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 |
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 |
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 |
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.
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RESULTS |
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.
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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.
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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.
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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.
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DISCUSSION |
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.
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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.
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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 |
1.
Ashworth, US.
Rapid method for the determination of casein in milk by the dye binding method and for the detection of mastitis.
J Dairy Sci
48:
537-543,
1965[ISI].
2.
Bligh, EG,
and
Dyer WJ.
A rapid method for total lipid extraction and purification.
Can J Biochem Physiol
37:
911-917,
1959[ISI].
3.
Brown-Grant, K.
Extrathyroidal iodide concentrating mechanisms.
Physiol Rev
41:
189-213,
1961[Free Full Text].
4.
Carrasco, N.
Iodide transport in the thyroid gland.
Biochim Biophys Acta
1154:
65-82,
1993[ISI][Medline].
5.
Cho, JY,
Leveille R,
Kao R,
Rousset B,
Parlow AF,
Burak WE, Jr,
Mazzaferri EL,
and
Jhiang SM.
Hormonal regulation of radioiodide uptake activity and Na+-I
symporter expression in mammary glands.
Endocrinology
85:
2936-2943,
2000.
6.
Dai, G,
Levy O,
and
Carrasco N.
Cloning and characterization of the thyroid iodide transporter.
Nature
379:
468-460,
1996.
7.
Fugazzola, L,
Cerutti N,
Mannavola D,
Vannucchi G,
and
Beck-Peccoz P.
The role of pendrin in iodide regulation.
Exp Clin Endocrinol Diabetes
109:
18-22,
2001[ISI][Medline].
8.
Rillema, JA.
Early actions of prolactin on uridine metabolism in mammary gland explants.
Endocrinology
92:
1673-1679,
1973[ISI][Medline].
9.
Rillema, JA,
and
Rowady DL.
Characteristics of the prolactin stimulation of iodide uptake into mouse mammary gland explants.
Proc Soc Exp Biol Med
215:
366-369,
1997[Abstract].
10.
Rillema, JA,
and
Yu TX.
Prolactin stimulation of iodide uptake into mouse mammary gland explants.
Am J Physiol Endocrinol Metab
271:
E879-E882,
1996[Free Full Text].
11.
Rillema, JA,
Yu TX,
and
Jhiang SM.
Effect of prolactin on sodium iodide symporter expression in mouse mammary gland explants.
Am J Physiol Endocrinol Metab
279:
E769-E772,
2000[Abstract/Free Full Text].
12.
Royaux, IE,
Suzuki K,
Mori A,
Katoh R,
Everett LA,
Kohn LD,
and
Green ED.
Pendrin, the protein encoded by the Pendred Syndrome gene (PDS), is an apical porter of iodide in the thyroid and is regulated by thyroglobulin in FRTL-5 cells.
Endocrinology
141:
839-845,
2000[Abstract/Free Full Text].
13.
Scott, DA,
Wang R,
Kreman TM,
Sheffield VC,
and
Karnisk LP.
The Pendred syndrome encodes a chloride-iodide transport protein.
Nat Genet
21:
440-443,
1999[ISI][Medline].
14.
Shenan, DG.
Iodide transport in lactating rat mammary tissue via a pathway independent from the Na+-I
cotransporter: evidence for sulfate/iodide exchange.
Biochem Biophys Res Commun
280:
1359-1363,
2001[ISI][Medline].
15.
Spitzweg, C,
Heufelder AE,
and
Morris JC.
Thyroid iodine transport.
Thyroid
10:
321-330,
2000[ISI][Medline].
16.
Spitzweg, C,
Joba W,
Eisenmenger W,
and
Heufelder AE.
Analysis of human sodium iodide symporter gene expression in extrathyroidal tissues and cloning of its complimentary deoxyribonucleic acids from salivary gland, mammary gland and gastric mucosa.
J Clin Endocrinol Metab
83:
1746-1751,
1998[Abstract/Free Full Text].
17.
Tazebay, UH,
Wapnir IL,
Levy O,
Dohan O,
Zuckier LS,
Zhoa QH,
Deng HF,
Ameta PS,
Fineburg S,
Pestell RG,
and
Carrasco N.
The mammary gland iodide transporter is expressed during lactation and in breast cancer.
Nature Med
6:
871-878,
2000[ISI][Medline].
18.
Wolff, J.
Transport of iodide and other anions in the thyroid gland.
Physiol Rev
44:
45-90,
1964[Free Full Text].
Am J Physiol Endocrinol Metab 284(1):E25-E28
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