Prolactin regulation of the pendrin-iodide transporter in the mammary gland

James A. Rillema and Melissa A. Hill

Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201-1928


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Iodide is an essential constituent of milk that is present in concentrations more than an order of magnitude higher than in the maternal plasma. Earlier, a sodium-iodide symporter was identified in the mammary gland; this transporter is presumed to take iodide from the maternal plasma into the alveolar epithelial cells of the mammary gland. We now report the existence of a second iodide transporter, pendrin, which is also essential for iodide accumulation in milk. Via Western blotting methods, high levels of the transporter were detected in lactating tissues; lesser amounts were found in tissues from midpregnant and virgin mice. Prolactin, at physiological concentrations, stimulated the expression of the pendrin transporter in cultured mammary tissues taken from 12- to 14-day-pregnant mice. The prolactin effect on iodide uptake into cultured mammary tissues was abolished by pendrin transport inhibitors, including DIDS, furosemide, and probenecid. These studies suggest that the prolactin stimulation of pendrin activity is an essential element in the prolactin stimulation of iodide uptake into milk.

iodide transport; pendrin; prolactin


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IODIDE IN MILK is of critical importance for the development and growth of the neonate. The iodide in milk is essential for the synthesis of thyroid hormones, which in turn regulate developmental processes (3, 4, 18). We and a number of other investigators (5, 6, 9-11, 16, 17) have characterized a sodium-iodide symporter that actively transports iodide from the maternal plasma into the alveolar epithelial cells of the mammary gland. The results of our earlier studies, however, suggested the presence of an additional iodide transport mechanism (9). Recently, Shennan, via a series of elegant efflux experiments (14), has identified a DIDS-sensitive anion exchange mechanism that accepts iodide as a substrate; these studies clearly demonstrate a second iodide transporter in mammary tissues. Because the Pendred Syndrome is associated with a mutated iodide transporter that is DIDS sensitive, we carried out studies to determine whether the transporter identified by Shennan may be the pendrin-iodide transporter (7, 12, 13).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Virgin, midpregnant (10-14 days of pregnancy), Swiss-Webster mice were used in all experiments; they were purchased from Harlan Laboratories (Indianapolis, IN). Ovine prolactin (PRL; PS-19, National Institutes of Health) was a gift from the National Institutes of Health. Other substances were purchased from the following sources: cortisol from Charles Pfizer (New York); Hank's balanced salt solution (HBSS) and Medium 199-Earle's salts from GIBCO Laboratories (Grand Island, NY); porcine insulin, penicillin, and streptomycin from Eli Lilly (Indianapolis, IN); L-[4,5-3H(N)]leucine, [1-14C]acetate, [3H]OH, and [carboxyl-14C]inulin (405.8 mCi/g) from New England Nuclear (Boston, MA); 125I from Amersham (Arlington Heights, IL); and furosemide, probenecid, and 4,4'-diisothiocyanto-stilbene-2,2'-disulfonic acid (DIDS) from Sigma Chemical (St. Louis, MO). Pendrin antibody initially was a gift from Dr. Eric A. Green, NIH (Bethesda, MD); most experiments were carried out with rabbit anti-peptide antibodies raised against rat pendrin sequence 630-643 (PTKEIEIQUDWNSE; GenBank accession no. AF167412) as specified by Royauy et al. (13); the antibody was prepared by Zymed Laboratories (South San Francisco, CA).

Mammary gland explants were prepared, as previously described (8), from mice that had been pregnant for 12-14 days. Mice were killed by cervical dislocation, and mammary glands were removed and placed in HBSS. Explants (3-6 mg each) from each of 8-10 animals were prepared and placed on siliconized lens paper in 60-mm petri dishes containing 6 ml of Medium 199-Earle's salts with 1 µg/ml of insulin, 10-7 M cortisol, and/or 1 µg/ml of PRL. Next, tissues were incubated for 48 h at 37°C under a humidified 95% air-5% CO2 atmosphere. All studies involving the preparation of mouse mammary gland explants were performed in compliance with the regulations of the Animal Care and Use Committee of Wayne State University.

For Western blotting studies, tissues were taken and processed directly from the mice. Alternatively, after incubations with PRL and/or drugs, the tissues were weighed and disrupted in 1:2 (wt/vol) lysis buffer with a ground-glass homogenizer; the lysis buffer contained 2% NP-40, 10 mM Tris, 50 mM NaCl, 30 mM sodium pyrophosphate, 2.5 mM EDTA, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml of leupeptin, at pH 7.6. After 30 min on a rocking platform, lysates were centrifuged (100,000 g) for 30 min at 4°C. The resulting supernatants, containing >95% of extractable protein, were separated by SDS-PAGE (8-20% linear gradient) under reducing conditions and transferred to polyvinylidine difluoride membranes (Schleicher and Schuell). Membranes were probed with 1:2,500 rat anti-pendrin for 2 h, followed by treatment with anti-rabbit IgG horseradish peroxidase conjugate (Amersham NA934; 25 ml at 1:3,000 dilution for 1.5 h). Detection was accomplished by incubation with enhanced chemiluminescence reagents (Amersham) and exposure to photographic film. The bands (molecular mass 70 kDa) were quantitated by laser densitometry. Results are expressed on the basis of the relative density of the bands. Statistical comparisons were made with Student's t-test. All values represent the means ± SE of three or more experimental observations.

For iodide uptake determinations after the specified hormone treatments, the tissues were transferred to vessels containing 125I (0.25 µCi/ml; 0.3 ng/ml iodide) in 4 ml of HBSS; incubations were carried out in a rotary water bath at 37°C (120 cycles/min). The tissues were then weighed and homogenized in 2 ml of 10% trichloroacetic acid (TCA) containing 0.1 mM NaI; the samples were centrifuged at 2,000 g for 10 min, and radioactivity in the TCA-insoluble fraction was determined. The intracellular accumulation of radiolabeled iodide was calculated by subtracting the amount of radiolabel in the extracellular space from the total radioactivity in the tissue homogenates (10). For these calculations, the total water content (51.0%) and extracellular space (32.5%) were determined by the volume of distribution of [3H]OH and [14C]inulin (1 mM), respectively. In time course studies, equilibration was achieved with [3H]OH and [14C]inulin by 15 min after their addition. PRL had no effect on the volumes of distribution of these substances under the conditions employed in these experiments. Results of the iodide uptake studies are expressed as a distribution ratio that represents the ratio of the intracellular specific activity divided by the extracellular specific activity of the radiolabeled iodide.

For experiments in which the rate of casein synthesis was determined, [3H]leucine (1.0 µCi/ml) was added to culture medium for the final 2 h of incubation. Quantities of [3H]leucine incorporated into the casein-rich phosphoprotein fraction were determined in a protein fraction isoelectrically precipitated at pH 4.6 (1). Finally, in experiments to determine the rate of lipid synthesis, tissues were exposed to [14C]acetate (0.5 µCi/ml) for the last 2 h of incubation. Tissues were then weighed and homogenized in 400 µl of distilled water, and the lipids were extracted by the method of Bligh and Dyer (2). Radioactivity in the organic layer was then quantitated. Results are expressed as disintegrations per minute per milligram wet tissue weight.


    RESULTS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Figure 1 shows a Western blot of the pendrin transporter in mammary glands taken from virgin (lanes 1 and 2), pregnant (lanes 3 and 4), and lactating mice (lanes 5 and 6); fifteen micrograms of protein (Bradford determination) were electrophoresed on each lane. One major band appears at a molecular mass of ~90 kDa. Another major band appears at ~120 kDa; this latter band is most prominent in the proteins from the lactating mice. The quantitative results from several determinations of the 90-kDa band are presented in the bar graph. Clearly, the lactating tissue has the highest concentration of the pendrin protein.


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Fig. 1.   Pendrin protein concentration in mammary glands of virgin, midpregnant, and lactating mice. Protein extracts (15 µg) of mammary tissues were subjected to Western blot analysis, as specified in MATERIALS AND METHODS. Values are means ± SE of 3 determinations. In the immunoblot, proteins in lanes 1 and 2 were from virgin mice, in lanes 3 and 4 from midpregnant mice, and in lanes 5 and 6 from lactating mice.

Figure 2 shows the 90-kDa pendrin concentration in mammary tissues that were taken from midpregnant mice and subsequently cultured for 2 days with the three lactogenic hormones (insulin, cortisol, and PRL). Only when the tissues were treated with all three hormones was there a significant increase in pendrin levels.


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Fig. 2.   Effect of lactogenic hormones on pendrin expression. Mammary gland tissues from midpregnant mice were cultured for 2 days with 1 µg/ml of insulin (I), 10-7 M cortisol (H), 1 µg/ml of prolactin (P), and all combinations of these hormones. Proteins were then subjected to Western blot analysis as specified in MATERIALS AND METHODS. Values are means ± SE of 3 observations.

There are three well-known nonspecific inhibitors of anion exchange, including DIDS, that have been reported to inhibit iodide transport in mammary tissue (14). Figure 3 shows that each of these drugs, at appropriate concentrations, abolishes the PRL stimulation of iodide uptake into cultured mammary tissues that had been treated with PRL for 1 day. The drugs were present only during the 2-h 125I uptake period. Figure 4 shows that the exposure of the tissues to the drugs is reversible and, hence, specific. The third and fourth bars above each drug represent 125I uptake in control and PRL-treated tissues, respectively, that were treated for 2 h with the drug, after which 125I uptake was determined in the absence of the drugs during a subsequent 2-h incubation. Clearly, the PRL effect again appears after the drug removal.


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Fig. 3.   Effect of pendrin transporter inhibitors on prolactin (PRL) stimulation of iodide uptake into cultured mammary gland tissues. Mammary tissues from midpregnant mice were cultured for 2 days with I + H (open bars) or I + H + PRL (closed bars). During a subsequent 2-h incubation, 125I uptake was carried out in the presence of the transport inhibitors as specified. Iodide accumulation was then calculated as a distribution ratio. Values are means ± SE of 6 observations. * Significantly greater than control, P < 0.01.



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Fig. 4.   Reversibility of pendrin transport inhibitor effects. Mammary tissues were treated as in Fig. 3, except that tissues in lanes 3 and 4 under each drug were control and PRL-treated tissues, respectively. After 48 h, tissues were cultured for 2 h with indicated drugs. 125I uptake was then assessed after an additional 2-h incubation in the absence of the drugs. Values are means ± SE of 6 observations. * Significantly greater than control, P < 0.01.

Figures 5 and 6 are additional specificity studies showing that the anion exchange inhibitors do not abolish the PRL effects on triglyceride synthesis (Fig. 5) or on casein synthesis (Fig. 6). Furosemide and probenecid, however, did significantly attenuate the magnitude of the PRL responses.


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Fig. 5.   Effect of pendrin transport inhibitors on prolactin stimulation of triglyceride synthesis. Tissues were treated as in Fig. 3, except that they were cultured for 2 h with 0.1 µCi/ml of [14C]acetate after the 48-h hormone treatment. Values are means ± SE of 6 observations. * Significantly greater than control, P < 0.01.



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Fig. 6.   Effect of pendrin transport inhibitors on prolactin stimulation of casein synthesis. Tissues were treated as in Fig. 3, except that they were cultured for 2 h with 1 µCi/ml of [3H]leucine after the 48-h hormone treatment. Values are means ± SE of 6 observations. * Significantly greater than control, P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These studies show that the pendrin transporter is expressed in the mouse mammary gland and that its expression is significantly increased in tissues from lactating animals. In addition, the hormone complex consisting of insulin, cortisol, and PRL enhances by threefold the pendrin content of cultured mammary tissues. The pendrin protein was identified and quantified via Western blotting by employing a pendrin antibody that was developed to a specific amino acid sequence that is present in both human and rat pendrin; in view of our Western blotting results, this amino acid sequence is likely also present in the mouse pendrin. The size of the pendrin protein (~90 kDa) is consistent in a number of tissues from several species (7). The identification of a larger protein (~120 kDa) that also binds to the pendrin antibody is consistent with a similar molecular weight species that has been identified in thyroid cells (12); the possible function of this protein has not yet been determined.

Although the pendrin protein was discovered as a mutated protein variant in the Pendred Syndrome, this iodide transporter is expressed in a variety of tissues and presumably has important functions in each of these tissues (7). In the mammary gland, the importance of these transporters is suggested by the experiments in which the three anion exchange inhibitors (DIDS, furosemide, and probenecid) were found to abolish the PRL stimulation of iodide transport into cultured mammary tissues. Specificity in these experiments was established by 1) a reversibility of the drug inhibition and 2) the failure of these drugs to abolish two other lactogenic effects of PRL. These studies thus suggest that the pendrin-iodide transporter and the sodium-iodide symporter are both hormone-regulated transporters for iodide accumulation in milk during lactation.

The sodium-iodide symporter has been located on the basolateral surface of alveolar epithelial cells in the mammary gland (5) and in a similar location on the thyroid follicular cells (15). The pendrin protein has been localized on the apical border of thyroid follicular cells (12). In mammary tissues taken from virgin, pregnant, and lactating mice, we carried out immunohistochemistry studies (J. A. Rillema, unpublished observations) employing the pendrin antibody used in the Western blotting experiments. Clear specific expression of pendrin occurs within the alveolar epithelial cells but not the stromal cells. Highest expression of pendrin was apparent in the lactating tissues, but the protein was not localized to the apical or basolateral surface of the alveolar epithelial cells. At the present time, therefore, we do not know precisely where the pendrin transporter is functioning in the mammary cells.


    ACKNOWLEDGEMENTS

This work was supported by funds from the Children's Hospital of Michigan and National Institutes of Health Division of Research Resources Grant RR-08167.


    FOOTNOTES

Address for reprint requests and other correspondence: J. A. Rillema, Dept. of Physiology, Wayne State Univ. School of Medicine, Rm. 5374 Scott Hall, 540 E. Canfield Ave., Detroit, MI 48201-1928 (E-mail: jrillema{at}med.wayne.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

September 11, 2002;10.1152/ajpendo.00383.2002

Received 28 August 2002; accepted in final form 4 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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