Effect of prolactin on phosphate transport and incorporation
in mouse mammary gland explants
James A.
Rillema
Department of Physiology, Wayne State University School of
Medicine, Detroit, Michigan 48201
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ABSTRACT |
Inorganic phosphate is present in milk at a
concentration that is severalfold higher than in maternal plasma. In
cultured mammary tissues from 12- to 14-day-pregnant mice, the
intracellular concentration of 32PO4 was six
times higher than in the culture medium after a 4-h treatment with
32PO4. Of the principal lactogenic hormones
[insulin (I), cortisol (H), and prolactin (PRL)], only I and PRL (in
the presence of H and I) stimulated 32PO4
uptake into cultured mammary tissues; H, by itself or in the presence
of I or PRL, inhibited 32PO4 uptake. All three
lactogenic hormones together effected the greatest stimulation of
32PO4 uptake. Similar hormone effects were
observed with regard to 32PO4 incorporation
into lipids and trichloroacetic acid-insoluble molecules. In a time
course study, the onset of the PRL stimulation of
32PO4 uptake and incorporation occurred
8-12 h after PRL addition; in dose-response studies, the PRL
effect was manifested with PRL concentrations of 50 ng/ml and above.
From kinetic studies, the apparent maximal velocity of PO4
uptake was determined to be ~7.7 mM · h
1 · l cell water
1;
the apparent Michaelis-Menten constant was ~3-5 mM. The PRL effect on 32PO4 uptake was abolished when
sodium was absent from the uptake medium. These studies thus
demonstrate a complex interaction of three hormones (I, H, and PRL) in
the regulation of 32PO4 uptake and
incorporation into macromolecules in cultured mouse mammary tissues.
insulin; cortisol
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INTRODUCTION |
PHOSPHATE IS AN ESSENTIAL
COMPONENT of milk for the nourishment of the neonate. In milk, it
is present in several forms, including inorganic phosphate, colloidal
phosphate, and casein phosphate and in phospholipids (5,
12). Because of the high concentration of phosphate in milk that
meets the nutritional demands of the suckling neonate, copious amounts
of phosphate are transferred from the maternal plasma into milk. One
author (2) reported that all of the plasma inorganic
phosphate is replaced every 10 min in lactating rats.
Even when only inorganic phosphate is considered, its concentration in
milk is 5- to 10-fold higher than in the maternal plasma. A highly
efficient transport mechanism is therefore obviously present in the
alveolar epithelial cells of the mammary gland. Shillingford et al.
(13) recently characterized one phosphate transport
mechanism in lactating rat mammary glands. Phosphate uptake was
predominantly via a sodium-dependent transport mechanism, presumably
located on the basolateral membrane of the alveolar epithelial cells; a
sodium-phosphate symporter was postulated. Phosphate uptake occurred
via a saturable mechanism with an apparent Michaelis-Menten constant
(Km) of 1.13 mM.
The experiments reported in this study were carried out to characterize
the effects of three lactogenic hormones [insulin, cortisol, and
prolactin (PRL)] on phosphate uptake and incorporation in mouse
mammary gland explants. The specific focus is on the prolactin
regulation of phosphate uptake.
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MATERIALS AND METHODS |
Midpregnant (10-14 days of pregnancy) Swiss-Webster mice
were used in all experiments. They were purchased from Harlan
Laboratories (Indianapolis, IN). Ovine PRL (National Institutes of
Health PS-19) was a gift from the National Institutes of Health. Other
substances were purchased from the following sources: cortisol from
Charles Pfizer (New York, NY); choline chloride, Hanks' balanced salt solution (HBSS), and medium 199 (M-199)-Earle's salts from Sigma Chemical (St. Louis, MO); 32PO4,
3HOH, and [carboxy-14C]inulin
(405.8 mCi/g) from New England Nuclear (Boston, MA); porcine insulin,
penicillin, and streptomycin from Eli Lilly (Indianapolis, IN).
Explants of mouse mammary tissues were prepared and cultured as
described earlier (8). The explants were cultured on
siliconized lens paper floating on 6 ml of M-199-Earle's salts
containing 1 µg/ml insulin plus 10
7 M cortisol and/or
PRL (0-1 µg/ml); all incubations were carried out in 60 × 15-mm petri dishes maintained at 37°C in an atmosphere of 95%
O2-5% CO2. In experiments where the effects of
PRL on phosphate uptake and incorporation were to be determined, the
tissues were initially cultured for 24-36 h with insulin plus
cortisol, after which PRL was added and incubations continued for the
times specified for each experiment. Unless specified otherwise, for
the final 2 h of culture, the tissues were transferred to vessels
containing 32PO4 (0.5 µCi/ml) in 4 ml of
M-199; incubations were carried out in a rotary water bath at 37°C
(120 cycles/min). The tissues were then weighed and homogenized in 4 ml
of 5% trichloroacetic acid (TCA). One-milliliter samples were employed
to assess the extent of 32P incorporation into the lipid
fraction (3). The remaining 3 ml were centrifuged at 2,000 g for 10 min. Radioactivity in 1-ml aliquots of the
TCA-soluble fraction was determined by liquid scintillation techniques.
After the pellet was washed with an additional 5 ml of 5% TCA,
radioactivity in the TCA-insoluble fraction was determined after
solubilization in 2 ml 1 N NaOH. The intracellular accumulation
of radiolabeled, unincorporated PO4 was calculated by
subtracting the amount of radiolabel in the extracellular space from
the total TCA-soluble radioactivity in the tissue homogenates (8,
11). For these calculations, the total water content (51.0%)
and extracellular space (24.6%) were determined by the volume of
distribution of 3HOH and [14C]inulin (1 mM),
respectively. In time course studies, equilibration was achieved with
3HOH and [14C]insulin by 15 min after their
addition. PRL had no effect on the volumes of distribution of these
substances under the conditions employed by these experiments. Results
of the phosphate uptake studies are expressed as a distribution ratio,
which represents the ratio of the intracellular specific activity
divided by the extracellular specific activity of the radiolabeled
phosphate. The results of the incorporation studies are expressed as
disintegrations per minute per milligram wet weight of tissues.
Statistical comparisons were made with Student's t-test
when two means were compared or by an analysis of variance followed by
Dunnett's test for multiple comparisons. Means are considered significantly different (*) when P < 0.05. Results are
expressed as means ± SE.
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RESULTS |
Figure 1 shows a time course of
32PO4 uptake in mouse mammary gland explants
that were cultured in the presence or absence of PRL for 24 h.
Uptake increased progressively over a 4-h labeling period, and the
uptake was twice as great in the PRL-treated tissues. Distribution
ratios of ~6 were achieved in the PRL-treated tissues, suggesting
that an energy-requiring mechanism is involved in phosphate uptake into
mammary cells. Because inorganic phosphate uptake was calculated from
the 32PO4 present in TCA-soluble tissue
fractions, an experiment was carried out to determine the actual
percentage of 32PO4 in this fraction that was
present as inorganic phosphate. By use of the inorganic phosphate
precipitation method of Willard et al. (14), aliquots of
the TCA supernatants from Fig. 1 were subjected to
precipitation and subsequent quantitation; 81% of the
32PO4 in the PRL-treated TCA extracts and 86%
of the 32PO4 in the control TCA extracts
precipitated as inorganic phosphate. Most of the
32PO4 in the TCA-soluble tissue extracts is
thus present as inorganic phosphate.

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Fig. 1.
Time course of 32PO4 accumulation
in cultured mammary gland explants. Tissues were cultured for 1 day
with insulin (I; 1 µg/ml) plus cortisol (H; 10 7 M).
Tissues were then cultured for 1 more day with I + H or I + H + 1 µg/ml prolactin (PRL). 32PO4 (0.5 µCi/l) was present during the final times indicated in the figure.
Intracellular accumulation in a 5% trichloroacetic acid (TCA)-soluble
tissue fraction is expressed as a distribution ratio of the means ± SE of 6 observations. *Greater than control with P < 0.05.
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Figures 2 and
3 show the time course of
32PO4 labeling of lipids (primarily
phospholipids) and TCA-insoluble molecules (primarily casein) in the
tissues described in Fig. 1. The 32PO4
incorporation into each of these tissue fractions increases progressively with time, and PRL causes about a twofold stimulation of
incorporation in each of these fractions; this likely reflects the
50-100% stimulation of casein and phospholipid synthesis that occurs in response to PRL (5, 10).

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Fig. 2.
Time course of 32PO4
incorporation into lipids in cultured mammary gland explants.
Experimental details are the same as in Fig. 1, except that
32PO4 incorporation into lipids was determined
(see MATERIALS AND METHODS). Results are expressed as
means ± SE of 6 observations.
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Fig. 3.
Time course of 32PO4
incorporation into a TCA-insoluble fraction from cultured mammary gland
explants. Experimental details are the same as in Fig. 1, except that
32PO4 incorporation into a 5% TCA-insoluble
fraction was determined. Results represent means ± SE of 6 observations.
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Figures 4-6 report the results of an
experiment wherein explants were cultured for 2 days with all possible
combinations of three lactogenic hormones (insulin, cortisol, and PRL);
the tissues were then pulse labeled for 2 h with
32PO4. When the hormones were tested
individually (Fig. 4), insulin stimulated 32PO4
uptake, PRL had no effect, and cortisol (10
7 M) inhibited
uptake. When the hormones were tested in combinations, the only
combination in which PRL stimulated 32PO4
uptake was when all three hormones were tested in concert. In the
associated incorporation data as presented in Figs.
5 and 6,
only insulin by itself stimulates 32PO4
incorporation into lipids and the TCA-insoluble fraction, whereas PRL
has a stimulatory effect only when combined with insulin and
cortisol.

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Fig. 4.
Effects of lactogenic hormones on
32PO4 accumulation in cultured mammary gland
explants. Explants were cultured for 2 days with or without all
combinations of 3 lactogenic hormones: 1 µg/ml insulin (I),
10 7 M cortisol (H), and/or 1 µg/ml PRL.
32PO4 (0.5 µCi/ml) was present for the final
2 h. C, control. Numbers represent means ± SE of 6 observations of 32PO4 accumulation in a 5%
TCA-soluble tissue fraction. *Greater than control, P < 0.05; **greater than I + H, P < 0.05.
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Fig. 5.
Effects of lactogenic hormones on
32PO4 incorporation into lipids in mouse
mammary explants. Tissues were treated as in Fig. 4, except that
incorporation into lipids was determined. Results are expressed as
means ± SE of 6 observations.
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Fig. 6.
Effects of lactogenic hormones on
32PO4 incorporation into a TCA-insoluble
fraction from cultured mammary gland explants. Experimental details are
the same as in Fig. 4, except that 32PO4
incorporation into a 5% TCA-insoluble fraction was determined. Results
represent means ± SE of 6 observations.
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A time course for the PRL stimulation of 32PO4
uptake and incorporation is presented in Fig.
7. Tissues employed in this experiment were initially cultured with insulin plus cortisol; PRL was then added
for the times indicated. Effects of PRL on phosphate uptake and
incorporation were first detected 8-12 h after PRL was added to
the cultured tissues; the responses were maintained through 30 h.
In dose-response studies (Fig. 8), PRL
effects were expressed with all PRL concentrations of 50 ng/ml and
above; these concentrations are physiological in that they are similar
to plasma concentrations in mice and other species.

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Fig. 7.
Time course of PRL stimulation of
32PO4 uptake and incorporation into lipids and
a TCA-insoluble fraction in cultured mammary gland explants. Tissues
were cultured for 1 day with 1 µg/ml insulin + 10 7
M cortisol. PRL (1 µg/ml) was then added to the culture medium, and
incubations were continued for the times specified.
32PO4 (0.5 µCi/ml) was added to the culture
medium for the final 2 h of culture. 32PO4
uptake and incorporation were then assessed. Results are presented as
means ± SE of 6 observations.
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Fig. 8.
Effect of PRL concentration on
32PO4 uptake and incorporation into lipids and
a TCA-insoluble fraction in cultured mammary gland explants. Tissues
were cultured for 1 day with 1 µg/ml insulin + 10 7
M cortisol. PRL at the concentrations indicated was then added, and
incubations were continued for 1 additional day.
32PO4 (0.5 µCi/ml) was present during the
final 2 h of culture. 32PO4 uptake and
incorporation were then assessed. Results are expressed as means ± SE of 6 observations.
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With control and 24-h PRL-treated tissues, phosphate uptake was
quantitated with culture medium phosphate concentrations of 1-10
mM (Fig. 9). The intracellular phosphate
concentration was calculated from the TCA-soluble
32PO4 that was taken up from the culture
medium; the total amount of phosphate in the cells was not determined
but is likely higher than the calculated concentrations presented in
Fig. 9. Saturation kinetics for uptake are clearly apparent. Because a
large fraction of the inorganic phosphate is rapidly incorporated into
lipids and proteins, a precise estimation of transport kinetics
(Km and maximum velocity,
Vmax) is not possible. Regardless, however, if
the data in Fig. 9 are plotted as the reciprocal of the velocity vs.
the phosphate concentration, an apparent Km of
~4 mM and an apparent Vmax of 7.5 mM · h
1 · l cell water
1
were determined. These values compare favorably with those published for rat mammary tissues (4):
Km = 1.13 mM,
Vmax = 13.4 mM · h
1 · l cell water
1.

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Fig. 9.
Phosphate concentration vs. 32PO4
uptake and incorporation in cultured mammary gland explants. Tissues
were treated with hormones as in Fig. 1. Phosphate (0.5 µCi/ml
32PO4) at the concentrations indicated was
present during the final 2 h of culture. The
intracellular-to-media phosphate concentration ratio was then
calculated and expressed as the mean ± SE of 6 observations.
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The experiments in Figs.
10-12
were carried out to determine the sodium dependence of the PRL effects
on 32PO4 uptake and incorporation. In these
studies, explants were cultured for 24 h in the absence (control)
or presence of PRL; the tissues were then cultured for 2 additional
hours with 32PO4 contained in M-199, KRB, or
KRB with the NaCl substituted for with choline chloride. In the final
experimental combination, the tissues were cultured for 2 h with
the sodium-free KRB, after which they were treated for 2 h with
sodium-KRB containing 32PO4. Several
interesting observations evolved from these experiments. First, the PRL
effects on 32PO4 uptake and incorporation into
lipids were abolished when these determinations were made with
sodium-free medium. The PRL effect on 32PO4
incorporation into TCA-precipitable molecules (primarily proteins) was
attenuated but not abolished with the sodium-free medium. The 2-h
exposure of the tissues to the sodium-free medium was not detrimental
to the functioning of the tissues, since the magnitude of the PRL
responses was fully restored in tissues that were first cultured in
sodium-free medium for 2 h, after which
32PO4 uptake was assessed in normal KRB.
Another interesting observation was that the magnitude of
32PO4 uptake and incorporation was ~50% when
uptake was determined with 32PO4 contained in
M-199 vs. KRB. Because the phosphate concentrations in M-199 and KRB
are the same (1 mM), one or more of the components of M-199 must be
impairing phosphate uptake and incorporation; this component(s) has yet
to be identified. A final observation in these studies is that
distribution ratios greater than three were generated when
32PO4 uptake was determined in the absence of
sodium. This may suggest that another energy-requiring transporter for
phosphate may exist in mammary tissues; this transporter is clearly not
affected by PRL treatment.

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Fig. 10.
Effect of sodium on 32PO4 uptake
in cultured mammary gland explants. Tissues were treated with hormones
as in Fig. 1. During the final 2 h of culture, 0.5 µCi/ml
32PO4 was present in medium consisting of
medium 199 (M-199), Krebs-Ringer bicarbonate buffer (KRB, containing
sodium), or KRB containing choline chloride substituted for sodium
chloride. In far right, the tissues were cultured for 2 h in sodium-free medium before the terminal 2-h culture with
32PO4 in sodium-containing KRB. Results are
expressed as means ± SE of 6 observations.
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Fig. 11.
Effect of sodium on 32PO4
incorporation into lipids in cultured mammary gland explants.
Experimental details are the same as in Fig. 10, except that
32PO4 incorporation into lipids was determined
(see MATERIALS AND METHODS). Results are expressed as
means ± SE of 6 observations.
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Fig. 12.
Effect of sodium on 32PO4
incorporation into a TCA-insoluble fraction from cultured mammary gland
explants. Experimental details are the same as in Fig. 10, except that
32PO4 incorporation into a 5% TCA-insoluble
fraction was determined. Results are expressed as means ± SE of 6 observations.
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 |
DISCUSSION |
PRL as well as insulin and cortisol is vitally important in the
regulation of phosphate metabolism in the mammary gland. Because copious amounts of phosphate are secreted into milk during lactation, the hormone regulation of this process is of utmost importance. PRL at
physiological concentrations (50-1,000 ng/ml) functioned in
concert with insulin and cortisol to stimulate phosphate uptake as well
as its incorporation into lipids and TCA-insoluble materials. The onset
of these PRL responses was 8-12 h after PRL addition to the
cultured tissues; this correlates well with the onset of PRL's effect
on the synthesis of a number of other milk products, including lactose,
casein, and triglycerides; the PRL stimulation of iodide, amino acid,
and glucose transport follows a similar time course (1, 3, 4,
6-11). Precisely how all the signaling pathways for PRL,
insulin, and cortisol integrate to effect a maximum stimulation of milk
product synthesis remains to be explained.
The enhanced uptake of phosphate across the basolateral surface of the
alveolar epithelial cells is likely the primary mechanism accelerating
the accumulation of inorganic phosphate in milk (12, 13).
In addition, the increased phosphate uptake likely enhances the
substrate provision for phosphate incorporation into casein, as well as
combining with calcium in casein micelles. Clearly a
sodium-dependent phosphate transporter is present in mammary cells;
this is the only transporter that is stimulated by PRL, since the
phosphate uptake effect is abolished when uptake is determined in the
absence of sodium. Phosphate distribution ratios greater than three
maintain, however, when phosphate uptake is determined in the absence
of sodium. This suggests that a sodium-independent phosphate
transporter may also exist in the mammary gland. In addition, this
transporter is likely an energy-dependent, active transporter to
achieve a distribution of ratio greater than unity; clearly this
transporter is not regulated by PRL. In the future, it remains for the
phosphate transporter(s) to be isolated and their molecular structure determined.
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ACKNOWLEDGEMENTS |
This work was sponsored by funds from the Children's Hospital of Michigan.
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FOOTNOTES |
Address for reprint requests and other correspondence:
J. A. Rillema, Dept. of Physiology, Wayne State Univ. School
of Medicine, 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.
First published December 18, 2001;10.1152/ajpendo.0409.2001
Received 17 September 2001; accepted in final form 14 December 2001.
 |
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