Biomedical subjects
Publications and source records attributed to D A Ontjes.
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We have solubilized adenylate cyclase in a relatively stable form from rat adrenal membranes. The solubilized enzyme elutes on a column of Sepharose 4BR as a distinct peak with a higher molecular weight than the soluble fractions which bind 125I-ACTH. Both the soluble and membrane bound enzymes are activated by NaF and Gpp(NH)p, and both have similar affinities for MgATP. While the membrane bound enzyme is activated similarly by either Mg2+ or Mn2+, the soluble enzyme is more fully activated by Mn2+. Pretreatment of adrenal membranes with NaF or Gpp(NH)p before the addition of detergent enhances recovery of soluble enzyme activity, while recovery of activity in the unsolubilized membrane pellet is unchanged. In contrast, addition of ACTH prevents solubilization of the enzyme and greatly increases its recovery in the pellet. This observation is consistent with the theory that action of the hormone on a receptor subunit leads to an association between the receptor and a catalytic subunit. Such an association might make it more difficult to remove the enzyme from the surrounding lipid matrix of the membrane.
The complete amino acid sequence of rat thyrocalcitonin has been determined by automated Edman degradations of the intact molecule, a cyanogen bromide fragment, and by degradations of mixtures of peptides produced by hydrolysis of the hormone with trypsin and chymotrypsin. The sequence determined was H2N-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Leu-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ser-Ile-Gly-Val-Gly-Ala-Pro-NH2. This sequence differs in only two positions from that found in the human hormone, i.e. leucine-16 in the rat vs phenylalanine-16 in the human, and serine-26 in the rat vs alanine-26 in the human. These similarities and differences are consistent with the previously reported immunological properties of the hormones isolated from these two species.
The clinical characteristics, methods of diagnosis, and treatment of MEN-II have been described. Attention in the future should be focused on diagnosing MCT at the earliest possible time in MEN-II family members. The development of chemotherapeutic or other antitumor agents for the treatment of metastitic MCT should prove a fertile field for investigation.
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We have compared the effects of oral and intravenous ethanol on the secretion of both thyrocalcitonin and gastrin in five patients with medullary carcinoma of the thyroid. Ethanol caused a moderate rise in plasma thyrocalcitonin to 316% +/- 343% of baseline when given intravenously and to 197% +/- 106% of baseline when given orally. Only oral ethanol caused a measurable rise in serum gastrin levels. Serum calcium did not change significantly from baseline during either oral or intravenous administration. The results suggest that stimulation of thyrocalcitonin secretion by ethanol is not secondary to increased secretion of gastrin nor to the induction of hypercalcemia. Neither oral nor intravenous ethanol appears to be as effective as intravenous pentagastrin in testing for the presence of medullary carcinoma.
Pentagastrin is a potent stimulator of thyrocalcitonin secretion from "C" cells. Since medulllary carcinoma of the thyroid gland (MCT) is a tumor composed of "C" cells, pentagastrin was used to screen a large kindred with multiple endocrine neoplasia type II (MCT, pheochromocytoma (s), and parathyroid hyperplasia). Four children with no thyroid abnormalities evident on physical examination, with negative thyroid scans, and with normal levels of plasma thyrocalcitonin both before and after calcium infusion, were found to have elevated peripheral levels of this hormone following pentagastrin injection. All four children were found to have very small foci of MCT, in both thyroid lobes at the time of total thyroidectomy. Pentagastrin stimulation used conjointly with selective catheterization of the inferior thyroid vein provided even greater diagnostic accuracy in detecting elevations in thyrocalcitonin secretion. This test has great diagnostic utility, especially in screening patients with multiple endocrine neoplasia type II.
Thyrocalcitonin (TCT) is useful as a diagnostic and therapeutic agent in selected human diseases. Elevated plasma levels of TCT occur in patients with medullary carcinoma of the thyroid gland. Asymptomatic relatives of these patients harboring microscopic foci of tumor may have abnormal plasma levels of TCT in the basal state or after provocative stimuli. In both instances the plasma levels of TCT can be used in the diagnosis and management of this thyroid neoplasm. Elevated plasma levels of TCT have also been described recently in subjects with certain extrathyroidal neoplasms or renal failure. Moderate elevations are seen during normal pregnancy and in the neonatal period. Although exogenous TCT has actions on several organs, including bone, the kidneys and the gastrointestinal tract, its physiological role in man, if any, is still unknown. The recently reported measurements of TCT in normal subjects should facilitate the clarification of this issue. TCT has been used as a therapeutic agent in Paget's disease of bone, hypercalcemia of diverse etiologies, osteopenia and several other skeletal disorders. The dramatic improvement of patients with Paget's disease has been a unique therapeutic action of TCT. The therapeutic responses in hypercalcemic subjects given TCT are encouraging but more information is needed about the pharmacology of the hormones in these subjects before conclusions can be formed. At present the therapeutic role of TCT in osteopenia is hypothetical and the results of ongoing and future studies are needed to determine its effects.
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.
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