epithelia. Examination of the ENT1 promoter identified a hypoxia inducible factor 1 (HIF-1)–dependent repression of ENT1 during hypoxia. Using in vitro and in vivo models of Ado signaling, we revealed that decreased Ado uptake promotes vascular barrier and dampens neutrophil tissue accumulation during hypoxia. Moreover, epithelial Hif1 α mutant animals displayed increased epithelial ENT1 expression. Together, these results identify transcriptional repression of ENT as an innate mechanism to elevate extracellular Ado during hypoxia.

Equilibrative nucleoside transporters (ENTs) are a recently characterized and poorly understood group of membrane proteins that are important in the uptake of endogenous nucleosides required for nucleic acid and nucleoside triphosphate synthesis. Despite their central importance in cellular metabolism and nucleoside analog chemotherapy, no human ENT gene has been described and nothing is known about gene structure and function. To gain insight into the ENT gene family, we used experimental and in silico comparative genomic approaches to identify ENT genes in three evolutionarily diverse organisms with completely (or almost completely) sequenced genomes, Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster. We describe the chromosomal location, the predicted ENT gene structure and putative structural topologies of predicted ENT proteins derived from the open reading frames. Despite variations in genomic layout and limited ortholog protein sequence identity (< or =27.45%), predicted topologies of ENT proteins are strikingly similar, suggesting an evolutionary conservation of a prototypic structure. In addition, a similar distribution of protein domains on exons is apparent in all three taxa. These data demonstrate that comparative sequence analyses should be combined with other approaches (such as genomic and proteomic analyses) to fully understand structure, function and evolution of protein families.

A monoclonal antibody raised against adenovirus E1A-associated cellular proteins recognized Nek9, a NimA-related protein kinase. Subcellular fractionation and immunofluorescence indicated that Nek9 was primarily cytoplasmic with a small portion located in the nucleus whereas E1A was primarily nuclear. Although co-immunoprecipitation experiments indicated that nuclear Nek9 interacted, directly or indirectly, with E1A, the major effect of E1A was to diminish the amount of Nek9 in the nucleus suggesting that E1A alters the subcellular distribution of Nek9 and that the interaction is transient. A Nek9 deletion mutant lacking a central RCC1-like domain interacted stably with E1A and accumulated in the nucleus in the presence of E1A, possibly representing an intermediate stage of the normally transient Nek9/E1A interaction. The interaction of Nek9 with E1A was dependent on the N-terminal sequences of E1A. Attempts to stably overexpress either Nek9 or the kinase-inactive mutant in various cell lines were unsuccessful; however, the presence of E1A allowed stable overexpression of both proteins. These results suggest that E1A disrupts a nuclear function of Nek9.

Equilibrative nucleoside transporters (ENTs) are membrane proteins that transport nucleosides, nucleobases and analogs across membranes. ENT genes and the regulation of their expression are poorly understood. Therefore, we isolated and functionally characterized the promoter of the prototypic human ENT, hENT1. A single transcriptional initiation site 58 bp downstream of the TATA box and 272 bp upstream of the translation initiation site is present. Limited sequence similarity exists between the hENT1 and mouse ENT1 (mENT1) promoters suggesting conservation of ENT1 transcriptional regulators in mammals. Putative consensus sites for transcription factors exist within the hENT1 promoter. Reporter assays revealed similar but not identical transcriptional activity profiles in human cells.

Machado J, Abdulla P, Hanna WJB, Hilliker AJ, Coe IR. Genomic analysis of nucleoside transporters in Diptera and functional characterization of DmENT2, a Drosophila equilibrative nucleoside transporter. Physiol Genomics 28: [337][338][339][340][341][342][343][344][345][346][347] 2007. First published November 7, 2006; doi:10.1152/physiolgenomics.00087.2006.-The recent completion of genome sequencing projects in a number of eukaryotes allows comparative analysis of orthologs, which can aid in identifying evolutionary constraints on protein structure and function. Nucleoside transporters (NTs) are present in a diverse array of organisms and previous studies have suggested that there is low protein sequence similarity but conserved structure in invertebrate and vertebrate NT orthologs. In addition, most taxa possess multiple NT isoforms but their respective roles in the physiology of the organism are not clear. To investigate the evolution of the structure and function of NTs, we have extended our previous studies by identifying NT orthologs in the Dipteran Anopheles gambiae and comparing these proteins to human and Drosophila melanogaster (Dm) NTs. In addition, we have functionally characterized DmENT2, one of three putative D. melanogaster ENTs that we have previously described. DmENT2 has broad substrate specificity, is insensitive to standard nucleoside transport inhibitors and is expressed in the digestive tract of late stage embryos based on in situ hybridization. DmENT1 and DmENT2 are expressed in most stages during development with the exception of early embryogenesis suggesting specific physiological roles for each isoform. These data represent the first complete genomic analysis of Dipteran NTs and the first report of the functional characterization of any Dipteran NT.concentrative nucleoside transporters; evolution; Anopheles NUCLEOSIDE TRANSPORTERS (NTs) are integral membrane proteins responsible for movement of nucleosides across cell membranes (4, 13, 26). Nucleosides play key roles in eukaryote physiology, acting as signaling molecules, neuromodulators and in the regulation of cardiovascular activity (2, 4, 6). Nucleosides are also precursors of nucleic acids and are either synthesized de novo or salvaged from the extracellular environment via NTs. These salvage pathways are needed when de novo pathways are lacking, for example in protozoans, such as the malarial parasite Plasmodium, that cannot synthesize purines (20) and in specialized eukaryotic cells, such as in enterocytes, bone marrow, and certain brain cells (10).NTs have been primarily studied in mammalian systems and are divided into two main categories based on their mechanism of transport. The equilibrative nucleoside transporters (ENTs) facilitate the movement of nucleosides down their concentration gradients, while the concentrative nucleoside transporters (CNTs) actively transport nucleosides against their concentration gradient by cotransport of a cation, usually Na ϩ , down its gradient (13). In mammals, ENTs have broad permeant selec...