3-Iodothyronamine (T1AM) is a naturally occurring derivative of thyroid hormone that can potentially activate the orphan G protein-coupled receptor (GPCR), known as trace amine-associated receptor 1 (TAAR1). Significant functional effects have been observed after administration of exogenous T1AM: in the isolated heart, a negative inotropic and chronotropic action was produced, and the resistance to ischemic injury was increased, possibly as a consequence of an action on intracellular calcium homeostasis. In the present study we investigated the uptake and catabolism of exogenous T1AM in cardiac preparations using liquid chromatography tandem mass spectrometry (HPLC-ESI-MS-MS) analysis. Isolated working rat hearts were perfused with T1AM (50 nM) and analysis was performed in the recirculating buffer and in cardiac homogenate. Similar experiments were performed in isolated cardiomyoblats (H9c2 cells). In the latter model both incubation medium and cell lysate were collected at different times and submitted to analysis. The analytical method included reverse phase HPLC coupled to tandem mass spectrometry (ESI-MS-MS), and enabled contemporary detection of T1AM, 3-iodothyroacetic acid (TA1), thyronamine (T0AM) and thyroacetic acid (TA0). Experiments were repeated in the presence of 0.1 mM iproniazid, an inhibitor of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO). In both models T1AM concentration in the perfusion buffer decreased exponentially over time (the half life was on the order of 20 min in isolated heart perfused with 200 ml of recirculating buffer). T1AM could be detected in cardiac homogenate and in cell lysate and total cellular or tissue concentration was approximately 20 fold higher than extracellular concentration, showing that significant uptake and/or protein binding occurred. We also detected TA1, a product of T1AM oxidative deamination, which significantly accumulated in cell lysate and cardiac homogenate. Pretreatment of H9c2 cells or isolated hearts with iproniazid abolished T1AM conversion to TA1. Deiodinated derivates (i.e. T0AM and TA0) were not detected in any model. We concluded that T1AM is taken up by cardiomyocytes and can be catabolized to TA1 through iproniazid-sensitive amine oxidases. HPLC-ESI-MS-MS proved to be an effective and quantitative technique to elucidate T1AM metabolism.
3-Iodothyronamine cardiac metabolism: new insights resulting from liquid chromatography tandem mass spectrometry analysis
CHIELLINI, GRAZIA;GHELARDONI, SANDRA;SABA, ALESSANDRO;ZUCCHI, RICCARDO
2010-01-01
Abstract
3-Iodothyronamine (T1AM) is a naturally occurring derivative of thyroid hormone that can potentially activate the orphan G protein-coupled receptor (GPCR), known as trace amine-associated receptor 1 (TAAR1). Significant functional effects have been observed after administration of exogenous T1AM: in the isolated heart, a negative inotropic and chronotropic action was produced, and the resistance to ischemic injury was increased, possibly as a consequence of an action on intracellular calcium homeostasis. In the present study we investigated the uptake and catabolism of exogenous T1AM in cardiac preparations using liquid chromatography tandem mass spectrometry (HPLC-ESI-MS-MS) analysis. Isolated working rat hearts were perfused with T1AM (50 nM) and analysis was performed in the recirculating buffer and in cardiac homogenate. Similar experiments were performed in isolated cardiomyoblats (H9c2 cells). In the latter model both incubation medium and cell lysate were collected at different times and submitted to analysis. The analytical method included reverse phase HPLC coupled to tandem mass spectrometry (ESI-MS-MS), and enabled contemporary detection of T1AM, 3-iodothyroacetic acid (TA1), thyronamine (T0AM) and thyroacetic acid (TA0). Experiments were repeated in the presence of 0.1 mM iproniazid, an inhibitor of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO). In both models T1AM concentration in the perfusion buffer decreased exponentially over time (the half life was on the order of 20 min in isolated heart perfused with 200 ml of recirculating buffer). T1AM could be detected in cardiac homogenate and in cell lysate and total cellular or tissue concentration was approximately 20 fold higher than extracellular concentration, showing that significant uptake and/or protein binding occurred. We also detected TA1, a product of T1AM oxidative deamination, which significantly accumulated in cell lysate and cardiac homogenate. Pretreatment of H9c2 cells or isolated hearts with iproniazid abolished T1AM conversion to TA1. Deiodinated derivates (i.e. T0AM and TA0) were not detected in any model. We concluded that T1AM is taken up by cardiomyocytes and can be catabolized to TA1 through iproniazid-sensitive amine oxidases. HPLC-ESI-MS-MS proved to be an effective and quantitative technique to elucidate T1AM metabolism.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
