Colin G. Thomas

Current management of the patient with autonomously functioning nodular goiter.

Autonomously functioning thyroid nodules (AFTNs) are presumably independent of TSH for growth and function and appear hot on scintiscan because they selectively concentrate radionuclide to a greater extent than the remaining thyroid gland, which is controlled by the normal TH-TSH feedback mechanism. Such autonomously functioning tissue may occur in patchy areas, as a solitary nodule, or as multiple nodules (classic Plummers disease), with the mass of hyperfunctioning tissue and the related secretion of thyroid hormones determining whether the patient is euthyroid or hyperthyroid. Important diagnostic tests include a 99mTc thyroid scan, T4 RIA, T3 uptake, FTI, TSH RIA, and occasionally T3 RIA (T3 thyrotoxicosis). Solitary autonomous nodules in adult patients characteristically progress slowly over many years, with toxicity rarely developing in nodules less than 2.5 cm in diameter and occurring primarily in nodules 3 cm or larger and in older patients. The decision to treat a solitary nodule depends upon the size and degree of function of the nodule and the patients age. Surgery and radioactive iodine are effective therapies. Hyperfunctioning thyroid nodules in children and adolescents (under age 18) have a more rapidly progressive course than those in adults and should be treated by thyroid lobectomy at the time of diagnosis. Subtotal thyroidectomy is the preferred treatment for most patients with toxic multinodular goiter, because it achieves prompt control of the hyperthyroidism and removes the goiter. Radioiodine therapy and long-term antithyroid drug therapy are alternative forms of treatment for patients who are poor surgical risks or who develop recurrent hyperthyroidism following thyroid surgery.

Stimulation of inositol phosphate formation in FRTL-5 rat thyroid cells by catecholamines and its relationship to changes in 45Ca2+ efflux and cyclic AMP accumulation

Catecholamines specifically stimulated the rapid formation of inositol phosphates, bisphosphates and trisphosphates in a concentration-dependent manner in FRTL-5 thyroid cells. Further analysis by high performance liquid chromatography revealed the presence of two isomers of inositol trisphosphate, 1,4,5- and 1,3,4-trisphosphate, suggesting that the 1,4,5-trisphosphate of inositol is further metabolized to the 1,3,4-trisphosphate isomer. The alpha 1-adrenoreceptor antagonist, prazosin, inhibited the effects of epinephrine, while the alpha 2-adrenoreceptor antagonist, yohimbine, was without effect. Treatment of FRTL-5 cells with pertussis toxin (to inhibit Ni) did not abolish the epinephrine effect on inositol trisphosphate formation. Carbachol, N6-[L-2-phenylisopropyl]-adenosine and forskolin were without effect on phosphoinositide metabolism. Both epinephrine and the calcium ionophore A23187 stimulated 45Ca2+ efflux from 45Ca2+-loaded FRTL-5 cells. The time-course of the epinephrine effect indicates that inositol 1,4,5-trisphosphate formation (t1/2 approximately 1 s) precedes both the efflux of 45Ca2+ (t1/2 approximately 30 s) as well as the reduction of cyclic AMP levels (t1/2 approximately 90 s) in response to epinephrine. These results strongly suggest that inositol 1,4,5-trisphosphate has the appropriate properties to act as a second messenger by which alpha 1-adrenergic hormones, through mobilization of intracellular Ca2+ and activation of cyclic AMP phosphodiesterase, reduce cyclic AMP levels in FRTL-5 cells.

Regeneration of splenic remnants after partial splenectomy in rats

Splenic regeneration in the rat was measured after removal of 25, 50, or 75% of the spleen, 50% of the spleen with autotransplantation of the excised portion, and splenectomy with autotransplantation of 50% of the spleen. Splenic growth in rats undergoing sham splenectomies served as a control. Splenic mass at 6 weeks and 4 months after surgery was directly related to the remnant size. “Normalized” spleen weights (measured as grams of splenic tissue per 100 grams of rat weight) after 25, 50, and 75% splenectomy were 57, 41, and 38% of controls at 6 weeks, and 77, 71, and 44% of controls at 4 months. All differences were significant at P < 0.03 except those between 50 and 75% splenectomy at 6 weeks, and between 25 and 50% splenectomy at 4 months. A comparison of autotransplanted splenic mass after total splenectomy with that after 50% splenectomy (0.042 ± 0.005 and 0.025 ± 0.004, respectively, at 6 weeks) demonstrated that an intact subtotal spleen inhibited significantly regeneration of the autotransplanted spleen. The effect of autotransplanted splenic tissue on regeneration of a splenic remnant was little to none at 4 months.

Partial splenectomy and overwhelming infection in rats.

Susceptibility to overwhelming sepsis in rats was measured by intravenous Streptococcus pneumoniae challenge 5 weeks after removal of 25, 50, or 75% of the spleen, with sham splenectomy and total splenectomy groups included for comparison. The LD50 (given in organisms per animal) for total splenectomy was 1.02 × 103, for 75% splenectomy, 1.79 × 104, for 50% splenectomy, 4.69 × 104, for 25% splenectomy, 4.90 × 105, and for sham splenectomy, 8.04 × 106. All differences were significant at P < 0.05 except for that between 50 and 75% splenectomy. Thus sham, 25, 50, and 75% splenectomy were all associated with a higher LD50 than total splenectomy, and the LD50 increased in proportion to the size of the splenic remnant. No threshold for protection against overwhelming infection was noted, but a rapid fall in LD50 from 75% to total splenectomy may be indicative of a critical splenic mass within that range.

Surgery of the thyroid.

The sophistication of current surgical treatment of thyroid disorders reflects a better understanding of the pathophysiology. The development of more methods of therapy with antithyroid drugs, beta-adrenergic blocking agents, thyroid hormones, and radioactive isotopes as well as external irradiation has had an important effect on the role that surgery plays in the management of diseases of the thyroid. Iodine, which was use for many disorders, is now probably contraindicated except for preparation of the hyperthyroid patient for surgical treatment. Surgery has now taken its place as one form of therapy for an endocrine organ that is subject to a variety of diseases. It is necessary for the surgeon who undertakes operations on the thyroid to have an understanding of thyroid physiology as well as its pathophysiology.

Covalent crosslinking of thyrotropin to thyroid plasma membrane receptors: subunit composition of the thyrotropin receptor

The subunit composition of the thyrotropin (TSH) receptor has been characterized using the bifunctional crosslinking agent, disuccinimidyl suberate (DSS), to covalently link [125I]TSH to its receptor. Purified thyroid membranes were labeled with [125I]TSH, and the hormone-receptor complex was crosslinked by incubation with 0.1 mM DSS. Analysis of this crosslinked complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions indicated the presence of a specifically labeled hormone-receptor complex, corresponding to a Mr of 68,000 +/- 3000 before correction for the relative molecular mass of TSH. When reducing agents were absent during SDS solubilization, the mobility of the band increased slightly, suggesting the presence of intramolecular disulfide bonds. The labeling of the 68,000 band was specifically inhibited by TSH, but not by other glycoprotein hormones. Specific labeling occurred only in thyroid, and not in liver or muscle plasma membranes. Protease-free immunoglobulin G, isolated from sera of patients with Graves disease and capable of competing with TSH for binding to its receptor, inhibited the labeling of the 68,000 complex. When the hormone-receptor complex was crosslinked with higher concentrations of DSS (greater than 0.3 mM), a second specifically labeled band was observed, with a Mr of 80,000 +/- 5000. This complex exhibited hormone, tissue, and immunologic specificities similar to those of the 68,000 band. Continuous sucrose density gradient analysis indicated that the intact solubilized receptor possessed a sedimentation coefficient of 10.5 S prior to correction for detergent binding. However, this value increased to 16 S when determined under conditions which took into account the change in hydrodynamic properties attributable to bound Triton X-100. These data suggest that the 80,000 and 68,000 bands represent binding components of the TSH receptor and that the receptor molecule most likely contains multiple subunits, linked by noncovalent forces.

Apparent "negative cooperativity" kinetics in the absence of a nonlinear Scatchard plot of thyrotropin-receptor interaction in a human thyroid adenoma.

A human thyroid adenoma (benign nodule) was identified which exhibited a linear Scatchard plot of 125I-TSH binding, characteristic of a single class of binding site with high affinity (Kd = 0.5±0.1 nM) and low binding capacity (0.8±0.2 pmol/mg protein). In contrast, Scatchard analysis of binding to adjacent normal thyroid was nonlinear, suggesting the presence of high and low-affinity binding sites with Kds of 0.4±0.2 and of 27.9±11.0 nM and capacities of 0.7±0.3 and 1.8±1.0 pmol/mg protein, respectively. Dissociation of bound 125I-TSH from membranes of both adenoma and normal tissue revealed identical enhancement of dissociation in the presence of excess native hormone, thought to be evidence for the “negative cooperativity” model of hormone-receptor interaction. Furthermore, adenylate cyclase from both tissues was equally responsive to TSH. Thus, a thyroid adenoma which contains TSH-responsive adenylate cyclase still exhibited enhanced dissociation by native hormone, even though Scatchard analysis yielded a single, non-cooperative class of binding sites. This suggests that enhanced dissociation of bound hormone does not provide a demonstration of negatively-cooperative site-site interaction. Furthermore, nonlinear Scatchard plots, typical of TSH binding in normal thyroid, represent two classes of binding sites, of which the high affinity type is responsible for stimulation of adenylate cyclase.

α1-adrenergic regulation of TSH-stimulated cyclic AMP accumulation in rat thyroid cells

Abstract Addition of epinephrine to cultured FRTL-5 rat thyroid cells led to a concentration-dependent reduction of TSH- and forskolin-stimulated cAMP accumulation. Clonidine, which preferentially activates the α 2 -adrenoreceptor, had no effect on cAMP levels. The reduction of cAMP levels by epinephrine was selectively blocked by prazosin, an α 1 -adrenoreceptor antagonist, but not by yohimbine, an α 2 -adrenoreceptor antagonist. Pretreatment of FRTL-5 cells with pertussis toxin failed to abolish the inhibitory effect of epinephrine on cAMP accumulation. The bioactivity of the pertussis toxin preparation in this cell line was verified by its ability to ADP-ribosylate the α-subunit of the inhibitory guanine nucleotide regulatory protein, Ni, as well as its ability to abolish the inhibitory effect of N 6 -[ l -2-phenylisopropyl]-adenosine on TSH-stimulated cAMP formation. The inhibitory effect of epinephrine on cAMP levels was dependent on Ca 2+ and was reversed by 3-isobutyl-1-methylxanthine. Taken together, these results suggest that epinephrine reduces cAMP levels via α 1 -adrenoreceptors. The failure of pertussis toxin to abolish this α-adrenergic effect is consistent with the conclusion that epinephrine-induced attenuation of cAMP accumulation occurs through activation of a Ca 2+ -calmodulin-sensitive phosphodiesterase and does not involve Ni or Ni-like proteins.

Further studies on the covalent crosslinking of thyrotropin to its receptor: Evidence that both the α and β subunits of thyrotropin are crosslinked to the receptor

Abstract Highly purified α and β-subunits of thyrotropin were individually radioiodinated and, subsequently, recombined with their unlabeled complementary subunits. This procedure resulted in the formation of [ 125 I]thyrotropin(TSH) hybrid molecules which were labeled on only one hormone subunit. Characterization of the binding properties of these two hybrid molecules demonstrated that both yielded nonlinear Scatchard plots with K d and B max values similar to those obtained with radioiodinated native TSH and that both were capable of interaction with the high- and low-affinity binding components of the TSH receptor. The recombined [ 125 I]TSH molecules were then crosslinked to the TSH receptor using disuccinimidyl suberate. Following electrophoresis and autoradiography, two labeled TSH-receptor complexes with M r of 68,000 and 80,000 were observed. These two complexes exhibited hormone specificity and electrophoretic mobility identical to those previously observed using native [ 125 I]TSH. Crosslinking with increasing concentrations of disuccinimidyl suberate suggested that the formation of the 68,000 and 80,000 complexes was sequential with the 68,000 appearing before the 80,000. Furthermore, the two bands were labeled regardless of which TSH subunit of the hybrid TSH was radioiodinated. These data strongly suggest that the 68,000 and 80,000 TSH-receptor complexes are the result of crosslinking to the TSH α-β dimer and not to one subunit in the case of the 68,000 complex and to the TSH α-β dimer in the case of the 80,000 complex, as had been hypothesized previously.

Resolution of radioiodinated thyrotropin into receptor active and inactive components by column chromatography

Following radioiodination by the lactoperoxidase method and subsequent purification on Sephadex G100, it was found that [125I]TSH exhibited varying degrees of binding activities to the thyrotropin receptor. In order to further purify the radiolabeled hormone, the [125I]TSH preparation was chromatographed on Sepharose 6B. Two peaks of radioactive material (Peaks I and II) were recovered, containing approx. 60% of the applied radioactivity. Upon elution with Mg2+, the remainder of the radiolabeled material was recovered as a single peak (Peak III). Characterization of these 3 peaks by radioimmunoassay demonstrated that all 3 were immunocompetent, although Peaks I and III were 3-4-fold more immunoreactive than Peak II. Analysis by radioreceptor assay indicated that Peak III showed an increase in receptor-binding capacity (in comparison with the [125I]TSH preparation purified by Sephadex G100 alone), while both Peaks I and II exhibited significantly reduced binding activity. In contrast, human TSH (NIH) chromatographed mainly as a receptor inactive peak, although it was fully immunocompetent. Scatchard analysis of receptor binding to bovine [125I]TSH from Peak III yielded a curvilinear plot with affinities similar to those we have previously reported for [125I]TSH purified by Sephadex G100 chromatography. The total number of binding sites, however, increased proportionally with the active fraction of the [125I]TSH preparation. Since the mass of bound hormone is calculated from the percent bound of total radioactivity and only a fraction of the measured total participates in the binding, it is therefore necessary to correct for the inactive fraction when calculating the total receptor number.