The Na+/I- symporter mediates active iodide uptake in the intestine

Abstract

Absorption of dietary iodide, presumably in the small intestine, is the first step in iodide (I(-)) utilization. From the bloodstream, I(-) is actively taken up via the Na(+)/I(-) symporter (NIS) in the thyroid for thyroid hormone biosynthesis and in such other tissues as lactating breast, which supplies I(-) to the newborn in the milk. The molecular basis for intestinal I(-) absorption is unknown. We sought to determine whether I(-) is actively accumulated by enterocytes and, if so, whether this process is mediated by NIS and regulated by I(-) itself. NIS expression was localized exclusively at the apical surface of rat and mouse enterocytes. In vivo intestine-to-blood transport of pertechnetate, a NIS substrate, was sensitive to the NIS inhibitor perchlorate. Brush border membrane vesicles accumulated I(-) in a sodium-dependent, perchlorate-sensitive manner with kinetic parameters similar to those of thyroid cells. NIS was expressed in intestinal epithelial cell line 6, and I(-) uptake in these cells was also kinetically similar to that in thyrocytes. I(-) downregulated NIS protein expression and its own NIS-mediated transport both in vitro and in vivo. We conclude that NIS is functionally expressed on the apical surface of enterocytes, where it mediates active I(-) accumulation. Therefore, NIS is a significant and possibly central component of the I(-) absorption system in the small intestine, a system of key importance for thyroid hormone biosynthesis and thus systemic intermediary metabolism.

Fig. 1.

Functional expression of Na⁺/I⁻ symporter (NIS) in mouse and rat small intestine. Small intestine was harvested and immunohistochemistry was carried out with 6.7 nM anti-NIS antibody as described in materials and methods. In all investigated sections, which included the entire small intestine of each animal, NIS-specific staining was visible in the apical membrane of the epithelial layer (arrows). Mouse (A, duodenum; B, jejunum; and C, ileum) and rat (D, duodenum; E, jejunum; and F, ileum). Magnification ×40 except in F (×100). G: NIS mediates intestinal ^99mTcO₄⁻ uptake in vivo. ^99mTcO₄⁻ alone (open bars) and ^99mTcO₄⁻ and NaClO₄ (100 to 580 nmol; solid bars) were administered at 2-h intervals to 4 rats via a duodenal catheter, and blood samples were collected as described in materials and methods. Shown is the 9-min time point. **P < 0.01 in unpaired t-test.

Fig. 2.

NIS mRNA and protein expression along the villus-crypt axis. A: total RNA was extracted from enterocytes sequentially isolated, in nine fractions, from the small intestine along the villus-crypt axis as described in materials and methods. Differential expression of NIS mRNA was analyzed by RT-PCR and standardized with respect to β-actin mRNA expression. Purity of the villus-crypt fraction separation was confirmed by analysis of the expression of alkaline phosphatase (ALP; a villus marker) and PCNA (a crypt marker) mRNAs. Densitometric ratios of NIS, PCNA, and ALP over β-actin expression are shown. B: ALP activity from villus-tip epithelial cells in fractions A (homogenate), B (nonpurified apical membranes), and C (enriched apical membranes). C: NIS immunoblot: lane 1, FRTL-5 cell membranes (10 μg); lanes 2-4, fraction A, B, or C (50 μg). Bottom: ezrin immunoblot as loading control after stripping anti-NIS antibodies. Boxes indicate different gels.

Fig. 3.

I⁻ uptake in rat small intestine brush border membrane vesicles (BBMV). A: steady-state I⁻ uptake assays (5-min time points; see inset for time course) in BBMV (50 μg) were carried out with 20 μM I⁻/140 mM Na⁺ (white bar), 20 μM I⁻/140 mM Na⁺/40 μM ClO₄⁻ (black bar), and 20 μM I⁻/140 mM choline (gray bar) as described in materials and methods. I⁻ transport displayed NIS-specific characteristics, i.e., Na⁺ dependence and ClO₄⁻ sensitivity. Inset: time course of I⁻ uptake in BBMV. Transport saturated at 5 min. B: initial rates (5-s time points) of I⁻ uptake were determined at the indicated I⁻ concentrations and a constant concentration of Na⁺ (140 mM) in the absence (solid line) or presence (dotted line) of 40 μM ClO₄⁻ as described in materials and methods. Data were processed using the equation v = V_max[I⁻]/[K_m + (I⁻)]. Data were fitted by nonlinear least squares using Gnuplot software. The background, corresponding to time point 0 for each concentration of I⁻, was subtracted. Nonspecific I⁻ binding was 38.4 ± 3.0% of the transport value. NIS exhibited a K_m value for I⁻ of 13.4 ± 2.0 μM and a V_max of 22.0 ± 0.9 pmol I⁻/mg protein. All kinetic parameters were determined at least in triplicate and are expressed as means ± SE.

Fig. 4.

Functional expression of NIS in epithelial cell line 6 (IEC-6) cells. NIS expression was assessed by immunoblot A: total protein extracts (10 μg) from FRTL-5 and IEC-6 cells; proteins were electrophoresed and electroblotted as described previously (15) and probed with 2.2 nM polyclonal anti-NIS antibody. B: NIS expression in IEC-6 cells analyzed by indirect immunofluorescence. Permeabilized IEC-6 cells were incubated with rabbit anti-NIS antibody (top), mouse anti-α₁-subunit of the Na⁺-K⁺-ATPase antibody (middle), and subsequently with Alexa-594-tagged anti-mouse IgG and Alexa-488-tagged anti-rabbit IgG antibodies as described in materials and methods. Overlay of the two images is shown at bottom. NIS is clearly localized at the plasma membrane of IEC-6 cells and colocalized with the Na⁺/K⁺ ATPase signal. C: steady-state (40 min) ¹²⁵I⁻ transport assay in FRTL-5 and IEC-6 cells performed with 20 μM I⁻/140 mM Na⁺ (open bars) or 20 μM I⁻/140 mM Na⁺/80 μM ClO₄⁻ (solid bars). D: initial rates (2-min time points) of NIS-mediated I⁻ transport in IEC-6 (solid line) or FRTL-5 (dotted line) cells were determined at varying concentrations of I⁻ and a constant concentration of Na⁺ (140 mM). Data were processed using the equation v = V_max[I⁻]/[K_m + (I⁻)]. Background values obtained in the presence of ClO₄⁻ were subtracted. Data were fitted by nonlinear least squares using Gnuplot software. All kinetic parameters were determined at least in triplicate and are expressed as means ± SE.

Fig. 5.

A high-I⁻ diet reduces intestinal I⁻ transport and NIS protein in vivo. Rats were provided water (control) or 0.05% KI-supplemented water. After the indicated times, BBMV were purified as described in materials and methods, and a steady-state I⁻ uptake assay was performed (A) with 50 μg protein and 20 μM ¹²⁵I⁻ alone (gray bars) or in the presence of 80 μM ClO₄⁻ (dark bars). B: BBMV (100 μg) were also used for immunoblot and probed with anti-NIS and, after stripping the nitrocellulose, anti-ezrin antibodies. Ezrin was probed as a loading control, as described in materials and methods. Boxes indicate different gels. C: quantitation of the NIS/ezrin densitometric signal was done with ImageJ software (National Institutes of Health, Bethesda, MD).