Nucleoside uptake by red blood cells from a primitive vertebrate, the pacific hagfish (Eptatretus stouti), is mediated by a nitrobenzylthioinosine-insensitive transport system

Fincham, Daron A.; Wolowyk, Michael W.; Young, James D.

doi:10.1016/0005-2736(91)90112-l

Cited by 7 publications

(3 citation statements)

References 27 publications

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“…Previous studies of nucleoside transport in hagfish have been limited to red blood cells, which possess an equilibrative nitrobenzylthioinosine (NBMPR)-insensitive (ei-type) nucleoside transport process (13). The goal of the present study was to use recombinant DNA technology in combination with heterologous expression in Xenopus oocytes to attempt the cDNA cloning and functional characterization of a hagfish concentrative nucleoside transport protein.…”

Section: Resultsmentioning

confidence: 99%

“…As such, hagfish represent a unique research resource in molecular studies of early vertebrate evolution. Hagfish plasma is in approximate osmotic equilibrium with sea water (500 mM NaCl), and studies with their red blood cells have revealed a number of novel membrane transport characteristics (11,13,41,(52)(53)(54). Although these investigations have provided functional insights into hagfish membrane transport biology, there is little known structurally about hagfish transport proteins.…”

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confidence: 99%

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An ancient prevertebrate Na⁺-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti)

Yao¹,

Ng²,

Loewen³

et al. 2002

American Journal of Physiology-Cell Physiology

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The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1, hCNT2, and hCNT3 are pyrimidine nucleoside-selective (system cit), purine nucleoside-selective (system cif), or broadly selective for both pyrimidine and purine nucleosides (system cib), respectively. All have orthologs in other mammalian species and belong to a gene family (CNT) that has members in insects, nematodes, pathogenic yeast, and bacteria. Here, we report the cDNA cloning and functional characterization of a CNT family member from an ancient marine prevertebrate, the Pacific hagfish (Eptatretus stouti). This Na+-nucleoside symporter, designated hfCNT, is the first transport protein to be characterized in detail in hagfish and is a 683-amino acid residue protein with 13 predicted transmembrane helical segments (TMs). hfCNT was 52, 50, and 57% identical in sequence to hCNT1, hCNT2, and hCNT3, respectively. Similarity to hCNT3 was particularly marked in the TM 4-13 region. When produced in Xenopus oocytes, hfCNT exhibited the transport properties of system cib, with uridine, thymidine, and inosine apparent K(m) values of 10-45 microM. The antiviral nucleoside drugs 3'-azido-3'-deoxythymidine, 2',3'-dideoxycytidine, and 2',3'-dideoxyinosine were also transported. Simultaneous measurement of uridine-evoked currents and radiolabeled uridine uptake under voltage-clamp conditions gave a Na+-to-uridine coupling ratio of 2:1 (cf. 2:1 for hCNT3 and 1:1 for hCNT1/2). The apparent K50 value for Na+ activation was >100 mM. A 50:50 chimera between hfCNT and hCNT1 (TMs 7-13 of hfCNT replaced by those of hCNT1) exhibited hCNT1-like cation interactions, establishing that the structural determinants of cation stoichiometry and binding affinity were located within the carboxy-terminal half of the protein. The high degree of sequence similarity between hfCNT and hCNT3 may indicate functional constraints on the primary structure of the transporter and suggests that cib-type CNTs fulfill important physiological functions.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

An ancient prevertebrate Na⁺-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti)

Yao¹,

Ng²,

Loewen³

et al. 2002

American Journal of Physiology-Cell Physiology

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“…A volume-sensitive amino acid channel found in elasmobranchs and teleosts is not present in hagfish erythrocytes (Fugelli and Thoroed 1986;Fincham et al 1987Fincham et al , 1990Goldstein and Brill 1991;Brill et al 1992). Other transport systems characterised in hagfish erythrocytes include a Na + -dependent KCl-cotransport system (Ellory and Wolowyk 1991) and a nitrobenzylthioinosine-insensitive nucleoside transporter (Fincham et al 1991). In contrast to erythrocytes from teleosts, hagfish erythrocytes also have a high permeability to glucose (Ingermann et al 1984;Young et al 1994).…”

Section: Introductionmentioning

confidence: 94%

Na⁺-dependent transport of pyruvate in erythrocytes of the Pacific hagfish (Eptatretus stouti)

Tiihonen¹,

Yao²,

Nikinmaa³

et al. 2000

Can. J. Zool.

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We investigated the mechanisms of pyruvate transport in the erythrocytes of an ancient marine agnathan, the Pacific hagfish (Eptatretus stouti), and a sedentary euryhaline teleost, the starry flounder (Platichthys stellatus). Uptake of [ 14 C]pyruvate (50 µM, 10°C) by flounder erythrocytes was slow (t 1/2 (half-life)~30 min), nonconcentrative, and mediated by the band 3 Cl -/HCO 3 − exchanger in combination with a process similar to the H + /monocarboxylate symporter present in freshwater teleosts and mammalian erythrocytes. In contrast, pyruvate uptake by hagfish erythrocytes (50 µM, 10°C) was rapid (t 1/2~1 .5 min) and, in 10 min, reached an intracellular concentration more than 20-fold higher than that present in the extracellular medium. Pyruvate accounted for almost 90% of the accumulated intracellular radioactivity, the remaining label being incorporated into tricarboxylic acid cycle intermediates and glutamate. Influx of pyruvate was saturable (apparent K m = 12 mM) and inhibited by p-chloromercuriphenylsulphonate (PCMBS) (K i = 71 µM) and 4,4′-diisothiocyanatostilbene-2,2′-disulphonate (DIDS) (K i = 0.49 mM). Transport was inhibited poorly by α-cyano-4-hydroxycinnamate (CIN) (K i > 4 mM) and was not coupled to the movement of protons. Instead, the influx of pyruvate was Na + dependent. A sigmoidal relationship between pyruvate transport and extracellular Na + concentration was observed, suggesting a Na + :pyruvate coupling ratio greater than 1:1. In contrast with previously described Na + -dependent monocarboxylate transport activities in mammalian renal and intestinal epithelia, the hagfish erythrocyte system did not transport lactate. Résumé: Nous avons étudié les mécanismes du transport du pyruvate dans les érythrocytes chez une très ancienne espèce d'agnathe, la Myxine brune (Eptatretus stouti) et chez un téléostéen sédentaire euryhalin, le Flet étoilé (Platichthys stellatus). L'entrée du [ 14 C]pyruvate (50 µM, 10EC) dans les érythrocytes du Flet étoilé est lente (t 1/2 (demi-vie)~30 min) et le pyruvate ne s'y accumule pas. L'entrée se fait par l'intermédiaire de l'échangeur Cl -/HCO 3 − (bande 3) par un méca-nisme semblable à celui du symporteur H + /monocarboxylates des érythrocytes de mammifères et de téléostéens d'eau douce. En revanche, l'absorption du pyruvate dans les érythrocytes de la myxine (50 µM, 10EC) est rapide (t 1/2~1 ,5 min) et, en 10 min, atteint une concentration intracellulaire plus de 20 fois plus élevée que celle du milieu extracellulaire d'où il provient. Le pyruvate est responsable de plus de 90 % de la radioactivité accumulée dans les cellules et le reste du carbone marqué est incorporé dans les intermédiaires du cycle de l'acide tricarboxylique et sous forme de glutamate. L'influx du pyruvate est saturable (K m apparent = 12 mM) et inhibé par le p-sulfonate de chloromercuriphényle (PCMBS) (K i = 71 µM) et le disulphonate de 4,4N-diisothiocyanatostilbène-2,2N (DIDS) (K i = 0,49 mM). Le transport est peu inhibé par l'α-cyano-4-hydroxycinnamate (CIN) (K i > 4 mM) et n'est pas c...

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Enhancement of proliferation in cultures of Chinook salmon embryo cells by interactions between inosine and bovine sera

Ganassin

Tran

Rabgey

et al. 1994

Journal Cellular Physiology

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The influence of inosine on DNA synthesis by Chinook salmon embryo cells (CHSE-214) was investigated because previously cell number was shown to increase from six- to thirtyfold if inosine was added to the basal medium (L-15) supplemented with either dialyzed fetal bovine serum (dFBS), calf serum (CS), or dCS. Relative to L-15, 3H-thymidine incorporation was inhibited by these sera alone but elevated in nondialyzed (intact) FBS. Inosine at 10 microM stimulated 3H-thymidine incorporation from ten- to seventyfold in dFBS, CS, and dCS but was only slightly stimulatory in FBS and in L-15 alone. As well as inosine, hypoxanthine, cIMP, IMP, IDP, and ITP were just as stimulatory, but the nonsalvageable purines (xanthine, xanthosine, and XMP) were not. The stimulatory action of inosine was highest in low density cultures. Dipyridamole and S-(p-nitrobenzyl)-6-thioinosine (NBTI), inhibitors of facilitated nonconcentrative nucleoside transport, did not completely block the enhancement of cell number by inosine and by themselves increased proliferation in CS and dCS. Overall, these results suggest that exogenous inosine promoted CHSE-214 proliferation by overcoming factors in the nondialyzable fraction of sera that led to purine loss and by raising intracellular purine nucleotides to levels necessary for cells to respond to growth factors in the nondialyzable fraction of sera.

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Nucleoside uptake by red blood cells from a primitive vertebrate, the pacific hagfish (Eptatretus stouti), is mediated by a nitrobenzylthioinosine-insensitive transport system

Cited by 7 publications

References 27 publications

An ancient prevertebrate Na⁺-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti)

An ancient prevertebrate Na⁺-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti)

Na⁺-dependent transport of pyruvate in erythrocytes of the Pacific hagfish (Eptatretus stouti)

Enhancement of proliferation in cultures of Chinook salmon embryo cells by interactions between inosine and bovine sera

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Nucleoside uptake by red blood cells from a primitive vertebrate, the pacific hagfish (Eptatretus stouti), is mediated by a nitrobenzylthioinosine-insensitive transport system

Cited by 7 publications

References 27 publications

An ancient prevertebrate Na+-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti)

An ancient prevertebrate Na+-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti)

Na+-dependent transport of pyruvate in erythrocytes of the Pacific hagfish (Eptatretus stouti)

Enhancement of proliferation in cultures of Chinook salmon embryo cells by interactions between inosine and bovine sera

Contact Info

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Resources

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An ancient prevertebrate Na⁺-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti)

An ancient prevertebrate Na⁺-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti)

Na⁺-dependent transport of pyruvate in erythrocytes of the Pacific hagfish (Eptatretus stouti)