Onno Bakker

DESETHYLAMIODARONE IS A COMPETITIVE INHIBITOR OF THE BINDING OF THYROID HORMONE TO THE THYROID HORMONE ALPHA 1-RECEPTOR PROTEIN

Desethylamiodarone (DEA), the major metabolite of the potent antiarrythmic drug amiodarone, is a non-competitive inhibitor of the binding of thyroid hormone (T3) to the beta 1-thyroid hormone receptor (T3R). In the present study, we investigated whether DEA acts in a similar way with respect to the alpha 1-T3R. The chicken alpha 1-T3R, expressed in an E. coli system, was incubated in the presence or absence of DEA with [125I]T3 in buffer containing 0.05% Triton X-100, 0.05% BSA and 1% ethanol (v/v) in order to solubilise DEA. DEA, but not amiodarone, inhibited T3 binding in a dose-dependent manner; the IC50 value was 3.5 x 10(-5) M. Scatchard analyses in the presence of DEA demonstrated a dose-dependent decrease in Ka values, but no change in MBC. Lineweaver-Burk plots clearly indicated competitive inhibition by DEA. Pre-incubation of the alpha 1-receptor with DEA decreased maximal [125I]T3 binding, which was independent of the duration of pre-incubation. In conclusion, in contrast to the beta 1-T3R, where DEA acts as a non-competitive inhibitor, we now report as a new finding the competitive action of DEA to the alpha 1-T3R.

Thyrotropin-releasing hormone gene expression in the human hypothalamus

We studied the distribution of mRNA coding for thyrotropin-releasing hormone (TRH) in the human hypothalamus by means of in situ hybridization. In 10% formalin-fixed paraffin-embedded tissue sections of five hypothalami, TRH mRNA-containing cells were found in several nuclei and areas. Numerous TRH mRNA-containing cells were detected in the medial region of the caudal part of the paraventricular nucleus. These neurons were heavily labeled and mainly small to medium-sized. Few, lightly- and medium-labeled, small cells were detected in the suprachiasmatic nucleus. In addition, heavily labeled single cells were found in the perifornical area and the anterior- and lateral hypothalamic regions. In the latter region, occasional heavily labeled cells were found just dorsal to the supraoptic nucleus. Neither in the supraoptic nucleus nor in the sexually dimorphic nucleus of the preoptic area were TRH mRNA-containing cells found. This is the first description of TRH mRNA containing cells in the human hypothalamus.

Amiodarone‐induced hypercholesterolemia is associated with a decrease in liver LDL receptor mRNA

Amiodarone decreases plasma and tissue triiodothyronine (T3) and increases plasma cholesterol levels resembling changes seen during hypothyroidism. To elucidate the mechanism of amiodarone‐induced hypercholesterolemia we investigated gene expression of three key proteins in cholesterol metabolism (cholesterol 7α‐hydroxylase, LDL receptor, HMG‐CoA reductase) in livers of rats. Animals were treated with amiodarone or propylthiouracil (to induce mild hypothyroidism). The LDL receptor mRNA was downregulated (≈50%) in both amiodarone‐treated and hypothyroid animals, while the other mRNA remained unchanged after 14‐day treatment. The results suggest that amiodarone‐induced hypercholesterolemia is associated with decreased LDL receptor mRNA levels.

Specific detection of type III iodothyronine deiodinase protein in chicken cerebellar purkinje cells

Because iodothyronine deiodinases play a crucial role in the regulation of the available intracellular T3 concentration, it is important to determine their cellular localization. In brain, the presence of type III iodothyronine deiodinase (D3) seems to be important to maintain homeostasis of T3 levels. Until now, no cellular localization pattern of the D3 protein was reported in chicken brain. In this study polyclonal antisera were produced against specific peptides corresponding to the D3 amino acid sequence. Their use in immunocytochemistry led to the localization of D3 in the Purkinje cells of the chicken cerebellum. Both preimmune serum as well as the primary antiserum exhausted with the peptide itself were used as negative controls. Extracts of chick cerebellum and liver were made in the presence of Triton X-100 to solubilize the membrane-bound deiodinases. Using these extracts in Western blot analysis, a band of the expected molecular weight (∼30 kDa) could be detected in both tissues. Using a full-...

Amiodarone decreases gene expression of low-density lipoprotein receptor at both the mRNA and the protein level

Amiodarone, a potent antiarrhythmic drug, decreases plasma and tissue triiodothyronine (T3) and increases plasma cholesterol levels, resembling changes seen during hypothyroidism. The increase of serum cholesterol during amiodarone medication is associated with a decreased expression of the hepatic low-density lipoprotein (LDL) receptor mRNA. To further elucidate the mechanism of amiodarone-induced hypercholesterolemia, we investigated whether the decreased mRNA levels are the result of decreased transcription or increased degradation or both, and whether protein expression is decreased accordingly. Relative to pair-fed controls, amiodarone treatment increased plasma cholesterol by 69% and decreased expression of the mRNA encoding for the hepatic LDL receptor by 45%. To study this decrease in mRNA, we performed a run-on assay, from which it appears that amiodarone acts by decreasing LDL receptor mRNA expression 2.5-fold at the transcriptional level. The decay rate of liver LDL receptor mRNA, measured at different time points after injecting actinomycin D, was not different between amiodarone-treated and control animals (116+/-32 minutes and 84+/-10 minutes, P=.44). Hepatocytes in primary culture isolated from amiodarone-treated and control animals were used to determine specific binding of [125I]-LDL to hepatic LDL receptors. Amiodarone decreased specific LDL binding and Scatchard analysis demonstrated that amiodarone treatment reduced the number of LDL receptors by 69%, without affecting the dissociation constant (Kd). In conclusion, amiodarone-induced hypercholesterolemia can be explained by decreased transcription of the LDL receptor gene, resulting in lower mRNA and protein levels.

Effect of amiodarone and dronedarone administration in rats on thyroid hormone-dependent gene expression in different cardiac components

OBJECTIVE In view of their different actions on thyroid hormone receptor (TR) isoforms we set out to investigate whether amiodarone (AM) and dronedarone (Dron) have different and/or component-specific effects on cardiac gene expression. DESIGN Rats were treated with AM or Dron and the expression of TRalpha 1, TRalpha 2, TRbeta 1 and several tri-iodothyronine (T3)-regulated genes was studied in different parts of the heart, namely the right atrium (RA), left ventricular wall (LVW) and apex. METHODS Rats were treated for 14 days with 100 mg/kg body weight AM or Dron. The expression of TRalpha 1, TRalpha 2, TRbeta 1 and T3-regulated genes was studied using real-time PCR and non-radioactive in situ hybridisation. RESULTS AM and Dron affected TR expression in the RA similarly by decreasing TRalpha 1 and beta 1 expression by about 50%. In the LVW, AM and Dron decreased TRbeta 1 and, interestingly, AM increased TRalpha 1. In the apex, AM also increased TRalpha 2. The changes seen in T3-dependent gene expression are reminiscent of foetal reprogramming. CONCLUSION Taken together, our results indicate that AM and Dron have similar effects on the expression of TR isoforms in the RA, which could partly contribute to their ability to decrease heart rate. On the other hand, the more profound effect of AM appears on TR- and T3-dependent gene expression in the left ventricle suggests foetal reprogramming.

Desethylamiodarone interferes with the binding of co-activator GRIP-1 to the β1-thyroid hormone receptor

Ligand binding to the thyroid hormone nuclear receptor β1 (TRβ1) is inhibited by desethylamiodarone (DEA), the major metabolite of the widely used anti‐arrhythmic drug amiodarone. Gene expression of thyroid hormone (triiodothyronine, T3)‐regulated genes can therefore be affected by amiodarone due to less ligand binding to the receptor. Previous studies have indicated the possibility of still other explanations for the inhibitory effects of amiodarone on T3‐dependent gene expression, probably via interference with receptor/co‐activator and co‐repressor complex. The binding site of DEA is postulated to be on the outside surface of the receptor protein overlapping the regions where co‐activator and co‐repressor bind. Here we show the effect of a drug metabolite on the interaction of TRβ1 with the co‐activator GRIP‐1 (glucocorticoid receptor interacting protein‐1). The T3‐dependent binding of GRIP‐1 to the TRβ1 is disrupted by DEA. A DEA dose experiment showed that the drug metabolite acts like an antagonist under ‘normal’ conditions (at 10−7 M T3 and 5×10−6→10−3 M DEA), but as an agonist under extreme conditions (at 0 and 10−9 M T3 and >10−4 M DEA). To our knowledge, these results show for the first time that a metabolite of a drug which was not devised for this purpose can interfere with nuclear receptor/co‐activator interaction.

Hyper and hypothyroidism change the expression and diurnal variation of thyroid hormone receptor isoforms in rat liver without major changes in their zonal distribution.

We investigated the effect of hypothyroidism or hyperthyroidism on mRNA and protein expression, diurnal variation and zonal distribution of thyroid hormone receptor (TR) isoforms TRalpha1, TRalpha2 and TRbeta1 in rat liver. Hypothyroidism results in increased isoform mRNA and protein expression whereas hyperthyroidism shows a decreased TRalpha1 and TRalpha2 mRNA and protein expression. During hyperthyroidism no change is seen in TRbeta1 mRNA, but TRbeta1 protein is upregulated in the light period and downregulated in the dark period. Diurnal changes (measured at 13:30 and 19:30 h) in the TR isoform proteins are abolished in hypothyroidism and hyperthyroidism, with the exception of a reversal in diurnal changes of TRbeta1 in hyperthyroidism. Zonal distribution of the isoforms is not affected by hypo- or hyperthyroidism. We therefore conclude that thyroid hormone influences both the levels and the diurnal expression of its receptor isoforms but not the zonal distribution.

Effect of mutations in the β1‐thyroid hormone receptor on the inhibition of T3 binding by desethylamiodarone

Desethylamiodarone (DEA) acts as a competitive inhibitor of triiodothyronine (T3) binding to the α1‐thyroid hormone receptor (TRα1) but as a non‐competitive inhibitor with respect to TRβ1. To gain insight into the position of the binding site of desethylamiodarone on TRβ1 we investigated the naturally occurring mutants Y321C, R429Q, P453A, P453T and the artificial mutants L421R and E457A in the ligand binding domain of human TRβ1. The IC50 values (in μM) of DEA for P453A (50±11) and P453T (55±16) mutant TRβ1 are not different from that for the wild type TRβ1 (56±15), but the IC50 values of R429Q (32±7; P<0.001) and E457A (17±3; P<0.001) are significantly lower than of the wild type. Scatchard plots and Langmuir analyses indicate a non‐competitive nature of the inhibition by DEA of T3 binding to all four mutant TRβ1s tested. Mutants P453A and P453T do not influence overall electrostatic potential, and also do not influence the affinity for DEA compared to wild type. Mutant E457A causes a change from a negatively charged amino acid to a hydrophobic amino acid, enhancing the affinity for DEA. Mutant R429Q, located in helix 11, causes an electrostatic potential change from positive to uncharged, also resulting in greater affinity for DEA. We therefore postulate that amino acids R429 and E457 are at or close to the binding site for DEA, and that DEA does not bind in the T3 binding pocket itself, in line with the non‐competitive nature of the inhibition of T3 binding to TRβ1 by DEA.

Housekeeping Genes: A Gold Standard?

Gene expression studies that aim at precision require normalization to an internal control or “housekeeping” gene. The greatest challenge in choosing an appropriate housekeeping gene is maintaining expression consistency during treatment.