To understand the roles that nucleoside transporters play in the in vivo distribution of clinically important nucleoside analogs, the substrate specificity of each transporter isoform should be determined. In the present work, we studied the substrate specificities of the human and rat orthologs of the Na+-dependent purine-selective nucleoside transporter (SPNT; concentrative nucleoside transporter 2), for nucleosides, nucleobases, and base- and ribose-modified nucleoside analogs. The two-electrode voltage-clamp technique in Xenopus laevisoocytes expressing these transporters was used. Purine nucleosides and uridine induced currents in oocytes expressing rat SPNT (rSPNT) or human SPNT1 (hSPNT1). The rank order of magnitude of nucleoside-induced currents was guanosine > uridine > adenosine > inosine and guanosine > uridine > inosine > adenosine for rSPNT- and hSPNT1-expressing oocytes, respectively. Uridine analogs (modified at the 5-position of the base) induced little or no current, suggesting that these compounds are only poorly transported by either transporter. Cladribine induced currents in oocytes expressing rSPNT (K 0.5 = 57 ± 12 μM) but not hSPNT1. The ribose-modified nucleoside analogs, adenine arabinoside, and 2′,3′-dideoxyadenosine induced currents in rSPNT-expressing, but not in hSPNT1-expressing, oocytes. These data suggest that there are notable species differences in the specificity of SPNT for synthetic nucleoside analogs.
- two-electrode voltage clamp
- sodium dependent
nucleoside analogs are clinically important in the treatment of various neoplasms and viral infections. Many of these compounds are hydrophilic and thus exploit endogenous nucleoside transporters to cross cell membranes. In particular, concentrative or Na+-dependent purine-selective nucleoside transporters [SPNT; or concentrative nucleoside transporters (CNTs)] present in intestinal, hepatic, and renal epithelia may play a role in the absorption or elimination of nucleoside analogs. Several subtypes of concentrative nucleoside transport activity have been characterized in the kidney. The N1 subtype prefers purine nucleosides as substrates but also transports uridine; N2 prefers pyrimidine nucleosides but also transports adenosine; N3 is broadly selective and transports both purine and pyrimidine nucleosides (26). Transporters with activities corresponding to the N1, N2, and N3 subtypes have been cloned from various species and include CNT1 (N1 subtype), CNT2 or SPNT (N2 subtype), and CNT3 (N3 subtype) (2, 5, 7, 11, 14, 16, 19, 20, 27,30). Rat CNT1 and rat SPNT (rSPNT) were recently localized to the brush-border membrane (BBM) of a stably transfected kidney cell line (13). This work corroborated earlier work using BBM vesicles prepared from kidneys of several species in which concentrative nucleoside transport processes were localized to the BBM (10, 28, 29). Localization of these concentrative transporters to the BBM suggests a role of reabsorption of nucleosides and nucleoside analogs in the kidney.
To understand the roles of the individual CNTs in drug absorption or reabsorption as well as tissue-specific distribution, it is necessary to determine the substrate specificity of each transporter. Because these transporters are electrogenic, the two-electrode voltage-clamp method can be used to assess their specificities (1, 3, 12,15). This method has two major advantages over conventional isotopic uptake methods. First, the method clearly distinguishes between inhibitors of transport and actual substrates (9). Second, radiolabeled compounds are not required; therefore, specificity can be assessed for a wide array of compounds.
The goal of this study was to determine the substrate specificities of the rat and human orthologs of SPNT. This transporter is particularly interesting because it is located in the intestine, liver, and kidney as well as in a variety of other tissues, suggesting that it may play a role in the absorption, elimination, and tissue-specific targeting of nucleoside analogs (2, 27). Furthermore, many nucleoside analogs are purine nucleoside derivatives and may interact with the transporter. For these studies, we focused on three aspects of specificity. First, we determined the specificity of the SPNT orthologs for naturally occurring nucleosides and nucleobases. Second, we assessed specificity for base-modified nucleoside analogs. Finally, we evaluated the specificity for ribose-modified analogs. Our findings suggest that there are profound interspecies differences in the specificities of the transporters for both naturally occurring nucleosides and synthetic analogs. In general, the rat ortholog accepted a wider array of substrates compared with the human ortholog.
MATERIALS AND METHODS
Expression in Xenopus laevis oocytes.
Oocytes were isolated from oocyte-positive X. laevis. The oocytes were dissected and treated with collagenase D (Boehringer Mannheim) in Ca2+-free ORII medium to remove the follicular layer. Healthy, defolliculated stage V and VI oocytes were maintained at 18°C in Barth's medium. The cDNA coding for rSPNT or human SPNT1 (hSPNT1) was subcloned into an amphibian high-expression vector, pOX (8), and linearized with NotI. cRNA was transcribed and capped in vitro with T3 RNA polymerase by using an mCAP RNA capping kit (Stratagene). Fifty nanoliters of cRNA (∼1 μg/μl) were injected into healthy stage V or VI oocytes (6).
Two-electrode voltage clamp.
Electrophysiological analysis using a two-electrode voltage clamp was carried out as described previously (3). Briefly, healthy oocytes injected with cRNA for rSPNT or hSPNT1 or uninjected were voltage clamped (GeneClamp 500B) at a membrane potential of −50 mV unless otherwise noted. The oocyte was superfused with Na+buffer until a stable baseline current was achieved. The oocyte was then superfused for ∼30–60 s with Na+ buffer containing a test compound. After exposure to a compound, the oocyte was washed with Na+-free buffer (NaCl replaced with choline chloride) followed by Na+ buffer until a stable baseline was obtained.
Currents from a given oocyte were normalized to the guanosine current induced in that same oocyte. Normalized data from at least two oocytes from 2–3 donor frogs were pooled and plotted. Values are expressed as means ± SD. Data at varying concentrations of substrate from individual oocytes were fit to the Michaelis-Menten equation [I = (I max × S)/(K 0.5 + S), in whichI is equal to the current, I max is the maximal current, K 0.5 is the concentration at the half-maximal current, and S is substrate concentration]. Statistical comparisons were made with a Student's unpairedt-test. Results were considered statistically significant with a probability of P < 0.05.
Figure 1 shows representative currents induced in oocytes expressing either rSPNT or hSPNT1 or in an uninjected control oocyte. Significant currents were induced by saturating concentrations (500 μM) of guanosine, inosine, adenosine, and uridine, but not cytidine and thymidine (125 μM), in Na+ buffer in both hSPNT- and rSPNT-expressing oocytes. No currents were induced by any of the nucleosides in uninjected oocytes. The currents observed in each oocyte were normalized to the guanosine currents in that oocyte. Data pooled from 6–8 oocytes are shown in Fig. 2 and are consistent with the currents shown for the single oocyte in Fig. 1. Guanosine-induced currents were voltage dependent in oocytes expressing rSPNT (data not shown). Only small currents (∼10% of the currents induced in the presence of Na+) were induced by guanosine when Na+ was replaced with Li+ or choline in the superfusion buffer (data not shown).
A series of naturally occurring adenosine analogs, including adenine, AMP, ADP, and ATP, as well as 2′-deoxyadenosine were studied (Fig.3). Adenine, a nucleobase, did not induce a current in oocytes expressing either rSPNT or hSPNT1, suggesting that it is not a substrate of either of these transporters. 2′-Deoxyadenosine induced a current similar in magnitude to that of adenosine in oocytes expressing hSPNT1 and greater in magnitude than adenosine in oocytes expressing rSPNT. Interestingly, 5′-AMP was found to induce significant currents in oocytes expressing rSPNT but not in those expressing hSPNT1. Only very small currents were induced by 5′-ADP and 5′-ATP. No currents were induced by any of these compounds in uninjected oocytes.
A series of base-modified and ribose-modified nucleoside analogs were studied. Uridine, which is a substrate of SPNT, and thymidine, which is not a substrate, are structural analogs of each other; thymidine is deoxyuridine methylated at the 5-position of the base. We therefore tested whether any moieties other than hydrogen were allowable at this position by measuring the currents induced by a series of analogs with different halogen groups or a hydroxy-methyl at this position (Fig.4). 5-Chloro-deoxyuridine and 5-bromo-deoxyuridine induced small currents relative to the currents induced by uridine, suggesting that they are only poorly transported by both rSPNT and hSPNT1. 5-Fluoro-deoxyuridine, 5-iodo-deoxyuridine, and 5-hydroxymethyl-deoxyuridine induced no detectable current in oocytes expressing either rSPNT or hSPNT1, suggesting that none of these compounds are substrates. The base-modified adenosine analog 2-chloro-2′-deoxyadenosine (2CdA) induced large currents in rSPNT-expressing oocytes. In contrast, only very small currents were induced by 2CdA in oocytes expressing hSPNT1. TheK 0.5 of 2CdA in interacting with rSPNT was 57 ± 12 μM (Fig. 5).
Several ribose-modified nucleoside analogs were studied to determine whether modifications to the ribose were tolerated (Fig.6). 2′,3′-Dideoxyinosine and 2′,3′-dideoxyadenosine (ddA) induced modest currents (∼15 and 30%, respectively, of the current induced by guanosine) in oocytes expressing rSPNT. In contrast, ddA induced no detectable current in oocytes expressing hSPNT1, and only small currents were induced by 2′,3′-dideoxyinosine (∼5% of the current induced by guanosine). Interestingly, 2′-deoxyadenosine induced substantial currents in both rSPNT- and hSPNT1-expressing oocytes. The currents induced by 2′-deoxyadenosine in rSPNT-expressing oocytes were larger (approximately double) than those induced by adenosine but similar to those induced by adenosine in hSPNT1-expressing oocytes. Adenine arabinoside (Ara-A), another ribose-modified adenosine analog, differs from adenosine by virtue of its 2′ and 3′ hydroxyl groups being in atrans configuration rather than a cisconfiguration. In our studies, Ara-A produced a current of ∼70% of that induced by guanosine and about equal to adenosine in rSPNT-expressing oocytes, but it produced no detectable current in hSPNT1-expressing oocytes or in uninjected control oocytes.
In this study, we employed electrophysiological methods to determine the specificities of rSPNT and hSPNT1 with respect to1) naturally occurring nucleosides, 2) base-modified nucleoside analogs, and 3) ribose-modified nucleoside analogs. Our results demonstrating that both rSPNT and hSPNT1 preferred purine nucleosides and uridine corroborate previous studies using isotopic uptake methods (2, 21, 22, 27). Interestingly, we observed that guanosine was the preferred substrate of both orthologs of SPNT (Fig. 2). Guanosine is important for the cell through its role as a precursor to GTP, which provides the driving force for signal transduction pathways via G protein-coupled receptors. Salvage of guanosine from extracellular sources for GTP synthesis is much less costly in terms of energy compared with de novo synthesis of GTP, which involves multistep pathways (32). Guanosine may be preferred over inosine and adenosine because de novo GTP synthesis requires more energy than de novo synthesis of ATP or ITP. Interestingly, adenosine has a higher turnover rate for rSPNT compared with inosine, and the converse is true for hSPNT1.
Our data suggest that 5′-AMP is a substrate of rSPNT but not hSPNT1, whereas 5′-ADP and 5′-ATP are not significantly transported by either SPNT ortholog. ATP and ADP are ligands of the P2 subtype of purine receptors, whereas AMP and adenosine are not (18,31). Ecto-nucleotidases (ecto-ATPase, ecto-ADPase, and 5′-nucleotidase) terminate the signal of P2 receptors by converting ATP to adenosine in three steps with the conversion of AMP to adenosine being rate limiting (4, 23, 24). rSPNT and the P2Y receptor are both expressed in several tissues, including heart, liver, and spleen; thus rSPNT may play a role in the recycling of AMP and adenosine produced by ecto-nucleotidases.
Our laboratory has previously shown that antiviral uridine analogs with substitutions in the 5-position of the base (e.g., floxuridine) are substrates of the pyrimidine-selective nucleoside transporter from rat (rCNT1) (3). In another study, it was demonstrated that one of these base-modified pyrimidine nucleosides, floxuridine, moderately inhibited inosine uptake in human intestinal BBM vesicles; however, because inhibition was used, it was not known whether floxuridine was a substrate. Our data clearly indicate that floxuridine is not a substrate but only an inhibitor. In contrast to the previous work with rCNT1, we observed that substitutions at the 5-position of uridine result in compounds that are poorly translocated by both rSPNT and hSPNT1. These data, when taken with the previous rCNT1 study, suggest that in tissues in which both CNT1 and SPNT are present, CNT1 preferentially transports 5-uridine analogs, whereas both transporters transport uridine.
Cladribine, important in the treatment of hairy cell leukemia, has been previously shown in isotopic uptake studies to be a good substrate and potent inhibitor of rSPNT (IC50 of 13.8 μM in inhibition of uptake of 1 μM inosine) and a poor substrate and inhibitor of hSPNT1 (IC50 of 371 μM in inhibition of uptake of 1 μM inosine) (21, 22). In addition, cladribine, at high concentrations (1 mM), only weakly inhibited inosine uptake by human intestinal BBM vesicles (17). Our studies using electrophysiological methods are in excellent agreement with the prior work (Fig. 4) and suggest that hSPNT1 does not play a role in the intestinal absorption, renal reabsorption, or tissue disposition of cladribine.
Many important antiviral agents are ribose-modified nucleoside analogs, several of which were studied here to determine whether any are substrates of rSPNT or hSPNT1. Our data suggest that hSPNT1 is not important in the renal recovery or the delivery of antiviral ribose-modified nucleoside analogs to their target tissues. The studies with the adenosine analogs provide insight into the optimal configuration of the ribose group for transport. First, a 3′-hydroxyl group is necessary for transport by both SPNT orthologs. That is, no detectable currents were induced by ddA. Second, a 2′-hydroxyl group is accepted by both orthologs, but rSPNT prefers 2′-deoxyribose. Finally, the α-position of the 2′ hydroxyl is preferred by hSPNT1 (adenosine is transported whereas Ara-A is not) but not by rSPNT (both adenosine and Ara-A are transported equally well).
Our data suggesting that there are differences in the substrate selectivities of rSPNT and hSPNT1 may help to predict species differences in the disposition of nucleoside analogs. Namely, our data indicate that rSPNT is more permissive of base or ribose modifications than is hSPNT1 (e.g., rSPNT transports Ara-A and 2CdA whereas hSPNT1 does not). An alignment of the transmembrane domain 7–8 region (amino acids 296–358 of hSPNT1; Fig.7), shown previously to be critical in substrate discrimination (25), shows that there are five differences in amino acid sequence between these two transporters. It is possible that one or several of these in combination may be responsible for the selectivity differences between the SPNT orthologs.
This work was supported by National Institute of General Medical Sciences Grant GM-42230.
Address for reprint requests and other correspondence: K. M. Giacomini, 513 Parnassus, Rm. S926, Univ. of California, San Francisco, CA 94143 (E-mail:).
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February 19, 2002;10.1152/ajprenal.00274.2001
- Copyright © 2002 the American Physiological Society