Temporal effect of prolactin on the activities of lactose synthetase, alpha-lactalbumin, and galactosyl transferase in mouse mammary gland explants

The onset of the prolactin (PRL) stimulation of lactose synthesis is between 4 and 8 hr after adding PRL to cultured mouse mammary tissues. The synthesis of lactose is catalyzed by the enzyme lactose synthetase, which is composed of two parts, alpha-lactalbumin and galactosyl transferase. In time-sequence studies, it was found that the activity of galactosyl transferase is enhanced by PRL in concert with the onset of the PRL stimulation of lactose synthesis. In contrast, the earliest detectable effect of PRL on alpha-lactalbumin activity occurred 24 hr after adding PRL to the cultures. It is, therefore, apparent that the rate-limiting component for the PRL stimulation of lactose synthesis in cultured mouse mammary tissues is galactosyl transferase activity.     

Observations on the early actions of prolactin on RNA and casein synthesis in mouse mammary gland explants

The action of prolactin on RNA synthesis in cultured mouse mammary gland explants becomes manifest when the tissues are exposed only briefly (1 h or less) to prolactin. In contrast, the action of prolactin on casein synthesis only becomes apparent when the tissues are cultured for 6 h or more with prolactin. Once the actions of prolactin on RNA and casein synthesis are initiated, however, these effects persist for hours or days, even when the tissues are subsequently cultured in the absence of prolactin.     

Sequence of prolactin effects on phospholipid synthesis in mouse mammary gland explants

In cultured mouse mammary gland explants derived from 12-14 day pregnant mice, the effect of prolactin (PRL) on the rate of incorporation of several precursors into neutral lipids and phospholipids was determined. Employing [14C]-acetate as a substrate, PRL stimulates its incorporation into a) neutral lipids by 4-6 hours, b) phosphatidyl choline (PC) and phosphatidyl inositol-phosphatidyl serine (PI-PS) by 1-2 hours, and c) phosphatidyl ethanolamine (PE) by 2-4 hours. Using [3H]-glycerol as a substrate, the temporal response to PRL for its incorporation into the neutral lipids was the same as that for [14C]-acetate, however, PRL did not enhance the rate of [3H]-glycerol incorporation into the phospholipids at any time through 16 hours. PRL similarly had no effect on the rates of [3H]-choline, [3H]-serine, [3H]-ethanolamine, or [32P]O4 incorporation into the phospholipids at hormone exposure periods of 8 hours or more. And finally, PRL had no effect on the rates of [3H]-arachidonate or [14C]-linoleate incorporation into neutral lipids or phospholipids at culture periods up to 18 hours. These data suggest that the early effect of PRL on [14C]-acetate incorporation into the phospholipids is due to either the insertion of newly synthesized fatty acids and/or the extension of fatty acids contained in the phospholipids.     

Effects of cyclic nucleotides on lipid biosynthesis in mouse mammary gland explants

The effects of dibutyryl cAMP, 1-methyl-3-isobutyl xanthine (MIX), cGMP, dibutyryl cGMP, and 8-bromo cGMP on the rate of lipid synthesis in mouse mammary gland explants were studied. Dibutyryl cAMP at 10(-4) M selectively inhibited the effect of prolactin on the rate of [14C]acetate incorporation into lipids. At 10(-3) M, dB-cAMP inhibited the effects of insulin, insulin plus cortisol, and prolactin. The phosphodiesterase inhibitor, MIX, inhibited both basal and prolactin-stimulated incorporation rates in a concentration-dependent fashion. These data suggest an inhibitory role for cAMP in the regulation of lipogenesis in the mammary gland. Cyclic GMP, db-cGMP, and 8-bromo cGMP were all without effect on either basal or prolactin-stimulated incorporation rates. Therefore, it appears that cGMP, by itself, is not involved in the regulation of lipogenesis in the mammary gland.     

Effect of prolactin on phosphate transport and incorporation in mouse mammary gland explants

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 (32)PO(4) was six times higher than in the culture medium after a 4-h treatment with (32)PO(4). Of the principal lactogenic hormones [insulin (I), cortisol (H), and prolactin (PRL)], only I and PRL (in the presence of H and I) stimulated (32)PO(4) uptake into cultured mammary tissues; H, by itself or in the presence of I or PRL, inhibited (32)PO(4) uptake. All three lactogenic hormones together effected the greatest stimulation of (32)PO(4) uptake. Similar hormone effects were observed with regard to (32)PO(4) incorporation into lipids and trichloroacetic acid-insoluble molecules. In a time course study, the onset of the PRL stimulation of (32)PO(4) 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 PO(4) uptake was determined to be approximately 7.7 mM x h(-1) x l cell water(-1); the apparent Michaelis-Menten constant was approximately 3-5 mM. The PRL effect on (32)PO(4) 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 (32)PO(4) uptake and incorporation into macromolecules in cultured mouse mammary tissues.     

Effect of prolactin on sodium iodide symporter expression in mouse mammary gland explants

Iodide accumulates in milk at a concentration that is more than an order of magnitude higher than the iodide concentration in maternal plasma. In earlier studies from our laboratory, we have shown that prolactin (PRL) enhances iodide accumulation by two- to threefold in cultured mammary tissues taken from pregnant mice. In the present studies, we demonstrate via Western blotting techniques that prolactin elevates the quantity of the sodium iodide symporter (NIS) in cultured mouse mammary tissues. In time-course studies, the onset of the PRL effect of NIS accumulation was found to be between 4 and 16 h after addition of PRL to the explants. The lowest PRL concentration that elicited a significant response was 1 ng/ml, and a maximum effect was elicited with PRL concentrations >100 ng/ml. Actinomycin D, cycloheximide, and thiocyanate abolished the PRL effect on NIS accumulation, whereas perchlorate was without effect. These studies suggest that the PRL stimulation of iodide accumulation in milk is mediated, at least in part, by the PRL stimulation of NIS accumulation in mammary gland tissues. These studies further demonstrate that the PRL effect on NIS accumulation occurs via an RNA protein synthesis-dependent mechanism.     

Phospholipases and the effect of prolactin on uridine incorporation into RNA in mammary gland explants of mice

The possible effects of phospholipase A and phospholipase C on the rate of uridine incorporation into RNA in mammary gland explants of mice were tested. Phospholipase C had no effect on the rate of uridine incorporation, but it did suppress the action of prolactin on this metabolic parameter. In contrast, phospholipase A was found to stimulate the rate of uridine incorporation into RNA in a manner similar to that of prolactin. The time-courses for the onset of the prolactin and phospholipase A effects were the same. Also, the phospholipase A effect was nonadditive to the effect produced by a maximally stimulatory concentration of prolactin. Finally it was observed that, like the prolactin effect, the phospholipase A effect was abolished by incubation with dibutyryl cyclic AMP, theophylline, quinine, indomethacin and prostaglandin E₁. Further, the phospholipase A effect was nonadditive to the prolactin-like effects produced by cyclic GMP, prostaglandin F_2α or arachidonic acid. These data therefore suggest that prolactin and phospholipase A stimulate RNA synthesis in mammary gland explants via similar processes.     

Phospholipases and the effect of prolactin on uridine incorporation into RNA in mammary gland explants of mice.

The possible effects of phospholipase A and phospholipase C on the rate of uridine incorporation into RNA in mammary gland explants of mice were tested. Phospholipase C had no effect on the rate of uridine incorporation, but it did suppress the action of prolactin on this metabolic parameter. In contrast, phospholipase A was found to stimulate the rate of uridine incorporation into RNA in a manner similar to that of prolactin. The time-courses for the onset of the prolactin and phospholipase A effects were the same. Also, the phospholipase A effect was nonadditive to the effect produced by a maximally stimulatory concentration of prolactin. Finally it was observed that, like the prolactin effect, the phospholipase A effect was abolished by incubation with dibutyryl cyclic AMP, theophylline, quinine, indomethacin and prostaglandin E1. Further, the phospholipase A effect was nonadditive to the prolactin-like effects produced by the cyclic GMP, prostaglandin F2alpha or arachidonic acid. These data therefore suggest that prolactin and phospholipase A stimulate RNA synthesis in mammary gland explants via similar processes.     

Possible interaction of cyclic nucleotides with the prolactin stimulation of casein synthesis in mouse mammary gland explants.

During a 10-h incubation, cyclic nucleotide phosphodiesterase inhibitors, viz. theophylline and quinine, were found to reduce by 40-50% the rate of [3H] leucine incorporation into casein in mammary gland explants from midpregnant mice. Further, dibutyryl cyclic AMP as well as the phosphodiesterase inhibitors were found to abolish the prolactin stimulation of leucine incorporation into casein. Elevated levels of cyclic AMP therefore appear to impair the functionality of the mammary gland. Although cyclic GMP was previously shown to stimulate RNA synthesis in the mammary gland in a prolactin-like manner, it had no effect on the rate of casein synthesis in mammary gland explants. Preincubation of explants with cyclic GMP did, however, attenuate the time required for the commencement of the prolactin stimulation of the rate of leucine incorporation into casein. A physiological role of cyclic GMP for the regulation of the rate of casein synthesis is thus suggested.     

Binding of mouse choriomammotropin to prolactin receptors in the mouse mammary gland.

The interaction between mouse choriomammotropin and mouse mammary glands was examined by radioreceptor assays using ovine prolactin (NIH-P-S9) iodinated by lactoperoxidase as a tracer. Mouse pituitary extracts and placental extracts were subjected to 10% acrylamide gel electrophoresis. Gels were cut into 2-mm segments after electrophoresis, and stored in 1 ml 0.05 M phosphate buffer (pH 7.4) containing 0.05 M NaCl overnight for elution. Lactating mammary tissues from D strain mice were incubated for 120 min in 1 ml Medium 199 containing 6 ng of 125I-prolactin and 0.1 ml of each eluate. Pituitary extracts displaced 125I-prolactin only at the position which coincides with the prolactin band. Displacement was observed at two positions of the gel when placental extracts were used. Relative mobilities (Rm) were 0.21 and 0.71, respectively. The slowly migrating component of choriomammotropin inhibited the binding of 125-I-prolactin more strongly that the rapidly migrating one. Neither of them was identified as a distinct band in stained gels. The molecular weight of ovine prolactin, mouse pituitary prolactin and the slowly migrating component of mouse choriomammotropin was estimated to be 23000 using disc electrophoresis but the ion charges of these hormones were considerably different.     

Hormonal induction and regulation of lactose synthetase in mouse mammary gland

The augmentation of lactose synthetase activity during late pregnancy and lactation was measured by using both a tissue-culture assay and a cell-free assay. The results indicated at least a 100-fold augmentation in specific activity between late pregnancy and lactation. The cell-free assay indicated that the activities of both subunits of this enzyme had increased to 20-30% of the value during lactation by the last day of pregnancy. The tissue-culture assay, however, showed activities only 3-4% of the maximum at the time of parturition. This suggests that not all the enzyme present in the tissue before lactation commenced was active. Since at all stages of pregnancy and lactation the B subunit, alpha-lactalbumin (which is also a milk protein), was rate-limiting, it is suggested that the rate of lactose synthesis may be linked to the rate of milk-protein synthesis. Both subunits of lactose synthetase could be induced in tissue culture by the hormones insulin+hydrocortisone+prolactin. Of the three hormones, prolactin appeared to be the ;trigger' that induced the synthesis of these proteins if the tissue had been stimulated previously by insulin+hydrocortisone.     

Actions of quinacrine on rna and casein syntheses in mouse mammary gland explants

Quinacrine at concentrations of 0.025 mM and greater significantly reduced or abolished the prolactin stimulation of RNA synthesis in mouse mammary gland explants. At concentrations of 0.01 mM or greater, the stimulatory action of prolactin on [3H]-leucine incorporation into a casein-rich protein fraction was also reduced or abolished. The possible relationship of these actions of quinacrine to its inhibition of phospholipase A2 and/or prostaglandin biosynthesis is discussed.     

Hormone regulation of choline uptake and incorporation in mouse mammary gland explants

Choline is a nutrient in milk that is essential for the nourishment and growth of newborns. In rat milk, choline is present in concentrations that are more than an order of magnitude higher than in maternal serum. Using cultured mammary tissues taken from 12-14-day pregnant mice, the effects of the three primary lactogenic hormones--prolactin (PRL), insulin (I), and cortisol (H)--on choline uptake and incorporation into lipids were determined. By itself or in the presence of H and/or PRL, I was the only hormone that increased the accumulation of choline in aqueous tissue fractions. In contrast, PRL, when present with I plus H, was the only hormone that stimulated the incorporation of choline into the lipid fraction of tissues. Choline uptake was found to be sodium and time dependent; maximum distribution ratios >18 were achieved after a 6-hr uptake time. In kinetic studies the apparent Km for choline uptake was calculated to be approximately 2.7 mM, whereas the Vmax was 7.4 mM intracellular water per 30 mins. These results suggest the existence of a sodium-dependent active transporter for choline in the mouse mammary gland that is specifically stimulated by I. PRL, in contrast, only stimulates the incorporation of choline into lipids.     

Polyamine biosynthesis and DNA synthesis in cultured mammary gland explants from virgin mice

The mammary cells in virgin mice are essentially non-proliferative, but they can be induced to undergo DNA synthesis in vitro in the presence of insulin. Time course studies on polyamine biosynthesis and DNA synthesis showed that insulin elicits sequential stimulation of the activity of the polyamine biosynthetic enzymes, ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase (SAMDC) and spermidine synthase, and an increase in the concentration of spermidine prior to the augmentation of DNA synthesis. At 48 to 72 hours of culture when DNA synthesis is maximal, the concentration of spermidine increased 2? to 3-fold, whereas the level of spermine remained unchanged. Addition of methyl glyoxal bis(guanylhydrazone) (5—10 μM), a potent inhibitor of SAMDC, to the medium at the onset of culture resulted in inhibition of spermidine formation and DNA synthesis, but when added at 24 hours or 48 hours of culture, the inhibitory effect on DNA synthesis was greatly reduced. The drug, however, produced little inhibition of RNA and protein synthesis. Inhibition of DNA synthesis by the drug can be reversed by addition of spermidine or other polyamines such as putrescine, cadaverine and spermine to the culture. Spermidine is, however, the only polyamine that is effective at physiological concentrations (100～150 pmoles/mg tissue). These results suggest a possibility that spermidine may play a key role in the regulation of mammary cell proliferation.