Arsenate transport by sodium/phosphate cotransporter type IIb

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Abstract

Arsenic is a metalloid that causes the dysfunction of critical enzymes, oxidative stress, and malignancies. In recent years several transporters of AsIII have been identified, including aquaglyceroporins (AQP) and multidrug resistance proteins (MRP). AsV transport, however, has not been sufficiently studied because it has been assumed that arsenate is taken up by mammalian cells through inorganic phosphate (Pi) transporters. In this paper we have analyzed the role of Pi transporters in the uptake of arsenate by directly using 73AsV as a radiotracer in phosphate transporter-expressing Xenopus laevis oocytes. The affinities of Pi transporters for H3AsO4 were lower than the affinities for Pi. NaPiIIa, NaPiIIc, Pit1, and Pit2 showed a Km for arsenate that was > 1 mM (i.e., at least ten times lower than the affinities for Pi). The NaPiIIb isoform showed the highest affinity for AsV in mouse (57 μM), rat (51 μM), and human (9.7 μM), which are very similar to the affinities for Pi. Therefore, NaPiIIb can have a prominent role in the toxicokinetics of arsenic following oral exposure to freshwater or food contaminated with AsV.

Introduction

Arsenic is a major public health problem and a major concern as an environmental contaminant. Chronic arsenic exposure causes skin lesions, vascular disease, hypertension, and cancer (Sams et al., 2007). Exposure to inorganic arsenic occurs most frequently in contaminated freshwater. Worldwide, millions of people are exposed to arsenic in drinking water that exceeds the World Health Organization standard of 10 μg/l.

Arsenic is a metalloid with two biologically important oxidation states, AsV and AsIII. In water at neutral pH, arsenic is present as arsenious acid As(OH)3 (Oremland & Stolz, 2003, Ramírez-Solís et al., 2004) and as arsenic acid H3AsO4 (iAsV), an analogue of inorganic phosphate.

The cellular pathways for metabolizing and transporting inorganic arsenic are complex and incompletely defined (Kumagai and Sumi, 2007). The fate of AsIII and AsV in the body involves biotransformation by glutathione conjugation, reduction and methylation (Scott et al., 1993, Lin et al., 2002, Hayakawa et al., 2005), and excretion of the parent compound and metabolites into urine and bile (Gregus et al., 2000).

In recent years, the mechanisms for trivalent arsenic uptake into cells have been described. The Escherichia coli glycerol facilitator GLpF, a member of the aquaporin superfamily, was the first transporter of As(OH)3 to be identified (Meng et al., 2004). Later, the GLpF homolog Fps1p (Wysocki et al., 2001) and several hexose permeases (Liu et al., 2004a) were shown to be additional routes of As(OH)3 uptake in Saccharomyces cerevisiae. More recently, it has also been shown that the glucose transporter GLUT1 and that the aquaglyceroporins AQP7 and AQP9 are also involved in the uptake of As(OH)3 and of methylarsonous acid (CH3As(OH)2) in mammalian cells (Liu et al., 2002, Liu et al., 2004b, Liu et al., 2006a, Liu et al., 2006b).

The transport of arsenic metabolites has also been studied. For example, multidrug resistance proteins 1 (MPR1/ABCC1) and 2 (MRP2/cMOAT) extrude arsenic triglutathione [As(GS)3] outside the mammalian cell (Kala et al., 2000, Leslie et al., 2004). ArsB in E. coli (Meng et al., 2004) and Acr3p in S. cerevisiae are also arsenite extrusion systems, while the ABC transporter Ycf1p catalyzes the uptake of As(GS)3 into the vacuole (Ghosh et al., 1999).

With respect to pentavalent arsenic, several transport systems have been proposed in both prokaryotes and eukaryotes, all of them related to inorganic phosphate carriers. In E. coli the phosphate specific transporter, Pst, catalyzes arsenate uptake (Willsky and Malamy, 1980). Similarly, Pho86p participates in H3AsO4 uptake in the eukaryote S. cerevisiae (Bun-ya et al., 1996). Based on the molecular similarities between phosphate and arsenate, it has been generally assumed that arsenate is also taken up by inorganic phosphate transporters in mammalian cells (Rosen, 2002, Virkki et al., 2007), although this has not been fully demonstrated (Csanaky & Gregus, 2001, Villa-Bellosta & Sorribas, 2008, Villa-Bellosta & Sorribas, 2009).

In this paper, we have studied 73AsV uptake by mammalian phosphate transporters using the Xenopus laevis oocyte expression system. Five inorganic phosphate (Pi) transporters are known to catalyze the uptake of inorganic phosphate through the plasma membrane in mammalian cells. They are grouped into two families, namely type II and type III sodium/phosphate cotransporters (Virkki et al., 2007, Villa-Bellosta & Sorribas, 2008). Type II cotransporters include the major Pi transporters in the epithelia of the kidney (NaPiIIa and NaPiIIc) and the intestine, liver, lung, osteoblasts, etc. (NaPiIIb) (Villa-Bellosta & Sorribas, 2008, Villa-Bellosta & Sorribas, 2010). At a subcellular level, type II transporters are expressed in the apical, brush-border membrane of proximal tubular cells (NaPiIIa and NaPiIIc), enterocytes and cholangiocytes (NaPiIIb), as well as the canalicular membrane of hepatocytes (NaPiIIb) (Frei et al., 2005). Type III transporters, Pit-1 and Pit-2, are also retroviral receptors with ubiquitous expression, and they have a structure that is similar to the phosphate transporters of all kingdoms of life (Villa-Bellosta & Sorribas, 2008, Villa-Bellosta & Sorribas, 2010, Villa-Bellosta et al., 2007). In epithelial cells, type III transporters are also expressed in the apical membrane of enterocytes (Villa-Bellosta & Sorribas, 2008, Giral et al., 2009) and of proximal tubular cells, as well as in the basolateral membrane of hepatocytes (Frei et al., 2005).

Section snippets

Chemicals

All chemicals used in this study were obtained from Sigma (Saint Louis, MO) or Fluka (St. Gallen, Switzerland). The H332PO4 radionuclide was from Perkin-Elmer (Waltham, MA), and the H373AsO4 (73AsV) was from Oak Ridge National Laboratory (Oak Ridge, TN). The cloning plasmid pPCR-Script was from Stratagene (La Jolla, CA). The ChargeSwitch Total RNA Cell kit, the Superscript III First-Strand Synthesis kit, and the Platinum Taq DNA Polymerase High Fidelity were all from Invitrogen. An in vitro

Characterization of iAsV transport in Xenopus oocyte-expressing rat sodium–phosphate cotransporters NaPiIIa, NaPiIIb, NaPiIIc, Pit-1, and PiT-2

10 ng of rat in vitro transcribed sodium/phosphate cotransporter (NaPi) cRNA was injected into X. laevis oocytes. After three days of expression-incubation, the oocytes were incubated in the presence of 50 µM 73iAsV for several time points. For all five rat Pi transporters, the uptake of arsenate was linear for at least 3 h (Fig. 1A). Uptake was strictly Na-dependent, given that no increase of 73iAsV transport was observed in the absence of Na+ in comparison to the water-injected oocytes (data not

Discussion

Based on the physicochemical similarities between inorganic arsenate (iAsV, H3AsO4) and inorganic phosphate (Pi, H3PO4), it has been generally assumed that arsenate crosses mammalian plasma membranes using Pi transporters (Rosen, 2002, Virkki et al., 2007). In addition, arsenate has been used traditionally as an experimental inhibitor of phosphate transport in biochemical assays. In all experimental models, however, the Ki values of arsenate on Pi transport were 5–40 times higher than the

Acknowledgments

This work was supported by a grant from the Spanish Ministry of Education and Science (BFU2006-06284/BFI to VS) and a predoctoral fellowship from the Government of Aragón, Spain (B086/2007 to RVB).

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