Nucleoside Transport in Erythrocytes from Bottle-Nosed Dolphin (Tursiops truncatus)

Comp. Biochem. Physiol. Vol. 117A, No. 1, pp. 127–134, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00256-3

Nucleoside Transport in Erythrocytes from Bottle-Nosed Dolphin (Tursiops truncatus) James D. Craik,1 James D. Young,2 and Christopher I. Cheeseman2 1

Chemistry Department, Bishop’s University, Lennoxville, Quebec J1M 1Z7, Canada, and 2 Membrane Transport Group, Physiology Department, Faculty of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada ABSTRACT. Entry of adenosine, and thymidine, into erythrocytes from adult dolphins was rapid, showed saturation at higher substrate concentrations, and was strongly inhibited by low concentrations of nitrobenzylthioinosine (NBMPR). Kinetic parameters were estimated from the concentration dependence of initial rates of tracer entry at 21°C, as Km 0.14 6 0.05 mM and Vmax 24.4 6 1.9 µ mol/litre cell water/sec for zero trans entry of adenosine, and Km 0.96 6 0.21 mM and Vmax 25.4 6 1.7 µmol /litre cell water/sec for thymidine. Adenosine, and thymidine, entry were inhibited by both purine and pyrimidine nucleosides. Mass law analysis of a saturable component of nitrobenzylthioinosine binding to dolphin red cell membranes gave values of Bmax 65.4 6 1.2 pmol/mg protein, and Kd of 1.53 6 0.08 nM for a single class of sites. Photo-irradiation of dolphin red cell membranes in the presence of tritiated nitrobenzylthioinosine led to radioactive labeling of polypeptides Mr 52, 500–58,000, on SDS-PAGE. comp biochem physiol 117A;1:127–134, 1997.  1997 Elsevier Science Inc. KEY WORDS. Nucleoside transport, nitrobenzylthioinosine, NBMPR, red blood cell, odontocetes

INTRODUCTION Red cells from adult mammals show a remarkable diversity in their permeability toward nucleosides. Species such as human, rabbit, pig, and mouse show rapid transport of nucleosides, while red cells from dogs, and most sheep, appear to be impermeable to these compounds (12). In human red cells, rapid entry of nucleosides is mediated by a facilitated diffusion pathway notable for a broad substrate specificity that includes both purine and pyrimidine nucleosides. This pathway is characterized by an acute sensitivity toward stoichiometric inhibition by nanomolar concentrations of nitrobenzylthioinosine (NBMPR) [see reviews by Paterson and Cass (19), and Plagemann and Wohlhueter (20)]. Red cells from adults from some other mammalian species demonstrate more complex transport phenotypes. Erythrocytes from the rat show a low permeability for nucleosides, and part of the saturable equilibrative nucleoside transport capacity exhibits a low sensitivity toward inhibition by NBMPR (requiring micromolar concentrations of NBMPR for an inhibitory effect). The sensitivity of equilibrative nucleoside transport systems to NBMPR forms the basis of a functional classification of nucleoside transporters in mammalian cells [es equilibrative and NBMPR-sensitive, ei equi-

Address reprint requests to: Dr. James D. Craik, Faculty Box 78, Chemistry Department, Bishop’s University, Lennoxville, Quebec J1M 1Z7. Tel. (819) 822-9600; Fax (819) 822-9661; E-mail: [email protected].

librative, and NBMPR-insensitive, in the nomenclature of Belt and co-workers (27)]. These es and ei transporters differ in their sensitivity to inhibitors other than NBMPR, for example, organomercurial compounds (13), and although polypeptides associated with high-affinity NBMPR binding have been identified in many species by photoaffinity labeling using the intrinsic photoreactivity of the NBMPR molecule (7,10), the molecular identity of ei transporters remains unknown. The great diversity in nucleoside transport capacity in erythrocytes from adult mammals suggests that nucleoside permeation may serve a variety of different physiological roles in different species. Red cells from adult bottle-nosed dolphins (Tursiops truncatus) show a very high permeability for d-glucose, and possess large amounts of the GLUT-1 isoform of the facilitated glucose transporter [Craik et al. (4) and unpublished results]. However, catabolism of d-glucose by dolphin erythrocytes incubated in vitro is markedly slower than in human red cells incubated under equivalent conditions, and a substantial portion of d-glucose degradation (about 9%) is via the pentose phosphate shunt rather than the glycolytic pathway (8). The closest terrestrial relatives of the odontocetes are thought to be the artiodactyls (ruminants, camels, pigs) (23) and erythrocytes from adult individuals from some of these species may use substrates other than d-glucose to support erythrocyte energy metabolism in vivo. Erythrocytes from adult pigs lack glucose transporter polypeptides in the plasma membrane (3) are unable to metabolize extracellular

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d-glucose (14), and rely on inosine, supplied by the liver, as their major source of metabolic energy (29,30). Nucleoside transport in red cells from bottle-nosed dolphins was examined in order to determine if a metabolic pathway analogous to that demonstrated in pig erythrocytes could contribute to the maintenance of dolphin red cell function through catabolism of plasma nucleosides. It was found that both purine and pyrimidine nucleosides can rapidly enter dolphin red cells, and that nucleoside entry is readily inhibited by NBMPR. However, dolphin red cell lysates showed only a very low activity of purine nucleoside phosphorylase which implies that inosine is unlikely to be a major source of metabolic energy in vivo for erythrocytes from T. truncatus. MATERIALS AND METHODS Chemicals and Solutions Nitrobenzylthioinosine, nucleosides, hypoxanthine, l-glucose, xanthine oxidase (grade III, from buttermilk), phenylmethylsulfonyl fluoride (PMSF) and Drabkin reagent were from Sigma Chemical Co. (St Louis, MO). [3H(G)] S(p-nitrobenzyl)-6-thioinosine (1.33 TBq/mmol), [2,8-3H] adenosine (1.44 TBq/mmol), [methyl-3H] thymidine (2.66 TBq/mmol) were from Moravek Biochemicals (Brea, CA). l-[1-14C] glucose (2.03 MBq/mmol) was from Amersham Canada (Oakville, Ontario, Canada). Phosphate-buffered saline solution (PBS) pH 7.4, contained 136.8 mM NaCl, 2.68 mM KCl, 4.29 mM Na2HPO4, and 1.47 mM KH2PO4. Cell lysis buffer was prepared from ice-cold 5 mM phosphate buffer, pH 8.0 by the addition of 0.1 mM PMSF from a stock solution (100 mM PMSF in anhydrous isopropanol) immediately before use. Zero Trans Entry of Nucleosides Uptake of adenosine and thymidine was determined by conventional isotopic tracer techniques. Intracellular radioactivity was measured following exposure of intact erythrocytes to solutions containing predetermined concentrations of d-adenosine or thymidine, and tracer amounts (about 1 µCi per ml) of tritiated nucleoside for short periods of time. Blood samples (in 7 ml heparinized tubes, 100 usp units of lithium heparin, Vacutainer (Beckton Dickinson, Rutherford, NJ), were washed five times (by centrifugation, 300 3 g, 5 min, and resuspension) with about 10 volumes of phosphate buffered saline, pH 7.4 to remove blood plasma and white cells, and to deplete the erythrocytes of endogenous nucleosides. Uptake was initiated by rapid addition of 100 µl of labeled nucleoside solution to 50 µl of red cell suspension in phosphate-buffered saline (PBS) (about 20% hematocrit) layered over an immiscible oil layer (butyl pthalate) in a 1.5 ml microcentrifuge tube. Influx was terminated after a predetermined incubation time by rapid addition of 250 µl of an inhibitor solution (100 µM dilazep in

PBS, ice cold) and immediate centrifugation in a benchtop microcentrifuge (Fisher model 235A) (12,000 3 g, 10 sec). Zero time points were obtained by addition of inhibitor solution immediately prior to addition of labeled nucleoside solution. The supernatant aqueous layer was removed by aspiration and the inside of the microcentrifuge tube was washed with distilled water. The walls of the tube were wiped dry and the oil layer was removed using absorbent cotton buds. The erythrocyte pellet was dispersed by the addition of 0.5 ml 0.5% (v/v) triton X-100 in distilled water. Protein was precipitated by addition of 0.25 ml 5% (w/ v) trichloroacetic acid solution, and pelleted by centrifugation (12,000 3 g, 30 sec). A 0.5 ml portion of the clear supernatant fluid was taken for scintillation counting after addition of 4 ml Ecolight (ICN Biochemicals) scintillation fluid. Water spaces in the pellet were determined from separate incubations of erythrocytes in which 3H2O (for total water space) or 14C-l-glucose (extracellular water space) replaced tritiated nucleoside tracer. Quench corrections were performed using an external standard method (Beckman LS 6500 scintillation counter). Initial rates of nucleoside entry were estimated from progress curves using a polynomial curve fit procedure, and transport parameters (and standard errors) were estimated from the concentration dependence of initial rates of entry using ENZFITTER nonlinear regression data analysis program (Biosoft, Cambridge, U.K.). Competition Assays The protocol described for zero trans entry was modified to include a high concentration (15 mM, except for guanosine, 3 mM) of a non-radioactive nucleoside in a medium containing a low concentration (50 µM) of adenosine, or thymidine, and radioactive tracer. Tracer uptake was determined in triplicate samples after 5-sec incubations. The limited aqueous solubility of guanosine prevented use of 15 mM concentrations in this assay. Preparation of Erythrocyte Plasma Membranes (Ghost Membranes) from Dolphin Erythrocytes Erythrocytes were collected and washed as described for the transport experiments. The loose red cell pellet was added to approximately 100 volumes of ice-cold lysis buffer, and stirred for about 3 min. This suspension was subjected to centrifugation (10,000 rpm, 15 min, 6°C, Sorvall SA-600 rotor) and the loose red cell membrane pellet washed four times with about 20 volumes of ice-cold 5 mM phosphate buffer, pH 8.0. Protein content of the membrane suspension was estimated by a dye-binding method, using protocols and reagents supplied by Bio-Rad Laboratories Inc. The membranes were stored in small portions at 270°C. Human red cell membranes were prepared from human blood using an identical protocol.

Nucleoside Transport in Dolphin Erythrocytes

NBMPR Binding Assays Reversible binding of tritiated nitrobenzylthioinosine (3HNBMPR) to saturable high-affinity sites associated with dolphin erythrocyte membranes was assayed in the presence, or absence, of a high concentration (4.5 µM) of unlabeled NBMPR. Portions (20 µl) of red cell membrane suspensions (1.6–2.6 mg protein/ml) were placed in 5 mm 3 20 mm polyallomer ultracentrifuge tubes (Beckman Airfuge, Beckman Instruments, Palo Alto, CA) together with 50 µl of PBS, or 16.6 µM NBMPR in PBS, and a small volume (5 µl) of 100 mM l-glucose solution containing 14C-l-glucose tracer was added. Graded amounts of 3H-NBMPR solution in distilled water, and distilled water, were added to give a final assay volume of 185 µl. The mixture was incubated for 90 min at room temperature before ultracentrifugation (Beckman Airfuge, 28 p.s.i.g., 15 min). Supernatant fluids were sampled (140 µl portions) and the concentration of free 3H-NBMPR estimated by scintillation counting (Ecolite scintillation fluid, ICN Biochemicals). Quench corrections were performed using an external standard method. The remaining supernatant liquid was carefully removed by aspiration, and the radioactivity associated with the membrane pellet was determined by scintillation counting, following incubation of the tubes in Ecolite scintillation fluid for 48 h. 14C-l-glucose tracer permitted correction of pellet counts for trapped incubation medium. Nonspecific binding of 3HNBMPR was estimated from tubes in which a large excess of unlabeled NBMPR was included, in addition to the tritiated ligand. Binding data was analyzed using ENZFITTER nonlinear regression data analysis program (Biosoft). Photolabeling of Erythrocyte Membranes with 3H-NBMPR Dolphin and human red cell membrane suspensions (approximately 1 mg protein per ml) in phosphate buffered saline medium containing 5.7 mM EDTA and 95 nM 3HNBMPR (with, or without, 4.75 µM unlabeled NBMPR), were incubated at room temperature for 40 min, then chilled on ice for 15 min. Samples were irradiated in disposable plastic semimicro cuvettes (DAL2022205, Fisher Scientific, Edmonton, Alberta) oriented so that light shone through a 5-mm light path at a distance of 7 cm from a water-jacketed 450-watt mercury lamp (Conrad Hanovia, NY) for 5 min. Following irradiation, dithiothreitol solution (0.5 M) was added to a final concentration of 10 mM, and the red cell membranes were recovered by centrifugation (90 sec, microcentrifuge), solubilized in SDS-PAGE sample buffer (incubation at 37°C for 2 min), then subjected to electrophoresis. SDS-PAGE and Blot Protocols Polyacrylamide slab gels (10% acrylamide) were prepared using standard protocols and reagents supplied by Bio-Rad

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Laboratories Ltd. (Mississauga, Ontario, Canada). Gels were run using the buffer system of Laemmli (15). Prestained molecular weight standards (Rainbow Markers, Amersham Inc., Canada), and unstained molecular weight standards (high molecular weight, Sigma Chemical Co., or low molecular weight, Bio-Rad Laboratories Ltd.) were included in separate lanes. For fluorography, gels were treated with Enhance fluorography cocktail (NEN Research Products, DuPont Canada Inc., Markham, Ontario, Canada) according to the manufacturer’s directions. Exposures of the treated and dried gels were performed at 270°C using Kodak XAR-5 x-ray film. In an alternative protocol, polypeptides were transferred to polyvinylidine difluoride membranes (Immobilon, Millipore Canada Inc., Napean, Ontario, Canada) or to nitrocellulose membranes (0.45 micron pore size, Bio-Rad Laboratories) using the buffer system of Towbin et al. (1979) (25 mM Tris, 192 mM glycine), with 5% (v/v) methanol. Transferred proteins were visualized using Ponceau S or Amido black stains. Radioactivity was detected by fluorography, as described above, after treating the polyvinylidine difluoride membrane with Enhance fluorography spray reagent (NEN Research Products) according to the manufacturer’s directions, or, in the case of proteins transferred to nitrocellulose membranes, the membrane was sectioned into 2-mm slices, dried, and dissolved by the addition of 0.3-ml dry acetone before counting in 5.0 ml Ecolite scintillation fluid (ICN Biochemicals). Red Cell Purine Nucleoside Phosphorylase Activity The relative activities of purine nucleoside phosphorylase in lysates derived from dolphin, human, rat, and rabbit erythrocytes, were assayed using a coupled xanthine oxidase spectrophotometric assay to follow the formation of uric acid (measured as an increase in absorbance at 293 nm) from inosine, as described by Duhm (5). Red cells were washed three times with PBS solution then disrupted by the addition of five volumes of 2 mM β-mercaptoethanol in icecold distilled water to loose-packed red cell pellets. After brief vortex mixing, membranes were sedimented from the hemolysate by centrifugation and purine nucleoside phosphorylase activity in the clear supernatant was measured at 20–22°C, pH 7.4 in a reaction mixture containing 90 mM KCl, 50 mM sodium phosphate, 4 µg/ml xanthine oxidase (1.3 units/mg protein) and 0.5 mM inosine. The reaction was started by addition of 2–5 µl of hemolysate, and the course of the reaction was followed for 2–6 min. The rate of uric acid production, from hypoxanthine, in assay mixtures from which inosine was omitted was not changed by addition of 2–5 µl volumes of red cell hemolysate. This confirmed that uricase was not active in these assay mixtures. Hemoglobin concentrations were estimated from the absorbance (measured at 540 nm) after 8-µl portions of the hemolysates were mixed with 2-ml volumes of Drabkin reagent.

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FIG. 1. Progress curve for the entry of thymidine into dolphin red cells at 21°C. Extracellular thymidine concentration was 0.172 mM. The broken line shows the initial rate of thymidine entry calculated from the fitted curve (solid line). In the presence of 4.4 mM NBMPR, entry of thymidine was 3.95% of the rate in the absence of the inhibitor.

RESULTS Zero Trans Entry of Nucleosides Figure 1 shows a progress curve for thymidine entry into dolphin erythrocytes and the inhibition of uptake by micromolar concentrations of NBMPR in the extracellular medium. Michaelis-Menten parameters of Km 0.14 6 0.05 mM and Vmax 24.4 6 1.9 µmol/l cell water/sec, and Km 0.96 6 0.21 mM and Vmax 25.4 6 1.7 µmol/l cell water/sec were estimated from the concentration dependence of initial rates of entry of adenosine and thymidine respectively (shown in Fig. 2). Competition Assays The results, shown in Table 1, demonstrated a broad specificity of the nucleoside permeation pathway towards inhibition by both purine and pyrimidine nucleosides. Inhibition of Nucleoside Transport by NBMPR

FIG. 2. Concentration dependence of zero trans entry of

Figure 3 shows the effect of graded concentrations of NBMPR on entry of thymidine into dolphin red cells. This

adenosine Km 0.14 6 0.05 mM and Vmax 24.4 6 1.9 mmol/l cell water/sec (panel A), and of thymidine Km 0.96 6 0.21 mM and Vmax 25.4 6 1.7 mmol/l cell water/sec (panel B) into dolphin erythrocytes at 21°C.

TABLE 1. Inhibition of adenosine and thymidine entry by

extracellular nucleosides Competing nucleoside (15 mM) Uridine Thymidine Adenosine Inosine Cytidine Guanosine (3 mM)

Adenosine uptake (% control)*

Thymidine uptake (% control)*

2.4 2.5 1.2 2.1 4.8 11.6

2.6 3.4 3.2 3.6 3.8 10.7

*No addition (control) was defined as 100%.

TABLE 2. Red cell purine nucleoside phosphorylase activity

(normalized to the activity of human red cell lysate) Species

Purine nucleoside phosphorylase (% human red cell activity)

Rabbit Dolphin* Rat* *Mean of three determinations.

115.1 7.4 10.6

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FIG. 3. Dose-response curve for the inhibition of thymidine entry into dolphin erythrocytes by NBMPR. Extracellular thymidine concentration was 0.172 mM.

dose-response curve shows that entry of thymidine is sensitive to inhibition by low concentrations of NBMPR. Adenosine entry was also readily inhibited by NBMPR (data not shown). NBMPR Binding Binding of tritiated NBMPR to isolated dolphin red cell membranes was consistent with presence of a single class of high affinity saturable binding sites. Mass law analysis of saturable binding allowed estimation of values for Bmax 65.4 6 1.2 pmol/mg protein, and K d of 1.53 6 0.08 nM for interaction of this ligand to a single class of sites (Fig. 4). Photolabeling of Erythrocyte Membranes with 3H-NBMPR Dolphin red cell membrane polypeptides of Mr 52,500– 58,000 showed specific labeling with tritiated NBMPR with minor labeling in the Mr 80,000–100,000 and 150,000 regions. Human red cell membranes, run in parallel lanes, showed labeling extending from Mr 52,000–70,000, with weaker labeling in the Mr 150,000 region (Fig. 5). Fluorography of blots confirmed that NBMPR-labeled polypeptides from dolphin red cells migrate as a narrow band on SDSPAGE, while labeled polypeptides from human erythrocytes migrate as a diffuse cluster of bands in the Band 4.5 region of the gel (Fig. 6). It is not known if the difference in radioactive label observed in human and dolphin red cell preparations was caused by differences in the efficiency of photolabeling, or resulted from differences in the efficiency of protein transfer during blotting. Purine Nucleoside Phosphorylase Activity Purine nucleoside phosphorylase activities (activity measured per unit of hemoglobin) were measured in lysates from

FIG. 4. Binding curve for saturable component of NBMPR

binding to a single class of high affinity sites associated with dolphin red cell membranes. Fitted curve is for Bmax 65.4 6 1.2 pmol/mg protein, and Kd of 1.53 6 0.08 nM. Binding was estimated for a range of NBMPR concentrations from 0.03 nM to 6.99 nM.

human, rabbit, dolphin, and rat erythrocytes. Table 2 shows red cell enzyme activities, normalized to the activity of a human erythrocyte lysate under these assay conditions. Purine nucleoside phosphorylase activity of dolphin red cells was markedly lower than that found in human or rabbit erythrocytes, and similar to that found in rat red cells. DISCUSSION This study demonstrates the rapid entry of a purine nucleoside, adenosine, and a pyrimidine nucleoside, thymidine, into dolphin erythrocytes. Entry was saturable and readily inhibited by low concentrations of nitrobenzylthioinosine (NBMPR), suggesting that much of this transport is mediated by an es transporter [nomenclature of Vijayalakshmi and Belt (27)]. The Km for adenosine entry into dolphin red cells under zero trans conditions [nomenclature of Stein (22)] agrees with a value of 0.126 mM determined for the entry of adenosine into sealed human red cell ghost membranes at 20°C using manual techniques (6) but is higher than that obtained for human erythrocytes at 22°C (Km range from 14–56 µM, for six individual donors) using a quenched flow method to directly determine initial rates of transport (18). The apparent Kd of saturable NBMPR binding to dolphin red cell membranes of 1.53 6 0.08 nM is close to that reported for similar studies of red cell membranes from human erythrocytes (Kd 1.0 nM (2)), from adult pigs (Kd of saturable NBMPR binding 1.3 nM for domestic pigs, 2 nM for

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FIG. 5. Profile of radioactivity found in dolphin erythrocyte

membranes (top panel), or human erythrocyte membranes (lower panel), following photoirradiation with tritiated NBMPR in the presence (hatched bars) or absence (clear bars) of a large excess of unlabeled NBMPR. Proteins were separated by SDS-PAGE and transferred to nitrocellulose, as described in Materials and Methods, before scintillation counting. Positions of molecular weight markers run in adjacent lanes are indicated; Mr 200,000 (A), 97,000 (B), 67,000 (C), 45,000 (D), 29,000 (E).

the Yucatan miniature pig (28,29)), and higher than that found in many cultured mammalian cells, and some other studies of red cell membranes (0.1–1.0 nM, reviewed by Cass (1)). The density of these binding sites, Bmax 65.4 6 1.2 pmol/mg erythrocyte membrane protein, is comparable to that found in red cells from adults of other mammalian species that show high permeability towards nucleosides (approximately twice the 20–40 pmol/mg protein found for pigs, (29)), and close to the number of binding sites on human red cell membranes (9,12). Values for Vmax for entry of adenosine and thymidine under zero trans conditions suggest that the turnover number for nucleoside transporters in dolphin red cells may be similar to those found in erythrocytes from other mammals under similar conditions (11). Photoaffinity labeling of dolphin red cell membranes

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FIG. 6. Fluorogram showing the distribution of radioactivity on a blot of human erythrocyte membranes (lanes A and B) and dolphin erythrocyte membranes (lanes C and D) following photoirradiation with tritiated NBMPR in the presence (lanes B and C) or absence (lanes A and D) of a large excess of unlabeled NBMPR. Proteins were separated by SDSPAGE, transferred to an immobilon membrane, and subjected to fluorography, as described in Materials and Methods. Positions of molecular weight markers (run in adjacent lanes of the SDS-PAGE gel) were determined by staining the blot with amido black, after removal of fluors by briefly soaking the membrane in ethanol.

with tritiated NBMPR demonstrated that dolphin erythrocyte NBMPR-binding polypeptides are less heterogeneous in their mobility on SDS-PAGE than the equivalent polypeptides from human red cells. A similar phenomenon has been observed for GLUT-1 equilibrative glucose transporter polypeptides in human and dolphin red cells (Craik et al., unpublished results) and could indicate a general difference in the glycosylation of integral membrane proteins in erythrocytes from these species. Although dolphin red cells show maximal velocities for nucleoside entry close to those reported for human red cells, purine nucleoside phosphorylase activity in dolphin red cell hemolysates was found to be low, comparable to that seen in rat erythrocytes, which show only a low transport capac-

Nucleoside Transport in Dolphin Erythrocytes

ity for exogenous nucleosides (11,13). Pig red cells, which rely on inosine as a source of metabolic energy, show high purine nucleoside phosphorylase activity, similar to that found in human erythrocytes [Sandberg et al. (21), Duhm (5)]. Sheep erythrocytes have a low permeability to dglucose (17) and show polymorphism of both nucleoside transport (red cells from most sheep are virtually impermeable to nucleosides) and purine nucleoside phosphorylase [an enzyme activity that shows no genetic linkage to nucleoside permeability, Tucker and Young (25,26)]. Red cells from individual sheep that demonstrated both low nucleoside transport capacity and low nucleoside phosphorylase activity showed decreased cellular adenosine triphosphate (ATP) levels, indicating that inosine may supplement glucose as an energy substrate in ovine erythrocytes in vivo (26). The high glucose permeability and relatively low purine nucleoside phosphorylase activity of dolphin erythrocytes suggest that inosine may have only a limited role as a substrate for dolphin red cell energy metabolism. However, adenosine production by the liver could be important for the maintenance of red cell purine nucleotide levels in dolphins, as suggested for the rabbit (16). This study demonstrates that dolphin red cells possess a nucleoside permeation pathway with functional properties that closely resemble those of the es transporter characterized in human erythrocytes. The high nucleoside transport capacity of dolphin red cells may represent a cellular adaptation that serves to optimize transfer of nucleosides between tissues, rather than a reflection of metabolic needs of the red cells themselves. This conjecture is difficult to test since the broad substrate specificity of the erythrocyte transport pathway precludes any simple prediction of the identity of the nucleosides involved, or of the physiological role(s) of inter-organ movements of these compounds in vivo. It remains to be seen if a high red cell nucleoside transport capacity is a physiological trait shared with other aquatic mammals. We are pleased to acknowledge the assistance of Mr. Mark Norman, Mr. Jerry Holik, and the staff of the Dolphin Lagoon, West Edmonton Mall, Edmonton, Alberta, Canada, in providing blood from samples taken in the course of routine veterinary checks on dolphins in their care. We thank Hoffmann-La Roche Ltd., Mississauga, Ontario, for a generous donation of dilazep hydrochloride (to JDC) and Dolores Mowles for assistance in the purification of nucleoside radiotracers by hplc. J.D.C. and J.D.Y. are grateful to the Natural Sciences and Engineering Research Council of Canada for financial support (Project Grants). JDY is a Medical Scientist of the Alberta Heritage Foundation for Medical Research.

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