Somatostatin-like immunoreactivity in transplantable medullary carcinoma of rat thyroid: partial chromatographic and biological characterization

Somatostatin-like immunoreactivity (SRIF-LI) has previously been demonstrated immunohistochemically in rat thyroid parafollicular cells. Studies were therefore performed to determine whether SRIF-LI was present in a transplantable medullary carcinoma of the thyroid (MCT) in the WAG/Rij strain of rat. SRIF-LI was found in MCT in significantly higher concentrations than in normal thyroid tissue. Thyroid and MCT SRIF-LI showed parallelism with the synthetic SRIF and RIA displacement curves and coeluted with synthetic SRIF on immunoaffinity chromatography. On gel filtration, thyroid SRIF-LI and the major peak of MCT SRIF-LI coeluted with synthetic SRIF. SRIF-LI of a larger molecular size was also present in the MCT. MCT and thyroid SRIF-LI coeluted with synthetic SRIF on high pressure liquid chromatography. MCT SRIF-LI purified by affinity chromatography was equipotent to synthetic SRIF in inhibiting dibutyryl cAMP-stimulated GH release by rat pituitary cells in monolayer culture. Serum SRIF-LI was elevated in tumor-bearing rats and showed characteristics similar to those of MCT SRIF-LI and synthetic SRIF on affinity and high pressure liquid chromatography (HPLC). Tumor-bearing rats showed diminished secretion of insulin after orally administered glucose and impaired secretion of GH in response to pentobarbital compared to normal control rats. The results indicate that SRIF-LI is produced in excessive quantities by a transplantable rat MCT and impairs the secretion of GH and insulin. The immunological, chromatographic, and biological properties of MCT SRIF-LI suggest that it is indistinguishable from synthetic SRIF.     

Somatostatin-like immunoreactivity in the retina.

A substance with somatostatin-like immunoreactivity (SLI) was found in extracts of goldfish, frog, and cow retina. Dilutions of retinal SLI parallel the standard curve for radioimmunoassay obtained with synthetic somatostatin. Chromatography of goldfish retinal extract on Sephadex G-50 revealed two peaks of SLI, one that coeluted with synthetic somatostatin and one that eluted as a larger molecule. Incubation in 8 M urea did not alter the chromatographic pattern of the extract. SLI was present in extracts of frog optic nerve and tectum in concentrations higher than those found in the retina. In goldfish retina, SLI was localized by immunofluorescence to four types of processes in the inner plexiform layer; their origins could be traced to three classes of SLI-containing cell bodies in the proximal row of the inner nuclear layer and one class in the ganglion cell layer. Localization of SLI to cells of the retina and characterizations of the molecular forms of retinal SLI suggest that the retina is a promising model system for studies on the potential neurotransmitter function of somatostatin.     

Somatostatin-like immunoreactivity in man. Measurement in peripheral plasma

A radioimmunoassay for the determination of somatostatin-like immunoreactivity (SLI) in plasma has been developed using antibodies raised in rabbits against synthetic somatostatin conjugated to hemocyanin and 125I-Tyr1-somatostatin. The detection limit was 2 pg/ml. A combination of EDTA and aprotinin (0.15 M and 80 Kallikrein Inhibitor Units = KIU/ml) inhibited the tracer degradation, but did not eliminate it completely. In addition somatostatin immunoreactivity in unextracted plasma showed a strong dilution effect and the estimates depended on the antiserum used (e.g. a plasma sample (1 : 5) assayed with antiserum R 141 apparently contained 150 pg/ml, whereas antiserum K 5615 yielded a value of 300 pg/ml). When plasma was extracted with acid ethanol, the values obtained with the two different antisera were practically the same, recovery of somatostatin added to plasma was approx. 80% and dilution of specimens gave proportional readings. Consequently, extraction of plasma is regarded as obligatory to ensure valid results. Mean basal level was 26.5 +/- 1.2 pg/ml (mean +/- SEM). When plasma extracts were subjected to chromatography on Sephadex G-25 at neutral pH, approx. 30% of the SLI was recovered in the MW1600 fraction; whereas the major part of the remaining immunoreactivity (37-60%) eluted earlier in one fraction and the residual immunoreactivity in the void volume.     

Digoxin-like immunoreactivity: occurrence in three molecular forms and partial characterization

Digoxin-like immunoreactivity (DLI) has been found in serum from subjects with a variety of physiological and pathological conditions, including uremia, liver disease, pregnancy, and the neonatal period. The physicochemical nature of this material is still not known. Using gel filtration chromatography, extraction with methylene chloride, and treatment with beta-glucuronidase, we determined that serum DLI exists in three forms: protein-bound, glucuronidated, and free, with respective mol wt of 5000, 400, and 230. In serum, DLI exists in the protein-bound and free forms, while in urine, DLI is found in the glucuronidated and free states.     

Solubilization, purification, and partial characterization of thyrotropin receptor from bovine and human thyroid glands

In an attempt to purify the thyroid receptor for TSH and to study interaction with the thyroid-stimulating antibody (TSAb) of graves' disease, we used both bovine and human thyroid glands. With either tissue, the 10,000 X g pellet of homogenized material was solubilized with 0.5% Triton N-101; excess Triton was removed by Amberlite XAD-2, and purification was effected by TSH affinity chromatography, followed by gel filtration on Sepharose 6B. Greatest purification was achieved with bovine tissue; this receptor preparation was 183-fold concentrated over the starting material, but contained only 6% of the original TSH-binding activity, due in part to spontaneous loss over the 4 days required for processing. On polyacrylamide gel electrophoresis, there were at least three protein bands, one of which was probably a subunit of thyroglobulin. Purified immunoglobulin G with thyroid-stimulating antibody activity inhibited the binding of TSH at all stages of purification of the receptor.     

Description and partial characterization of thyroid hormone-specific and thyroid hormone-dependent rat liver nuclear proteins

A fraction of readily elutable rat liver nuclear proteins (nuclear globulins), analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, contains two proteins which reflect the thyroid status of the animal. The larger (n-band) protein is abundant in normal rat nuclei but diminished in hypothyroidism by thyroidectomy or hypophysectomy and restored with T3 treatment. It is also diminished in starvation, during which time T3 treatment of concurrent hypothyroxinemia is ineffective in tis restoration. Refeeding restores the n-band protein in starved normal rats but not in starved thyroidectomized (Tx) rats. GH does not restore this protein in either Tx or hypophysectomized (Hx) rats. The dual requirements of euthyroidism and adequate nutrition imply that the n-band protein is thyroid hormone dependent but not thyroid hormone specific. The smaller of the two nuclear globulins, the t-band protein, is prominent in Tx or Hx rats and is not altered by additional hypometabolic factors (starvation) or by nonthyroid hormone related hypermetabolic stimuli (refeeding or GH treatment of Hx rats). It is reduced in normal or T3-treated Tx or Hx rats regardless of simultaneous hypometabolic states (starvation or nonthyroid hormone-related deficiencies of hypopituitarism) or hypermetabolic states (refeeding, liver regeneration, or hyperthyroidism). The t-band protein thus appears to be inversely thyroid hormone specific in its concentration. Electrophoretic and chromatographic analysis suggest that the n-band protein is a 125,000 mol wt subunit of a 290,000 mol wt holoprotein. The t-band protein is a 70,000 mol wt peptide which exists, in part, as a monomer but largely as a subunit of a protein complex with a mol wt greater than 100,000.     

Purification, characterization and radioimmunoassay of thyrocalcitonin from rat thyroid glands.

A highly purified preparation of rat thyrocalcitonin (TCT) has been obtained from lyophilized thyroid glands by gel chromatography following acid-acetone extraction. Biological activity of Sephadex G-50 eluates appeared in two peaks. The TCT in the major peak was concentrated, and applied to a Bio-Gel P-6 column, and a major protein peak was eluted which coincided with TCT activity. Potency, estimated by bioassay in rats, increase approximately 3500-fold from 0.075 MRC U/mg lyophilized glands to 250-400 MRC U/mg in the final product. The overall yield of TCT activity was about 36%. The purified product was characterized by chemical procedures and evaluated for its antigenic properties and use for radioimmunoassay. The purified rat TCT was used both labeled with 125I and as unlabeled standard. The following results were obtained: 1) Guinea pig antisera to either human or rat TCT were capable of binding 125I-rat TCT or 125I-human TCT; 2) Using either 125I-human or 125I-rat TCT and antisera to either TCT, pg amounts of rat and human TCT reacted in the assay while ng to mug amounts of salmon calcitonin or porcine TCT failed to react; 3) Using 125I-rat TCT and antisera to human or rat TCT, synthetic C-terminal (10-32 or 22-32) fragments of human TCT reacted well, while N-terminal (1-18) or desamide (1-32) derivatives reacted poorly or not at all; 4) Rat TCT was easily detected in normal thyroid venous plasma (5-10 ng/ml) and thyroid gland extracts (similar to 1 mug/gland) but not in peripheral blood; 5) Bioassay and radioimmunoassay of rat thyroid extracts (N equals 18) showed good agreement (r equals 0.86, p less than 0.001). The results support the idea that rat TCT is closely related to human TCT, indicate that major antigenic determinants reside in the C-terminal portion of the molecule, and show that antisera to either human or rat TCT can be used to measure rat TCT.     

Molecular cloning and partial characterization of a new autoimmune thyroid disease-related antigen

To clone and characterize antigens to autoantibodies in Hashimoto's thyroiditis we constructed a cDNA library in the expression vector lambda gt11 using mRNA prepared from Grave's thyroid tissue. This library was screened using serum from a patient with Hashimoto's thyroiditis which had an antimicrosomal antibody titer greater than 1:10(6). Five positive recombinants were identified and cloned. Of these, 3 reacted with 7 of 17 normal serum samples. The 2 other clones (IL-28 and IL-33) reacted with none of the 17 normal serum samples. IL-28 reacted with 4 of 15 and IL-33 with 2 of 15 Hashimoto's thyroiditis serum samples (antimicrosomal antibody titers, greater than 1:6400). The specificity of the interaction between the Hashimoto's thyroiditis samples and the fusion protein was demonstrated by Western blot analysis. In addition, neither 10(-6) M human thyroglobulin nor 100 mU/ml bovine TSH inhibited binding of the serum samples to these 2 clones. Lysate from clones IL-28 and IL-33 did not reduce the antimicrosomal antibody titer in a hemagglutination assay. Absorption of Hashimoto's thyroiditis serum with purified thyroid microsomes reduced the serum antimicrosomal antibody titer, but not binding to these 2 clones. The cDNA inserts of clones IL-28 and IL-33 were approximately 0.6 and 0.4 kilobases (kb), respectively. The 0.6-kb IL-28 insert was used to probe human thyroid and human liver poly(A)+ mRNA. A single band of 3.3 kb was evident only with the thyroid mRNA. The IL-28 insert was subcloned into M13 and sequenced in both directions by the dideoxy technique and found to be 572 basepairs in length. When tested against the GenBank and Dayhoff gene banks, no significant homology with any known sequence was determined. In summary, a cDNA fragment of a previously unrecognized gene coding for an autoimmune thyroid disease-related antigen has been cloned and partly characterized; and the protein produced by this clone is not thyroglobulin, the thyroid microsomal antigen, or the TSH-binding site of the TSH receptor. We have, therefore, identified a new autoimmune thyroid disease-related antigen, the pathogenetic significance of which remains to be determined.     

Somatostatin-like immunoreactivity in the pituitary and brain of three teleost fish species: somatostatin as a potential regulator of prolactin cell function

Somatostatin-like immunofluorescence occurs in the hypothalamus and neurohypophysis of three euryhaline teleosts: tilapia, killifish, and mudsucker. This immunofluorescence was eliminated by incubating the primary antibody with excess somatostatin or somatostatin-28 but not with urotensin II, a partial analogue of somatostatin. In all three fishes, the strongest reaction was seen in the proximal pars distalis and parts of the pars intermedia. Strongly fluorescing processes from cells of the preoptic nucleus extend toward the pituitary. Distinct fluorescence was also associated with the neurohypophysis penetrating into the rostral pars distalis in the tilapia but not in the killifish or mudsucker. In the tilapia, an extensive network of immunofluorescent fibers and small cells were present in the anterior dorsolateral telencephalon, in addition to a moderately fluorescing group of cells anterolateral to the preoptic nucleus. A small area of diffuse fluorescence was also seen in the anterior dorsolateral midbrain tegmentum. Previous physiological studies have implicated somatostatin as a regulator of prolactin cell activity in tilapia. The present study demonstrates the route by which somatostatin may be delivered to the rostral pars distalis to inhibit prolactin secretion.     

Somatostatin-like immunoreactivity in duck plasma, hypothalamus and neural lobe during post-hatch growth: comparison with plasma and pituitary growth hormone concentrations

Variations in the concentrations of plasma and pituitary GH were determined in ducks for 66 and 87 days after hatch, and compared with somatostatin-like immunoreactivity (SLI) in the plasma, hypothalamus and neural lobe. Plasma GH levels gradually decreased during growth, while pituitary GH content increased. The concentration of pituitary GH increased during the first 3 weeks of age and remained relatively constant thereafter. The decline in plasma GH concentration was paralleled by a similar fall in the level of plasma SLI. While the content of hypothalamic SLI increased during development, the SLI concentration was maximal at 14 days of age and lowest in adults. The content and concentration of SLI in the neural lobe, in contrast, increased progressively during development. Gel filtration of hypothalamic and neural lobe extracts demonstrated that both young and older birds had two main peaks of SLI, corresponding to somatostatin-14 and somatostatin-28, and a third, larger form. The elution pattern of plasma SLI was similar in young and older birds and was principally composed of a large molecular species ('big' somatostatin), although an additional small peak eluting between somatostatin-28 and somatostatin-14 was eluted from a large pool of plasma from 90-day-old ducks. These results suggest that increased plasma GH levels in young birds do not result from a hypothalamic somatostatin deficiency nor from variations in molecular forms of SLI, and that the age-related decline in plasma GH concentration is not due to a deficiency in pituitary GH content. The decline in the circulating GH level during growth is probably due to an increase in hypothalamic somatostatin release.     

Altered immunoreactivity of thyroglobulin in thyroid disease

We prepared 3 samples of 19S thyroglobulin (Tg), 1 from a patient with Graves' disease, another from a patient with nontoxic goiter, and the third from a pool of Tg from normal subjects, and used each Tg preparation to produce a polyvalent antiserum in rabbits. The 3 antisera were similar to each other in their reactivity with thyroid Tg samples from 25 patients with various thyroid disorders and from 10 normal subjects. However, the immunoreactivity of the 35 individual Tg samples varied considerably. Decreased reactivity was associated with proteolysis during Tg preparation, iodination in vitro with 20 or more atoms of iodine/molecule Tg, the 27S species of Tg, Tg from 3 patients with thyroid cancer, and Tg from several patients with Graves' disease. The antiserum to Graves' Tg contained some antibodies that did not bind normal Tg on an affinity column, and these antibodies reacted more with Tg from patients with Graves' disease than with Tg from normal subjects or patients with nontoxic goiters. Thus, Tg from patients with Graves' disease may contain antigenic sites that are not present or exposed in Tgs from other subjects. We conclude that thyroid Tgs from patients with Graves' disease and from those with thyroid cancer may be different in structure from the Tgs of normal subjects. This conclusion is important to an understanding of Tg structure in thyroid disease and to the use of thyroid Tg for preparation of antisera and standards for measuring serum Tg concentrations.     

Somatostatin-like immunoreactivity in the cerebrospinal fluid of aged patients with Parkinson's disease. The effect of dopatherapy.

Cerebrospinal fluid (CSF) somatostatin concentrations were measured in 35 aged patients with Parkinson's disease (mean age, 79.5 years) and 11 control subjects (mean age, 82.3 years). In patients with Parkinson's disease the levels of somatostatin-like immunoreactivity were lower than in controls (P less than .02); these values were lowest in the untreated group. Somatostatin-like immunoreactivity levels in the CSF tended to increase with treatment but not significantly (P = .11). Somatostatin values were not correlated to age, sex, or duration of the disease. Somatostatin concentrations tended to be lower in more severely affected patients with higher scores on the Hoehn and Yahr (P = .13) and Webster staging scales (P = .13) and lower scores on Mini-Mental State (P = .10), but without statistical significance for these correlations.     

Neuropeptide Y in bovine adrenal glands: distribution and characterization

Neuropeptide Y (NPY) distribution in bovine adrenal glands was studied by RIA and immunohistochemical technique. NPY content (picomoles per mg protein +/- SEM) of chromaffin cells, medulla, cortex, and whole glands was 4.2 +/- 0.16, 2.7 +/- 0.28, 0.19 +/- 0.02, and 0.94 +/- 0.14, respectively, while the chromaffin granule NPY content was 53. Immunohistochemically, NPY immunoreactivity was detected in norepinephrine containing chromaffin cells and also in nerve fibers crossing through adrenal cortex and medulla. NPY and enkephalin immunoreactivities were found in distinct chromaffin cells. Biochemical characterization by HPLC revealed two major NPY immunoreactive peptides. The most abundant molecular form was identified as authentic NPY.