Objective: 3-iodothyronamine (T1AM) derives from thyroid hormone through decarboxylation anddeiodination, and binds to plasma membrane receptors known as trace amine-associated receptors. Since we have previously observed that T1AM produces a negative inotropic effect in rat heart (IC50close to 20 microM), in this work we determined the effect of T1AM on calcium homeostasis. Methods: Isolated adult rat cardiomyocytes were used to evaluate the calcium transient by the fluo-3technique and to record L-type calcium current, by whole cell patch clamp. In additional experiments sarcoplasmic reticulum (SR) vesicles were obtained from isolated rat hearts perfused in the absence orin the presence of T1AM. After vesicle loading with 45Ca, the rate constant of calcium release was determined by quick filtration technique. Ryanodine binding was also assayed, since ryanodine is aspecific ligand of the SR calcium channel, and interventions affecting channel function usually modify ryanodine binding. Results: 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), while sarcolemmal calcium current density was unchanged. In SR experiments the release rate measured in the presence of 10 microM unlabeled calcium was not significantly modified after perfusion with T1AM. However perfusion with T1AM determined a significant increase in 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 decreased after perfusion with T1AM, due to reduced Bmax (265±19 vs 381±19 fmol/mg of protein, P<0.05) with unchanged Kd. Conclusions: We conclude that T1AM affects calcium homeostasis and suggest that its negative inotropic action is due to a diminished pool of SR calcium as a result of increased diastolic leak.
Effect of 3-iodothyronamine on myocardial calcium homeostasis
GHELARDONI, SANDRA;CHIELLINI, GRAZIA;ZUCCHI, RICCARDO
2009-01-01
Abstract
Objective: 3-iodothyronamine (T1AM) derives from thyroid hormone through decarboxylation anddeiodination, and binds to plasma membrane receptors known as trace amine-associated receptors. Since we have previously observed that T1AM produces a negative inotropic effect in rat heart (IC50close to 20 microM), in this work we determined the effect of T1AM on calcium homeostasis. Methods: Isolated adult rat cardiomyocytes were used to evaluate the calcium transient by the fluo-3technique and to record L-type calcium current, by whole cell patch clamp. In additional experiments sarcoplasmic reticulum (SR) vesicles were obtained from isolated rat hearts perfused in the absence orin the presence of T1AM. After vesicle loading with 45Ca, the rate constant of calcium release was determined by quick filtration technique. Ryanodine binding was also assayed, since ryanodine is aspecific ligand of the SR calcium channel, and interventions affecting channel function usually modify ryanodine binding. Results: 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), while sarcolemmal calcium current density was unchanged. In SR experiments the release rate measured in the presence of 10 microM unlabeled calcium was not significantly modified after perfusion with T1AM. However perfusion with T1AM determined a significant increase in 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 decreased after perfusion with T1AM, due to reduced Bmax (265±19 vs 381±19 fmol/mg of protein, P<0.05) with unchanged Kd. Conclusions: We conclude that T1AM affects calcium homeostasis and suggest that its negative inotropic action is due to a diminished pool of SR calcium as a result of increased diastolic leak.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
