EFFECT OF PROLACTIN ON THE TADPOLE TAIL FIN. III. EFFECT OF PROLACTIN ON THE HYDROXYLATION OF PROTOCOLLAGEN-PROLINE OF THE TAIL FIN

The fact that bovine prolactin stimulates the collagen synthesis of the tadpole tail fin was confirmed by the present experiments, in which specific radioactivity of ¹⁴C-hydroxyproline hydroxylated from the incorporated ¹⁴C-proline was measured by the method different from that of the previous report (Y oshizato and Y asumasu , 1970). Stimulatory effect of prolactin on the incorporation of ¹⁴C-proline into the collagen fraction did not disappear even under the conditions, where the activity of protocollagen-proline hydroxylase was inhibited by α,α'-dipyridyl. In fact prolactin had not a significant effect on the activity of the emzyme. It, therefore, is concluded that the hormone promotes the incorporation of ¹⁴C-proline into collagen without affecting the hydroxylation step of the protein synthesis.     

EFFECT OF PROLACTIN ON THE TADPOLE TAIL FIN. I. STIMULATORY EFFECT OF PROLACTIN ON THE COLLAGEN SYNTHESIS OF THE TADPOLE TAIL FIN

Bovine prolactin stimulated the growth of connective tissues both in the tail fins and in other regions of the tadpole tail. Correlated with the morphological effect of the hormone on the tadpole tails, protein synthesis in tail fins was promoted about 2 times by prolactin. Experiments performed to determine the kind of protein, the synthesis of which was stimulated by prolactin, revealed that the hormone specifically enhanced collagen synthesis about 40 folds as compared to untreated animals.     

EFFECT OF PROLACTIN ON THE TADPOLE TAIL FIN. V. STIMULATORY EFFECT OF PROLACTIN ON THE INCORPORATION OF 3H-THYMIDINE INTO DNA OF THE TADPOLE TAIL FIN

DNA content of the tadpole tail fin was increased appreciably by the treatment of animals with bovine prolactin. Incorporation of ³H-thymidine into DNA in the hormone-treated tail fins was remarkably stimulated as compared to the control ones. From the experiment on the effect of prolactin on the DNase activity, it is concluded that prolactin not only stimulates the synthetic parthway, but also suppresses significantly the catabolic one of the DNA metabolism.     

EFFECTS OF THYROXINE AND PROLACTIN ON COLLAGEN BREAKDOWN IN THE THIGH BONE AND TAIL FIN OF THE RANA CATESBEIANA TADPOLE

Injection of ¹⁴C-proline into the tadpole causes labeling of protein in the collagen fraction of the thigh bone and tail fin. The radioactivity of the ¹⁴C-hydroxyproline residue is about 26% of the total radioactivity in the ¹⁴C-labeled protein of the collagen fraction in the thigh bone as well as in the tail fin. In ¹⁴C-proline-loaded tadpoles into which prolactin has been injected, the radioactivity in the collagen fraction in these tissues is markedly higher than that in control animals. In thyroxine-treated tadpoles, the ¹⁴C-radioactivity of the collagen fraction in the thigh bone is always higher than that of the controls, but it is markedly low in the tail fin. During the incubation of thigh bone and tail fin isolated from ¹⁴C-proline-loaded tadpoles, low molecular weight materials containing ¹⁴C-hydroxyproline are released from the ¹⁴C-labeled protein of these tissues. The rate of ¹⁴C-hydroxyproline release, which represents the rate of collagen breakdown, is higher in thigh bone and tail fin isolated from thyroxine-treated tadpoles and is markedly lower in these tissues isolated from prolactin-treated tadpoles than in those isolated from controls. In these tissues, the high rate of collagen breakdown in thyroxine-treated tadpoles is reduced by prolactin injection.     

EFFECTS OF THYROXINE AND PROLACTIN ON THE RATES OF PROTEIN SYNTHESIS IN THE THIGH BONES OF THE TADPOLE OF RANA CATESBEIANA

In thigh bones isolated from a Rana catesbeiana tadpole which has been kept in a 5 × 10⁻⁸ M thyroxine solution for several days, the rate of ¹⁴C-leucine incorporation into protein becomes higher than that in the thigh bones of control animals. Intraperitoneal injection of prolactin also results in an increase in the rate of ¹⁴C-leucine incorporation into protein in the thigh bones at a rate very similar to that in thyroxine-treated animals. In the thigh bones of the thyroxine-treated tadpoles, the rate of ¹⁴C-proline incorporation into protein is markedly higher than that of control animals. Prolactin treatment of the tadpoles also causes an increase in the rate of ¹⁴C-proline incorporation, but the rate is lower than that found in thyroxine-treated animals. The injection of prolactin into thyroxine-treated tadpoles fails to cause further increase in the rates of incorporation of these amino acids into protein. In the thigh bones of tadpoles at the climax of metamorphosis, prolactin injection does not cause any increase in the rates of ¹⁴C-labeled proline and leucine incorporation, whereas both rates become slightly higher in the thigh bones of thyroxine-treated tadpoles at this stage. The thigh bones probably become insensitive to prolactin when they are exposed to thyroxine.     

ON THE MECHANISM OF THYROID MEDIATED MITOGENESIS IN ADULT ANURA. I. PRELIMINARY ANALYSIS OF GROWTH KINETICS AND MACRO-MOLECULAR SYNTHESES, IN LENS EPITHELIUM, UNDER THE INFLUENCE OF EXOGENOUS TRIIODOTHYRONINE

Injection of triiodothyronine into adult frogs leads to an increase in the mitotic index of a number of tissues. The work reported here shows that in one such tissue, lens epithelium, the mitogenic effect is chiefly due to an increase in growth fraction. Practically all of the cells in the population enter DNA synthesis (although not simultaneously) as a consequence of treatment. The administration of hormone also leads to an increase in intranuclear ³H actinomycin D binding. In addition ³H uridine and ³H amino acid incorporation rates rise as does microphotometrically detectable RNA and protein content.     

HORMONES CONTROLLING COLLAGEN SYNTHESIS DURING METAMORPHOSIS IN THE THIGH BONE OF THE TADPOLE OF RANA CATESBEIANA

The rate of ¹⁴C-proline incorporation into collagen in the thigh bone of the Rana catesbeiana tadpole was determined in vitro. Intraperitoneal injection of bovine prolactin caused an increase in the rate of collagen synthesis during the premetamorphic stages (stages 12–16) and the early metamorphic stage (stage 18), but it exerted no effect on collagen synthesis in the metamorphic stages (stages 20–25). On the other hand, injection of growth hormone stimulated the rate of collagen synthesis in the metamorphic stages and caused a slight increase in the premetamorphic stages. When a tadpole in the early premetamorphic stages (stages 12–14) was kept in 5 × 10₋₈ M thyroxine solution for several days, the rate of collagen synthesis became higher than that in the bone of the control animal. The rate of collagen synthesis was not enhanced by prolactin in the thyroxine-treated tadpole, but was stimulated by growth hormone, even when the thyroxine-treated animal remained in the premetamorphic stages. With the treatment of the tadpole by thyroxine, prolactin-sensitivity seems to be reduced, and growth hormone-sensitivity becomes apparent.     

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.     

Changes in amino acid uptake and protein synthesis of bullfrog tadpole liver and tail tissues during triiodothyronine-induced metamorphosis

The effect of triiodothyronine (T₃′) on the uptake of several amino acids into the amino acid pools and into proteins of Rana catesbeiana tadpole liver and tail muscle and tail fin has been studied. Labeling of the alanine and glycine pool was stimulated in the liver more than the leucine pool. After exposure to T₃ for 3 days, uptake of α-aminoisobutyric acid (a transport model substrate) into liver was stimulated about 55%. In tail tissues uptake of leucine was stimulated but uptake of alanine was depressed by T₃. Incorporation of leucine and alanine into tissue protein was stimulated in the liver but inhibited in tail tissues after T₃ injection.Changes in other macromolecules and ATP and ADP levels in liver and tail muscle were also investigated during induced metamorphosis. In the liver, the total DNA content did not change, but the RNA and protein content per liver increased significantly. The increase in RNA/DNA and protein/DNA ratios, suggested that liver cells underwent hypertrophy during induced metamorphosis. The ATP level showed a transient decrease after 3 days of T₃ treatment. In tail muscle, protein and RNA content decreased as the muscle regressed, but the DNA content and ATP level remained unchanged throughout the experimental period.     

THE EFFECT OF ACTIDIONE ON MITOSIS IN THE SLIME MOLD PHYSARUM POLYCEPHALUM

Actidione, reportedly a specific inhibitor of protein synthesis, was found to reduce the incorporation of labeled amino acids into proteins of the slime mold Physarum polycephalum without drastically inhibiting the incorporation of nucleic acid precursors into RNA. This inhibitor was found to completely block the ensuing mitosis if it was added at any time between telophase and late prophase. Plasmodia given Actidione in late prophase (about the time of nucleolar dissolution) went on through telophase to reconstruction even though nuclear amino acid incorporation was drastically reduced during that period.     

PURIFICATION AND BIOLOGICAL CHARACTERIZATION OF BACTERIALLY EXPRESSED RECOMBINANT BUFFALO PROLACTIN

Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22 mg/L from 100 mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells.     

THE EFFECT OF EXTRACELLULAR POTASSIUM CONCENTRATION ON PROTEIN SYNTHESIS IN GUINEA-PIG HIPPOCAMPAL SLICES1,2

Abstract— We have measured the effect of small variations in extracellular potassium concentrations ([K⁺]) upon the incorporation of radioactively labelled amino acid into the protein of the isolated guinea-pig hippocampal slice. The slice is super-perfused with glucose fortified buffer and maintains an ATP concentration of 33–36 nmol/mg protein and incorporates lysine into protein at a rate of 0.82 pmol/(ig protein/h. Within the range of extracellular K⁺ from 1.3 to 8.1 mil the change in the rate of lysine incorporation into protein is proportional to the logarithm of the extracellular K⁺ concentration. Incorporation increases by about 100% over this range. Measurements of the specific activity of the presumed intracellular amino acid pool indicate that the effect of changes in extracellular [K⁺] is to alter the rate of protein synthesis rather than alter the availability of radioactively labelled precursor. Altering extracellular [K⁺] does not affect tissue levels of ATP or creatine phosphate, indicating that the effect on amino acid incorporation does not result from an effect upon energy metabolism. It is suggested that this effect of extracellular [K.⁺] may be a means by which changes in cerebral electrical activity lead to changes in the rate of protein synthesis in brain.     

Polyamine requirement for prolactin action on macromolecular synthesis in the MCF-7 human mammary epithelial cell line

The actions of insulin, hydrocortisone, prolactin and growth hormone on the synthesis of macromolecules in MCF-7 cells was determined in a serum-free defined medium. The inclusion of the polyamine spermidine in the medium was shown to enhance the insulin stimulation of the rate of [3H]uridine incorporation into RNA in a manner similar to that demonstrated for hydrocortisone. Spermidine, in addition to insulin and hydrocortisone, was also essential for prolactin to manifest a stimulation of the rate of [3H]uridine incorporation; this effect of spermidine was optimal with spermidine concentrations between 1 and 5 mM. Prolactin also stimulated the rate of [3H]leucine incorporation into total cellular protein and into an isoelectrically precipitable (pH 4.6) phosphoprotein fraction. The actions of prolactin on total protein and phosphoprotein synthesis were only expressed if spermidine, in addition to insulin and hydrocortisone, was contained in the culture medium. All of the prolactin responses were observed employing physiological concentrations of prolactin. Specificity of the prolactin responses was established by demonstrating that porcine growth hormone had no effects on RNA or phosphoprotein synthesis in the MCF-7 cells.     

Lactating goat mammary gland cells in culture.

1. Isolated mammary gland cells were cultured embedded in collagen gels or as monolayers on floating collagen gels. Under these conditions the cells were able to grow for at least 6 weeks during five passages. Growth was sustained in M199/F12 (1:1) supplemented with insulin, hydrocortisone, epidermal growth factor, tri-iodothyronine, estradiol and bovine serum albumin. 2. The cells secreted lactose into the medium in significant amounts throughout the culture period. 3. Prolactin had a slightly stimulatory effect as had fetal bovine serum on growth and protein synthesis, but none of these factors were obligatory in this respect. Insulin-like growth factor I (Somatomedin C) could replace high concentrations of insulin whereas bovine growth hormone had no detectable effect. 4. Depending on the hormone content of the medium and the age of the culture, different labelling patterns of the arachidonic acid-containing phospholipids were observed. The effect of prolactin on phosphatidyl inositol and arachidonic acid metabolism was studied.     

Studies on the mechanism by which prolactin regulates protein, RNA, and DNA synthesis in Nb2 node lymphoma cells

Specific aspects of the prolactin stimulation of RNA, DNA and protein synthesis in the Nb2 node lymphoma cell line were determined. In time sequence studies the onset of the prolactin stimulation of the incorporation of radiolabeled precursors into these macromolecules was found to be 0.5-1 h for [3H]uridine incorporation into RNA, 1-2 h for [3H]leucine incorporation into protein, and 4-8 h for [3H]thymidine incorporation into DNA. The total DNA content of the cell cultures was increased by 12-18 hours after addition of prolactin. Amiloride, an inhibitor of the plasma-membrane-bound Na+/H+ antiporter, was found to inhibit the mitogenic effects of prolactin. Amiloride was also found to inhibit the prolactin stimulation of DNA, RNA and protein synthesis, thus suggesting that the initial regulation of the Na+/H+ antiporter may initiate these responses as well as the mitogenic effect of prolactin. In contrast, H-7, a drug which inhibits protein kinase C, had no effect on the magnitude of the prolactin stimulation of DNA, RNA or protein synthesis at a drug concentration (100 muM) that abolished the mitogenic effect of prolactin. The early effects of prolactin on RNA, DNA and protein synthesis would therefore appear not to involve an activation of protein kinase C.     

催乳素对培养的绵羊睾丸间质细胞睾酮分泌的影响

在绵羊睾丸间质细胞体外无血清长期培养的条件下,研究了催乳素对睾丸间质细胞睾酮分泌的调节作用。实验结果表明,催乳素可增强细胞对人绒毛膜促性腺激素(hCG)刺激的反应。催乳素的这种作用呈双相调节。睾酮分泌量显著高于hCG和催乳素单独作用时的总和。在hCG存在下,不同的底物转化为睾酮的量不同。其中雄烯二酮和孕酮转化为睾酮的方式存在着双相性。脱氢表雄酮转为睾酮的量少,不存在双相性,而与其剂量成正比。催乳素在hCG存在下可调节底物转化为睾酮。低剂量的催乳素(1ng/ml)可使一定剂量的孕酮(10～30ng/ml)转化为睾酮的量明显增加,而高剂量的催乳素(>10ng/ml)却明显地抑制孕酮转化为睾酮。催乳素可明显地抑制雄烯二酮转化为睾酮,与剂量无关。可见催乳素对于孕酮和雄烯二酮这两个关键底物转化为睾酮的调节是不同的。催乳素增强hCG刺激睾酮分泌的作用可能部分是通过其促进孕酮转化为睾酮来实现的。     

Prolactin-induced stimulation of H(3)-proline incorporation into the basement lamella of tadpole skin: light and electron microscope study

Radioautographic studies revealed that prolactin markedly stimulates the incorporation of H(3)-proline into the basement lamella of the tail fin of frog tadpole. Radioactivity in fibroblasts reached a peak at 3 hours. Thereafter, the number of silver grains in fibroblasts was reduced with concomitant increase in the basement lamella. Fibroblasts of the prolactin animals also showed extensively developed rough endoplasmic reticulum and Golgi elements. Epidermis of control animals was labelled as in the prolactin animals, although the basement lamella was sparsely labelled. Fibroblasts, not the epidermis, seem to secrete collagen into the basement lamella.     

THE EFFECT OF CORTISONE ON DNA CONTENT OF RAT HEPATOCYTES

Rats were treated with cortisone, x-radiation, and both agents in combination, and the effect noted on the DNA content of hepatocytes. Nuclei were enumerated both in whole liver homogenates and following isolation. The incorporation of P³² into DNA was also studied in relation to these agents. The following observations were made:— 1.The DNA content of nuclei fell both during cortisone administration and following x-radiation. In the former instance, the fall was progressive with continuing administration of hormone; in the latter instance, there was a return to normal 5 days after radiation. 2. Cortisone administration to x-radiated rats caused a fall in DNA/nucleus and prevented the return to normal at 5 days. 3. There was no evidence that the effects of cortisone and x-rays were additive in reducing DNA/nucleus. 4. These data indicate an alteration in DNA/nucleus, but simple changes in ploidy cannot be excluded. Either explanation requires that the agents used affect the DNA of non-regenerating nuclei. 5. Cortisone interfered with the incorporation of P³² into the DNA of regenerating liver. Only a small effect on DNA synthesis in resting liver was observed with cortisone or x-radiation. 6. DNA content of nuclei returned to normal 5 days after x-radiation and 3 days after discontinuance of cortisone. Slight increase in the incorporation of P³² by DNA was observed during recovery phases. 7. The hypothesis is proposed that the apparent losses and increases in DNA/nucleus were due to depolymerization and repolymerization of DNA. Following x-radiation and/or cortisone administration, it is proposed that some DNA is depolymerized and becomes cold acid-soluble and dissociated from organized chromatin. Later, conditions are such that this degraded DNA is repolymerized. 8. These data might be interpreted to indicate that a portion of the DNA is not essential to cell integrity; alternatively, there may be two or more species of DNA, one of which is more readily affected by the agents investigated in the present report.     

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.