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.     

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.     

Hormonal control of casein synthesis in mammary explants from pregnant goats

The effects of insulin, cortisol, prolactin, 3,3',5-triiodo-L-thyronine (L-T3) and progesterone on the synthesis of total protein and casein in mammary explants from pregnant goats were studied. In the absence of hormones and in the presence of insulin plus cortisol the rate of incorporation of 14C-leucine into proteins that were precipitated with the anti-casein antibody decreased during culture. The addition of prolactin to hormonal combination of insulin and cortisol caused large stimulation of rates of casein synthesis. Maximum incorporation of leucine was attained between 3 and 5 days of culture in the presence of 0.5 microgram ml-1 of prolactin. Prolactin stimulated-casein and total protein synthesis were not consistently affected by the addition of L-T3 or progesterone. The inhibition of DNA synthesis by hydroxyurea or cytosine-arabinofuranoside had no effect on casein synthesis in mammary explants from pregnant goats.     

Casein turnover in rabbit mammary explants in organ culture

1. Explants of mammary gland from mid-pregnant rabbits were cultured in medium 199 containing insulin, prolactin and cortisol, and specific anti-casein immunoglobulin G was used to measure the amount, rate of synthesis and rate of degradation of casein in the explants in the presence of hormones and after removal of hormones from previously stimulated tissue. 2. The amount of casein in particle-free supernatants prepared from mammary explants was measured by ;rocket' immunoelectrophoresis. 3. The rate of incorporation of l-[4,5-(3)H]leucine into casein was measured after isolation of the casein by immunoadsorbent chromatography and polyacrylamide-gel electrophoresis in the presence of urea and sodium dodecyl sulphate. 4. Casein accumulates in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in a decrease in the rate of accumulation of casein in the explants. 5. Casein-synthetic rate increases in mammary explants in the presence of insulin, prolactin and cortisol, but not in the absence of hormones. Removal of hormones after 24h in culture results in continued casein synthesis at approx. 30% of the rate in the presence of hormones. The synthetic rate does not decrease to values observed in explants cultured throughout in the absence of hormones. 6. Casein is not degraded in mammary explants during a phase of rapid casein accumulation (36-72h) in the presence of hormones. Furthermore casein is not degraded when hormones are removed from the tissue after between 36 and 72h in culture. 7. Casein is glycosylated in mammary explants; the extent of glycosylation parallels the rate of synthesis. The glycosylated protein is rapidly secreted from the tissue. 8. The results are consistent with the notion that after hormonal stimulation mammary explants from mid-pregnant rabbits synthesize, glycosylate and rapidly secrete casein. Removal of hormones decreases the synthetic rate of casein, but does not cause the accumulation of a pool of degradable casein in the lobuloalveolar cells.     

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.     

Prolactin-like actions of phospholipase C on RNA and casein synthesis in mouse mammary gland explants

Phospholipase C stimulated the rate of [3H]-uridine incorporation into RNA in cultured mouse mammary gland explants. This effect was similar to that elicited by prolactin in that the time-course and magnitude of response were the same. In addition, the effects of prolactin and phospholipase C were non-additive when these agents were tested together. Although phospholipase C, by itself, had no effect on the rate of [3H]-leucine incorporation into casein, it was found to stimulate casein synthesis when the explants were simultaneously exposed to 0.5 mM spermidine. These observations are compatible with the idea that at least certain of the actions of prolactin in the mammary gland may be carried out via phospholipase C.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [3H]leucine or [3H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [3H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [3H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [3H]mannose and [14C]leucine confirmed this. The decreased incorporation of [3H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Regulation of enzyme turnover during tissue differentiation. Interactions of insulin, prolactin and cortisol in controlling the turnover of fatty acid synthetase in rabbit mammary gland in organ culture

1. Explants of mammary gland from mid-pregnant rabbits were cultured in Medium 199 containing combinations of insulin, prolactin and cortisol. With hormone combinations which included prolactin, a sustained increase in the apparent rate of synthesis and in the amount of fatty acid synthetase was measurable immunologically. Maximum increase was produced with insulin, prolactin and cortisol present together. 2. With prolactin present alone, synthetase activity in the explants decreased to undetectable values after 1 day in culture, whereas the incorporation of l-[U-(14)C]leucine into immunodetectable material increased. Prolactin may therefore direct the synthesis of immunologically cross-reactive precursors of fatty acid synthetase which are enzymically inactive. 3. Culture with dibutyryl cyclic AMP plus theophylline in the presence of insulin, prolactin and cortisol delayed the increase in the rate of synthesis and accumulation of the synthetase. These compounds may also prevent the apparent decrease in the rate of degradation of the synthetase which occurs on day 2 of culture. 4. A large decrease in the apparent rate of degradation of the synthetase on day 2 of culture occurs during culture with hormone combinations which include prolactin. The protein obtained by centrifugation of explant homogenates for 6min at 14000g(av.) is degraded continuously throughout the culture period. 5. This decrease in the apparent rate of degradation of the synthetase was measured by radio-immunological precipitation. It is probably part of a regulated programme of enzyme degradation and not a reflexion of the reutilization of radioactive amino acids for the following reasons. (a) The calculated increase in the amount of the synthetase in explants on day 2 of culture with insulin, prolactin and cortisol was approximately equal to the measured increase of the enzyme complex which accumulates in the explants. This suggests little or no enzyme degradation has occurred. (b) Explants were cultured for 24h with insulin, prolactin and cortisol. They were then incubated with l-[U-(14)C]leucine, washed and incubated again for up to 4(1/2)h. l-[U-(14)C]Leucine rapidly equilibrated with the intracellular amino acid pool. Within 10min of incubation after washing explants to remove endogenous l-[U-(14)C]leucine the previously linear incorporation of l-[U-(14)C]-leucine into total explant protein ceased. This suggests that protein is synthesized from an amino acid pool which rapidly equilibrates with amino acids in the culture medium. (c) Explants were cultured for 24h as described in (b) but after washing they were cultured with insulin, prolactin and cortisol for 24h. Approx. 90% of the radioactivity lost from the ;free' intracellular amino acid pool and from amino acids derived from the degradation of explant protein in this period was detected in the culture medium. This suggests that the ;free' intracellular amino acids and amino acids derived from protein degradation can equilibrate with amino acids in the medium. A residual ;free' radioactive amino acid pool was present in the tissue. (d) Casein represents approx. 20% of the protein synthesized after 1 day in culture with insulin, prolactin and cortisol. Histological evidence suggests that on day 2 of culture, casein is unlikely to be degraded in the tissue. No increase in the radioactivity incorporated into casein can be measured in the 23h after incubation of explants with l-[U-(14)C]leucine as described in (b). This suggests that the incorporation of radioactivity into proteins during culture after incubation with l-[U-(14)C]leucine is minimal. (e) Inhibition of protein synthesis in explants by cycloheximide after incubation with l-[U-(14)C]leucine does not reveal a latent continuous degradation of fatty acid synthetase on day 2 of culture which might have been masked by the high rates of protein synthesis and therefore the accumulation of the enzyme. 6. The conclusion is discussed that there is a real decrease (or even cessation) in the rate of degradation of fatty acid synthetase during the period when the enzyme accumulates in explants cultured with hormone combinations which contain prolactin.     

Hormonal activation of adenylate cyclase and its role in cyclic AMP mediated regulation of RNA synthesis in the mouse mammary gland.

The activity of adenylate cyclase (EC 4.6.1.1) in the mouse mammary gland increases during late pregnancy and reaches its maximum value at one day pre partum. In the mouse mammary gland explant culture the adenylate cyclase activity is stimulated by a cooperative action of insulin, prolactin and hydrocortisone. The effect of these hormones can be demonstrated in intact cells, but not in a cell-free system. In the explants, RNA synthesis is stimulated by dibutyryl cyclic AMP, insulin and prolactin. The effects of both protein hormones and cyclic AMP are additive. The results obtained suggest that insulin and prolactin in cooperation with hydrocortisone are involved in the regulation of RNA synthesis in the mammary gland by activation of the adenylate cyclase system, independently of their effect on this process not mediated by cyclic AMP.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [³H]leucine or [³H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [³H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [³H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [³H]mannose and [¹⁴C]leucine confirmed this. The decreased incorporation of [³H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Prolactin actions on casein and lipid biosynthesis in mouse and rabbit mammary gland explants are abolished by p-bromphenacyl bromide and quinacrine, phospholipase A2 inhibitors

p-Bromphenacyl bromide (BPB) at concentrations of 50 microM and above and quinacrine (50 microM) abolished the actions of prolactin (PRL) on casein and lipid biosynthesis in cultured mouse mammary gland explants. In cultured rabbit mammary gland explants, 100 microM BPB or quinacrine abolished the PRL stimulation of casein synthesis, while 50 microM BPB or 250 microM quinacrine abolished the PRL stimulation of lipid biosynthesis. Since BPB and quinacrine are known to inhibit the enzyme phospholipase A2 (PLA2), it is possible that ongoing PLA2 activity is essential for prolactin to express its actions on at least certain lactogenic processes.     

Cyclic guanosine 3',5'-monophosphate and phosphodiesterase activity in mitogen-stimulated human lymphocytes.

The cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase from human leukemic lymphocytes differes from the normal cell enzyme in having a much higher activity and a loss of inhibition by cyclic guanosine 3′,5′-monophosphate (cyclic GMP). In an effort to determine the mechanism of these alterations, we have studied this enzyme in a model system, lectin-stimulated normal human lymphocytes. Following stimulation of cells with concanavalin A (con A) the enzyme activity gradually becomes altered, until it fully resembles the phosphodiesterase found in leukemic lymphocytes. The changes in the enzyme parallel cell proliferation as measured by increases in thymidine incorporation into DNA. The addition of a guanylate cyclase inhibitor preparation from the bitter melon prevents both the changes in the phosphodiesterase and the thymidine incorporation into DNA. This blockage can be partially reversed by addition of 8-bromo cyclic guanosine 3′,5′-monophosphate (8-bromo cyclic GMP) to the con A-stimulated normal lymphocytes. These results indicate a possible role of cyclic GMP in a growth related alteration of cyclic AMP phosphodiesterase.     

Inhibition by purine compounds of cyclic GMP-stimulated cyclic AMP phosphodiesterase activity from a particulate fraction of rat striatum

Cyclic AMP phosphodiesterase activity in a particulate fraction of rat striatum is stimulated two fold by cyclic GMP. An investigation of the effects of various purine compounds on basal and cyclic GMP-stimulated cyclic AMP phosphodiesterase activity as measured at a low substrate concentration (3 uM) was carried out. Adenosine inhibits cyclic GMP-stimulated cyclic AMP phosphodiesterase activity with an IC₅₀ of 400 uM while inhibiting basal cyclic AMP phosphodiesterase activity with an IC₅₀ of 2.4 mM. Adenosine blocks cyclic GMP stimulation of cyclic AMP hydrolysis with an IC₅₀ of 80 uM. Inosine and hypoxanthine have a similar profile of action but are less effective with IC₅₀'s of 200 and 400 uM respectively on cyclic GMP stimulation of phosphodiesterase activity and only 20–40% inhibition of basal enzyme activity up to 2.4 mM. Adenine, guanosine and guanine block cyclic GMP stimulation of cyclic AMP phosphodiesterase activity with IC₅₀'s of 100–200 uM. Classical phosphodiesterase inhibitors of the alkylxanthine type are also selective for the stimulated enzyme with IC₅₀'s of 200 and 25 uM for theophylline and IBMX on cyclic GMP-stimulated cyclic AMP hydrolysis and IC₅₀'s of 500 and 50 uM respectively on basal phosphodiesterase activity. Theophylline and IBMX are potent inhibitors of cyclic GMP stimulation of cyclic AMP phosphodiesterase activity with IC₅₀'s of 50 and 5 uM. These findings suggest a role for physiologically available purine compounds and alkylxanthines in the regulation of cyclic nucleotide metabolism through interaction with cyclic GMP stimulation of cyclic AMP phosphodiesterase activity.     

Effect of kinase C inhibitor, gossypol, on the actions of prolactin in cultured mouse mammary tissues

Gossypol, a drug which has been shown to be an inhibitor of kinase C activity in mouse mammary tissues, is shown to abolish several of the actions of prolactin in cultured mouse mammary gland explants. The prolactin effects that are abolished include its stimulatory effects on ornithine decarboxylase activity, the rate of [3H]uridine incorporation into RNA, the rate of [3H]leucine incorporation into a casein-rich phosphoprotein fraction, and the rate of [14C]acetate incorporation into lipids. Since the inhibitory concentrations of gossypol employed in these studies correspond well with the gossypol concentrations required to inhibit kinase C activity, we conclude that ongoing kinase C activity is essential for prolactin to express its differentiative actions in mammary tissues.     

Effect of lithium ions on RNA synthesis in mammary gland explants of mice.

LiC1 was found to stimulate RNA synthesis in the mammary gland in a manner similar to that of prolactin. Since LiC1 is known to inhibit adenyl cyclase and thus to reduce levels of cyclic AMP, it is concluded that a reduced level of cyclic AMP may be one step in the mechanism whereby prolactin regulates the metabolism of the mammary gland.     

Phorbol myristate acetate stimulates RNA and casein synthesis in cultured mouse mammary gland tissues

Prolactin and phorbol myristate acetate (TPA) stimulate the rate of [3H]uridine incorporation in cultured mouse mammary gland explants in a similar fashion. Both the time-courses and magnitude of responses were the same; in addition, maximum stimulatory concentrations of TPA and prolactin elicited a nonadditive response when tested together. Nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, abolished both the TPA and prolactin effects on [3H]uridine incorporation. TPA also effected an enhanced rate of [3H]leucine incorporation into a casein-rich phosphoprotein fraction, but only if the explants were also cultured with spermidine.     

Substrate specificity of a high-affinity, monovalent cation-dependent amino acid carrier.

When mammary gland explants from mid-pregnant rats were incubated with insulin (5 μg/ml) and [³H]cortisol (5 μg/ml) for one day, the tissue accumulated 1.69 μg cortisol/g wet tissue. During a second incubation with insulin and prolactin (5 μg/ml), only 20% of the steroid was lost per day. Such retention of glucocorticoid had an important biological consequence: the tissue exposed for one day to insulin and cortisol showed a transient stimulation of casein synthesis during a subsequent, five-day incubation with insulin and prolactin. No casein synthesis was detected, if the first culture medium contained only insulin. In conclusion, mammary gland explants from mid-pregnant rats require a glucocorticoid for casein synthesis, but this requirement may be obscured if the explants are initially incubated in medium containing cortisol, since they are capable of accumulating and retaining this steroid. Similar interpretative difficulties may arise in studies on other steroid-tissue relationships.     

Cyclic nucleotide phosphodiesterases in the cricket, Acheta domesticus.

Exceptionally high levels of guanosine 3'-5'-cyclic monophosphate (cyclic GMP) in the accessory reproductive gland of the male house cricket, Acheta domesticus, led to an investigation of cyclic nucleotide phosphodiesterase (EC 3.1.4.--) as a possible regulatory enzyme. Cricket cyclic nucleotide phosphodiesterase activity with cyclic GMP or cyclic AMP as substrate had a pH optimum around 9.0, required Mg2+ or Mn2+ for maximal activity, and was inhibited by EDTA and methylxanthines. Cyclic GMP phosphodiesterase occurred mainly in the soluble fraction of homogenates of accessory glands or whole crickets, but cyclic AMP phosphodiesterase in the accessory gland was primarily particulate. Kinetic analysis indicated three forms of cyclic GMP phosphodiesterase, with Km values at 2.9 muM, 71 muM and 1.5 mM. Chromatography of whole cricket or accessory gland extracts on DEAE cellulose gave an initial peak having comparable activity with either cyclic GMP or cyclic AMP, and a second peak specific for cyclic AMP. There were no appreciable changes in the specific activity or kinetic properties of accessory gland cyclic GMP phosphodiesterase during a developmental period over which cyclic GMP levels rise more than 500-fold. Thus, the accumulation of cyclic GMP in the accessory gland is probably not associated with concomitant developmental modulation of phosphodiesterase activity.     

Effects of hormones on protein and amino acid metabolism in mammary-gland explants of mice.

The effects of insulin, cortisol and prolactin on amino acid uptake and protein biosynthesis were determined in mammary-gland explants from mid-pregnant mice. Insulin stimulated [3H]leucine incorporation into protein within 15 min of adding insulin to the incubation medium. Insulin also had a rapid stimulatory effect on the rate of aminoiso[14C]butyric acid uptake, but it had no effect on the intracellular accumulation of [3H]leucine. Cortisol inhibited the rate of [3H]leucine incorporation into protein during the initial 4h of incubation, but it had no effect at subsequent times. [3H]Leucine uptake was unaffected by cortisol, but amino[14C]isobutyric acid uptake was inhibited after a 4h exposure period to this hormone. Prolactin stimulated the rate of [3H]leucine incorporation into protein when tissues were exposed to this hormone for 4h or more; up to 4h, however, no effect of prolactin was detected. At all times tested, prolactin had no effect on the uptake of either amino[14C]isobutyric acid or [3H]leucine. Incubation with actinomycin D abolished the prolactin stimulation of protein biosynthesis, but this antibiotic did not affect the insulin response. A distinct difference in the mechanism of action of these hormones on protein biosynthesis in the mammary gland is thus apparent.     

Prolactin stimulation of iodide uptake and incorporation into protein is polyamine-dependent in mouse mammary gland explants

Previous studies have demonstrated that the prolactin stimulation of most lactational processes (casein, lactose, and triglyceride synthesis) requires an earlier stimulating effect of prolactin on the synthesis of the polyamines. Spermidine appears to be the specific polyamine required for prolactin to enhance milk product synthesis. Inorganic iodide is present in milk at more than an order of magnitude higher concentration than that of the maternal plasma. Since prolactin stimulates iodide accumulation in milk, the goal of these studies was to determine the role of the polyamines in this hormone response. Two drugs were employed in these studies: DFMO (difluoromethylornithine), which inhibits ornithine decarboxylase, and MGBG [methylglyoxal bis(guanyl-hydrazone)], which inhibits S-adenosyl methionine decarboxylase. In mammary gland explants from midpregnant (10-14 days of pregnancy) mice, MGBG at 100 microM abolished the prolactin stimulation of iodide uptake and incorporation into milk proteins, whereas DFMO caused a concentration-dependent inhibition of the PRL response. Selected sensitivity of the MGBG and DFMO inhibitions was validated by a reversal of the drug inhibitions with the addition of 1 mM spermidine to the culture medium. These data suggest that the polyamine signaling pathway is involved in the prolactin stimulation of iodide uptake into milk.