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- Drvota, Viktor
(författare)
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Molecular pharmacology of amiodarone
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amiodarone (Am) is a potent class III antiarrhythmic drug. Its use is limited due to serious side effects and it was therefore used only when other therapeutic interventions proved to be ineffective. The CAST study in 1989, showing an increased mortality in patients treated with Class I drugs, changed this scenario and resulted in an increased use of amiodarone. The mechanism underlying the electrophysiological effects of amiodarone remains uncertain. An intriguing possibility is that Am, as indicated by earlier studies, exerts its action through antagonism of thyroid hormone (T3). The actions of T3 are mediated through nuclear receptors, the thyroid hormone receptors (ThR), which regulate gene expression essential for the cellular integrity and the electrical stability of the cardiomyocyte. In addition, the interaction between the thyroid hormone- and the B-adrenergic signaling pathways is well known and there is a possibility that the antiadrenergic effect of Am is mediated through the T3 system. AIMS OF THE STUDY: 1) To characterize the interaction between Am and ThR; to determine whether other antiarrhytbmic drugs have any specific affinity for the ThR; 2) To establish a suitable cell culture model for studies of the molecular effects of Am on B-adrenergic receptors (B-Ar); 3) To investigate the effects of Am and of cathecholamine stress on ThR levels; 4) To characterize the antiadrenergic mechanism of Am; and 5) To investigate if the prolongation of the repolarisation induced by desethylamiodarone (DEA) is dependent of gene expression and if levels of ThR subtypes are affected. DEA is the major metabolite of Am with similar pharmacodynamic profile. CONCLUSIONS: 1) Am inhibits T3 binding to human ThR B. Am acts as a non-competitive inhibitor at low concentrations (< 2 uLM) and as a competitive inhibitor at higher concentrations (2-8 uLM). Disopyramide, lignocaine, propafenone, metoprolol, dI-sotalol and verapamil do not bind to human ThR; 2) The AT-1 cardiomyocytes were found to be a suitable cell model. These cells express functional B adrenergic receptors as measured with radioligand binding and cAMP measurements Spontaneous cytosolic Ca2+ transients present in the AT-1 cardiomyocytes could be modulated by sympathomimetic drugs; 3) Thyroid hormone receptor mRNA levels in AT-1 cardiomyocytes are modulated by both Am and isoproterenol. This observation demonstrates that Am exerts its effect on cardiomyocytes at least in part via the ThRs Together with previous observations of T3 action on B-Ar levels, these findings suggest a bi-directional regulation between the thyroid hormone and the B-adrenergic systems; 4) In AT-1 cardiomyocytes Am downregulates cell surface B-Ar and cAMP generation without affecting the affinity for the radioactive ligand to the receptor. The downregulation of B-Ar was reversed by T3. The protein synthesis inhibitor cycloheximide blocked the Am induced down regulation of B-Ar. Western blot analysis revealed a decreased expression of B-Ar protein in Am treated cells as compared to control. The house-keeping protein a-tubulin remained unchanged. These findings indicate a new antiadrenergic mechanism mediated through gene expression; and 5) In Langendorff perfused guinea pig hearts, the prolongation of cardiac repolarisation induced by DEA could be totally blocked by the protein synthesis inhibitor cycloheximide. The action of DEA could be reversed by T3 indicating an antagonism between DEA and T3. In DEA treated hearts as compared to control hearts, Western blot analysis revealed an increased expression of ThR a, protein and a decreased expression of ThR B, protein. The house-keeping protein c-tubulin remained unchanged. These results suggest a new electrophysiologic mechanism dependent on gene expression, probably by modulation of ThR, rather than a direct effect on cell membrane receptors or channels.
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| 2. |
- Shahrara, Shiva, et al.
(författare)
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Characterization of AT-1 Cardiomyocytes as a Model for Studies of T3 Effects on Cardiac Cells
- 1997
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 237:2, s. 303-306
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Tidskriftsartikel (refereegranskat)abstract
- AT-1 cardiomyocyte derive from atrial tumors in transgenic mice. Earlier studies have indicated a highly differentiated, contracting, cardiac phenotype in primary cultures and the AT-1 cardiomyocyte may thus be an excellent in vitro model in cardiac research. Thyroid hormone (T3) has positive inotropic and chronotropic effects and is clinically known to be relevant in various pathological heart conditions. Thyroid Hormone Receptors (TR) are ligand regulated transcriptional activators who mediate the effects of T3. The aim of this study was to determine whether AT-1 cardiomyocytes express TR. Regular binding competition assays showed a Kd of 370 +/- 105 for 125I-T3 binding to TR. Reverse transcription-PCR in mouse showed that TRalpha1, alpha2, beta1 and beta2 mRNA were expressed in AT-1 cardiomyocytes and mouse myocardium. Western blot with polyclonal rabbit antibodies against human TR revealed the presence of TRalpha1, beta2 and low levels of TRbeta1 while TRalpha2 was not detectable. Generally, for the detected subtypes the intensities of the bands were weaker for AT-1 cardiomyocytes in comparison to mouse heart. We conclude that the AT-1 cardiomyocytes express both protein and mRNA for TR and may provide a useful model for studying T3 effects in cultured cardiac myocytes.
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