Karen R. Hitchcock

Immunohistochemical localization of thyroid hormone in rat thyroid gland.

Direct and indirect immunofluorescence techniques were used to localize the thyroid hormones triidothyronine (T3) and thyroxine (T4) in adult rat thyroid gland. Optimum dilutions of the antisera were established and four tissue fixatives were investigated for usefulness in this technique. Use of antibodies specific for either T3 or T4 resulted in brilliant fluorescence in the colloid pools and apical cytoplasm of follicular cells. In all cases, the adjacent parathyroid gland was devoid of fluorescence. This report demonstrates that these dipeptide hormones can be localized by using immunofluorescence techniques.

Chapter 5 Maintenance of Human and Rat Pulmonary Type II Cells in an Organotypic Culture System

Publisher Summary This chapter discusses the maintenance of human and rat pulmonary type II cells in an organotypic culture system. The lungs of mammals and some inframammalian vertebrates contain a potent surface-active material that coats the alveolar surface, decreases the surface tension, and thus stabilizes the alveoli against collapse. The presence of this pulmonary surfactant is essential for normal pulmonary function, and its absence or diminution is an important etiological factor in respiratory distress syndrome. The mammalian pulmonary alveolus is lined with an endodermally derived epithelium consisting of two cell types: type I and type II pulmonary epithelial cells. The human fetal lung at 18–20 weeks of gestation contains epithelial tubules instead of pulmonary alveoli. The tubules are comprised of glycogen-rich epithelial cells containing very few lamellar bodies. The phospholipid composition of surfactant isolated from organotypic cultures is compared to that isolated from adult rat lung. Fatty acid analysis of surfactant phosphatidylcholine from organotypic cultures demonstrates that myristate, palmitate, and stearate are the major fatty acids present.

Thyroxine metabolism in cultured cells derived from fetal rat lung.

Summary: The role of thyroxine (T4) in fetal lung development has been demonstrated previously. Because triiodothyronine (T3), not T4, is thought to be the active hormone, and because T3 nuclear receptors appear to be present in fetal lung, the following studies were performed to examine peripheral metabolism of [125I]T4 by organotypic and mixed monolayer cultures of fetal lung cells. The cells were derived from fetal rat lungs obtained at 16 and 19 days of gestation.Short term (2 h) incubations of homogenized organotypic cultures and mixed monolayer cultures with [125I]T4 showed minimal amounts of [125]T3 generation expressed as % added T4 per mg protein (0.43 ± 0.32 and 0.56 ± 0.28, respectively). In addition, there appeared to be “excess iodide” (2.21) and substantial generation of rT3 (i.e., 2.67 ± 1.1 and 1.87 ± 1.0, respectively).In a subsequent group of experiments, deiodination of [125I]T4 was allowed to proceed by incubating intact organotypic cultures with [125IT4 for longer periods (36 h). Subsequent assessment and comparison of deiodination products in the subcellular fractions showed that the highest % of [125I]T3 (relative to total thyronines) was present in the fraction enriched with nuclei (7.7 ± 2.3). This was significantly different from the [125I]T3 in homogenized whole cells (4.54 ± 1.5), and markedly different from the medium which contained the lowest % of [125I]T3 (0.18 ± 0.2). Further confirmation was obtained to show that this high concentration of [125I]T3 in the nuclear fraction had in fact been generated de novo and did not merely result from redistribution of labeled hormones among the cellular compartments. Incubation of organotypic cultures with two different isotopically-labeled hormones ([131I]T3 and [125I]T4) permitted calculation of nuclear to medium ratios for [131I]T3 and [125I]T3; the ratio of these provided an index of intracellular conversion of [125I]T4 to [125I]T3. The mean of five such experiments demonstrated that of the total [125I]T3 attached to the nuclear fraction at the end of the incubation, 32% was newly generated and 68% was derived by influx from the medium. These results indicate that organotypic cultures derived from day 19 fetal rat lung are capable of deiodinating T4 and that both rT3 and T3 are generated. The generated T3 appears to be retained by the lung cells and is bound to the nuclear fraction. On the other hand, rT3 is distributed more evenly throughout the cell and in the medium and presumably is not bound.

387 Thyronine Metabolism in Fetal Pulmonary type II Cells

The importance of thyroid hormone in fetal lung development and surfactant production is well known. Yet, triiodothyronine (T3), the active hormone, is present in low concentrations in fetal rat serum. We therefore studied metabolism of 125I T4 in surfactant-producing type II cells maintained in organotypic culture (Douglas, W.H.J. et al. In Vitro 12:373-381, 1976). The type II cells were obtained from fetal rat lungs at 16 and 19 days of gestation (term, 22-23 days). Cultures were incubated for 36 hrs. in medium enriched with 125I T4 at 37°C. Products of 125I T4 metabolism in medium, cell homogenate and a subcellular fraction enriched with nuclear material were assayed by chromatography. Confirmation by radioautography was performed. The distribution of radioactive compounds expressed as % of 125I T4 added to 108 day 19 cells and corrected for spontaneous degradation, follows:The results obtained from day 16 cells were similar. This suggests that fetal type II cells can deiodinate T4. The high percentage of 125I T3 present in the cells suggests intracellular T3 generation. There is a total net gain of 125Iodide in the system. This excess iodide formation suggests pathways of thyronine metabolism which could result in formation and degradation of r2T3T SuchTphenolic ring deiodination may terminate in 3,3′-T2 3′-T or T0.