John W. Perry

Regulation of ornithine decarboxylase in cultured mouse mammary gland by the osmolarity in the cellular environment

The biphasic increase of ornithine decarboxylase activity in mouse mammary gland in organ culture occurs with a hormone-independent first peak and a hormone-dependent second peak. The data presented indicate that a change in the osmolarity of the cellular environment is the major contributing factor for the emergence of the hormone-independent ornithine decarboxylase activity in mammary explants. Thus, incubation of mammary explants for 3 h in a medium diluted 53% with distilled water results in approx. 1000-fold stimulation of enzyme activity over the initial level, whereas a similar dilution of the medium with 0.18 M NaCl or 0.3 M sucrose blocks the increase. The increase in enzyme activity is similarly affected by a reduction of the concentration of NaCl in the culture medium. The hypoosmotic stimulation of ornithine decarboxylase activity appears to be affected at a posttranscriptional level, and is enhanced further by the actions of insulin and prolactin. The hypoosmotic enhancement of ornithine decarboxylase activity produces a large increase in the intracellular concentration of putrescine in mammary explants. However, neither the concentration of spermidine and spermine nor the activity of S-adenosyl-L-methionine decarboxylase is affected. In addition, studies of putrescine transport in mammary explants show that hypotonicity causes an increase in the rate of influx and a decrease in efflux of putrescine with enhancement of intracellular putrescine accumulation. On the other hand, the uptake of spermidine, spermine, amino acids, sugar, and a lipophilic cation, triphenylmethylphosphonium is unaffected. These data suggest a possibility that osmotic alteration in cellular environment causes an incresed need for putrescine in mammary cells, resulting in stimulation of ornithine decarboxylase activity, which may represent a cellular mechanism for maintaining the homeostasis of the intracellular cationic environment.

Hormonal regulation of spermidine synthase during the development of mouse mammary epithelium in vitro.

The activity of spermidine synthase increases rapidly during the induction of mammary epithelial development by insulin, cortisol and prolactin in vitro. The increase is detectable 2–3 hours after the initiation of culture, and thereafter continues almost linearly up to 72 hours. The enzyme activity remains unchanged in the absence of the hormones during culture. The increase in enzyme activity is dependent only on the actions of insulin and cortisol which appear to act at both transcriptional and translational levels. Studies with cycloheximide suggest that spermidine synthase in mammary cells has a half-life of more than 12 hours.

Concentration-dependent differential effects of cortisol on synthesis of alpha-lactalbumin and of casein in cultured mouse mammary gland explants: importance of prolactin concentration.

SummaryCortisol was previously shown to elicit a concentration-dependent inhibition of α-lactalbumin accumulation in midpregnant mouse mammary gland cultured in medium containing optimal concentrations of 5 μg/ml prolactin and insulin. In contrast, casein accumulation under these conditions was progressively stimulated by addition of increasing amounts of cortisol (Ono, M.; Oka, T. Cell 19: 473–480; 1980). In the present study we found that in the presence of a suboptimal concentration of 0.5 μg/ml prolactin, 2.8×10−9M to 2.8×10−7M cortisol stimulated α-lactalbumin accumulation. Furthermore, higher concentrations of cortisol produced a smaller inhibition of α-lactalbumin accumulation as compared to that obtained in cultures containing 5 μg/ml prolactin. The maximal increase in α-lactalbumin accumulation attained in the presence of 1.4×10−8M cortisol, 0.5 μg/ml prolactin, and insulin was comparable to that observed in culture containing 5 μg/ml prolactin and insulin. Similar results were obtained in a cortisol concentration-response study of α-lactalbumin accumulation in cultures containing a suboptimal concentration of 0.5 μg/ml human placental lactogen. Measurement of the rate of α-lactalbumin synthesis in cultured tissue indicated that the opposing effects of low and high concentrations of cortisol on α-lactalbumin accumulation involved an alteration in the rate of synthesis of the milk protein. In contrast to α-lactalbumin, the synthesis of casein was stimulated in a concentration-dependent manner by addition of cortisol that acted synergistically with either 0.5 μg/ml or 5 μg/ml prolactin. The maximal increases were obtained in the presence of 2.8×10−6M cortisol. These results indicated that the action of cortisol on α-lactalbumin accumulation can be modulated by the concentration, of prolactin and suggest that the interplay between cortisol and prolactin in regulation of α-lactalbumin synthesis may be different from that involved in casein synthesis.

Glucocorticoid stimulation of choline kinase activity during the development of mouse mammary gland

Abstract Mouse mammary gland contains choline kinase activity that can be stimulated by polyamines. Developmental studies show that the activity of choline kinase in mammary gland is low in both virgin and nonpregnant primiparous animals but increases severalfold during pregnancy and reaches a maximal level during the lactation period. Similar increases in enzyme activity are observed by cultivation of tissue explants in the presence of insulin, cortisol, and prolactin, a combination of hormones which induces the ultrastructural and biochemical changes associated with the development of mammary gland during pregnancy and lactation. The increase in enzyme activity in cultured explants is dependent only on the actions of both insulin and cortisol and parallels the formation of rough endoplasmic reticulum, which is effected by the same combination of hormones. The hormonal stimulation of choline kinase activity appears to involve the action of spermidine, a polyamine which accumulates in the cells under the influence of cortisol and mimicks the effect of cortisol on milk-protein synthesis in cultured explants.

The study of differentiative potential of the lactating mouse mammary gland in organ culture

SummaryThe organ culture of the mammary gland of lactating mice was used to examine the response of the differentiated gland to lactogenic stimuli, insulin, cortisol, and prolactin. Time course studies showed that casein synthesis in cultured tissue decreased rapidly during the first 2 d despite the presence of the three hormones, but on the 3rd d tissue cultured with either insulin and prolactin or all three hormones regained the ability to synthesize milk proteins, casein, and α-lactalbumin: a greater increase occurred in the three hormone system. The delayed addition of prolactin on Day 2 to the culture system containing insulin and cortisol also stimulated casein synthesis. The addition of cytarabine, which inhibited insulin-dependent cell proliferation in cultured explants, did not block the rebound of milk protein synthesis. The results indicate that in the presence of insulin, cortisol, and prolactin mammary epithelial cells in culture first lose and then regain the ability of synthesizing milk protein without requiring the formation of new daughter cells.

Further characterization of the inhibition of casein production in a primary mouse mammary epithelial cell culture by epidermal growth factor: Antagonism by cyclic AMP

Epidermal growth factor (EGF) inhibited casein production and the accumulation of casein mRNA activity induced by insulin (I), cortisol (F) and prolactin (P) in a primary culture of mammary epithelial cells from pregnant mice. The inhibitory effects of EGF were blocked by 8-bromo cyclic AMP (8-br-cAMP) in a dose-dependent manner. The effect of 8-br-cAMP was observed at a concentration as low as 20 microM and was maximal at 500 microM. Dibutyryl cyclic AMP (db-cAMP), cAMP, and 3-isobutylmethylxanthine (IBMX), an inhibitor of phosphodiesterase, also antagonized the inhibitory effect of EGF on casein production. 8-Br-cAMP had, however, no effect on the mitogenic activity of EGF in this system. These results suggest a possible modulatory role of cAMP in EGF-induced inhibition of casein production in cultured mammary epithelial cells.

Polyamines in Normal and Neoplastic Growth of Mammary Gland

The diamine putrescine and the polyamines spermidine and spermine are small aliphatic nitrogenous bases which are present in all mammalian cells. The structures of these polycations are presented below:

The biosynthesis of spermidine as a key regulatory step in the hormonal induction of milk-protein synthesis in the organ culture of the mouse mammary gland

The diamine putrescine and the polyamine spermidine and spermine are aliphatic polycations which are synthesized by mammalian cells and are considered to play essential roles in cell growth and development. In the mouse mammary gland the concentration of spermidine remains low in the virgin state, when mammary cells are developmentally dormant (Oka et al., 1981). The concentration of spermidine begins to increase during pregnancy and reaches a maximal level during the lactation period when the development of the mammary gland culminates in the synthesis of milk-proteins. Such changes can be induced in vitro by cultivation of mammary tissue explants in a chemically-defined synthetic medium containing appropriate combination of hormones (Oka et al., 1981). This organ culture system has been used to examine the possible physiological function of polyamines in the development of the mammary gland.