Genomic analysis of nucleoside transporters in Diptera and functional characterization of DmENT2, a Drosophila equilibrative nucleoside transporter

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.

Inhibition of glucose uptake in murine cardiomyocyte cell line HL-1 by cardioprotective drugs dilazep and dipyridamole

Inhibition of adenosine reuptake by nucleoside transport inhibitors, such as dipyridamole and dilazep, is proposed to increase extracellular levels of adenosine and thereby potentiate adenosine receptor-dependent pathways that promote cardiovascular health. Thus adenosine can act as a paracrine and/or autocrine hormone, which has been shown to regulate glucose uptake in some cell types. However, the role of adenosine in modulating glucose transport in cardiomyocytes is not clear. Therefore, we investigated whether exogenously applied adenosine or inhibition of adenosine transport by S-(4-nitrobenzyl)-6-thioinosine (NBTI), dipyridamole, or dilazep modulated basal and insulin-stimulated glucose uptake in the murine cardiomyocyte cell line HL-1. HL-1 cell lysates were subjected to SDS-PAGE and immunoblotting to determine which GLUT isoforms are present. Glucose uptake was measured in the presence of dipyridamole (3–300 μM), dilazep (1–100 μM), NBTI (10–500 nM), and adenosine (50–250 μM) or the nonmetabolizable adenosine analog 2-chloro-adenosine (250 μM). Our results demonstrated that HL-1 cells possess GLUT1 and GLUT4, the isoforms typically present in cardiomyocytes. We found no evidence for adenosine-dependent regulation of basal or insulin-stimulated glucose transport in HL-1 cardiomyocytes. However, we did observe a dose-dependent inhibition of glucose transport by dipyridamole (basal, IC50= 12.2 μM, insulin stimulated, IC50= 13.09 μM) and dilazep (basal, IC50= 5.7 μM, insulin stimulated, IC50= 19 μM) but not NBTI. Thus our data suggest that dipyridamole and dilazep, which are widely used to specifically inhibit nucleoside transport, have a broader spectrum of transport inhibition than previously described. Moreover, these data may explain previous observations, in which dipyridamole was noted to be proischemic at high doses.

Selective transport of adenosine into porcine coronary smooth muscle

Adenosine (ADO), an endogenous regulator of coronary vascular tone, enhances vasorelaxation in the presence of nucleoside transport inhibitors such as dipyridamole. We tested the hypothesis that coronary smooth muscle (CSM) contains a high-affinity transporter for ADO. ADO-mediated relaxation of isolated large and small porcine coronary artery rings was enhanced 12-fold and 3.4-fold, respectively, by the transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI). Enhanced relaxation was independent of endothelium and was selective for ADO over synthetic analogs. Uptake of [3H]ADO into freshly dissociated CSM cells or endothelium-denuded rings was linear and concentration dependent. Kinetic analysis yielded a maximum uptake ( V max) of 67 ± 7.0 pmol · mg protein−1 · min−1 and a Michaelis constant ( K m) of 10.5 ± 5.8 μM in isolated cells and a V max of 5.1 ± 0.5 pmol · min−1 · mg wet wt−1and a K m of 17.6 ± 2.6 μM in intact rings. NBTI inhibited transport into small arteries (IC50 = 42 nM) and cells. Analyses of extracellular space and diffusion kinetics using [3H]sucrose indicate the V max and K m for ADO transport are sufficient to clear a significant amount of extracellular adenosine. These data indicate CSM possess a high-affinity nucleoside transporter and that the activity of this transporter is sufficient to modulate ADO sensitivity of large and small coronary arteries.