Activation and inactivation of thyroxine by cultured rat hepatoma cells

The metabolism of thyroxine, 3,3′,5-triiodothyronine and 3,3′,5′-triiodothyronine was investigated in rat hepatoma cell cultures (R117-21B). These iodothyronines were labeled with ¹²⁵I in the phenolic ring and the metabolites were analyzed by ion-exchange column chromatography.When thyroxine was incubated with the cells at 37°C, its glucuronide was the major product and a little increase in ¹²⁵I^? was detected. Although 3,3′,5-triiodothyronine was not observed in the incubation medium, this metabolite was clearly identified in the ethanol extract obtained from the cell homogenates after 24 h incubation.This cell line also metabolized labeled 3,3′,5-triiodothyronine added to culture medium. After 24 h incubation, 3,3′,5-triiodothyronine glucuronide was the major metabolite and iodothyronine sulfates were also formed. The sulfates contained, 3,3′,5-triiodothyronine and 3,3′-diiodothyronine sulfates and an unknown component.In the metabolism of 3,3′,5′-triiodothyronine, the cells were very active in carrying out glucuronidation and phenolic ring deiodination, and this metabolism yielded 3,3′,5′-triiodothyronine and 3,3′-diiodothyronine glucuronides. The iodide fraction contained a small amount of 3,3′-diiodothyronine sulfate.These results show that the R117-21B rat hepatoma cells metabolize the thyroid hormones and their analogs by phenolic and nonphenolic ring deiodinations, by glucuronidation and by sulfation.     

Uptake and metabolism of thyroid hormones by cultured monkey hepatocarconoma cells Effects of potassium cyanide and dinitrophenol

Cultured monkey hepatocarcinoma cells (NCLP-6E) were used to investigate the uptake and metabolism of thyroid hormones. Intracellular accumulation was shown by the failure to acutely release hormone from cells subsequently exposed to serum proteins, and by the metabolic transformation of the hormones to deiodinated products and their sulfates. When hepatocarconoma cell monolayers were studied at hormone concentrations below 10^?10 M, neither KCN nor dinitrophenol inhibited uptake. Taken together with previous findings that uptake was neither saturable nor reduced at low temperature, these results indicate that this process was not active transport. Deiodination of both the phenolic and non-phenolic rings, however, was partially inhibited by KCN but not by dinitrophenol. Sulfation of 3,3′-diiodothyronine and 3′-monoiodothyronine was strongly inhibited by both KCN and dinitrophenol.Uptake of the hormones and their metabolites was also measured in suspended hepatocarcinoma cells and compared with the uptake by normal rat hepatocytes, human fibroblasts and human lymphocytes. In these experiments 1 μM triiodothyronine and 0.47 mM dinitrophenol were used to inhibit deiodination and sulfation, respectively. Uptake was similar in all cell types. Accumulation was highest with 3,5,3′-triiodothyronine, intermediate with other compounds having iodines in both rings, lowest with compounds iodinated in only one ring, and absent with iodothronine sulfates. These findings help to explain the relative rates of metabolism of the iodothyronines and their release from the cells.     

Evidence for a single enzyme in rat liver catalysing the deiodination of the tyrosyl and the phenolic ring of iodothyronines.

The enzymic 5'-deiodination of 3',5'-di-iodothyronine and 5-deiodination of 3,3',5-tri-iodothyronine by rat liver microsomal fractions were found to be characterized by apparent Km values of 0.77 and 17.4 microM respectively, 3',5'-Di-iodothyronine was a competitive inhibitor of 3,3',5-tri-iodothyronine 5-deiodination (Ki 0.65 microM) and 3,3',5-tri-iodothyronine was a competitive inhibitor of 3',5'-di-iodothyronine 5'-deiodination (Ki 19.6 microM). In addition, several radiographic contrast agents and iodothyronine analogues inhibited both reactions competitively and with equal potencies (r = 0.999). These results strongly suggest the existence of a single hepatic deiodinase acting on both the tyrosyl and phenolic ring of iodothyronines.     

Phenolic and nonphenolic ring deiodinations of iodothyronines in cultured hepatocarcinoma cell hemogenate from monkey

Iodothyronine monodeiodinase activities in homogenates of cultured monkey hepatocarcinoma cells were measured by the deiodination of [3,5-¹²⁵I]triido-l-thyronine or 3-[3′5′-¹²⁵I]triido-l-thyronine (phenolic ring-labeled ‘reverse’ triiodothyronine). The assay system utilized a small ion-exchange column (AG50W-X4, 0.9×～1 cm) to measure ¹²⁵I^?. Both deiodinases were destroyed by boiling for 1 min.Maximal nonphenolic ring deiodination was observed at pH 7.9 whereas maximal phenolic ring deiodination was at pH 6.3. Both reactions were enhanced strongly by dithiothreitol (0.1–5 mM), and slightly by 5 mM β-mercaptoethanol. Phenolic ring deiodination was strongly inhibited by 0.1 mM propylthiouracil. Nonphenolic ring deiodination was accelerated by EDTA (1.2 mM) and inhibited by Mg²⁺ (5 mM). Methylmercaptoimidazol and Mg²⁺, Ca²⁺ and Mn²⁺ (0.1–1.0 mM) had little or no effect on either reaction, but Zn²⁺ (0.1 mM) strongly inhibited both.Both reactions were inhibited by excess iodothyronine analogues at 10 mM to 10μM, and thyroxine was shown to be a competitive inhibitor in both cases. On the basis of relative affinities and inhibitory effects, it appears that the order of affinity for the phenolic ring deiodinase is 3,3′,5′-triiodo-l-thyronine-(rT₃) > l-thyroxine(T₄) > 3,4,3′-triido-l-thyronine(T₃), whereas for the nonphenolic ring deiodinase the order is T₃ > T₄ > rT₃. Diiodotyrosine did not affect their deiodination.     

Integrin participates in the effect of thyroxine on plasma membrane in immature rat testis

Background

The secretory activity of Sertoli cells (SC) is dependent on ion channel functions and protein synthesis and is critical to ongoing spermatogenesis. The aim of this study was to investigate the mechanism of action associated with a non-metabolizable amino acid [¹⁴C]-MeAIB (α-(methyl-amino)isobutyric acid) accumulation stimulated by T₄ and the role of the integrin receptor in this event, and also to clarify whether the T₄ effect on MeAIB accumulation and on Ca²⁺ influx culminates in cell secretion.

Methods

We have studied the rapid and plasma membrane initiated effects of T₄ by using ⁴⁵Ca²⁺ uptake and [⁴⁵C]-MeAIB accumulation assays, respectively. Thymidine incorporation into DNA was used to monitor nuclear activity and quinacrine to analyze the secretory activity on SC.

Results

The stimulation of MeAIB accumulation by T₄ appears to be mediated by the integrin receptor in the plasma membrane since tetrac and RGD peptide were able to nullify the effect of this hormone. In addition, T₄ increases extracellular Ca²⁺ uptake and Ca²⁺ from intracellular stocks to enhance nuclear activity, but this genomic action seems not to influence SC secretion mediated by T₄. Also, the cytoskeleton and ClC-3 chloride channel contribute to the membrane-associated responses of SC.

Conclusions

T₄ integrin receptor activation ultimately determines the plasma membrane responses on amino acid transport in SC, but it is not involved in calcium influx, cell secretion or the nuclear effect of the hormone.

General significance

The integrin receptor activation by T₄ may take a role in plasma membrane processes involved in the male reproductive system.     

Studies on the inhibition of glycogenolysis by D-galactosamine in rat liver hepatocytes.

Effect of galactosamine on glycogenolysis was studied in isolated hepatocytes. It was found that addition of galactosamine strongly inhibited glycogenolysis in normal hepatocytes. Galactosamine-inhibited glycogenolysis was not stimulated by epinephrine or glucagon. This inhibition was specific as no such inhibition was observed with galactose, 2-deoxy-glucose or glucosamine. The glucagon-stimulated cyclic AMP formation in galactosamine-treated hepatocytes was the same as in normal cells; Glc-1-P and Glc-6-P did not accumulate nor was lactate formation enhanced. The glucose production by hepatocytes from regenerating liver was only slightly inhibited by galactosamine and glucagon addition stimulated glycogenolysis in the presence of the amino sugar.     

The Effect of Mycorrhizal Fungi on the Fate of PCBs in Two Vegetated Systems

The objective of this study was to investigate the effects of mycorrhizal infection on vegetative uptake of polychlorinated biphenyls. Feltleaf willow (Salix alaxensis) and balsam poplar (Populus balsamifera) were grown in soil spiked with 6 mg/kg 3,3′,4,4′-tetrachlorobiphenyl-(UL-¹⁴C). The fungicide Daconil₂₇₈₇® was employed to suppress indigenous mycorrhizal infection. After 100 days of greenhouse incubation in semienclosed phytoreactors, mycorrhizal infection was found to be approximately threefold higher in the untreated willows vs. the fungicide-amended willows. Radio-label uptake was found to correlate most highly with mycorrhizal infection in the willow roots (R = 0.83). Over the same time period, mycorrhizal infection in the poplars was not significantly affected by fungicide addition. In the poplar phytoreactors, radiolabel uptake was most highly correlated with water use (R = 0.70). The overall vegetative radiolabel uptake was low (≈ 1%), but the limited uptake was attributed to soil sorption processes rather than vegetative limitations.     

Flavonoids of Gardenia cramerii and G. fosbergii bud exudates

From the bud exudates of Gardenia cramerii and G. fosbergii, two species endemic to Sri Lanka, a new flavonoid with an unusual B-ring oxidation pattern, 5,5′-dihydroxy-6,7,2′,3′-tetramethoxyflavone, was characterized. Two other rare flavonoids, 5,3′,5′-trihydroxy-3,6,7,4′-tetramethoxyflavone and 5-hydroxy-6,7,3′,4′,5′-pentamethoxyflavone were also isolated from both Gardenia species.     

Additive and/or synergistic action (downregulation) of androgens and thyroid hormones on the cellular distribution and localization of a true tissue kallikrein, mK1, in the mouse submandibular gland.

We investigated the effects of 5alpha-dihydrotestosterone (DHT), 3,5,3'-triiodo-l-thyronine (T(3)), and dexamethasone (Dex) on the expression of mK1 in the granular convoluted tubule (GCT) cells of the submandibular gland (SMG) of hypophysectomized (Hypox) male mice by indirect enzyme-labeled antibody and immunogold antibody methods for light and electron microscopy. Hypox resulted in considerable atrophy of the GCT cells, which were always immunoreactive for mK1, and the cells were characterized by apical small dense secretory granules labeled with gold particles suggesting the presence of mK1, small Golgi apparatus, sparse rough endoplasmic reticulum (RER), and developed basal infoldings. Each of the hormones, DHT, T(3), and Dex, enhanced the GCT phenotype to various degrees in Hypox male mice. Both DHT alone and T(3) alone moderately inhibited mK1 synthesis by increasing the number of mK1-immunonegative GCT cells in Hypox males, but Dex alone had no inhibitory effect on mK1 synthesis. A significant trophic effect on GCT cells was induced by combined injection of DHT and T(3) or of all three hormones, and was reflected in the appearance of abundant large secretory granules, well-developed Golgi apparatus and RER, and reduced basal infoldings. Only a few such GCT cells were immunopositive for mK1, and the pattern of immunopositive and immunonegative cells very closely resembled the mosaic pattern seen in normal male GCTs. These findings suggested that the sexual dimorphism of mK1 expression and the morphological appearance of GCT cells can be induced by treatment with two hormones, DHT and T(3), but not by either of them alone. T(3) appears to have a permissive effect on committed GCT cells that results in downregulation of mK1 expression in these cells.     

Subcellular location of adenylate cyclase in rat cardiac muscle

Crude homogenates of rat cardiac muscle were fractionated in order to examine the subcellular location of adenylate cyclase in this tissue. The fractionation procedure employed differential centrifugation of homonized material, followed by collagenase treatment, centrifugation on a discontinuous sucrose density gradient and extraction with 1 M KCl. The particulate fraction obtained by this procedure contained a high specific activity and yield of adenylate cyclase, moderate levels of mitochondria and low levels of sarcoplasmic reticulum and contractile protein as judged by marker enzyme activities. Adenylate cyclase was purified 20-fold with a 33% yield from the crude homogenate, while mitochondrial, sarcoplasmic reticulum and contractile protein yields were 5, 0.4 and 0.7% respectively. The membrane fractions prepared in this manner were examined by sodium dodecyl sulfate · gel electrophoresis.Adenylate cyclase copurified with ouabain-sensitive (Na⁺ + K⁺)-ATPase, a plasma membrane marker enzyme, and not with Ca²⁺-accumulating activity, which is associated with the sarcoplasmic reticulum. The distribution of marker enzyme activities indicates that heart adenylate cyclase is not located in the sarcoplasmic reticulum but is localized predominantly, if not exclusively, in the plasma membrane.     

Paradoxical stimulation by 1-methyl-3-isobutylxanthine of rat liver cyclic AMP phosphodiesterase activity

DNA newly synthesized in UV irradiated Escherichia coli B/r Hcr⁺ was 2 min pulse-labeled at various periods, then denatured and analysed by sucrose gradient centrifugation either in neutral or in alkaline conditions. Data indicate that in DNA of damaged cells alkali-labile sites are produced. In cells saturated with inducible proteins production of alkali-labile sites disappears in ～1 h. In the absence of inducible proteins production of alkali-labile sites continues.