3-iodothyronamine (T1AM) is a thyroid hormone derivative which binds to a new class of plasma membrane receptors known as trace amine-associated receptors. We have shown that in isolated rat hearts and in adult rat cardiomyocytes T1AM produces a negative inotropic effect, with IC50 on the order of 20 microM. This work was aimed at determining the effect of T1AM on calcium homeostasis. Isolated adult rat cardiomyocytes were used to evaluate the calcium transient by the flura-3 technique and to record the L-type calcium current by whole cell patch clamp technique. In additional experiments sarcoplasmic reticulum (SR) vesicles were obtained from hearts perfused in the absence or in the presence of T1AM. After vesicle loading with 45Ca, the rate constant of calcium release was determined through the quick filtration technique, using an authomatic apparatus with 10 msec resolution. The binding of ryanodine was also determined: ryanodine is a specific ligand of the SR calcium channel, and in general interventions affecting channel function modify ryanodine binding. In adult rat cardiomyocytes acute exposure to 20 microM T1AM decreased the amplitude of the calcium transient (0.06±0.01 vs 0.10±0.02 arbitrary units, P<0.05) and reduced its decay time (86±26 vs 176±40 msec, P<0.01). In patch clamped cardiomyocytes exposed to 20 microM T1AM sarcolemmal L-type calcium current density was unchanged while current facilitation by membrane depolarization was abolished: under control conditions, the current evoked from a holding potential of -45 mV was 30% greater than that evoked from a holding potential of -75 mV and T1AM completely abolished this phenomenon. Since facilitation is known to be abolished by depletion of the SR calcium pool, calcium release was evaluated in SR vesicles obtained from hearts perfused in the absence or in the presence of 20 microM T1AM. When calcium release was induced by exposure to 10 microM unlabeled calcium, no significant change was observed after perfusion with T1AM. However perfusion with T1AM determined a significant increase of the rate constant of calcium release measured under conditions promoting channel closure, i.e. exposure to 0.3 mM tetracaine and 1 mM EGTA (0.32±0.03 vs 0.13±0.04 sec-1, P<0.01). Ryanodine binding was decreased after perfusion with T1AM, due to reduced Bmax (265±19 vs 381±19 fmol/mg of protein, P<0.05) with unchanged Kd. We conclude that T1AM affects calcium homeostasis and suggest that its negative inotropic action is due to a diminished SR calcium pool as a result of increased diastolic leak.

Modulation of cardiac calcium homeostasis by 3-iodothyronamine

CHIELLINI, GRAZIA;GHELARDONI, SANDRA;ZUCCHI, RICCARDO
2009

Abstract

3-iodothyronamine (T1AM) is a thyroid hormone derivative which binds to a new class of plasma membrane receptors known as trace amine-associated receptors. We have shown that in isolated rat hearts and in adult rat cardiomyocytes T1AM produces a negative inotropic effect, with IC50 on the order of 20 microM. This work was aimed at determining the effect of T1AM on calcium homeostasis. Isolated adult rat cardiomyocytes were used to evaluate the calcium transient by the flura-3 technique and to record the L-type calcium current by whole cell patch clamp technique. In additional experiments sarcoplasmic reticulum (SR) vesicles were obtained from hearts perfused in the absence or in the presence of T1AM. After vesicle loading with 45Ca, the rate constant of calcium release was determined through the quick filtration technique, using an authomatic apparatus with 10 msec resolution. The binding of ryanodine was also determined: ryanodine is a specific ligand of the SR calcium channel, and in general interventions affecting channel function modify ryanodine binding. In adult rat cardiomyocytes acute exposure to 20 microM T1AM decreased the amplitude of the calcium transient (0.06±0.01 vs 0.10±0.02 arbitrary units, P<0.05) and reduced its decay time (86±26 vs 176±40 msec, P<0.01). In patch clamped cardiomyocytes exposed to 20 microM T1AM sarcolemmal L-type calcium current density was unchanged while current facilitation by membrane depolarization was abolished: under control conditions, the current evoked from a holding potential of -45 mV was 30% greater than that evoked from a holding potential of -75 mV and T1AM completely abolished this phenomenon. Since facilitation is known to be abolished by depletion of the SR calcium pool, calcium release was evaluated in SR vesicles obtained from hearts perfused in the absence or in the presence of 20 microM T1AM. When calcium release was induced by exposure to 10 microM unlabeled calcium, no significant change was observed after perfusion with T1AM. However perfusion with T1AM determined a significant increase of the rate constant of calcium release measured under conditions promoting channel closure, i.e. exposure to 0.3 mM tetracaine and 1 mM EGTA (0.32±0.03 vs 0.13±0.04 sec-1, P<0.01). Ryanodine binding was decreased after perfusion with T1AM, due to reduced Bmax (265±19 vs 381±19 fmol/mg of protein, P<0.05) with unchanged Kd. We conclude that T1AM affects calcium homeostasis and suggest that its negative inotropic action is due to a diminished SR calcium pool as a result of increased diastolic leak.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11568/134037
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