Insulin receptor down-regulation, prevention at a post-receptor site.

Nicotinamide (50mM) prevented insulin-mediated down-regulation of insulin receptors in IM-9 lymphoblastoid cells. Half-maximum effectiveness was between 10 and 33mM. Nicotinamide did not influence insulin binding to the cells, cell viability, insulin degradation or protein synthesis. A variety of inhibitors of ADP-ribosylation reactions besides nicotinamide, most of them pyridine analogues, similarly prevented insulin-induced receptor loss. Spermine decreased the number of insulin receptors in IM-9 cells, but this effect was not inhibited by nicotinamide.

Studies on the bioactivity of radioiodinated highly purified bovine thyrotropin: analytical polyacrylamide gel electrophoresis.

Highly 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.

Differential effect of protein synthesis inhibition on TSH desensitization at different stages of primary thyroid cell culture.

In 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.

Evidence for species specificity in the interaction between thyrotropin and thyroid-stimulating immunoglobulin and their receptor in thyroid tissue.

The 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 dog than in human thyroid cells. Relative bioactivity of bTSH and TSI aslo 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.

Prevention by nicotinamide of desensitization to thyrotropin stimulation in cultured human thyroid cells.

The 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.

High performance gel permeation chromatography of bovine thyrotropin (TSH): effect of column stability and mobile phase variation.

Because of previously observed variability in the elution patterns of radiolabeled bovine TSH (bTSH) and bTSH bioactivity (in a particular bTSH preparation) on gel permeation high performance liquid chromatography (GPC), studies were conducted to examine the effect of different conditions on the elution of this material. Continued use of a Waters' I-125 column at pH 7.2 demonstrated progressive retardation in the elution of bioactivity and radioactivity in this highly-purified bTSH preparation, with progressive but incomplete separation of these two functions. With decreasing mobile phase pH the elution positions of TSH bioactivity and radioactivity advanced to a coincident peak near to the void volume (pH 4). Addition to the mobile phase of the ion pair reagents tetrabutyl-ammonium phosphate (PIC-A) and pentane sulfonic acid (PIC B5) produced marked alterations in the elution positions of TSH radioactivity and bioactivity, with separation of these functions in the presence of the former reagent. These data indicate the present limitations of GPC for the purification of TSH.

Modulation by thymus-derived (T) cells of thyroid cell-stimulated prostaglandin E release by human peripheral blood mononuclear cells.

Cultures of plastic-adherent, human peripheral blood mononuclear cells generated prostaglandin E (PGE). Culture of the adherent cells (predominantly monocytes) with human thyroid cells enhanced PGE accumulation in the medium, although to a lesser degree than occurs with unseparated blood mononuclear cells. Recombination of the adherent cells with monocyte-depleted, nonadherent cells restored both basal and thyroid cell-stimulated PGE generation to the levels seen with unseparated cells. Thymus-derived (T) cells, obtained by rosetting with sheep erythrocytes, similarly enhanced both the adherent cell basal PGE production as well as the increased PGE accumulation that occurs in the presence of thyroid cells (50-120% augmentation by the T cells). Significant augmentation of thyroid cell-stimulated PGE release by 10(5) adherent cells occurred with the addition of as few as 5 x 10(4) T cells. Culture medium transfer experiments and separation of cell types during culture by a semipermeable membrane provided evidence against the possibility that the adherent cells were releasing a factor that stimulated T-cell PGE generation or that T cells were releasing a factor that enhanced adherent cell PGE generation. The results suggest instead that this T-cell effect requires direct contact with the adherent cells. These data demonstrate the importance of human T cells in the release by adherent cells of PGE, a mediator of suppressor function by some immune cells.