Cadmium transport by human Nramp 2 expressed in Xenopus laevis oocytes

doi:10.1016/S0041-008X(02)00078-9

Toxicology and Applied Pharmacology

Volume 187, Issue 3, 15 March 2003, Pages 162-167

https://doi.org/10.1016/S0041-008X(02)00078-9 Get rights and content

Abstract

Using the Xenopus oocyte expression system, human Nramp2, a human intestinal iron transporter, was shown to work as a cadmium transporter. An 1824-bp human Nramp2 cDNA was constructed by PCR cloning from reverse transcription products of human kidney mRNA. When the pH of the extracellular solution was 6.0, human Nramp2 transported ¹⁰⁹Cd²⁺. Substitution of external Cl⁻ with NO3− had no effect on human Nramp2-dependent cadmium uptake. The concentration-dependent Cd²⁺ transport of human Nramp2 indicated Michaelis–Menten type transport with an average K_m value of 1.04 ± 0.13 μM and an average V_max of 14.7 ± 1.9 pmol/oocyte/h (n = 3). Cd²⁺ transport via human Nramp2 was inhibited significantly by Cd²⁺, Fe²⁺, Pb²⁺, Mn²⁺, Cu²⁺, and Ni²⁺, while it was not inhibited by Hg²⁺ and Zn²⁺. Transport of 0.1 μM Cd²⁺ by human Nramp2 was inhibited by metallothionein (IC50 = 0.14 μM). Therefore, human Nramp2 is suggested to function as a pH-dependent cadmium absorption transporter on the luminal membrane of human intestinal cells.

Introduction

Cadmium (Cd) is a highly toxic metal and has been recognized as an environmental contaminant that may enter the food chain. Thus, the mechanisms of intestinal cadmium absorption need to be elucidated for understanding cadmium toxicity to the human body. Since it is assumed that the mammalian organism has not developed specialized intestinal absorption mechanisms for nonessential metals, it is generally considered that uptake of cadmium occurs via pathways also used by essential trace metals. Intestinal uptake of cadmium is inhibited by zinc (Foulkes, 1991) and iron (Sullivan and Ruemmler, 1987). Natural resistance-associated macrophage protein 2 (Nramp2) is thought to be the main transporter that absorbs iron at the brush-border membrane of mammalian intestine (Andrews, 2000). Rat Nramp2, also designated as DCT1 or DMT1, was identified as a proton-coupled metal ion transporter, which demonstrated Fe²⁺, Zn²⁺, and Cd²⁺ transport electrophysiologically (Gunshin et al., 1997). Iron transport by human Nramp2 expressed in Chinese hamster ovary (CHO) cells has been measured using the fluorescent, metal-sensitive dye calcein and was inhibited by Cd²⁺ (Picard et al., 2000). Human Nramp2 was reported to be localized to the brush border of human duodenum by immunohistochemistry (Griffiths et al., 2000). Up-regulated Nramp2 expression in iron-deficiency status (Han et al., 1999) is coincident with increased cadmium absorption in humans with iron deficiency (Flanagan et al., 1978). Therefore, human Nramp2 is a candidate for the cadmium transporter at the brush border of human duodenum.

Elisma and Jumarie (2001) reported that the specific uptake of ¹⁰⁹Cd by human enterocyte-like Caco-2 cells increased fourfold as extracellular pH was lowered from 7.5 to 5.5. Furthermore, this ¹⁰⁹Cd transport was characterized as a saturable system (K_m = 1.1 ± 0.1 μM, V_max = 87 ± 3 pmol/3 min/mg protein). An excess of Fe²⁺ failed to affect ¹⁰⁹Cd uptake when the extracellular pH was 7.4, whereas a strong inhibition was observed when the extracellular pH was 5.5. In contrast, the maximal inhibitory effect of Zn²⁺ was observed when extracellular pH was 7.4. Therefore, the authors concluded that Fe²⁺ might compete with Cd²⁺ for Nramp2, whereas Zn²⁺ competes for a different system of cadmium transport in the Caco-2 cell.

The purpose of the present study is to confirm and characterize ¹⁰⁹Cd transport by human Nramp2 molecule in acidified extracellular conditions using the Xenopus oocyte expression system. The results suggest that human Nramp2 is one of the transporters that absorb cadmium at the brush-border membrane of the human intestine.

Section snippets

Construction of human Nramp2 cDNA

Oligonucleotide primers (5′-GGC GGC GTG TCA GGT GGT TGC GGA GCT GGT AAG AAT CAT AT-3′ and 5′-GCA GGT AGC CAT CAG AGC CAG TGT GTT TCT ATG GTT TAC TGT GTG-3′) based on the human Nramp2 sequence (Genbank Accession no. NM000617) were used for RT–PCR amplification with the Advantage-HF2 PCR kit (BD Biosciences Clontech, Palo Alto, CA) from human kidney poly(A)⁺ RNA (BD Biosciences Clontech), which had been reverse transcribed ahead by the Advantage-2 reverse transcription kit (BD Biosciences

pH dependency and influence of NO₃ substitution of human Nramp2-dependent cadmium transport

Figure 1 indicates the influence of extracellular pH and NO3− anion on cadmium transport using human Nramp2 RNA-injected and noninjected X. laevis oocytes. When the pH of the extracellular solution was 7.4, the ¹⁰⁹Cd transport by human Nramp2 RNA-injected oocytes did not differ from the intrinsic ¹⁰⁹Cd transport by noninjected oocytes. Intrinsic ¹⁰⁹Cd transport by noninjected oocytes was attenuated at an extracellular pH of 6.0, whereas ¹⁰⁹Cd transport by human Nramp2 RNA-injected oocytes was

Discussion

Cadmium transport by human Nramp2 was not observed when extracellular pH was 7.4. Therefore, cadmium uptake by Caco-2 cells (Elisma and Jumarie, 2001) at pH 7.4 did not respond to human Nramp2-mediated Cd²⁺ uptake. Orally administrated cadmium chloride was found to be deposited in the duodenum of rats (Phillpotts, 1979) and mice (Sorensen et al., 1993). Since intraluminal pH of human duodenum is reported to be between 5.0 and 6.0 at the duodenojejunal junction (Ovesen et al., 1986), human

Acknowledgements

The authors thank Ms Akie Toki for her technical assistance. This research was supported by Comprehensive Research on Health Effects of Heavy Metals and Arsenic.

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Regular articleCadmium transport by human Nramp 2 expressed in Xenopus laevis oocytes

Abstract

Introduction

Section snippets

Construction of human Nramp2 cDNA

pH dependency and influence of NO3 substitution of human Nramp2-dependent cadmium transport

Discussion

Acknowledgements

Dig Liver Dis.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Gastroenterology

Toxicology

Toxicology

Eur. J. Pharmacol.

J. Nutr.

Gastroenterology

Toxicology

Regular article
Cadmium transport by human Nramp 2 expressed in Xenopus laevis oocytes

pH dependency and influence of NO₃ substitution of human Nramp2-dependent cadmium transport