Thyroxine (T(4)) is the predominant form of thyroid hormone (TH). Hyperthyroidism, a condition associated with excess TH, is characterized by increases in metabolic rate, core body temperature and cardiac performance. In target tissues, T(4) is enzymatically deiodinated to 3,5,3'-triiodothyronine (T(3)), a high-affinity ligand for the nuclear TH receptors TR alpha and TR beta, whose activation controls normal vertebrate development and physiology. T(3)-modulated transcription of target genes via activation of TR alpha and TR beta is a slow process, the effects of which manifest over hours and days. Although rapidly occurring effects of TH have been documented, the molecules that mediate these non-genomic effects remain obscure. Here we report the discovery of 3-iodothyronamine (T(1)AM), a naturally occurring derivative of TH that in vitro is a potent agonist of the G protein-coupled trace amine receptor TAR1. Administering T(1)AM in vivo induces profound hypothermia and bradycardia within minutes. T(1)AM treatment also rapidly reduces cardiac output in an ex vivo working heart preparation. These results suggest the existence of a new signaling pathway, stimulation of which leads to rapid physiological and behavioral consequences that are opposite those associated with excess TH.

3-Iodothyronamine T1AM is a novel endogenous thyroid hormone derivative that activates the G protein-coupled receptor known as trace anime-associated receptor 1 (TAAR1). In the isolated working rat heart and in rat cardiomyocytes, T1AM produced a reversible, dose-dependent negative inotropic effect (e.g., 27+/-5, 51+/-3, and 65+/-2% decrease in cardiac output at 19, 25, and 38 microM concentration, respectively). An independent negative chronotropic effect was also observed. The hemodynamic effects of T1AM were remarkably increased in the presence of the tyrosine kinase inhibitor genistein, whereas they were attenuated in the presence of the tyrosine phosphatase inhibitor vanadate. No effect was produced by inhibitors of protein kinase A, protein kinase C, calcium-calmodulin kinase II, phosphatidylinositol-3-kinase, or MAP kinases. Tissue cAMP levels were unchanged. In rat ventricular tissue, Western blot experiments with antiphosphotyrosine antibodies showed reduced phosphorylation of microsomal and cytosolic proteins after perfusion with synthetic T1AM; reverse transcriptase-polymerase chain reaction experiments revealed the presence of transcripts for at least 5 TAAR subtypes; specific and saturable binding of [125I]T1AM was observed, with a dissociation constant in the low micromolar range (5 microM); and endogenous T1AM was detectable by tandem mass spectrometry. In conclusion, our findings provide evidence for the existence of a novel aminergic system modulating cardiac function.

Receptors for growth hormone secretagogues have been identified in cardiac tissue, but their functional role is unknown. We have investigated the effect of different growth hormone secretagogues on contractile performance and on the susceptibility to ischemic injury, in isolated working rat hearts. In particular, we tested the endogenous secretagogue ghrelin and the synthetic secretagogues hexarelin and MK-0677. Under aerobic conditions, none of these substances produced any significant hemodynamic effects. In hearts subjected to 30 minutes of ischemia followed by 120 minutes of reperfusion, the synthetic peptidyl secretagogue hexarelin (1 microM) significantly reduced infarct size, as estimated on the basis of triphenyltetrazolium chloride staining, while the non-peptidyl secretagogue MK-0677 was ineffective. The endogenous peptidyl secretagogue ghrelin (20 nM) was also protective, while desacylated ghrelin, which is devoid of biological effects, did not modify ischemic injury. The protection provided by hexarelin was partly abolished by the protein kinase C inhibitor chelerythrine. We conclude that ghrelin and hexarelin have a specific cardioprotective effect, which is independent of growth hormone secretion, and might be related to protein kinase C activation.

We investigated the effect of ischemia and reperfusion on the cardiac ryanodine receptor, which corresponds to the sarcoplasmic reticulum Ca2+ channel. Isolated working rat hearts were subjected to 10 to 30 minutes of global ischemia, followed or not by reperfusion. Ischemia produced significant reduction in the density of high-affinity 3H-ryanodine binding sites, determined either in whole-heart homogenate (Bmax, 220 +/- 22, 203 +/- 12, and 228 +/- 14 fmol/mg protein after 10, 20, and 30 minutes of ischemia versus 298 +/- 18 fmol/mg protein in the control condition; P < .01) or in a fraction enriched in sarcoplasmic reticulum (Bmax, 1.08 +/- 0.15 pmol/mg protein after 20 minutes of ischemia versus 1.69 +/- 0.08 pmol/mg protein in the control condition; P < .01). The Kd (1.5 +/- 0.1 nmol/L) and the Ca2+ dependence of high-affinity 3H-ryanodine binding were not affected by ischemia. The density of low-affinity 3H-ryanodine binding sites was also reduced after 20 minutes of ischemia (14.0 +/- 2.3 versus 34.0 +/- 8.2 pmol/mg protein in the sarcoplasmic reticulum fraction, P < .05), without significant changes in Kd (4.7 +/- 1.2 versus 2.4 +/- 1.0 mumol/L). All these changes persisted after 20 minutes of reperfusion. Analysis of tissue fractions showed that 55% of the ryanodine binding sites were retained in the pellet of a low-speed centrifugation ("nuclear pellet") and that the effects of ischemia concerned only the receptors released in the supernatant ("postnuclear supernatant"). In parallel experiments, we evaluated the effect of ryanodine on oxalate-supported Ca2+ uptake, which represents sarcoplasmic reticulum Ca2+ uptake. As expected, we found that high concentrations of ryanodine stimulated Ca2+ uptake, owing to channel blockade. The response to 900 mumol/L ryanodine was slightly reduced in crude homogenate and significantly reduced in postnuclear supernatant obtained from ischemic hearts. In conclusion, the number of ryanodine receptors is reduced after ischemia; this effect concerns a subpopulation of the receptors, persists after reperfusion, and might contribute to modify sarcoplasmic reticulum function.

Ghrelin, the natural ligand of the GH secretagogue (GHS) receptor, was originally isolated from the stomach and detected in several tissues, but a systematic study of its tissue distribution has not been performed. In the present investigation, we evaluated ghrelin gene expression (by RT-PCR technique) and ghrelin protein concentration (by enzyme immunoassay technique) in tissues obtained from control rats as well as in rats subjected to 48-h fasting. The ghrelin gene was expressed in stomach, small intestine, brain, cerebellum, pituitary, heart, pancreas, salivary gland, adrenal, ovary and testis, with maximum expression occurring in the stomach, while no significant expression was detected by standard RT-PCR in liver, lung, kidney and skeletal muscle. Ghrelin protein was detected in stomach, small intestine, brain, cerebellum, pituitary, lung, skeletal muscle pancreas, salivary gland, adrenal, ovary and testis, at concentrations ranging from 0.05 to 1.43 ng/mg of homogenate protein (the highest concentration occurred in the lung, followed by the brain). Ghrelin was not detectable in the heart, liver and kidney. Therefore, gene and protein expression were dissociated. Fasting did not produce significant changes in ghrelin gene expression, while the distribution of ghrelin between different tissues was significantly modified: protein concentration increased in the brain, cerebellum, lung and salivary gland, while it decreased in the stomach.