The gene expression of thyroid hormone receptors (TR) in ECRF24 immortalized human umbilical vein endothelial cells (HUVECs) was investigated at both the mRNA and the protein level. Endothelin-1 (ET-1) and von Willebrand factor (vWF) production were measured in response to triiodothyronine (T(3)) administration. A real-time PCR technique was used to quantify the presence of mRNAs encoding for the different isoforms of the TR. The binding of T(3) to nuclear TRs was studied in isolated endothelial cell nuclei by Scatchard analysis. Expression of TR at the protein level was investigated by immunocytochemistry and Western blotting using TR-isoform-specific polyclonal rabbit antisera. ET-1 and vWF were measured in cell supernatants with a two-site immunoenzymatic assay. Scatchard analysis yielded a maximum binding capacity of 55 fmol T(3)/mg DNA (+/-200 sites/cell) with a K(d) of 125 pmol/l. Messenger RNAs encoding for the TRalpha1 and the TRalpha2 and the TRbeta1 were observed. The approximate number of mRNA molecules per cell was at least 50 molecules per cell for TRalpha1, five for TRalpha2 and two for TRbeta1. Immunocytochemistry revealed (peri)nuclear staining for TRbeta1, TRalpha1 and TRalpha2. ET-1 and vWF secretion did not increase upon addition of T(3) (10(-10)-10(-6) M). Immortalized ECRF24 HUVECs express TR, but at low levels. The number of TRs per endothelial cell is probably too low to be functional and no change in ET-1 or vWF production was found after addition of T(3). Therefore we conclude that the genomic effects of T(3) are unlikely to occur in these immortalized HUVECs.

Many metabolic processes occur simultaneously in the liver in different locations along the porto-central axis of the liver units. These processes are often regulated by hormones, one of which is thyroid hormone which for its action depends on the presence of the different isoforms of the thyroid hormone receptor (TR). These are encoded by two genes: c-erbA-alpha encoding TRalpha1 and TRalpha2 and their respective Delta isoforms, and c-erbA-beta which encodes TRbeta1, TRbeta2 and TRbeta3. We recently found a zonal (pericentral) expression of and a diurnal variation in the TRbeta1 isoform in rat liver. We were therefore also interested to see whether TRalpha1 and TRalpha2 expression showed similar characteristics. For this reason we raised both polyclonal and monoclonal antibodies against TRalpha1 and TRalpha2 isoforms and characterised these. Antibody specificity was tested using Western blots and immunohistochemistry in liver of TR isoform-specific knockout animals. Using these antibodies we found that the TRalpha1 and TRalpha2 isoforms are zonally expressed around the central vein in rat liver. The experiments show that the portal to central gradient of TRalpha1 is broader than that of TRbeta1. Moreover, the expression of the TRalpha2 protein showed a diurnal variation with a peak in the afternoon when the animals are least active whereas no such variation was found for the TRalpha1 protein.From our data it appears that both the TRalpha1 and TRalpha2 isoforms show a zonal distribution in liver. This finding, together with the observed diurnal rhythm, has major implications for interpreting and timing experiments concerning the TR and its downstream actions in liver.

Nuclear thyroid hormone (T₃) receptors (TR) play a critical role in mediating the effects of T₃ on development, differentiation and normal physiology of many organs. The heart is a major target organ of T₃, and recent studies in knockout mice demonstrated distinct effects of the different TR isoforms on cardiac function, but the specific actions of TR isoforms and their specific localization in the heart remain unclear. We therefore studied the expression of TRα1, TRα2 and TRβ1 isoforms in the mouse heart at different stages of development, using monoclonal antibodies against TRα1, TRα2 and TRβ1. In order to identify distinct components of the embryonic heart, in situ hybridization for cardiac-specific markers was used with the expression pattern of sarcoplasmic reticulum calcium-ATPase 2a as a marker of myocardial structures, while the pattern of expression of connexin40 was used to indicate the developing chamber myocardium and peripheral ventricular conduction system. Here we show that in the ventricles of the adult heart the TRβ1 isoform is confined to the cells that form the peripheral ventricular conduction system. TRα1, on the other hand, is present in working myocardium as well as in the peripheral ventricular conduction system. In the atria and in the proximal conduction system (sinoatrial node, atrio-ventricular node), TRα1 and TRβ1 isoforms are co-expressed. We also found the heterogeneous expression of the TRα1, TRα2 and TRβ1 isoforms in the developing mouse heart, which, in the case of the TRβ1 isoform, gradually revealed a dynamic expression pattern. It was present in all cardiomyocytes at the early stages of cardiogenesis, but from embryonic day 11.5 and into adulthood, TRβ1 demonstrated a gradual confinement to the peripheral ventricular conduction system (PVCS), suggesting a specific role of this isoform in the formation of PVCS. Detailed knowledge of the distribution of TRα1 and TRβ1 in the heart is of importance for understanding not only their mechanism of action in the heart but also the design and (clinical) use of TR isoform-specific agonists and antagonists.