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.     

Effect of iodide on glucose oxidation and 32P incorporation into phospholipids stimulated by different agents in dog thyroid slices

Since iodide (I-) inhibits TSH stimulation of cAMP formation, which mediates most of the effects of the hormone, it has been assumed that this accounts for the inhibitory action of iodide on the thyroid. However, TSH stimulation of 32P incorporation into phospholipids and stimulation of thyroid metabolism by other agonists, such as carbachol, phorbol esters, and ionophore A23187, is not cAMP mediated. The present studies examined the effect of iodide on stimulation of glucose oxidation and 32P incorporation into phospholipids by TSH and other agonists to determine if the inhibition of cAMP formation was responsible for the action of iodide. Preincubation of dog thyroid slices for 1 h with iodide (10(-4) M) inhibited TSH-, (Bu)2cAMP-, carbachol-, methylene blue-, 12-O-tetradecanoyl phorbol-13-acetate-, ionophore A23187-, prostaglandin E1-, and cholera toxin-stimulated glucose oxidation. I- also inhibited the stimulation by TSH, 12-O-tetradecanoyl phorbol-13-acetate, carbachol, and ionophore A23187 of 32P incorporation into phospholipids. The inhibition was similar whether iodide was added 2 h before or simultaneously with the agonist. I- itself sometimes stimulated basal glucose oxidation, but had no effect on basal 32P incorporation into phospholipids. The effects of iodide on basal and agonist-stimulated thyroid metabolism were blocked by methimazole (10(-3) M). When dog thyroid slices were preloaded with 32PO4 or [1-14C]glucose, the iodide inhibition of agonist stimulation disappeared, suggesting that the effect of iodide involves the transport process. In conclusion, I- inhibited stimulation of glucose oxidation and 32P incorporation into phospholipids by all agonists, indicating that the effect is independent of the cAMP system and that iodide autoregulation does not only involve this system. Oxidation and organification of iodide are necessary for the inhibition. The ability of iodide to decrease glucose and 32PO4 transport may play an important role in thyroid autoregulation.     

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.     

Insulin-stimulated glucose uptake,leucine incorporation into protein,and uridine incorporation into RNA in skin fibroblast cultures from patients with diabetes mellitus

Summary Since intrinsic cellular abnormalities have previously been reported in diabetes mellitus, skin fibroblasts from patients with insulin-dependent diabetes and non-insulin-dependent diabetes, and from age-matched young and old controls, were examined for stimulation by insulin (4–4000 ng/ml) of glucose uptake, leucine incorporation into protein, and uridine incorporation into RNA. No differences in insulin-stimulated glucose uptake were seen between donor types. At 40 and 400 ng/ml, insulin did not stimulate as much leucine incorporation into protein in insulin-dependent diabetics as in young controls (p<0.05) and at 4000 ng/ml, less insulinstimulated leucine incorporation was seen in insulindependent diabetics than in young controls or noninsulin dependent diabetics (p<0.01). Lower insulin-stimulated uridine incorporation into RNA in old controls than in other cell lines appeared to be largely secondary to a two-fold increase in basal incorporation in these old controls. These results provide additional evidence for intrinsic cellular abnormalities in diabetes mellitus. Whether the differences in basal or insulin-stimulated response between fibroblasts of different donor types are attributable to alterations in protein or RNA synthesis, metabolite pool size or turnover have yet to be determined.     

Action of thyrotropin on phosphate incorporation into thyroid proteins in vitro.

It is now well established that cAMP is the intracellular mediator of many effects of thyrotropin on the thyroid. Greengard has postulated that all the effects of cAMP inside the cell are secondary to the phosphorylation of proteins by cAMP activated protein kinase(s). The purpose of our work is to define, in an intact cell system, the nature of the thyroid proteins, the phosphorylation of which is stimulated by cAMP and TSH.     

Effect of lactate on glucose incorporation into fetal lung phospholipids

Fetal rabbit lung explants were incubated with 3.0 mM glucose and varying levels of lactate. An increase in lactate concentrations resulted in a decrease in glucose incorporation into total disaturated phosphatidylcholine. Glucose utilization for surfactant phosphatidylcholine synthesis was also reduced by approximately 35% in the presence of 5.0 mM lactate. The decreased incorporation of glucose occurred in the fatty acid portion of both total tissue disaturated phosphatidylcholine and surfactant phosphatidylcholine. The effect of lactate on glucose incorporation into pulmonary phospholipids was not affected by the presence of pyruvate in concentrations up to 500 microM. Pyruvate alone produced only a slight decrease in glucose utilization for lung phospholipid production. These data indicate that glucose and lactate are competitive substrates for late gestation surfactant phospholipid fatty acid synthesis, and that lactate is potentially a very important substrate for fetal lung development.     

Gastric cytoprotection by prostaglandins, ranitidine, and probanthine in rats. Role of endogenous prostaglandins

Intragastric administration of aspirin (ASA) plus 0.15 M HCl to fasted rats produced typical gastric ulcers accompanied by almost complete disappearance of mucosal prostaglandins (PGs). Pretreatment with various exogenous PGs that were biologically inactive (e.g. 6-keto-PGF1 alpha or PGF2 beta) or active (PGE2 and PGI2) but used in non-antisecretory doses prevented the formation of these gastric lesions ('cytoprotection'). Besides PGs, antisecretory compounds such as ranitidine, a new H2-receptor antagonist, and probanthine were also found to be cytoprotective, even when given in non-antisecretory doses. Mucosal generation of PGs in animals treated with ASA and HCl plus ranitidine or probanthine was very low and not significantly different from those receiving only ASA and HCl. Thus, the cytoprotection appears to be the property not only of PGs but also of conventional gastric antisecretory compounds such as H2-receptor antagonists or anticholinergics. This cytoprotection can be demonstrated under conditions excluding any role of gastric secretory inhibition and in the absence of endogenous PGs.     

Determination of cyclic 3',5'-AMP accumulation in resting and stimulated thyroid slices: Heterogeneity of the ATP precursor pool.

cAMP accumulation in dog thyroid slices has been estimated by two methods: from the radioactivity of cAMP in tissues prelabeled with ³H-adenine, ³H-adenosine or ³²P-phosphate, and by the protein binding assay of Gilman et al. In the first method, the pitfalls in measuring cAMP accumulation in radioactivity per weight of tissue as a conversion ratio from its precursors (³H-cAMP/³H of the TCA soluble pool, or ³H-cAMP/³H-ATP), or from the specific radioactivity of the precursors used have been demonstrated. It is shown that the measurement of labeled cAMP accumulation may provide reliable estimates of relative increases in concentration but not of the absolute concentrations of cAMP in the slices. The specific radioactivity of cAMP was higher than the specific radioactivity of ATP in slices prelabeled with ³H-adenine and ³H-adenosine which suggests the existence of a specific precursor ATP pool involving less than 50% of tissue ATP.TSH, even when it enhances by a factor of 20 the accumulation of cAMP, does not decrease cell ATP levels which shows that the drain of ATP by this synthesis is easily compensated by the energy providing metabolic pathways of the tissue.     

Selective incorporation of influenza virus RNA segments into virions

The genome of influenza A virus is comprised of eight viral RNA (vRNA) segments. Although the products of all eight vRNA segments must be present for viral replication, little is known about the mechanism(s) responsible for incorporation of these segments into virions. Two models have been proposed for the generation of infectious virions containing eight vRNA segments. The random-incorporation model assumes a common structural feature in all the vRNAs, enabling any combination of vRNAs to be incorporated randomly into virions. The selective-incorporation model predicts the presence of specific structures in each vRNA segment, leading to the incorporation of a set of eight vRNA segments into virions. Here we demonstrate that eight different vRNA segments must be present for efficient virion formation and that sequences within the coding region of (and thus unique to) the neuraminidase vRNA possess a signal that drives incorporation of this segment into virions. These findings indicate a unique contribution from individual vRNA segments and thus suggest a selective (rather than random) mechanism of vRNA recruitment into virions. The neuraminidase vRNA incorporation signal and others yet to be identified should provide attractive targets for the attenuation of influenza viruses in vaccine production and the design of new antiviral drugs.     

Helodermin-like peptides in thyroid C cells: stimulation of thyroid hormone secretion and suppression of calcium incorporation into bone.

Helodermin is a vasoactive intestinal peptide-like peptide in the salivary gland venom of the lizard Heloderma suspectum. Helodermin-like immunofluorescence was observed in the parafollicular (C) cells in several mammals and in the C cell homologues of the chicken ultimobranchial gland. Thus, helodermin-like peptides coexist with calcitonin. The results of radioimmunoassay agreed with the immunocytochemical findings. HPLC of rat thyroid extracts revealed one major peak of helodermin-like immunoreactivity, which eluted in a position close to that of lizard helodermin. Helodermin stimulated basal thyroid hormone secretion and colloid droplet formation in conscious mice. The effect of large doses of helodermin was quite long-lasting and the maximal response occurred after 2-6 hr. In addition, helodermin suppressed the incorporation of calcium into bone in conscious rats. The findings suggest that helodermin-like peptides in C cells may be involved in the local regulation of thyroid hormone secretion and in the maintenance of calcium homeostasis.