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Evidence that organic iodine attenuates the adenosine 3',5'-monophosphate response to thyrotropin stimulation in thyroid tissue by an action at or near the adenylate cyclase catalytic unit
No full textStudies were conducted to define more clearly the site in the thyroid adenylate cyclase complex at which iodine exerts its inhibitory effect on activation of this enzyme by TSH. Iodine- and TSH-induced desensitization were additive. Dissociation was observed between the rates of recovery from TSH- and iodine-induced desensitization. Cycloheximide (10-4 M) prevented recovery from the inhibitory effect of iodine on thyroid adenylate cyclase activation. Preincubation of freshly isolated dog thyroid follicles in 10-4 M iodide decreased the subsequent cAMP response to cholera toxin (0.5 μg/ml) stimulation. This effect of iodide was prevented by 3 mM methimazole. Thyroid adenylate cyclase regulatory protein (Ns) activity was assessed by the ability of detergent extracts of thyroid plasma membranes to reconstitute adenylate cyclase responsiveness to isoproterenol in N-deficient S49 cyc- plasma membranes. Thyroid Ns activities were similar in control and iodide-pretreated thyroid cells. The inhibitory effect of iodine on TSH activation of thyroid cAMP generation was additive to that of inhibition via the α2-adrenergic pathway and also additive to inhibition by 2',5'-dideoxyadenosine (an adenosine P-site agonist). Preincubation of freshly dispersed dog thyroid cells in 10-4 M NaI reduced the cAMP response to stimulation by 100 μM forskolin. These data provide evidence that in iodine-induced TSH desensitization in the thyroid; 1) TSH receptor function is normal, 2) the regulatory protein (Ns) in the adenylate cyclase stimulatory pathway is functionally unaltered, 3) iodine does not exert its effect via the regulatory protein (Ni) in the pathway that inhibits adenylate cyclase activation, 4) iodine does not act via the adenosine P-site inhibitory pathway, 5) the action of iodine is at or near the adenylate cyclase catalytic unit, and 6) new protein synthesis is necessary for recovery from iodine desensitization
Inhibitors of specific aminoacyl-tRNA synthetases prevent thyrotropin-induced desensitization in cultured human thyroid cells
No full text[No abstract available
Autoregulation by iodine of thyroid protein synthesis: Influence of iodine on amino acid transport in cultured thyroid cells
No full textStudies were conducted to determine whether the inhibitory effect of iodine on thyroid protein synthesis could be explained by a reduction in intracellular amino acid transport. The nonmetabolizable amino acid [14C]cycloleucine was used as a probe for the L system of neutral amino acid transport in dog thyroid cells during the initial 24 h of primary culture. Uptake of cycloleucine was linear for up to 30 min. At all time points, cycloleucine transport was reduced in cells preincubated for 3 h in NaI (10-4 M). Inhibition (in a typical experiment) by NaI of cycloleucine transport (10 min) was 19%, 42%, and 69% at 10-6, 10-5, and 10-4 M iodide, respectively. Methimazole (3 mM) together with iodide abolished the inhibitory effect of iodide on cycloleucine transport, implying the necessity of iodide organification. Methimazole itself did not significantly alter cycloleucine transport. T3, T4, MIT, and DIT similarly did not inhibit cycloleucine transport. NaI did not inhibit cycloleucine transport in cells lacking a mechanism for iodide organification. Double reciprocal plots of cycloleucine influx at different substrate concentrations indicated that NaI decreases the maximum velocity of cycloleucine transport (2.1 vs. 4.0 nmol min-1 mg protein-1) without affecting the K(m) (1 mM). In contrast to influx, iodine did not affect cycloleucine efflux. The inhibitory action of iodine on cycloleucine transport was reversible after removal of extracellular iodide, with full recovery occurring within 24 h. Iodine similarly inhibited the cellular uptake of [14C]α-methylaminoisobutyric acid as well as [14C]α-aminoisobutyric acid in the presence of 30 mM methyl-α-aminoisobutyric acid, that is of specific probes for the A and ASC transport systems of neutral amino acids, respectively. These data indicate that autoregulation by iodine of thyroid protein synthesis occurs, at least in part, by regulation of the maximum velocity of neutral amino acid uptake via the A, ASC, and L transport systems
Prevention by nicotinamide of desensitization to thyrotropin stimulation in cultured human thyroid cells.
No full textThe presence of 50 mM nicotinamide together with 100 milliunits/ml of TSH in the incubation medium prevented the decline in human thyroid cell cAMP from maximum, stimulated levels (15-30 min) that occurs when the cells are exposed to TSH alone. Nicotinamide in the absence of TSH did not increase thyroid cell cAMP content. TSH desensitization, and its prevention by nicotinamide, occurred in the presence or absence of 3-isobutyl-methylxanthine. 1-Methyl nicotinamide and N'-methyl nicotinamide similarly prevented TSH desensitization. Recovery from TSH desensitization was prolonged and incomplete after 72 h. The presence of 50 mM nicotinamide hastened recovery from desensitization. Desensitization of the cAMP response to 10(6) M prostaglandin E1 and 1 mM adenosine was unaffected by nicotinamide. Other inhibitors of poly(ADP-ribose) polymerase activity, 5-bromouridine, 5-bromo-2'-deoxyuridine, and thymidine (all at 50 mM) completely or partially prevented TSH desensitization. Pyridoxine (50 mM) similarly prevented this phenomenon. As with dog thyroid cells, 10(-4) M cycloheximide blocked TSH desensitization. The combination of 10(-4) M cycloheximide and 50 mM nicotinamide had a synergistic effect in augmenting the thyroid cell cAMP response to TSH stimulation
Differential effect of protein synthesis inhibition on TSH desensitization at different stages of primary thyroid cell culture.
No full textIn contrast to our previous experience with cultured thyroid cells, cycloheximide, actinomycin D and nicotinamide did not prevent TSH-induced desensitization in dog thyroid cells in primary culture for only one day. With continued duration of culture prevention of TSH desensitization by these agents did emerge, but asynchronously. Thus on the second day of primary culture, while cycloheximide and actinomycin D prevented TSH desensitization, nicotinamide remained ineffective. On the third day of primary culture all three agents blocked TSH desensitization. Examination of precursor incorporation into newly synthesized DNA, RNA and protein revealed a temporal association between the appearance of susceptibility to inhibition of TSH desensitization and an increase in DNA and protein synthesis. These data provide an explanation for the discrepant reports regarding the effect of protein synthesis inhibitors on TSH desensitization
On the mechanism of 'escape' from desensitization of the cyclic AMP response to TSH in cultured human thyroid cells.
No full textStudies were conducted to examine the mechanism by which 'escape' from TSH desensitization of the cyclic AMP response to TSH (Endocrinology 109, 1156, 1981) occurs in confluent cultured thyroid cells. At confluence, cell replication and DNA synthesis are suppressed. An attempt was therefore made to reproduce escape in sparse thyroid cell monolayers using inhibitors of DNA synthesis. The concurrent presence of TSH and mitomycin C (5 micrograms/ml) did not influence the induction of desensitization to TSH after 6 h of stimulation, but cAMP levels then rebounded by 24 h; that is, mitomycin C reproduced escape in sparse cells. Hydroxyurea (10 mM) did not reproduce escape in sparse cells. Adenylate cyclase activity was unaltered in plasma membranes prepared from sparse thyroid cells treated with mitomycin C for 24 h. These data suggest that 'escape' from TSH desensitization is related to events occurring during the cell cycle associated with DNA synthesis, and is caused by an alteration in adenylate cyclase substrate or co-factor availability rather than in enzyme activity itself
Hormonal stimulation of eucaryotic cell ADP-ribosylation.
No full textThe effect of thyrotropin (TSH) on the ADP-ribosylation of endogenous thyroid cell acceptor proteins was examined. Cells were "permeabilized" at 4 degrees C in hypotonic medium and then exposed to [(32)P]- or [(3)H-adenine]NAD(+). The net incorporation of labeled ADP-ribose was measured by trichloroacetic acid precipitation. TSH (100 mU/ml) enhanced ADP-ribosylation with a maximum effect after 30-60 min in the majority of experiments. TSH stimulation was observed even when the incubation contained 1,000-fold more exogenous NAD(+) than the amount of NAD(+) contributed by the permeabilized cells, indicating an effect on enzymatic activity rather than an alteration in NAD(+) pool size or specific activity. No incorporation of radioactivity from labeled NAD(+) was observed in cells not rendered permeable to NAD(+) by hypotonic shock. TSH did not increase the rate of disappearance of trichloroacetic-precipitable radioactivity and did not contain intrinsic NAD(+) glycohydrolase activity. Alkali and snake venom phosphodiesterase, but not ribonuclease or deoxyribonuclease digestion of trichloroacetic acid precipitable thyroid cell radioactivity, revealed primarily 5'-AMP, consistent with an effect of TSH on mono-ADP ribosylation. Nicotinamide and thymidine (50 mM) inhibited both basal and TSH-stimulated ADP-ribosylation of thyroid cell protein. Dibutyryl cyclic (c)AMP (0.1 mM) inhibited endogenous ADP-ribosylation by approximately 35% but had no effect at lower concentrations. 0.5 mM isobutylmethylxanthine inhibited this reaction by approximately 60%. We suggest that TSH enhances thyroid cell ADP-ribosylation by a mechanism independent of cAMP as a second messenger, and that ADP-ribosylation plays a role in the expression of TSH
Evidence for species specificity in the interaction between thyrotropin and thyroid-stimulating immunoglobulin and their receptor in thyroid tissue
No full textThe cAMP response in cultured human and dog thyroid cells was used to examine the relationship between human TSH, nonprimate TSH, and thyroid-stimulating immunoglobulin (TSI) bioactivity in human and nonhuman thyroid tissue. The bovine TSH (bTSH) to human TSH potency ratio was approximately 6-fold greater in dogs than in human thyroid cells. Relative bioactivity of bTSH and TSI also differed in these cell types. Thus, four TSI samples produced approximately 6-fold greater stimulation relative to bTSH in human thyroid than in dog thyroid cells. It is discussed why these data suggest that the TSH receptor as well as TSH and TSI display species specificity as defined by the classical concept of this term
Influence of cell density on desensitization of the thyroid cell cyclic adenosine 3',5'-monophosphate response to thyrotropin stimulation.
No full textStudies on the bioactivity of radioiodinated highly purified bovine thyrotropin: analytical polyacrylamide gel electrophoresis.
No full textHighly purified bovine TSH (stored in solution at -70 C) was radioiodinated by the stoichiometric chloroamine-T method. The iodinated material ws subjected to analytical polyacrylamide disc gel electrophoresis. TSH was eluted from gel slices (1 mm width) and was analyzed for radioactivity and bioactivity. The latter was determined using the cultured thyroid cell cAMP response assay. Radioactivity in the TSH preparation migrated separately from bioactivity, but concordant with the protein bands observed in gels run in parallel. Further studies performed on bovine TSH purified in our laboratory, as well as on a different TSH preparation of exceptionally high potency (both stored as lyophilized powder) revealed a different pattern, with TSH bioactivity and radioactivity eluting concurrently. Iodination of TSH did not alter its electrophoretic migration on disc gel electrophoresis. In all preparations polymorphism of TSH bioactivity was observed, with at least four separate protein bands containing TSH bioactivity being present in our preparation. The relationship between the degree of iodination and retention of TSH bioactivity was examined. Incorporation of 125I into TSH was greatly different at two different concentrations of chloramine-T. Despite this, however, the progressive loss of TSH bioactivity was similar at both concentrations, indicating that incorporation of iodine into the TSH molecule is not itself responsible for the decrease in bioactivity. These studies indicate variability among different TSH preparations in terms of their retention of bioactivity. Significant loss of TSH bioactivity appears to occur during storage in solution. The damage to the biological activity of TSH during the iodination procedure is more likely related to the oxidation process than to the incorporation of iodine
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