Gamma-glutamyltransferase activity in mammary gland of pregnant rats and its regulation by ovarian hormones, prolactin and placental lactogen.

Ovariectomy and ovariectomy plus hysterectomy on day 18 of pregnancy increased gamma-glutamyltransferase activity in the mammary gland. The withdrawal of progesterone and the subsequent release of prolactin are responsible for the rise in enzyme activity. Rat placental lactogen in the absence of prolactin and progesterone is able to induce gamma-glutamyltransferase activity.     

Characterization of prolactin receptors in pig mammary gland.

Prolactin receptors present in the particulate fraction of lactating pig mammary gland were solubilized by 7.5mM-3-[(3-cholamidopropyl)dimethylammonio]-1-propane-su lph onic acid (Chaps) and purified by affinity chromatography on prolactin coupled to Affi-Gel 10. Nearly 30% of the particulate receptors were solubilized by the detergent and over a 1000-fold purification from homogenates was achieved. A water-soluble fraction rich in receptors was observed during the preparation of membranes, although this fraction has not yet been purified. Prolactin binding to the receptors was a time-dependent, reversible and saturable reaction in particulate, Chaps-solubilized and purified receptors. In all forms, receptors showed the same specificity to peptide hormones. Prolactin and human growth hormone bound to the same receptors, whereas bovine growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and insulin failed to bind. After solubilization, the dissociation constant (Kd) for prolactin was decreased 5-fold from 9.8 X 10(-11) M in the particulate receptors to 1.8 X 10(-11) M in solubilized and purified receptors, being due principally to an increase in the association rate constant from 1.0 X 10(9)M-1 X h-1 to (3.9-4.6) X 10(9)M-1 X h-1, respectively, with the dissociation rate constant remaining unchanged at (1.1-1.3) X 10(-2)h-1. Isoelectric focusing of the prolactin-receptor complex revealed two peaks, one at a pI of 5.5-5.6 and the other at 5.2-5.3. Microsomal receptors were covalently cross-linked to 125I-labelled ovine prolactin with ethylene glycol bis(succinimidyl succinate) and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Autoradiography of the gel revealed a major subunit of Mr 28 000-35 000 and a minor one of Mr 67 000-69 000. Anti-(prolactin receptor) antibodies raised against rabbit mammary gland prolactin receptors were equally effective in inhibiting prolactin binding to particulate, solubilized and affinity-purified receptors, suggesting that purified prolactin receptors have a structure indistinguishable immunologically from particulate receptors and rabbit mammary gland prolactin receptors. The present demonstration shows that particulate prolactin receptors from a domestic animal can be solubilized and purified without losing the original properties of high affinity and binding specificity for hormones.     

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.     

Role of steroid hormones and prolactin in canine mammary cancer

In several animal studies, prolactin has been found to be essential for mammary epithelial development, and its administration has been consistently shown to increase the rate of mammary tumours. High levels of steroid hormones have also been suggested to enhance mammary cancer development. The present study investigates the levels of the following hormones in serum and in tissue homogenates in dogs bearing canine mammary tumours: prolactin (PRL), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 17beta-estradiol (17beta-E2) and estrone sulfate (S04E1). Eighty mammary tumours (40 dysplasias and benign and 40 malignant tumours) from 32 female dogs, and 10 normal mammary glands from eight female dogs without history of mammary tumours, were analysed. Prolactin and steroid hormones in serum and tissue homogenates, were analysed by enzyme immunoassays (EIA) techniques, previously validated for this animal species. Levels of prolactin in tissue homogenates were significantly different between malignant and benign mammary tumours (p<0.01). Serum prolactin concentrations were lower in the control group as compared with the group of dogs with benign tumours and in dogs with malignant tumours (p=0.01). Serum prolactin levels in dogs with benign lesions were not significantly different than those obtained from dogs with malignant tumours. Levels of steroid hormones were significantly higher in malignant tumours compared with the benign tumours and normal mammary glands (p<0.01) both in serum and homogenate determinations. Our results suggest that the canine neoplastic mammary gland could be a source of prolactin. Our hypothesis is that both prolactin and steroid hormones are involved in the growth of canine mammary cancer, and that they might have an autocrine/paracrine role in the maintenance of this disease.     

Iodinated bovine prolactin. Characterization and binding to mammary gland cells.

Iodinated bovine prolactin (2.6 iodine atoms/molecule; labelled with a trace of 125I to give a specific activity of 0.041 muCi/mg) was prepared by the chloramine T method. It was active in two bioassays (pigeon crop sac and dispersed mouse mammary cell), though somewhat less active than the unmodified hormone. In an immunoassay, iodinated prolactin was more effective than the unmodified hormone at displacing 125I-prolactin from antibody. High specific activity 125I-prolactin (1 iodine atom/molecule; 70 muCi/microgram) was used for autoradiographic studies on the binding of prolactin to mouse mammary cells. In vivo the labelled hormone found in the mammary gland was associated with membranes of mammary epithelial cells and with alveolar lumen contents. In vitro 125I-prolactin was shown to bind to dispersed mouse mammary epithelial cells.     

Serum and insulin initiation of DNA synthesis in mammary gland epithelium in vitro

These experiments demonstrate that serum, like insulin, can initiate DNA synthesis in mouse mammary gland epithelium, resulting in a three- to four-fold increase in the rate of DNA synthesis and number of cells synthesizing DNA. Both serum and insulin also increase the tritiated thymidine counts in the acid soluble material of these cells, suggesting that each alters thymidine transport. When combined at any concentration, these agents produce an additive effect on DNA synthesis, number of cells synthesizing DNA and thymidine transport. The factor(s) in serum responsible for these effects is associated with high molecular weight material. These experiments suggest that DNA synthesis and thymidine transport are affected independently by serum and insulin in mammary gland epithelium.     

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.     

Activation of tyrosine kinases during induction of mammary gland tumors in GR mice by ovarian hormones

In GR mice, the induction of proliferative processes in mammary tumours with ovarian hormones (estrone and progesterone) is accompanied by the activation of phosphorylation of plasma membrane, cytosolic and nuclear proteins by endogenous protein kinases. The hormones stimulate tyrosine kinases of tumour cells whose activity is as high as 14.9-17.9% of the total phosphorylation in plasma membranes and 9.5-10.4% in cell nuclei. The ovarian hormones stimulate tyrosine kinases of tumour cells which phosphorylate proteins with Mr of 110-230 and 15 kD (plasma membranes), 170, 52 and 13 kD (cytosol) and 32 kD (nuclei) which are resistant to alkaline hydrolysis. Apart from tyrosine kinases, the ovarian hormones also stimulate serine and threonine protein kinases which seems to be due to the activation of protein kinase C and other protein kinases.