Action of KI, thyroxine and cyclic AMP on [3H]uridine incorporation into the RNA of thyroid slices.

Potassium iodide (KI) has been shown to impair thyroid protein biosynthesis both in vivo and in vitro. The present study was performed in order to clarify its mechanism of action. Ribonucleic acid (RNA) synthesis was studied in beef thyroid slices with either [32P] or [3H]-uridine as labelled precursors. Both KI and thyroxine (T4) at 10(-5) M significantly decreased RNA labelling under our conditions. In other experiments RNA degradation was examined in pulse-labelled and actinomycin D-treated slices. KI did not modify the degradation of the [3H]-RNA thus indicating that it interferes with the biosynthesis rather than with the degradation of RNA. Taking the perchloric acid soluble radioactivity as a rough index of the precursor pool the present results would indicate an action at this level. Both KC1O4 and methylmercapto-imidazole relieved the gland from the inhibitory action of KI, supporting the view that an intracellular and organified form of iodine is responsible for this action. Since T4 also reproduced the effects of KI on RNA synthesis we would like to propose iodothyronines as the intermediates of this action. Cyclic AMP has been shown to stimulate thyroid protein biosynthesis. The present results demonstrate an action at the RNA level. Cyclic AMP increased both the PCA-soluble and RNA-linked radioactivity, thus suggesting an effect at the RNA precursor pool. KI at 10(-5) M blocked the action of 2 mM cyclic AMP.     

Role of neurotransmitters, prostaglandins and glucose on precursor incorporation into the RNA of thyroid slices.

The present studies were performed in order to investigate the role of neurotransmitters, prostaglandins and glucose on [3H] uridine incorporation into total RNA in beef thyroid slices. Carbamylcholine strongly stimulated RNA labelling from [3H uridine; atropine abolished this effect. NaF, at 1 and 5 mM, progressively increased this parameter while norepinephrine caused a similar effect at 10(-3) but not at 10(-6) M. Phentolamine (1 mM) blocked the stimulatory action of TSH; propranolol and atropine did not. Glucose at concentrations between 4 and 24 mM caused a progressive increase in RNA labelling from [3H] uridine. This effect was inhibited by dinitrophenol. Prostaglandins (E1, A1, F1alpha and F2alpha) assayed in concentrations between 5 and 25 microgram/ml, with or without caffeine, had no effect on RNA labelling. Moreover, neither aspirin nor indomethacin inhibited TSH stimulation. Under similar experimental conditions, PGE1 did simulate PB125I formation.     

RNA synthesis in the Leydig cells of the mature rat: effect of oestradiol-17 beta

In view of the evidence that there may be an effect of high concentrations of oestradiol on testicular steroidogenic function, we have investigated the effect of this steroid on [3H]uridine incorporation into RNA by testicular cells. Our results have shown that oestradiol in vitro induced a marked dose-dependent inhibition of RNA synthesis by purified Leydig cells. The concentrations of oestradiol tested varied from 2 to 40 mumol/l; these concentrations also impaired net testosterone synthesis in vitro after human chorionic gonadotrophin (hCG) stimulation. Under the effect of oestradiol, the kinetics of [3H]nucleoside incorporation into RNA were impaired early and the inhibition of RNA synthesis was specific for oestrogenic compounds. It was concluded that, in Leydig cells, oestradiol, in addition to its known inhibitory action on the response of testosterone to hCG, triggers a more extensive response that also includes RNA synthesis in vitro.     

Insulin stimulates the incorporation of 32Pi into ribonucleic acid in cultured sertoli cells

Chronic treatment of cultured Sertoli cells with insulin, FSH, or testosterone does not affect the number of attached cells or the amount of total RNA or poly(A)+ RNA per culture dish. However, Sertoli cells cultured in the presence of insulin have a nearly 2-fold stimulated incorporation of 32Pi into poly(A)+ RNA. The other hormone treatments did not induce any stimulation over control values. The t1/2 of the total poly(A)+ RNA was determined in pulse-chase experiments. None of the hormone treatments affected the t1/2 (16 h) of the total poly(A)+ RNA. The incorporation of [3H]uridine and 32Pi into the total nucleotide pools and into some individual nucleotides was determined by high pressure liquid chromatography analysis of acid-soluble extracts. Insulin-treated cells had approximately a 1.5-fold increased specific activity of [3H]uridine or 32Pi into the total nucleotide pool and in ATP, GTP, and UTP pools. Insulin apparently stimulates the uptake of phosphorylation (or both) of free nucleosides.     

Regulation of Y-organ ecdysteroidogenesis by molt-inhibiting hormone in crabs: involvement of cyclic AMP-mediated protein synthesis

The crustacean neuropeptide, molt-inhibiting hormone (MIH), directly inhibits Y-organ ecdysteroidogenesis, an effect mediated by cyclic AMP (cAMP) and antagonized by calcium-calmodulin. We investigated regulation of Y-organ protein. RNA, and DNA syntheses by MIH, cAMP, and calcium in relation to steroidogenesis in vitro. Ecdysteroid production and [3H]leucine incorporation into protein were inhibited 50-60 and 80-90%, respectively, by MIH activity in eyestalk extracts (4 eyestalk equivalents), 10(-6) M forskolin, or a combination of 10(-2) M dibutyryl cAMP and 10(-4) M 3-isobutyl-1-methylxanthine (dbcAMP-IBMX). Calcium ionophore A23187 (10(-4) M) stimulated ecdysteroidogenesis two-fold, did not affect the relatively high basal (control) rate of protein synthesis, and reduced the inhibitory effects of forskolin on steroidogenesis and protein synthesis. Incorporation of [3H]uridine into RNA was unaffected by MIH, forskolin, or A23187 but was reduced 50% by dbcAMP-IBMX. Basal rates of [3H]thymidine incorporation into DNA were low and were not affected by treatments. The effects of MIH were specific; extracts of brain or muscle did not alter Y-organ steroidogenesis or protein synthesis, while muscle extract increased precursor incorporation into RNA. Eyestalk extract did not affect [3H]leucine incorporation into protein of brain, muscle, or gill. Cycloheximide (5 micrograms/ml) depressed protein synthesis 90% and steroidogenesis 60%, enhanced the inhibition induced by MIH, and blocked the stimulation of steroidogenesis induced by A23187; effects on basal steroidogenesis were evident after 1 hr. Actinomycin D (1 microgram/ml) depressed RNA synthesis 86% but did not alter basal, MIH-inhibited, or A23187-stimulated ecdysteroidogenesis during incubations. These results indicate that MIH suppresses Y-organ steroidogenesis in part by inhibiting protein synthesis at the translational level; the effect is mediated by cAMP. The stimulation of steroidogenesis by calcium, mediated by lowering cAMP, also appears to depend in part upon protein synthesis.     

Timing and localization of the stimulatory effect of follicle-stimulating hormone on uridine incorporation in the mouse testis in vivo

Human pituitary FSH increased the incorporation of [5-(3)H]uridine into RNA in vivo in the testes of intact 9-day-old mice and of hypophysectomized adults. In both groups the effect was greatest 8 h after administration of 5--7.5 i.u. FSH. Autoradiographs were prepared from the testes of hypophysectomized adult mice given subcutaneous injections of FSH or of 0.9% saline 6 h, and [5-(3)H]uridine 1.5 h, before death. Treatment with FSH caused statistically significant increases in the density of silver grains over the nuclei of Sertoli cells, type A and intermediate spermatogonia, and preleptotene and mid-pachytene primary spermatocytes. It was concluded that FSH has a generalized stimulatory action on RNA synthesis in the nuclei of Sertoli cells and those types of germinal cell which synthesize RNA most actively.     

Cytochalasin B: Lack of Effect on Mucopolysaccharide Synthesis and Selective Alterations in Precursor Uptake

Synthesis and secretion of mucopolysaccharide in mouse 3T3 fibroblasts and in embryonic submandibular glands are unaffected by amounts of cytochalasin B that alter the morphology of these cells and tissues. The drug markedly and reversibly inhibits incorporation of [³H]glucosamine into mucopolysaccharide by preventing cellular uptake of the precursor, but does not affect incorporation of radiosulfate. Cytochalasin does not alter DNA, RNA, or protein synthesis, but stimulates the uptake of orotic acid and markedly inhibits the uptake of glucose. These selective effects on the transport of small molecules suggest that the primary action of the drug may be on cell membranes. Since processes unrelated to microfilament disruption may be altered by cytochalasin, great caution must be exercised in interpreting studies with the drug.     

Isolation of cells from rat seminal vesicles and epididymis and their use in studying androgen action

Cells isolated enzymically from seminal vesicles and epididymides of normal and castrated rats were shown by electron microscopy to be intact and representative of the tissue. The cells synthesize and secrete tissue-specific proteins. Short-term incorporation of [3H]uridine and [35S]methionine was measured to determine the effects of castration on RNA and protein synthesis. Epididymal cells and tissue incorporated uridine at similar rates which were unaltered by castration. Similarly castration failed to diminish uridine incorporation by seminal vesicle cells and tissue. Therefore, androgens may principally control RNA degradation. A similar situation pertained to methionine incorporation by epididymal cells and tissue so here too control may be via protein degradation. In contrast, castration greatly decreased methionine incorporation by seminal vesicle tissue but not by isolated cells. Isolated cells were more active than in tissue, particularly those from castrated rats, and may be released from stromal-epithelial interactions and controls.     

Magnesium and calcium effects on uptake of hexoses and uridine by chick embryo fibroblasts.

Cultures of chick embryo fibroblasts were incubated for varying periods in media containing different concentrations of Ca2+ and Mg2+-Mg2+ deprivation produced a gradual decrease in the Vmax of the glucose transport system for the D-glucose analogues 3-O-[3H]methyl-D-glucose and 2-deoxy-D-[3H]glucose and a parallel decrease in the rate of production of lactate from glucose in the medium. It greatly reduced the rates of [3H]uridine uptake and incorporation by decreasing the Vmax of the uridine transport system. Addition of Mg2+ to Mg2+-deprived cultures rapidly increased the rate of [3H]uridine uptake without requiring protein synthesis and increased the rate of 2-deoxy-D-[3H]glucose uptake without requiring RNA synthesis. These effects of changes in Mg2+ concentration qualitatively reproduce the effects of such variables as cell density and serum and insulin concentrations. Ca2+ deprivation resulted in similar, though much smaller, changes in the activities of the two transport systems, but also greatly increased the "leakiness" of the cells to the nontransported hexose L-[3H]glucose.     

In vitro effects of estrogen on tgb and c-myc gene expression in normal and neoplastic human thyroids

The authors investigated the effects of 17 beta-estradiol (E) and thyroid-stimulating hormone (TSH) on tgb (coding for thyroglobulin), c-myc RNA levels, and [3H]thymidine (thy) incorporation in suspension cultures of normal, adenomatous and carcinomatous human thyroid follicles. The cultured follicles showed decreased tgb RNA and enhanced c-myc RNA levels. In the culture of normal and adenomatous samples E caused a significant increase of [3H]thy incorporation and tgb RNA levels, with no effect on c-myc RNA levels. No effect of E was observed in the carcinomatous thyroid culture. TSH induced a significant increase of [3H]thy incorporation and c-myc expression only in adenoma cultures and a significant increase of tgb RNA levels in both normal and adenomatous samples. TSH had no effect on the carcinoma. The results show that E, like TSH, stimulates in vitro the expression of the tgb gene in differentiated cells, without stimulating the expression of the c-myc proto-oncogene, suggesting a possible action of E on normal thyroid function and perhaps growth, even if not associated with increased c-myc expression.     

Androgenic regulation of rapidly synthesized RNA in the rat ventral prostate.

The influence of castration on the incorporation of nucleotide precursors into RNA of isolated prostatic tissue has been investigated. Castration brought about an apparent increase of incorporation by increasing the specific activity of the uridine nucleotide pool and not by an enhancement in synthesis of rapidly labelled RNA. An actual decrease in this reaction was shown by dividing the radioactivity incorporated into RNA by the specific activity of the uridine nucleotide. Administration of testosterone brought about enhanced synthesis of RNA in the prostates of rats castrated for 3 days as early as 4 h after injection, reaching maximum effect at 8 h. No significant difference was found between the turnover rates of rapidly labelled RNA from castrated, androgen-treated castrated and intact rats. The increased prostatic permeability to nucleosides as an early action of androgens in this organ is discussed.     

Pyrimidine nucleotide synthesis is preferentially supplied by exogenous cytidine in adult rat cultured cardiomyocytes.

The metabolism of pyrimidine nucleotides was studied in non-contracting myocytes isolated from adult rat hearts and compared to that observed in freshly prepared myocardium. The myocytes were cultured for up to 96 hrs in a commercial medium containing 50 microM cytidine, uridine, adenosine and adenine; 20 microM guanosine, thymidine and D-ribose; and 5 microM hypoxanthine, xanthine, guanine, thymine and uracil. Nucleotide pool sizes were measured by HPLC. Nucleotide and RNA labelling were followed by incorporation of [U-14C]-cytidine or [U-14C]-uridine added in trace amounts to the medium. The adenine nucleotide pool was 2.4-fold larger than in situ after 7 hrs of incubation and then returned to values 30% higher than that found in the myocardium after 25 hrs. Cytosine and uracil nucleotide pools after 25 hrs of culture were respectively 2 and 4-fold larger than in situ and remained at these levels thereafter. Intracellular cytidylate and uridylate equilibrated very rapidly with exogenous [U-14C]-cytidine but not with [U-14C]-uridine. We conclude that, under the experimental conditions used here, the synthesis of pyrimidine nucleotides in isolated myocytes is mainly supplied by exogenous nucleosides. Furthermore, extracellular cytidine is rapidly converted to both uracil and cytosine nucleotides while uridine serves only as the precursor for uracil nucleotide synthesis.     

T3 stimulation of 2-deoxy-D-glucose uptake in cultured chick embryo carcass derived cells, requires neosynthesis of proteins

T3 stimulated, in a dose dependent manner, the uptake of 2-deoxy-D-glucose (2-DOG) into cultured chick embryo carcass derived cells. A significant increase in sugar uptake was seen already at a T3 concentration of 1 pmol/l. The stimulation of 2-DOG uptake increased with time during the 6 h of exposure to T3. The hormone also stimulated, within 45 min, the incorporation of [3H]leucine and [3H]uridine into the trichloroacetic acid precipitable material of these cells. Actinomycin-D (100 micrograms/l) and cycloheximide (1 mg/l) each were capable of blocking the stimulatory effect of 10(-8) mol/l T3 on sugar uptake and on uridine and leucine incorporation. Thus, T3 in these cultured chick embryo cells stimulated sugar transport through processes dependent on neosynthesis of proteins. In this respect the effect of T3 is different from that seen in cultured chick embryo cardiomyocytes.     

Inhibition of cell growth by a hypothalamic peptide.

A fraction purified from acetic acid extracts of porcine hypothalami was found to contain significant antimitogenic activity when tested in normal and neoplastic cell lines. Addition of this hypothalamic material (1-100 micrograms/ml) to culture media significantly inhibited [3H]thymidine incorporation into cellular DNA in several cell lines. Amino acid incorporation into pituitary proteins and uridine incorporation into RNA were also significantly reduced by this factor(s). Addition to the culture media of this hypothalamic material at 5 micrograms/ml and 50 micrograms/ml per day decreased by 17% and 36%, respectively, cell numbers of 3T6 fibroblast cell cultures. Time-response curves showed that the inhibition of [3H]thymidine incorporation into DNA in 3T6 fibroblast cells begins within 2 hr after adding this fraction to the culture medium. The inhibitory action cannot be explained by a direct cytotoxic effect since 3T6 cells labeled with 51Cr and incubated for 6 hr in the presence of this hypothalamic fraction fail to show an increase in the release of 51Cr into the medium as compared with controls. Incubation with trypsin and chymotrypsin completely abolished the antimitogenic activity of this material and pepsin decreased it. This strongly suggests that the antimitogenic activity exhibited by this fraction is due to a polypeptide(s). These observations provide evidence for the presence in the mammalian hypothalamus of an antimitogenic peptide(s) that may be involved in the regulation of cell proliferation.     

The inhibitory effect of anti-tumor drugs on phosphatidylcholine synthesis and its reversal by geranylgeranylacetone in the isolated guinea pig gastric glands

To clarify the mechanism by which the administration of anti-tumor drugs, antibiotics or hypoglycemic agents causes gastric mucosal injury, the effects of these drugs on phosphatidylcholine synthesis in isolated guinea pig gastric glands were examined in vitro. Anti-tumor drugs such as tegafur, cyclophosphamide, and mitomycin C decreased [3H]choline incorporation into phosphatidylcholine. Furthermore, tegafur at 0.4 mg/ml decreased [3H]choline incorporation in the glands that had been pulsed with [3H]choline incorporation, suggesting that tegafur exerts its effect by inhibiting late step of phosphatidylcholine synthesis in the stomach. On the other hand, cefaclor and glibenclamide had no effect on [3H]choline incorporation. Geranylgeranylacetone, an anti-ulcer drug partially restored tegafur-induced reduction of [3H]choline incorporation into phosphatidylcholine. These results suggest that the anti-tumor drug-induced gastric mucosal injury may be due to drug-induced decrease in phosphatidylcholine synthesis, which the restoration of phosphatidylcholine synthesis by geranylgeranylacetone may explain its anti-ulcer action on drug-induced gastric mucosal lesions in vivo.     

Somatomedin and analogues of cyclic AMP increase the number of cells synthesizing DNA in cartilage from hypophysectomized rats

The effects of somatomedin and certain nucleotides on nuclear labelling of cartilage cells with [3H]thymidine were determined by autoradiography. Segments of costal cartilage from hypophysectomized rats were incubated for 24 h in a basal medium with or without additions and then pulsed for 2 h with [3H]thymidine in the basal medium. Both somatomedin (0.1 U/ml) and Bt2cAMP (10(-4)M) increased the number of labelled nuclei, and the combined effects were more than additive. A parallelism between the effects of these agents on nuclear labelling and their effects on total thymidine incorporation into DNA was demonstrated. The 8-bromated derivative of cAMP (10(-4)M) also enhanced chondrocyte nuclear labelling, but neither 8-Br-5'-AMP (10(-4)7) nor 8-Br-cGMP (10(-4)M) exhibited actions of the cAMP analogues. It is concluded that in cartilage obtained from hypophysectomized rats and incubated under the specified conditions (1) both somatomedin and cAMP analogues increase the number of cells synthesizing DNA as well as total thymidine incorporation into DNA, (2) the effects of the hormone and cyclic nucleotide in combination are synergistic, and (3) the increased incorporation of labelled thymidine into DNA reflects increased DNA synthesis and not merely an alteration of the specific activity of the intracellular thymidine nucleotide pool.     

Uptake,metabolism and action of triiodothyronine in calf-thyroid slices

T₃ and T₄ at 10^?6M caused a significant inhibition on [³H]uridine incorporation into RNA in calf-thyroid slices. This effect was not altered by addition of 10^?3M PTU or MMI. The metabolism of ¹²⁵I-T₃ was studied under the same conditions. There was a very slight dehalogenation after 60 min (less than 5%) which was inhibited by PTU.The time course of the uptake of labeled T₃ showed a temperature dependence, and this uptake was not altered by 10^?6M KI, 1-T₄, DIT or MIT, thus indicating specificity. The early phase of labeled T₃ entrance into the cell was inhibited by the addition of cold T₃ in a dose-dependent manner from 10^?9 to 10^?5M. Slices previously stimulated by cAMP or cGMP showed a significant increase in the uptake of labeled T₃. ATPase activity or ATP, protein or RNA synthesis does not seem to play a role in this process.The relationship between substitutions in the thyronine molecule and its biological action on RNA synthesis was also studied, and some preliminary conclusions were drawn.These studies demonstrate that T₃ has a direct action on the thyroid.