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.     

Inhibition of Replication in Functional Mouse Adrenal Tumor Cells by Adrenocorticotropic Hormone Mediated by Adenosine 3′:5′-Cyclic Monophosphate

Adrenocorticotropic hormone (ACTH) inhibited replication in functional adrenal tumor cells with a concomitant stimulation of steroidogenesis and a characteristic change of morphology from a flattened to a spherical type. [(3)H]Thymidine incorporation into DNA was inhibited by about 50% 6 hours after ACTH treatment. Both cyclic AMP and dibutyryl cyclic AMP inhibited [(3)H]thymidine incorporation and caused the characteristic morphological change noted with ACTH. The extent of stimulation of steroidogenesis and the amount of inhibition of [(3)H]thymidine incorporation in response to various doses of ACTH were closely related and both were in parallel with the concentration of cyclic AMP in the cells. Cyclic GMP and cyclic IMP did not inhibit [(3)H]thymidine incorporation significantly, and did not change the morphology of the cells. AMP inhibited [(3)H]thymidine incorporation into DNA and caused the characteristic morphological change. However, AMP did not increase the cyclic AMP content of the cells. CMP, GMP, and UMP showed a significant inhibition of [(3)H]thymidine incorporation into DNA, but the extent of the inhibition was much less than that with AMP. These nucleotides did not change the morphology of the cells.     

Action of KI and several iodocompounds on [3H]uridine incorporation into thyroid RNA.

The present studies were performed in order to further clarify the action of iodine and iodocompounds on the incorporation of labelled uridine into thyroid RNA. KI decreased RNA labelling but did not alter total [3H]uridine uptake or [3H]inulin distribution space. KI also inhibited the increase in RNA labelling produced by 8 mM glucose. T4 was more potent on a molar basis than KI in impairing uridine incorporation. TETRAC, TRIAC and isopropyl-T3 also decreased RNA labelling, while T2 and isopropyl-T2 were ineffective. KI did not alter the distribution of the uridine derivatives, UMP, UDP and UTP, as determined by the distribution of [3H]uridine in these compounds by paper chromatography, suggesting that the action of KI does not take place at the step of uridine phosphorylation. Like its effect on TSH, KI also impairs the stimulatory effect of exogenous cAMP and cGMP on RNA labelling, suggesting that its action is exerted beyond the step of cyclic nucleotide formation. Iodine and iodocompounds may exert their inhibitory action on RNA labelling at the step of nucleotide polymerization.     

Dibutyryl Adenosine Cyclic 3′:5′-Monophosphate Effects on Goldfish Behavior and Brain RNA Metabolism

Intraventricular administration of dibutyryl adenosine cyclic 3':5'-monophosphate into goldfish brains produced hyperactive animals. A study of the effects of the drug (25-50 mg/kg) on the incorporation of [5-(3)H] orotic acid, as a precursor of labeled uridine and cytidine, into newly synthesized RNA showed the formation of an RNA with a uridine to cytidine ratio 20-50% higher than that of the control. In double-labeling experiments with uridine as the labeled precursor, the synthesis of a nuclear RNA fraction (not produced in the absence of drug) was demonstrated. Some of this RNA was found to migrate into the cytoplasmic fraction and to become associated with polysomes. The results suggest that cyclic AMP might function as a "metabolic demand signal" for eliciting new RNA synthesis in goldfish brain.     

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.     

Regulation by guanosine 3'':5''-cyclic monophosphate of phospholipid methylation during chemotaxis in Dictyostelium discoideum.

In Dictyostelium discoideum, the chemoattractant cyclic AMP activates the enzyme guanylate cyclase, giving a brief up to 10-fold increase in the intracellular cyclic GMP content. The addition of physiological cyclic GMP concentrations to a homogenate of D. discoideum cells markedly increased the incorporation of the 3H-labeled methyl group from S-adenosyl-L-[methyl-3H]methionine into mono- and dimethylated phosphatidylethanolamine and phosphatidylcholine. Lipid methylation was inhibited by S-adenosyl-L-homocysteine, which inhibits transmethylation. When whole cells prelabeled with L-[methyl-3H]methionine were exposed to cyclic AMP, a rapid transient increase in the amount of [methyl-3H]phosphatidylcholine was observed. The time course of [methyl-3H]phosphatidylcholine formation agrees with its being mediated by the intracellular increase in cyclic GMP originating during chemotactic stimulation. Addition of the 8-Br derivative of cyclic GMP to whole cells also increased the levels of labeled phosphatidylcholine. It is therefore likely that cyclic GMP contributes to chemotaxis by regulating membrane function via phospholipid methylation.     

Sodium nitroprusside-induced protein phosphorylation in intact rat aorta is mimicked by 8-bromo cyclic GMP

The effects of sodium nitroprusside, 8-bromo cyclic GMP, 8-bromoguanosine 5′-monophosphate, 8-bromo cyclic AMP, dibutyryl cyclic AMP, and isoproterenol on incorporation of ³²P into proteins in intact rat thoracic aorta were studied. Aortas were incubated in [³²P]orthophosphate in order to label endogenous adenosine triphosphate. Agents were then added for various times and the tissues were homogenized and fractionated (100,000 × g for 60 min) into soluble and particulate fractions. Soluble and particulate fractions were subjected to isoelectric focusing followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiographs were made. Nitroprusside induced a concentration-dependent increase in incorporation of ³²P into nine proteins and a decrease in ³²P incorporation into two proteins. Some of these proteins appeared in both the soluble and particulate fractions of homogenates; others appeared only in the soluble fraction. The pattern of ³²P incorporation was identical after 2- or 15-min exposure to nitroprusside and was mimicked by exposure to 50-500 μM 8-bromo cyclic GMP. 8-Bromoguanosine 5′-monophosphate did not alter ³²P incorporation. Dibutyryl cyclic AMP at 50 μM had no effect upon ³²P incorporation whereas a higher concentration (0.5 mM) caused increased or decreased ³²P incorporation into some, but not all, of the same proteins. 8-Bromo cyclic AMP (5 mM) produced only small changes in ³²P incorporation. The pattern of ³²P incorporation induced by a relatively high concentration of isoproterenol 0.1 mM was similar but not identical to that seen with 0.5 mM dibutyryl cyclic AMP. The present study indicates that the incorporation of ³²P into endogenous proteins of intact rat aorta can be regulated by nitroprusside. These effects can be mimicked by cyclic GMP analogues and only partially by cyclic AMP analogues or isoproterenol. Presumably, these effects of nitroprusside are mediated through a cyclic GMP-dependent process (protein kinase or phosphatase) which may play a role in the relaxant properties of nitroprusside and cyclic GMP.     

Dibutyryl cyclic AMP, adenosine and guanosine blockade of the dopamine, ergocryptine and apomorphine inhibition of prolactin release in vitro.

Rat anterior hemipituitaries were incubated in Krebs-Ringer bicarbonate containing [3H]leucine. Newly synthesized [3H]prolactin and [3H]GH in the pituitary and incubation medium were assayed, as was the radioimmunoassayable prolactin released into the medium during a 5-h incubation. Dopamine (7.5 X 10(-8)M), ergocryptine (4 X 10(-10) M) and apomorphine (6 X 10(-8)M) all significantly inhibited both radioimmunoassayable prolactin release and newly-labeled [3H]prolactin release without affecting [3H]GH release. Conversely, dibutyryl cyclic AMP (2.5 mM) stimulated radioimmunoassayable prolactin release as well as [3H]prolactin and [3H]GH release. The addition of 2.5 mM dibutyryl cyclic AMP to media containing dopamine, ergocryptine or apomorphine completely restored both radioimmunoassayable prolactin release and [3H]prolactin release to at least control levels. Dopamine, ergocryptine and apomorphine all inhibited incorporation of [3H]leucine into prolactin but not into GH, whereas 2.5 mM dibltyryl cyclic AMP with any one of the inhibitors restored total incorporation into [3H]prolactin to levels insignificantly lower than the nucleotide-stimulated incorporation. Adenosine and guanosine at 2.5 mM also stimulated incorporation into [3H]prolactin and blocked the inhibitory effects of apomorphine upon [3H]prolactin synthesis and release. These nucleosides also stimulated [3H]GH release; and guanosine, but not adenosine, stimulated incorporation into [3H]GH. The ability of dibutryl cyclic AMP to block the effects of dopamine, ergocryptine and apomorphine upon prolactin release is consistent with these three inhibitors acting by a common mechanism. Cyclic AMP could be hypothesized as a second messenger for prolactin release, but the ability of adenosine and guanosine to mimic almost perfectly the effects of this cyclic nucleotide does not allow any conclusive interpretation.     

Alteration of Nucleoside Transport of Chinese Hamster Cells by Dibutyryl Adenosine 3′:5′-Cyclic Monophosphate

Cultured Chinese hamster ovary cells showed no significant change in generation time or fraction in the S-phase in the presence of 1 mM N(6),O(2')-dibutyryl adenosine 3':5'-cyclic monophosphate. Growth continued for at least two generations after expression of the morphological transformation induced by this cyclic AMP analog. Despite identical growth rates, apparent rates of DNA and RNA synthesis (incorporation of [(3)H]-thymidine or [(3)H]uridine) were reduced up to 15-fold in log phase by 1 mM cyclic nucleotide. [(3)H]Deoxycytidine incorporation was much less sensitive to dibutyryl cyclic AMP. Uptake studies with [(3)H]thymidine demonstrated an inhibition of transport rate dependent on the concentration of dibutyryl cyclic AMP in the growth medium. The rate of thymidine uptake at 1 degrees was decreased 21-fold by 1 mM cyclic nucleotide; half-maximal inhibition occurred at 6 muM. At 37 degrees , the pool size of acid-soluble thymidylate was strongly reduced by 1 mM cyclic nucleotide, and synergistic reduction of the pool size was found with 0.5 mM aminophylline. Phosphorylation of the acid-soluble intracellular label was unaffected by dibutyryl cyclic AMP. Inhibition of thymidine uptake is attributed to an observed decrease in thymidine kinase activity caused by growth in 1 mM dibutyryl cyclic AMP, and possibly to a simultaneous alteration in membrane permeability. Kinase-facilitated uptake of other metabolites may be regulated in a similar fashion by cyclic AMP.     

Specificity of the cyclic GMP-binding activity and of a cyclic GMP-dependent cyclic GMP phosphodiesterase in Dictyostelium discoideum

The nucleotide specificity of the cyclic GMP-binding activity in a homogenate of Dictyostelium discoideum was determined by competition of cyclic GMP derivatives with [8-3H]-cyclic GMP for the binding sites. The results indicate that cyclic GMP is bound to the binding proteins by hydrogen bonds at N2H2, N1-H and/or C6 = O, N7, 2(1)-OH and 3(1)-O and possibly via a charge-charge interaction with the phosphate moiety of cyclic GMP. Cyclic AMP only competes with cyclic GMP for binding at a 20,000-fold higher concentration. The same cyclic GMP derivatives were used to modify the hydrolysis of [8-3H]cyclic GMP by phosphodiesterase. The phosphodiesterase is activated by cyclic GMP. The nucleotide specificity of activation of the enzyme differs from the specificity of the enzyme for hydrolysis. This indicates that activation by cyclic GMP and hydrolysis of cyclic GMP occur at different sites of the enzyme. Cyclic AMP neither activates the cyclic GMP phosphodiesterase nor competes with cyclic GMP for hydrolysis. This indicates that intracellular cyclic AMP does not interfere with the action of intracellular cyclic GMP in D. discoideum.     

Regulation of casein synthesis by polyamines in mammary gland explants of mice.

Studies were carried out to determine whether the actions of prolactin on the metabolism of the mammary gland may involve polyamines. In mouse mammary gland explants that were preincubated for 2 days with insulin plus hydrocortisone, the rate of [3H]leucine incorporation into casein was enhanced in a prolactin-like manner during a further incubation with spermidine plus cyclic GMP or phospholipase A. Putrescine (0.5 mM) plus PGF2alpha, cyclic GMP or arachidonic acid also enhanced the rate of casein synthesis: but PGF2alpha plus 0.5 mM arginine, ornithine or spermine had no effect. Methyl GAG, an inhibitor of the enzyme S-adenosyl-L-methionine decarboxylase (which is required for the conversion of putrescine to spermidine), abolished the putrescine plus PGF2alpha stimulation of casein synthesis. Since this drug did not affect the action of spermidine plus PGF2alpha on casein synthesis, the specific action of spermidine on casein synthesis is suggested. Neither arginine, ornithine nor the polyamines, by themselves, affected the rate of [3H]uridine incorporation into RNA or the rate of [3H]leucine incorporation into casein. Spermidine levels were elevated within 4 h after adding prolactin to explants which were preincubated for 2 days with insulin plus hydrocortisone; this effect was apparent during incubation periods of up to 48 h with prolactin. Arginase and ornithine decarboxylase activities were also elevated in response to prolactin. Arginase activity was only elevated, however, during long incubation periods with prolactin, i.e., during incubation periods of longer than 2 days. In contrast, ornithine decarboxylase activity was elevated by prolactin within a 30 min incubation period; this effect was maximal after 2 h and persisted during exposure periods of up to 24 h.     

Acute antihypertrophic actions of bradykinin in the rat heart: importance of cyclic GMP

The antihypertrophic action of angiotensin (Ang)-converting enzyme (ACE) inhibitors in the heart is attributed in part to potentiation of bradykinin. Bradykinin prevents hypertrophy of cultured cardiomyocytes by releasing nitric oxide (NO) from endothelial cells, which increases cardiomyocyte guanosine 3'5'-cyclic monophosphate (cyclic GMP). It is unknown whether cyclic GMP is essential for the action of bradykinin, or whether findings in isolated cardiomyocytes apply in whole hearts, in the presence of other cell types and mechanical/dynamic activity. We now examine the contribution of cyclic GMP to the antihypertrophic action of bradykinin in cardiomyocytes and perfused hearts. In adult rat isolated cardiomyocytes cocultured with bovine aortic endothelial cells, the inhibitory action of bradykinin (10 micromol/L) against Ang II (1 micromol/L)-induced [3H]phenylalanine incorporation was abolished by the soluble guanylyl cyclase inhibitor [1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (10 micromol/L). In Langendorff-perfused rat hearts, Ang II (10 nmol/L)-induced increases in [3H]phenylalanine incorporation and atrial natriuretic peptide mRNA expression were prevented by bradykinin (100 nmol/L), the NO donor sodium nitroprusside (3 micromol/L), and the ACE inhibitor ramiprilat (100 nmol/L). The acute antihypertrophic action of bradykinin was accompanied by increased left ventricular cyclic GMP, and the ramiprilat effect was attenuated by HOE 140 (1 micromol/L, a B2-kinin receptor antagonist) or [1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (100 nmol/L). In conclusion, bradykinin exerts a direct inhibitory action against the acute hypertrophic response to Ang II in rat isolated hearts, and elevation of cardiomyocyte cyclic GMP may be an important antihypertrophic mechanism used by bradykinin and ramiprilat in the heart.     

Growth promoting effect of head activator in cultured chick embryo brain cells

To investigate the regulatory role of head activator (HA) and its synthetic analogue, (Arg1,Phe5)-HA(AHA) on brain cell growth, we measured serial uptakes of [3H]thymidine, [3H]uridine and [3H]leucine and changes in cyclic AMP content in cultured chick embryo brain cells. HA stimulated all of these uptakes at a concentration of 10(-10) M, while 10(-9) M AHA suppressed them. The stimulatory effect of HA on [3H]thymidine uptake was observed after 4 h of the treatment, reached a maximum of 200% of the initial value at 8 h and declined to the pre-treatment level at 14 h. [3H]uridine uptake began to increase after 6 h of HA treatment, and the effect lasted for 4 h. Increase in [3H]leucine followed after 12 h and sustained for 4 h. Prior to the initiation of HA stimulation, cyclic AMP also began to rise, reaching 170% of the pre-treatment level at 6 h. In contrast, depression of [3H]thymidine uptake by AHA was noted at 6 h and continued for 8 h. Uptake of [3H]uridine and [3H]leucine showed similar tendency. Cyclic AMP in AHA-treated cells at 6 h was significantly lower than that in non-treated cells. These results indicate that HA stimulates DNA, RNA and protein synthesis in an early stage of growing brain cells, in which cAMP may be involved as a regulator of nerve cell growth.     

Gonadotropin-releasing hormone-stimulated phosphoinositide hydrolysis in the anterior pituitary. Modulation by protein kinase C but not by cyclic nucleotides

Gonadotropin-releasing hormone (GnRH) produces a rapid and concentration-dependent hydrolysis of polyphosphoinositides in rat anterior pituitary cells in culture. Evaluation of the action of the decapeptide by measurement of [3H]-inositol phosphates and of prelabeled phosphoinositides demonstrated an effect on phosphatidylinositol-4,5-bis-phosphate and phosphatidylinositol-4-phosphate earlier than on phosphatidylinositol. The receptor antagonist [D-pGlu1,D-Phe2,D-Trp3,6]-luteinizing hormone-releasing hormone blocked the effect of GnRH on [3H]-inositol phosphate production. Protein kinase C activators attenuated GnRH-induced phosphoinositide hydrolysis, while neither cyclic AMP analogs nor cyclic GMP analogs were effective. These results indicate that phosphoinositide hydrolysis represents an important postreceptor transducing mechanism for GnRH action at the gonadotroph and that protein kinase C (but not cyclic nucleotides) may exert a negative feedback control on GnRH receptor-coupling mechanisms.     

GnRH action in rat anterior pituitary gland: regulation of protein, glycoprotein and LH synthesis.

The effect of synthetic GnRH on the synthesis of proteins and glycoproteins in the anterior pituitary and in vitro release of LH into the medium was studied. A maximal dose (25 ng/ml) of of synthetic GnRH caused optimum release of radioimmunoassayable LH into the medium after 2 h of incubation. A concomitant increase in cyclic AMP accumulation in the tissue and LH in the incubation medium was also observed under the influence of GnRH during different periods of incubation time. Incubation of the rat anterior pituitary with GnRH stimulated the incorporation of [3H] proline into acid precipitable proteins in a time- and dose-dependent manner, similar to radioimmunoassayable LH released into the medium. Similar results were obtained when pituitary was incubated with dibutyryl cyclic AMP. LH, in addition, enhanced the incorporation of [3H] glucosamine and [3H] amino acids mixture into acid-precipitable proteins suggesting that proteins including glycoproteins are synthesized by the rat anterior pituitary under the influence of GnRH. Approximately 10% of the radioactivity associated with proteins comigrated with radioimmunoassayable LH on the gels. GnRH also enhanced the incorporation of [3H] glucosamine and [3H] amino acid mixture into immunoprecipitable LH. The GnRH-induced incorporation of [3H] proline into anterior pituitary proteins was abolished by specific translation inhibitors.     

Antihypertrophic actions of the natriuretic peptides in adult rat cardiomyocytes: importance of cyclic GMP

Atrial natriuretic peptide (ANP) prevents hypertrophy of neonatal cardiomyocytes. However, whether this effect is retained in the adult phenotype or if other members of the natriuretic peptide family exhibit similar antihypertrophic properties, has not been elucidated. OBJECTIVE: Our objective was to examine whether the natriuretic peptides protect against adult cardiomyocyte hypertrophy in vitro. METHODS: Adult rat cardiomyocytes were incubated with angiotensin II (Ang II)+/-ANP, B-type (BNP) or C-type (CNP) natriuretic peptides for determination of [3H]phenylalanine incorporation, c-fos mRNA expression and cyclic GMP. The effects of 8-bromo-cyclic GMP (cyclic GMP analogue), HS-142-1 (particulate guanylyl cyclase inhibitor) and KT5823 (cyclic GMP-dependent protein kinase inhibitor) were also investigated. RESULTS: Ang II-stimulated increases in markers of hypertrophy, [3H]phenylalanine incorporation (to 136+/-3% of control, n=9) and c-fos mRNA expression (4.3+/-1.4-fold, n=5), were completely prevented by each of ANP, BNP or CNP. This protective action was accompanied by increased cardiomyocyte cyclic GMP. Inhibitory actions on [3H]phenylalanine incorporation were mimicked by 8-bromo-cyclic GMP, and were abolished by HS-142-1. KT5823 blocked the response to BNP and CNP, but not to ANP. CONCLUSION: ANP prevents hypertrophy of adult rat cardiomyocytes. This protective action is shared by BNP and CNP and involves activation of particulate guanylyl cyclase receptors. Antihypertrophic effects of BNP and CNP are mediated through cyclic GMP-dependent protein kinase, but ANP can activate additional pathways independent of cyclic GMP to prevent adult cardiomyocte hypertrophy. These novel findings are of interest particularly since BNP appears to exert antifibrotic rather than antihypertrophic actions in vivo, while CNP is thought to act at least in part via the endothelium.     

Pleiotypic Control by Cyclic AMP: Interaction with Cyclic GMP and Possible Role of Microtubules

Previous studies have shown that exogenous dibutyryl cyclic AMP inhibits the uptake of uridine, leucine, and 2-deoxyglucose by cultured mouse fibroblasts. 3':5'-cyclic GMP is shown here to counteract these inhibitory effects as well as the inhibition of precursor transport and leucine incorporation into proteins produced by prostaglandin E(1). We conclude, therefore, that cyclic GMP antagonizes the "pleiotypic" effects of cyclic AMP in these cells.Colcemid and vinblastine, but not cytochalasin B, reverse the transport inhibition caused by cyclic AMP without affecting the intracellular concentrations of cyclic AMP. These results suggest the possibility that cyclic AMP regulates the membrane transport of certain substrates by influencing the organization of microtubules.