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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.

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.

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.

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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

This work was sponsored by funds from the Children's Hospital of Michigan.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anderson, LD, and Rillema JA. Effects of hormones on protein and amino acid metabolism in mammary gland explants of mice. Biochem J 158: 355-359, 1976[ISI][Medline].

2.   Brommage, R. Measurement of calcium and phosphate fluxes during lactation in the rat. J Nutr 119: 428-438, 1988[ISI].

3.   Cameron, C, Linebaugh BE, and Rillema JA. Hormonal control of lipid metabolism in mouse mammary gland explants. Endocrinology 112: 1007-1011, 1983[Abstract].

4.   Golden, K, and Rillema JA. Studies on the mechanism by which prolactin stimulates alpha -aminoisobutyric acid uptake into cultured mouse mammary tissues. Horm Metab Res 25: 466-469, 1993[ISI][Medline].

5.   Holt, C, and Jennes R. Interrelationships of constituents and partition of salts in milk samples from eight species. Comp Biochem Physiol A Physiol 77: 275-282, 1984[ISI].

6.   Oppat, CA, and Rillema JA. Characteristics of the early effect of prolactin on lactose biosynthesis in mouse mammary gland explants. Proc Soc Exp Biol Med 188: 342-345, 1988[Abstract].

7.   Peters, BJ, and Rillema JA. Effect of prolactin on 2-deoxyglucose uptake in mouse mammary gland explants. Am J Physiol Endocrinol Metab 262: E627-E630, 1992[Abstract/Free Full Text].

8.   Rillema, JA. Early actions of prolactin on uridine metabolism in mammary gland explants. Endocrinology 92: 1673-1679, 1973[ISI][Medline].

9.   Rillema, JA, Foley KA, and Etindi RN. Temporal sequence of prolactin actions on phospholipids biosynthesis in mouse mammary gland explants. Endocrinology 116: 511-515, 1985[Abstract].

10.   Rillema, JA, Golden K, and Jenkins MA. Effect of prolactin on alpha -aminoisobutyric acid uptake in mouse mammary gland explants. Am J Physiol Endocrinol Metab 262: E402-E405, 1992[Abstract/Free Full Text].

11.   Rillema, JA, and Yu TX. Prolactin stimulation of iodide uptake into mouse mammary gland explants. Am J Physiol Endocrinal Metab 271: E879-E882, 1996.

12.   Shennan, DB, and Peaker M. Transport of milk constituents by the mammary gland. Physiol Rev 80: 925-951, 2000[Abstract/Free Full Text].

13.   Shillingford, JM, Calvert DT, Beechey RB, and Shennan DB. Phosphate transport via Na+-Pi cotransport and anion exchange in lactating rat mammary tissue. Exp Physiol 81: 273-284, 1996[Abstract].

14.   Willard, HH, Furman NH, and Bricker CE. Elements of Quantitative Analysis (4th edition). New York: D. Van Norstrand, 1956, p. 349-351.


Am J Physiol Endocrinol Metab 283(1):E132-E137
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society




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