Purines, and in particular hypoxanthine, are essential nutrients that the intracellular malaria parasite requires for growth and multiplication [17, 29]. Since parasites are only able to obtain these nutrients from the host milieu through salvage, availability of detailed information of the transporters involved in their uptake is crucial for the development of purine-based anti-malarial drugs. Much progress has been made in the last few years in identifying and characterizing purine transporters on the P. falciparum cell surface [1, 30, 31], and at least one of these transporters seems to be essential for growth at physiological purine concentrations .
The data presented in this manuscript clearly show that hypoxanthine enters P. falciparum-infected human erythrocytes overwhelmingly through a saturable, adenine-sensitive transport mechanism, which is compatible with the human FNT1 nucleobase transporter, but not with the properties reported for NPP [10, 11]. Adenosine similarly enters predominantly through a saturable transporter, consistent with hENT1. The presence of these transporters on the plasma membrane of non-infected RBC has previously been reported . Given the complexity of nutrient transport in the human erythrocyte, especially after Plasmodium invasion, it is imperative that a systematic approach be employed in order to obtain a genuine conclusion. Therefore, using classical transport inhibitors such as NPPB, furosemide and NBMPR, non-metabolized substrates such as uridine, and competitive inhibitors such as adenine for hypoxanthine uptake, the various pathways involved in purine uptake into P. falciparum-infected RBC were systematically dissected.
Prior to their use, the selectivity and IC50 values of these inhibitors was re-assessed to confirm which transporters are blocked and at what concentration. Uptake of sorbitol, a well-known substrate of the NPP [11, 23], into infected RBC was almost fully blocked by NPPB. This uptake could not have occurred through any of the endogenous transporters, as there was no sorbitol uptake in uninfected erythrocytes. Similarly, it was verified whether known NPP inhibitors affected transport through the endogenous nucleoside and nucleobase transporters. NPPB did not affect either carrier, whereas furosemide was a good inhibitor of hFNT1, but not of hENT1. Although the mechanism for the furosemide effect on hFNT1 was not further investigated, the finding is potentially important, particularly towards efforts in search of purine-based antiplasmodial compounds, as similar concentrations of furosemide would inhibit both the NPP and hFNT1. The furosemide inhibition of [3H]-hypoxanthine and [3H]-adenine uptake reported here was clearly not through inhibition of the NPP as it was equally observed in infected and uninfected RBC. In view of the above inhibitor profile, it is evident that purine uptake in infected erythrocytes, being saturable and NPPB-insensitive, is mostly mediated by the human transporters hENT1 and hFNT1.
There is no doubt that nucleosides and nucleobases can enter the infected RBC through the furosemide/NPPB-sensitive new permeation pathways (NPP). For example, Gero and co-workers showed residual uptake of d-adenosine in infected human erythrocytes in the presence of the hENT1 inhibitor NBMPR , estimated at 20% of the uninhibited uptake rate , although this was not observed in the present study (Figure 2A). Kirk and colleagues  similarly demonstrated the NBMPR-insensitive, but partly furosemide and NPPB-sensitive, influx of adenosine and thymidine. However, that study used a concentration of 1 mM extracellular nucleoside, whereas the present study used a more physiological concentration of 0.5 μM. Whilst the higher concentrations were useful to demonstrate that nucleoside uptake through NPP is possible, the present investigation has sought to address the question whether the intracellular parasite is dependent on the NPP for purine uptake at physiological concentrations, which range from 0.1 - 1 μM for both adenosine and hypoxanthine [33, 34]. Hypoxanthine uptake in P. falciparum-infected RBC was recently demonstrated, at 150 μM, to be ~50% inhibited by dantrolene, a newly discovered inhibitor of NPP, but the effect of dantrolene on hFNT1-mediated transport was not tested . As it is here reported that furosemide also inhibits both the hFNT1 and NPP transport pathways, the possibility of similar action by dantrolene should not be discounted.
Notwithstanding the above, some of the reports from the group of Gero reported the NBMPR-insensitive uptake of d-adenosine at 1 μM [8, 36]. They further showed uptake of l-adenosine and l-thymidine by P. falciparum-infected human erythrocytes, although these non-physiological enantiomers were not substrates for hENT1 [9, 36]. Instead, uptake of l-nucleosides uptake was non-saturable and sensitive to furosemide  and NPPB , indicative of NPP. The potential of the NPP to take up low levels of purines, perhaps indiscriminately, may be of pharmacological importance, allowing the entry of toxic enantiomers and analogues selectively into the infected erythrocytes only.
Gero and colleagues also observed  that nucleoside transport inhibitors, including NBMPR, dilazep and dipyridamole all display intrinsic activity against P. falciparum in vitro, at concentrations that inhibit hENT1 . This appears to show that inhibition of hENT1 leads to purine starvation of the developing parasite, which would be consistent with the observation that the rate of adenosine uptake is considerably higher than the rate of hypoxanthine uptake in infected RBC. Hypoxanthine is the preferred purine source for P. falciparum [17, 29], but since adenosine is rapidly converted to hypoxanthine in the cytosol of the infected erythrocyte , it may be that inhibitors of hENT1 should have at least a growth-delaying effect on P. falciparum in vivo and that a combination with an inhibitor of hFNT1 would be lethal. It must be emphasized, however, that such a strategy would also deprive the uninfected erythrocytes of a purine source and that, while erythrocytes do not need purines for nucleic acid synthesis, their energy balance would be affected. However, it is likely that infected erythrocytes will be far more severely affected as their intracellular purine stores would be rapidly depleted by the highly efficient P. falciparum purine salvage system . Yet, it is not certain that the above hENT1 inhibitors perform their anti-malarial activity by blocking the human adenosine transporter. For instance, Carter et al  report that the main P. falciparum nucleoside transporter, PfNT1, is 85% inhibited by 10 μM dipyridamole, although Parker et al  in a similar study did not find PfNT1 sensitive to dipyridamole. Furthermore, Gero and colleagues present clear evidence that NBMPR, in particular, is internalized and metabolized by the parasites . Thus, while not transported by hENT1, the transport inhibitors could enter the Plasmodium-infected cells through NPP, and either inhibit the parasite's own purine transporters on its plasma membrane, or attack an intracellular target.
The data presented in the current manuscript show that uptake of adenosine in infected erythrocytes was almost completely inhibited by NBMPR and fully saturable - features which are inconsistent with the properties of the NPP. It may therefore be concluded that the NPP plays only a minor role in the salvage of this nucleoside in infected RBC. The observation that the rate of adenosine uptake in infected RBC was identical in the presence or absence of 100 μM of the NPP-inhibitor furosemide further supports this conclusion. Owing to the high rate of adenosine transport in erythrocytes, the equilibrative nature of hENT1, and the rapid metabolism of adenosine inside both infected and non-infected RBC, it is extremely difficult to measure true initial rates of [3H]-adenosine transport accurately in this system , and the data presented here refer to uptake, being the sum of transport and metabolism, rather than transport. However, the authors believe the conclusion that influx of adenosine is mediated by hENT1 rather than NPP is completely justified. To further validate these observations, the experiment was repeated using uridine as permeant, which is a substrate of hENT1, but not metabolized by human erythrocytes  or salvaged by P. falciparum . The results show that, like adenosine, transport of uridine (25 μM) is overwhelmingly mediated by hENT1, being saturable and not inhibitable by NPPB.
This study clearly demonstrated that uptake of the nucleobases in infected human erythrocytes is also mainly through the exogenous hFNT1 transporter despite the presence of the NPP. As reported by Domin and colleagues, the non-infected human erythrocyte readily takes up adenine and hypoxanthine by facilitated diffusion through hFNT1 and, using ice-cold papaverine to instantly stop transport, it is possible to measure initial rates of transport over a brief but sufficiently long period . In Figure 5A, the highest level of hypoxanthine uptake corresponds to an intracellular concentration of 0.39 μM, using the estimated volume of 75 fL for infected human erythrocytes reported by Saliba and co-workers , compared to an extracellular concentration of 1 μM and had thus not reached equilibrium. The uptake was clearly saturable and the response to NPPB was variable, resulting in a small increase in the uptake rate (Figure 5A) or, no difference, or a minor inhibition (Figure 5B). In addition, the study shows that initial rates of transport were completely inhibited by adenine (Figure 6), which cannot be explained either in terms of [3H]-hypoxanthine transport through NPP, or through effects on hypoxanthine metabolism: adenine does not intersect with hypoxanthine metabolism either in the erythrocyte or inside the parasite [37, 39].
An interesting observation made in the current study is the apparent increase in the uptake rate of these nucleosides into infected RBC compared to uninfected erythrocytes. This was observed consistently for hypoxanthine, adenosine and uridine. While it is to be expected that infected RBC will take up more purines due to greater demand for nucleic acid synthesis from the intracellular parasite, these observations appear to reflect increased transport capacity, rather than increased usage. This is particularly obvious for uridine, which is not metabolized by Plasmodium. While this phenomenon has been reported previously for other nutrients, including tryptophan and choline (reviewed by Kirk and colleagues) , this had not yet been reported for purine transport across the infected erythrocyte membrane. The mechanism by which Plasmodium species increase nutrient uptake by host carriers is still a subject of intense debate.