Echocardiographic caudal vena cava measurements in healthy cats and in cats with congestive heart failure and non-cardiac causes of cavitary effusions.

BACKGROUND: Echocardiographic indices of the inferior vena cava have been associated with elevated right atrial pressures in humans. HYPOTHESIS/OBJECTIVES: Describe caudal vena caval (CVC) sonographic dimensions in healthy cats compared to cats with cardiogenic cavitary effusion (CCE), cardiogenic pulmonary edema (CPE), or non-cardiac causes of cavitary effusion (NCE). ANIMALS: 30 healthy control cats and 52 client-owned cats with CCE, CPE, or NCE examined at two university hospitals. METHODS: Sagittal 2-dimensional (2D) and M-mode CVC dimensions were acquired from the subxiphoid view. Caudal vena cava collapsibility index (CVC-CI) was calculated. Variables were compared between study groups using Kruskal-Wallis and Dunn's Bonferroni testing. Receiver operating characteristic curves were used to assess sensitivity and specificity for diagnostic categories. RESULTS: Healthy cats had sagittal 2D and M-mode (median, interquartile range) CVC maximal dimensions of 2.4 mm (1.3-4.0) and 3.4 mm (1.5-4.9) and CVC-CI of 52% (45.2-61.8) and 55% (47.8-61.3), respectively. The CVC maximal dimensions in healthy controls were smaller than in cats with cavitary effusions or pulmonary edema (all P<0.05). CVC-CI was different between CCE and NCE (P<0.0001) with cutoffs of CVC-CI ≤38% (2D) or ≤29% (M-mode) being 90.5% and 85.7% sensitive, and 94.4% and 100% specific for diagnosis of CCE, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Caudal vena cava measurements are larger in cats with cavitary effusions and cats with CPE than healthy cats. In cats with cavitary effusion, decreased CVC-CI, ≤38% (2D) or ≤29% (M-mode), was helpful in distinguishing between cardiogenic and noncardiogenic etiology.

Do modern types of wheat have lower quality for human health?

Wheat is the major staple food in Western Europe and an important source of energy, protein, dietary fibre, minerals, B vitamins and phytochemicals. Plant breeders have been immensely successful in increasing yields to feed the growing global population. However, concerns have been expressed that the focus on increasing yield and processing quality has resulted in reduced contents of components that contribute to human health and increases in adverse reactions. We review the evidence for this, based largely on studies in our own laboratories of sets of wheats bred and grown between the 18th century and modern times. With the exception of decreased contents of mineral micronutrients, there is no clear evidence that intensive breeding has resulted in decreases in beneficial components or increases in proteins which trigger adverse responses. In fact, a recent study of historic and modern wheats from the UK showed increases in the contents of dietary fibre components and a decreased content of asparagine in white flour, indicating increased benefits for health.

Temperature-Dependent Biomarkers of Estrogenic Exposure in a Piscivore Freshwater Fish.

The biological effects of endocrine-active compounds and increasing water temperatures as a result of climate change have been studied extensively and independently, but there is a dearth of research to examine the combined effect of these factors on exposed organisms. Recent data suggest that estrogenic exposure and rising ambient temperatures independently impact predator-prey relationships. However, establishing these connections in natural settings is complex. These obstacles can be circumvented if biomarkers of estrogenic exposure in resident fish can predict changes in predator-prey relationships. To test the effects of estrone and temperature, the piscivore bluegill sunfish (Lepomis macrochirus) was exposed for 30 days to estrone at concentrations (90 ± 17.6 ng/L [mean ± standard deviation] and 414 ± 146 ng/L) previously shown to reduce prey-capture success. Exposures were conducted at four temperatures (15 °C, 18 °C, 21 °C, 24 °C) to simulate breeding season ambient temperatures across the natural range of this species. A suite of morphological and physiological biomarkers previously linked to estrogenic exposures were examined. Biomarkers of estrone exposure were more commonly and severely impacted in male fish than in female fish. Notably, the gonadosomatic index was lower and gonads were less mature in exposed males. Additionally, temperature modulated the effects of estrone similarly in males and females with fish exposed at higher temperatures typically exhibiting a decreased morphological index. This study provides evidence that alterations in hepatic function and gonadal function may cause shifts in metabolism and energy allocation that may lead to declining prey capture performance.

Temperature and Estrogen Alter Predator-Prey Interactions between Fish Species.

A variety of environmental estrogens are commonly detected in human-impacted waterways. Although much is known about the effects of these environmental estrogens on the reproductive physiology and behavior of individuals within species, comparatively less is known about how these compounds alter the outcomes of interactions between species. Furthermore, few studies have considered how the effects of contaminants are modulated by natural variation in abiotic factors, such as temperature. To help fill this knowledge gap, we conducted a factorial experiment to examine the independent and combined effects of estrone (E1) and temperature on the outcome of predator-prey interactions between two common North American freshwater fishes, fathead minnows (Pimephales promelas) and bluegill sunfish (Lepomis macrochirus). Larval fathead minnows and adult sunfish were exposed to either a low (mean±standard deviation, 90.1 ± 18 ng/L; n = 16) or high (414 ± 147 ng/L; n = 15) concentration of E1 or to a solvent control for 30 days at one of four natural seasonal temperatures (15°C, 18°C, 21°C, and 24°C) before predation trials were performed. Exposure to E1 was associated with a significant increase in larval predation mortality that was independent of temperature. Across all temperature treatments, approximately 74% of control minnows survived; this survivorship significantly exceeded that of minnows exposed to either concentration of E1 (49% and 53% for minnows exposed to the low and high concentrations, respectively). However, exposure to E1 also impaired the prey-capture success of sunfish, partially mitigating predation pressure on exposed minnows. Overall prey-capture success by sunfish showed an inverted U-shaped distribution with temperature, with maximal prey consumption occurring at 21°C. This study illustrates the vulnerability of organismal interactions to estrogenic pollutants and highlights the need to include food web interactions in assessments of risk.

Estimation of the iron bioavailability in green vegetables using an in vitro digestion/Caco-2 cell model.

It is estimated that over 30% of the global population is anaemic, half of which is due to iron deficiency. The bioavailability of iron from vegetables is low and variable, and influenced by food composition and matrix. We have therefore determined the relative bioavailability of iron in five types of green vegetable, spinach, broccoli, savoy cabbage, curly kale and green pepper, by measuring the ferritin response in a simulated digestion/Caco-2 cell model. Savoy cabbage gave the highest ferritin response and analysis of the digest showed that the iron was present in low molecular weight fractions which contained glucose, fructose, organic acids and amino acids. The addition of fructose 1,6-biphosphate to the Caco-2 cells increased iron uptake 2-fold. These results demonstrate that cabbage was the best source of bioavailable iron out of the vegetables studied and suggest that the formation of complexes with fructose derivatives contribute to increase the iron bioavailability.

Temperature modulates estrone degradation and biological effects of exposure in fathead minnows.

Environmental pollutants, including estrogens, are widespread in aquatic environments frequently as a result of treated wastewater effluent discharged. Exposure to estrogens has been correlated with disruption of the normal physiological and reproductive function in aquatic organisms, which could impair the sustainability of exposed populations. However, assessing the effects of estrogen exposure on individuals is complicated by the fact that rates of chemical uptake and environmental degradation are temperature dependent. Because annual temperature regimes often coincide with critical periods of biological activity, temperature-dependent changes in estrogen degradation efficacy during wastewater treatment could modulate biological effects. We examined the interactions between ambient water temperature and degradation of estrone (E1) during wastewater treatment. In addition, we exposed mature fathead minnows (Pimephales promelas) to three environmentally relevant concentrations of E1 at four different water temperatures (15°C, 18°C, 21°C, and 24°C) to reflect natural seasonal variation. E1 degradation occurred with and without the support of robust nitrification at all temperatures; however, the onset of E1 degradation was delayed at cooler water temperatures. In addition, we observed significant interactive effects between temperature and E1 exposure. Female morphometric endpoints were more susceptible to temperature-modulating effects while physiological endpoints were more strongly affected in males. Collectively, the data demonstrate that natural seasonal fluctuations in temperature are sufficient to affect E1 degradation during wastewater treatment and induce sex-dependent physiological and anatomical changes in exposed fish.

Thermal modulation of anthropogenic estrogen exposure on a freshwater fish at two life stages.

Human-mediated environmental change can induce changes in the expression of complex behaviors within individuals and alter the outcomes of interactions between individuals. Although the independent effects of numerous stressors on aquatic biota are well documented (e.g., exposure to environmental contaminants), fewer studies have examined how natural variation in the ambient environment modulates these effects. In this study, we exposed reproductively mature and larval fathead minnows (Pimephales promelas) to three environmentally relevant concentrations (14, 22, and 65ng/L) of a common environmental estrogen, estrone (E1), at four water temperatures (15, 18, 21, and 24°C) reflecting natural spring and summer variation. We then conducted a series of behavioral experiments to assess the independent and interactive effects of temperature and estrogen exposure on intra- and interspecific interactions in three contexts with important fitness consequences; reproduction, foraging, and predator evasion. Our data demonstrated significant independent effects of temperature and/or estrogen exposure on the physiology, survival, and behavior of larval and adult fish. We also found evidence suggesting that thermal regime can modulate the effects of exposure on larval survival and predator-prey interactions, even within a relatively narrow range of seasonally fluctuating temperatures. These findings improve our understanding of the outcomes of interactions between anthropogenic stressors and natural abiotic environmental factors, and suggest that such interactions can have ecological and evolutionary implications for freshwater populations and communities.

Inhalation anesthesia induced by isoflurane alters penicillin disposition in swine tissues.

Twelve healthy swine were dosed with penicillin G intramuscularly. Fluids and tissues samples were collected at the end of two periods of general anesthesia, performed 24 h apart. Tissue samples were collected by minimally invasive laparoscopy under general anesthesia at 8 and 28 h postdose. Four nonanesthetized, penicillin-treated pigs were euthanized at 8 h postdose, and a second set of four similarly treated control pigs were sacrificed 28 h postdose. Liver penicillin tissue concentrations from animals that underwent anesthesia and laparoscopic tissue collection had tissue concentrations that were higher than nonanesthetized pigs at both time points. Urine, plasma, kidney, skeletal, and cardiac muscle showed no differences between the two groups. Laparoscopic tissue collection under general anesthesia in swine induces physiological changes that cause alterations in tissue pharmacokinetics not seen in conscious animals.

Triticum monococcum lines with distinct metabolic phenotypes and phloem-based partial resistance to the bird cherry-oat aphid Rhopalosiphum padi.

Crop protection is an integral part of establishing food security, by protecting the yield potential of crops. Cereal aphids cause yield losses by direct damage and transmission of viruses. Some wild relatives of wheat show resistance to aphids but the mechanisms remain unresolved. In order to elucidate the location of the partial resistance to the bird cherry-oat aphid, Rhopalosiphum padi, in diploid wheat lines of Triticum monococcum, we conducted aphid performance studies using developmental bioassays and electrical penetration graphs, as well as metabolic profiling of partially resistant and susceptible lines. This demonstrated that the partial resistance is related to a delayed effect on the reproduction and development of R. padi. The observed partial resistance is phloem based and is shown by an increase in number of probes before the first phloem ingestion, a higher number and duration of salivation events without subsequent phloem feeding and a shorter time spent phloem feeding on plants with reduced susceptibility. Clear metabolic phenotypes separate partially resistant and susceptible lines, with the former having lower levels of the majority of primary metabolites, including total carbohydrates. A number of compounds were identified as being at different levels in the susceptible and partially resistant lines, with asparagine, octopamine and glycine betaine elevated in less susceptible lines without aphid infestation. In addition, two of those, asparagine and octopamine, as well as threonine, glutamine, succinate, trehalose, glycerol, guanosine and choline increased in response to infestation, accumulating in plant tissue localised close to aphid feeding after 24 h. There was no clear evidence of systemic plant response to aphid infestation.

Outcome and survival in canine sick sinus syndrome and sinus node dysfunction: 93 cases (2002-2014).

INTRODUCTION: To evaluate the clinical presentation, diagnosis, treatment, and outcomes of a group of dogs with sinoatrial node abnormalities. ANIMALS: Ninety-three client-owned dogs at a referral institution. MATERIALS AND METHODS: Medical records were reviewed for clinical history, diagnostic testing, and medical or permanent artificial pacemaker (PAP) treatment. Owners or veterinarians were contacted for long-term follow-up. RESULTS: Sixty-one dogs were symptomatic for their bradyarrhythmia and were diagnosed with sick sinus syndrome (SSS). Thirty-two dogs were asymptomatic for their bradyarrhythmia and were diagnosed with sinus node dysfunction (SND). Miniature Schnauzers, West Highland White terriers, Cocker spaniels, and female dogs were overrepresented. Medical management with positive chronotropic drugs successfully controlled syncope long-term in 54% of SSS dogs, and acted as a bridge to PAP in 20%. Positive atropine response predicted medical treatment success. Forty-six percent of SSS dogs eventually underwent PAP implantation. Median survival time was approximately 18 months in SND and SSS dogs regardless of treatment strategy. Congestive heart failure (CHF) associated with progressive valvular heart disease occurred commonly in all groups, particularly in dogs with bradycardia-tachycardia syndrome. CONCLUSIONS: Sinus node dysfunction and SSS represent a spectrum of sinoatrial node disease, which for some dogs may also involve a component of autonomic dysfunction. Dogs with SND do not require treatment. Dogs with SSS often require treatment to reduce the frequency of syncope; medical management is often useful, particularly in atropine responsive dogs. Prognosis of SSS with treatment is good, though development of CHF does not appear to be mitigated by treatment.

Multi-platform metabolomics analyses of a broad collection of fragrant and non-fragrant rice varieties reveals the high complexity of grain quality characteristics.

The quality of rice in terms not only of its nutritional value but also in terms of its aroma and flavour is becoming increasingly important in modern rice breeding where global targets are focused on both yield stability and grain quality. In the present paper we have exploited advanced, multi-platform metabolomics approaches to determine the biochemical differences in 31 rice varieties from a diverse range of genetic backgrounds and origin. All were grown under the specific local conditions for which they have been bred and all aspects of varietal identification and sample purity have been guaranteed by local experts from each country. Metabolomics analyses using 6 platforms have revealed the extent of biochemical differences (and similarities) between the chosen rice genotypes. Comparison of fragrant rice varieties showed a difference in the metabolic profiles of jasmine and basmati varieties. However with no consistent separation of the germplasm class. Storage of grains had a significant effect on the metabolome of both basmati and jasmine rice varieties but changes were different for the two rice types. This shows how metabolic changes may help prove a causal relationship with developing good quality in basmati rice or incurring quality loss in jasmine rice in aged grains. Such metabolomics approaches are leading to hypotheses on the potential links between grain quality attributes, biochemical composition and genotype in the context of breeding for improvement. With this knowledge we shall establish a stronger, evidence-based foundation upon which to build targeted strategies to support breeders in their quest for improved rice varieties.

Complication rates associated with transvenous pacemaker implantation in dogs with high-grade atrioventricular block performed during versus after normal business hours.

BACKGROUND: Transvenous pacemaker implantation in dogs is associated with a relatively high complication rate. At our institution, pacemaker implantation in dogs with high-grade atrioventricular block (HG-AVB) frequently is performed as an after-hours emergency. HYPOTHESIS: Among dogs with HG-AVB, the rate of major complications is higher when pacemakers are implanted after hours (AH) compared to during business hours (BH). ANIMALS: Client-owned dogs with HG-AVB that underwent transvenous pacemaker implantation between January 2002 and December 2012 at the North Carolina State University Veterinary Teaching Hospital. METHODS: Retrospective medical record review. Two-year follow-up was required for complications analysis. RESULTS: Major complications occurred in 14/79 dogs (18%) and included lead dislodgement, lead or generator infection, lead or generator migration, and pacing failure. Incidence of major complications was significantly higher AH (10/36, 28%) compared to BH (4/43, 9%; P = .041), and all infectious complications occurred AH. Median survival time for all dogs was 27 months and did not differ between AH and BH groups for either all-cause (P = .70) or cardiac (P = .40) mortality. AH dogs were younger than BH dogs (P = .010), but there were no other clinically relevant differences between BH and AH groups in terms of demographic, clinical, or procedural variables. CONCLUSIONS AND CLINICAL IMPORTANCE: At our institution, AH transvenous pacemaker placement is associated with a higher rate of major complications (especially infections) compared to BH placement. This difference may be because of a variety of human factor differences AH versus BH.

Merits of random forests emerge in evaluation of chemometric classifiers by external validation.

Real-world applications will inevitably entail divergence between samples on which chemometric classifiers are trained and the unknowns requiring classification. This has long been recognized, but there is a shortage of empirical studies on which classifiers perform best in 'external validation' (EV), where the unknown samples are subject to sources of variation relative to the population used to train the classifier. Survey of 286 classification studies in analytical chemistry found only 6.6% that stated elements of variance between training and test samples. Instead, most tested classifiers using hold-outs or resampling (usually cross-validation) from the same population used in training. The present study evaluated a wide range of classifiers on NMR and mass spectra of plant and food materials, from four projects with different data properties (e.g., different numbers and prevalence of classes) and classification objectives. Use of cross-validation was found to be optimistic relative to EV on samples of different provenance to the training set (e.g., different genotypes, different growth conditions, different seasons of crop harvest). For classifier evaluations across the diverse tasks, we used ranks-based non-parametric comparisons, and permutation-based significance tests. Although latent variable methods (e.g., PLSDA) were used in 64% of the surveyed papers, they were among the less successful classifiers in EV, and orthogonal signal correction was counterproductive. Instead, the best EV performances were obtained with machine learning schemes that coped with the high dimensionality (914-1898 features). Random forests confirmed their resilience to high dimensionality, as best overall performers on the full data, despite being used in only 4.5% of the surveyed papers. Most other machine learning classifiers were improved by a feature selection filter (ReliefF), but still did not out-perform random forests.

Proteomic analysis of the temporal expression of bovine milk proteins during coliform mastitis and label-free relative quantification.

The discovery of biomarkers in milk indicative of local inflammation or disease in the bovine mammary gland has been hindered by the extreme biological complexity of milk, the dynamic range of proteins in the matrix that renders the identification of low-abundance proteins difficult, and the challenges associated with quantifying changes during disease in the abundance of proteins for which no antibody exists. The objectives of the current study were to characterize the temporal expression of milk proteins following Escherichia coli challenge and to evaluate change in relative abundance of identified proteins using a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) label-free semiquantitative approach. Liquid chromatography-MS/MS conducted on whey from milk samples collected just before infusion with E. coli and at 12, 18, 24, 36, 48, and 60h following infection resulted in the identification of the high- to medium-abundance proteins alpha(S1)-, alpha(S2)- beta-, and kappa-caseins and the whey proteins serum albumin, beta-lactoglobulin, and alpha-lactalbumin. Additionally, a select number of lower abundance markers of inflammation were also identified, including lactoferrin, transferrin, apolipoprotein AI, fibrinogen, glycosylation-dependent cell adhesion molecule-1, peptidoglycan recognition receptor protein, and cyclic dodecapeptide-1. Normalized peptide counts for each protein identified were used to evaluate temporal changes in milk proteins following infection. For comparison with relative protein abundance determined using proteomic-based methods, changes in serum albumin, lactoferrin, and transferrin in milk during disease were also measured using ELISA. Label-free, proteomic-based quantification revealed relative changes in milk proteins that corresponded to expression profiles generated by ELISA. The results indicate that label-free LC-MS/MS methods are a viable means of tracking changes in relative protein abundance in milk during disease. Despite the identification of primarily abundant milk proteins, the results indicate that, with further refinement, LC-MS/MS could be used to evaluate temporal changes in proteins related to host response for which no antibody or ELISA currently exists.

Proteomic analysis of differentially expressed proteins in bovine milk during experimentally induced Escherichia coli mastitis.

The objectives of the current study were to profile changes in protein composition using 2-dimensional gel electrophoresis on whey samples from a group of 8 cows before and 18 h after infection with Escherichia coli and to identify differentially expressed milk proteins by peptide sequencing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry post source decay. Only proteins present in whey fractions of all 8 cows were sequenced to avoid reporting a protein response unique to only a subset of infected cows. Despite the overwhelming presence of casein and beta-lactoglobulin, the low abundance proteins transthyretin, lactadherin, beta-2-microglobulin precursor, alpha-1-acid glycoprotein, and complement C3 precursor could be identified in whey samples from healthy cows. Whey samples at 18 h postinfection were characterized by an abundance of serum albumin, in spots of varying mass and isoelectric point, as well as increased transthyretin and complement C3 precursor levels. Also detected at 18 h postinoculation were the antimicrobial peptides cathelicidin, indolicidin, and bactenecin 5 and 7, and the proteins beta-fibrinogen, alpha-2-HS-glycoprotein, S100-A12, and alpha-1-antiproteinase. Most notable was the detection of the acute phase protein alpha-1-acid glycoprotein in mastitic whey samples, a result not previously reported. In contrast to methods used in previous proteomic analyses of bovine milk, the methods used in the current study enabled the rapid identification of milk proteins with minimal sample preparation. Use of a larger sample size than previous analyses also allowed for more robust protein identification. Results indicate that examination of the protein profile of whey samples from cows after inoculation with E. coli could provide a rapid survey of milk protein modulation during coliform mastitis and aid in the identification of biomarkers of this disease.

Comparison of bovine in vivo bioavailability of two sulfamethazine oral boluses exhibiting different in vitro dissolution profiles.

The bolus (or oblet) is a dosage form that can be used for the oral administration of pharmaceutical compounds to ruminating species. Unlike traditional tablets, oral boluses may contain quantities of drug on the order of grams rather than milligrams. Due to its size, it is only recently that USP-like in vitro dissolution methods have been developed for this dosage form. However, whether or not these dissolution tests can predict product in vivo performance has yet to be determined. The importance of this issue is apparent when the U.S. Food and Drug Administration Center for Veterinary Medicine is faced with the decision of whether to require additional in vivo bioequivalence study data to support the approval of changes in product chemistry or manufacturing method. The current study was undertaken to determine whether an in vivo/in vitro correlation can be established for bovine sulfamethazine oral boluses and to acquire insight into the magnitude of changes in in vitro product performance that can occur before corresponding changes are seen in in vivo blood level profiles. Based upon the results of this investigation, it is concluded that marked changes in in vitro sulfamethazine bolus performance can be tolerated before resulting in altered in vivo blood level profiles. However, the data also suggest that rumenal absorption may occur for some compounds. Therefore the degree to which variation in product in vitro dissolution profiles can be tolerated may be compound specific.

Characterization of nucleoside transport systems in cultured rat epididymal epithelium.

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na(+) dependent, while 30% was Na(+) independent. The Na(+)-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na(+)-dependent [(3)H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na(+)-dependent [(3)H]guanosine uptake was inhibited by thymidine by 20% and Na(+)-dependent [(3)H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na(+)-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.

Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells. Ent2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine.

We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosylate hENT1 to 37 kDa and hENT2 to 45 kDa. With hENT1 being more sensitive, there is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthioinosine (NBMPR) (IC(50), 0.4 +/- 0.1 nM versus 2.8 +/- 0.3 microM) and dipyridamole (IC(50), 5.0 +/- 0.9 nM versus 356 +/- 13 nM), respectively. [(3)H]NBMPR binds to ENT1 cells with a high affinity K(d) of 0.377 +/- 0.098 nM, and each ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/s for uridine. Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7. 7-, and 19.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1. The nucleobase hypoxanthine inhibits [(3)H]uridine uptake by hENT2 but has minimal effect on hENT1. Taken together, these results suggest that hENT2 might be important in transporting adenosine and its metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed.

Nucleoside transport in human colonic epithelial cell lines: evidence for two Na+-independent transport systems in T84 and Caco-2 cells.

RT-PCR of RNA isolated from monolayers of the human colonic epithelial cell lines T84 and Caco-2 demonstrated the presence of mRNA for the two cloned Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2, but not for the cloned Na+-dependent concentrative nucleoside transporters, CNT1 and CNT2. Uptake of [3H]uridine by cell monolayers in balanced Na+-containing and Na+-free media confirmed the presence of only Na+-independent nucleoside transport mechanisms. This uptake was decreased by 70-75% in the presence of 1 microM nitrobenzylthioinosine, a concentration that completely inhibits ENT1, and was completely blocked by the addition of 10 microM dipyridamole, a concentration that inhibits both ENT1 and ENT2. These findings indicate the presence in T84 and Caco-2 cells of two functional Na+-independent equilibrative nucleoside transporters, ENT1 and ENT2.