Prolactin,Cortisol,and Insulin Regulation of Nucleoside Uptake Into Mouse Mammary Gland Explants

Abstract

Nucleosides are essential components of milk that are used for the nourishment of newborns. Effects of the three primary lactogenic hormones, including prolactin (PRL), insulin (I), and cortisol (H), on nucleoside uptake and incorporation into cultured mammary tissues taken from 12- to 14-day pregnant mice were determined; most experiments focused on the regulation of uridine uptake. Insulin alone, as well as PRL in the presence of insulin and cortisol, was shown to stimulate uridine uptake and incorporation into RNA in mammary explants taken from 12- to 14-day pregnant mice. The PRL effects were expressed at concentrations of 25 ng/ml and above, which are physiological plasma concentrations. In the absence of sodium, uridine uptake and incorporation were diminished, suggesting the presence of a sodium-dependent uridine transporter. In kinetic studies the apparent K _m for uridine uptake was calculated to be 312 μM, and the V _max 2.90 μmol/hr/L cell water; PRL had no effect on the K _m but increased the V _max to 5.88 μmol/hr/L cell water. When assessing uridine uptake in the presence of the other nucleosides at 0.1 mM, only cytidine competed with uridine uptake. The fact that distribution ratios of greater than 15:1 were achieved with uridine indicates that uridine uptake may be via an active transporter. These studies show that PRL enhances uridine update in mammary tissues by stimulating the activity, and probably synthesis, of a sodium-dependent, active uridine and cytosine transporter.

Nucleosides are present in micromolar concentrations in milk, but they are important constituents for the nourishment of the newborn (1). To date, at least five major nucleoside transport processes have been identified in human and other mammalian cells and tissues; at least three of these are sodium-dependent processes (2, 3). Dietary nucleosides are known to have a number of profound effects on several physiological processes, including immune responses, iron absorption in the gut, and desaturation and elongation rates in fatty acid synthesis (1).

Uridine is a pyrimidine base nucleoside that is involved in several cellular biochemical processes, as well as being a specific base in RNA. Several years ago we reported a prolactin (PRL) stimulation of uridine uptake and incorporation into RNA in mouse mammary gland explants (4). This PRL effect was abolished by several antibiotics that inhibit RNA and protein synthesis, suggesting that the synthesis of new proteins, perhaps nucleoside transporters, are involved in the PRL response. The present studies were carried out to further characterize the effects of lactogenic hormones, including PRL, on uridine uptake and incorporation into RNA in cultured mammary tissues.

Materials and Methods

Midpregnant (12–14 days of pregnancy) Swiss–Webster mice were used in all experiments: they were purchased from Harlan Laboratories (Indianapolis, IN). Ovine prolactin (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, adenosine, cytosine, guanosine, thymidine, uridine, Hanks’ balanced salt solution (HBSS), and medium 199-Earle’s salts from Sigma Chemical Co. (St. Louis, MO); [³H]OH, [³H]-inulin and [³H]-uridine 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 (4). The explants were cultured on siliconized lens paper floating on 6 ml of medium 199-Earle’s salts containing 1 μg/ml insulin plus 10⁻⁷ M cortisol and/or PRL (0–1 μg/ml); all incubations were performed 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 uridine uptake and incorporation were to be determined, the tissues were initially cultured for 24–36 hr 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 1 or 2 hr of culture, the tissues were transferred to vessels containing [³H]-uridine (1 μCi/ml, 22 nM) in 6 ml of M-199; incubations were performed in a rotary water bath at 37°C (120 cycles/min). The tissues were then weighed and homogenized in 4 ml 5% trichloroacetic acid (TCA); the homogenates were centrifuged at 2000g for 10 min. Radioactivity in 1 ml aliquots of the TCA-soluble fraction was determined by liquid scintillation techniques. After washing the pellet with an additional 5 ml of 5% TCA, radioactivity in the TCA-insoluble fraction was determined after solubilization in 2 ml IN NaOH; this reflects the radioactive uridine incorporated into RNA. The intracellular accumulation of radiolabeled, unincorporated [³H]-uridine was calculated by subtracting the amount of radiolabel in the extracellular space from the total TCA-soluble radioactivity in the tissue homogenates (4). For these calculations the total water content (51.0%) and extracellular space (24.6%) were determined by the volume of distribution of [³H]OH and [³H]inulin (1 mM), respectively in tissues maintained in culture for 24 hr. In time course studies, equilibration was achieved with [³H]OH 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 uridine 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 uridine. The results of the incorporation studies are expressed as DPM/mg wet weight of tissues.

Statistical comparisons were made with Student’s t test when two means were compared, or an analysis of variance followed by Dunnet’s test for multiple comparisons. Means are considered significantly different (*) when P < 0.05. Results are expressed as the mean ± SE. In each experiment, tissues from 16–20 animals were pooled; each experiment was repeated at least 2 times.

Results

Figure 1 shows a time course of [³H]-uridine uptake in cultured mammary gland explants. Uptake into the 5% TCA-soluble tissue fraction increases progressively over a 4-hr labeling period using [³H]-uridine at 1 μCi/ml, 22 nM. After 4 hr, distribution ratios of greater than 15 were attained, suggesting the existence of an active transport mechanism. However, this is complicated by the fact that uridine is converted to a number of other variants after entering cells; this will be discussed later. [³H]-uridine incorporation into RNA is also linear with time over a 4-hr period (data not shown). For all subsequent experiments a 1- or 2-hr pulse-time with [³H]-uridine was employed.

Figure 2 reports the results of an experiment where 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 hr with [³H]-uridine. When the hormones were tested individually, insulin stimulated [³H]-uridine uptake, while PRL and cortisol (10⁻⁷ M) had no effects. When the hormones were tested in combinations, the only combination in which PRL stimulated [³H]-uridine uptake was when all three hormones were tested in concert. Similar results were observed when [³H]-uridine incorporation into RNA was determined (results not presented).

In a PRL time course study the PRL effect on [³H]-uridine uptake (Fig. 3 ) and incorporation (data not presented) was initially detectable by 10 hr after PRL was added to the tissues; this is consistent with our earlier studies (4). The magnitude of the response increases progressively through 24–30 hr. Dose-response effects of PRL on [³H]-uridine uptake (Fig. 4) and incorporation (data not included) indicate that the minimal PRL concentration that elicited a significant response was 25 ng/ml; maximum effects were achieved with PRL at concentrations of 100 ng/ml and above. These PRL concentrations correlate well with physiological plasma levels.

The experiments in Figures 5 and 6 were performed to determine the sodium-dependence of the PRL effects on [³H]-uridine uptake and incorporation. In these studies explants were cultured for 24 hr in the absence (control) or presence of PRL; the tissues were then cultured for 2 additional hours with [³H]-uridine contained in M-199, Kreb’s Ringer bicarbonate buffer (KRB), or KRB with the NaCl substituted for with choline chloride. In both the control and PRL-treated tissues, the extent of [³H]-uridine uptake and incorporation into RNA was reduced by about 50% in the absence of sodium. This clearly suggest that a significant fraction of uridine uptake into mammary cells occurs via a sodium-dependent mechanism.

With control and 24 hr PRL-treated tissues, uridine uptake was quantitated with culture media uridine concentrations of 1–25 mM (Fig. 7 ). The intracellular uridine concentration was calculated from the TCA-soluble [³H]-uridine that was taken up from the culture medium; the total amount of uridine in the cells was not determined, but is likely higher than the calculated concentrations presented in Figure 7 . Saturation kinetics for uptake are clearly apparent. Since a large fraction of the uridine is rapidly converted to other forms of uridine (4), a precise estimation of transport kinetics (apparent K _m and V _max) is not possible. However, if the data in Figure 7 is plotted as the reciprocal of the velocity versus the uridine concentration, an apparent K _m of about 312 μM and an apparent V _max of 2.90 μMol/hr/L cell water was determined. PRL had no effect on the apparent K _m, but increased the V _max to 5.88 μMol/hr/L cell water.

Figures 8 and 9 show the effects of the other nucleosides at 0.1 mM on [³H]-uridine uptake and incorporation into RNA. Thymidine, adenosine, and guanosine had no appreciable effects, while cytosine had a profound effect on decreasing [³H]-uridine uptake and incorporation. In addition, uridine distribution ratios were reduced to unity in the presence of excess cytosine, and the PRL effects on [³H]-uridine uptake and incorporation were abolished.

Discussion

During lactation the alveolar epithelial cells of the mammary gland transport numerous substances from the maternal plasma to milk. Examples of substances whose uptakes have been characterized include sodium, potassium, chloride, phosphate, iodide, calcium, citrate, choline, carnitine, glucose, and amino acids (5–9). Until recently, little was known about nucleoside uptake and its hormone regulation in mammary tissues. Very recently sodium-nucleoside symporters have been isolated from human mammary tissues (2, 10); each of these transporters were broad specificity purine and pyrimidine sodium-nucleoside transporters.

Our studies suggest that the prominent uridine transporter in the mouse mammary gland is a sodium symporter that is selective for uridine and cytosine. Excess levels (0.1 mM) of thymidine, adenosine or guanosine had no effect on uridine uptake into cultured mouse mammary tissues; the uridine concentration in these experiments was 22 nm. In contrast, cytosine had a profound effect on inhibiting uridine uptake. The molecular identification of this sodium-nucleoside transporter remains to be accomplished. This transporter could be one of five major nucleoside transporters that have been observed functionally in mammalian cells and tissues (2, 3).

The hormone studies indicate that the three lactogenic hormones are all involved in regulating uridine uptake for lactogenesis. Insulin alone stimulates uridine accumulation, and prolactin elicits an effect in concert with insulin and cortisol. The prolactin effect is likely elicited via the synthesis of additional transporters since the apparent V _max, but not K _m, of uridine uptake was increased by prolactin. In addition, protein and RNA synthesis inhibitors (4) abolished the prolactin stimulation of uridine uptake, suggesting that the synthesis of new proteins, perhaps transporters, is involved in the prolactin response. The prolactin effect also required a greater than 4-hr treatment time before the transport effect was expressed. All of these observations suggest that prolactin stimulates the synthesis of additional transporters.

The parallel effects of hormones on uridine uptake and incorporation are also of interest. Apparently the cells are programmed to up-regulate the mechanisms for providing the substrates for RNA synthesis at the same time that RNA synthesis is enhanced. A coordinated hormone regulation of multiple gene products for these responses is suggested from our experimental studies.

As pointed out by Schlimme and Meisel (1), the nucleosides have only recently become an object of nutritional research. Although they are present in only micromolar amounts in milk, they have a profound effect on several developmental processes. In addition, certain nucleoside drugs are widely employed for the treatment of cancer and viral diseases (2). The specific importance for the presence of the nucleosides in milk remains to be established.

Figure 1.

Time–course of [³H]-uridine accumulation in cultured mammary gland explants. Tissues were cultured for 1 day with insulin (1 μg/ml) plus cortisol (10⁻⁷ M). Tissues were then cultured with 1 μCi/ml [³H]-uridine for the times indicated. Intracellular accumulation of [³H]-uridine in a 5% TCA-soluble tissue fraction is expressed as a distribution ratio of the mean ± SE of six observations.

Figure 2.

Effect of lactogenic hormones on [³H]-uridine uptake into cultured mammary gland explants. Tissues were cultured for 2 days with no hormones (C), insulin (1 μg/ml, I), cortisol (10⁻⁷ M, H), prolactin (1 μg/ml, P), and all combinations of these hormones. After a 2-hr incubation with 1 μCi/ml [³H]-uridine, intracellular accumulation in a 5% TCA-soluble fraction was calculated and expressed as a distribution ratio. *Greater than control with P < 0.05. **Greater than * with P < 0.05.

Figure 3.

Time–course of PRL stimulation of [³H]-uridine uptake into cultured mammary gland explants. Tissues were initially cultured for 1 day with 1 μg/ml insulin plus 10⁻⁷ M cortisol. Incubation was then continued with 1 μg/ml PRL for the times indicated. [³H]-Uridine (1 μCi/ml) was present for the final 1 hr of culture. [³H]-Uridine uptake was then calculated as in Figure 1. Numbers represent the mean ± SE of four observations. *Greater than control with P < 0.05.

Figure 4.

Dose–response effect of PRL on [³H]-uridine uptake into cultured mammary gland explants. Tissues were initially cultured for 1 day with 1 μg/ml insulin plus 10⁻⁷ M cortisol. Incubation was then continued for 24 hr with the indicated concentrations of PRL. [³H]-Uridine (1 μCi/ml) was present for the final 1 hr of culture. [³H]-Uridine uptake was then calculated as in Figure 1 . Numbers represent the mean ± SE of four observations. *Greater than control with P < 0.05.

Figure 5.

Sodium dependence of [³H]-uridine uptake into cultured mammary gland explants. Mammary tissues were cultured for 1 day with 1 μg/ml insulin + 10⁻⁷ cortisol. Incubation was then continued for 24 hr with or without 1 μg/ml PRL present. During a subsequent 2 hr incubation, [³H]-uridine uptake was assessed in tissue cultured with Medium-199, KRB (containing sodium), or KRB containing choline chloride substituted for sodium chloride. Results are expressed as the mean ± SE of 4 observations. *Greater than control with P < 0.05. **Less than comparable control with P < 0.05.

Figure 6.

Sodium dependence of [³H]-uridine incorporation into RNA in cultured mammary gland explants. Experimental details are the same as in Figure 9 except that the [³H]-uridine incorporated into the TCA-precipitable fraction was determined.

Figure 7.

Lineweaver–Burke Plot for [³H]-uridine uptake in cultured mammary gland explants. Mammary tissues were cultured for 1 day with 1 μg/ml insulin plus 10⁻⁷ M cortisol. Incubation was continued for 24 hr in the presence or absence of 1 μg/ml PRL. Tissues were then cultured for 2 hr with [³H]-uridine (1 μCi/ml) at the concentrations indicated. The reciprocals of the substrate concentration and intracellular uridine accumulations were then plotted.

Figure 8.

Effect of other nucleosides on [³H]-uridine uptake in cultured mammary gland explants. Tissues were initially cultured for 1 day with 1 μg/ml insulin + 10⁻⁷ M cortisol. Incubation was then continued for 1 day in the presence or absence of 1 μg/ml PRL. [³H]-Uridine uptake (1 μCi/ml, 0.01 μM) was then determined during a 1-hr incubation with 0.1 mM nucleosides as specified. Numbers represent the mean ± SE of four observations. *Greater than control with P < 0.05.

Figure 9.

Effect of other nucleosides on [³H]-uridine incorporation into RNA in cultured mammary gland explants. Experimental details are the same as in Figure 8 , except that the [³H]-uridine incorporated into the TCA-precipitable fraction was determined.

Footnotes

This work was sponsored by funds from the Children’s Hospital of Michigan and the National Institutes of Health Grant RR08167.

1

To whom requests for reprints should be addressed at the Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. E-mail:

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