Simonetta Ronca-Testoni

3-Iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone

Thyroxine (T4) 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, T4 is enzymatically deiodinated to 3,5,3′-triiodothyronine (T3), a high-affinity ligand for the nuclear TH receptors TRα and TRβ, whose activation controls normal vertebrate development and physiology. T3-modulated transcription of target genes via activation of TRα and TRβ 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 (T1AM), a naturally occurring derivative of TH that in vitro is a potent agonist of the G protein–coupled trace amine receptor TAR1. Administering T1AM in vivo induces profound hypothermia and bradycardia within minutes. T1AM 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.

Cardiac effects of 3-iodothyronamine: a new aminergic system modulating cardiac function

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 μM 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 μM); 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.—Chiellini G., Frascarelli, S., Ghelardoni, S., Carnicelli, V., Tobias, S. C., DeBarber, A., Brogioni, S., Ronca‐Testoni, S., Cerbai, E., Grandy, D. K., Scanlan, T. S., Zucchi R. Cardiac effects of 3‐iodothyronamine: a new aminergic system modulating cardiac function. FASEB J. 21, 1597–1608 (2007)

Ghrelin tissue distribution: comparison between gene and protein expression

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.

Effect of ghrelin and synthetic growth hormone secretagogues in normal and ischemic rat heart

Abstract.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 µM) 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.

Modulation of sarcoplasmic reticulum function: a new strategy in cardioprotection?

This article reviews the experimental evidence suggesting that cytosolic Ca(2+) overload plays a major role in the development of myocardial injury during ischemia-reperfusion and that Ca(2+) release from the sarcoplasmic reticulum (SR) is of crucial importance in the early phase of ischemia. It is suggested that interventions able to deplete the SR Ca(2+) pool and/or to reduce the rate of SR Ca(2+) release should be cardioprotective. This thesis is supported by the review of experimental studies in which modulators of the SR Ca(2+)-ATPase or SR Ca(2+) release channel (ryanodine receptor) have been used. In addition, the role of the SR in ischemic preconditioning and in some instances of toxic myocardial injury (particularly, anthraquinone-induced injury) is discussed.

Postischemic Changes in Cardiac Sarcoplasmic Reticulum Ca2+ Channels : A Possible Mechanism of Ischemic Preconditioning

We investigated the modifications of cardiac ryanodine receptors/sarcoplasmic reticulum Ca2+ release channels occurring in ischemic preconditioning. In an isolated rat heart model, the injury produced by 30 minutes of global ischemia was reduced by preexposure to three 3-minute periods of global ischemia (preconditioning ischemia). The protection was still present 120 minutes after preconditioning ischemia but disappeared after 240 minutes. Three 1-minute periods of global ischemia did not provide any protection. In the crude homogenate obtained from ventricular myocardium, the density of [3H]ryanodine binding sites averaged 372 +/- 18 fmol/mg of protein in the control condition, decreased 5 minutes after preconditioning ischemia (290 +/- 15 fmol/mg, P < .01), was still significantly reduced after 120 minutes (298 +/- 17 fmol/mg, P < .05), and recovered after 240 minutes (341 +/- 21 fmol/mg). Three 1-minute periods of ischemia did not produce any change in ryanodine binding. The Kd for ryanodine (1.5 +/- 0.3 nmol/L) was unchanged in all cases. In parallel experiments, the crude homogenate or a microsomal fraction was passively loaded with 45Ca, and Ca(2+)-induced Ca2+ release was studied by the quick filtration technique. In both preparations, the rate constant of Ca(2+)-induced Ca2+ release decreased 5 and 120 minutes after preconditioning ischemia (homogenate values: 19.7 +/- 1.4 and 18.9 +/- 0.9 s-1 vs a control value of 25.4 +/- 1.7 s-1, P < .05 in both cases) and recovered after 240 minutes (23.0 +/- 1.9 s-1). The Ca2+ dependence of Ca(2+)-induced Ca2+ release was not affected by preconditioning ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac effects of trace amines: pharmacological characterization of trace amine-associated receptors

Trace amine-associated receptors, a novel class of G-protein coupled receptors which respond to trace amines but not to classical biogenic amines, have been found to be expressed in heart. Therefore, we investigated the cardiac effects of the trace amines p-tyramine, beta-phenylethylamine, octopamine, and tryptamine. Isolated rat hearts were perfused in the presence of trace amines, monitoring the hemodynamic variables. In addition, radioligand binding experiments with [3H]-p-tyramine and [125I]-3-iodothyronamine were performed in rat ventricular tissue. Octopamine, beta-phenylethylamine, and tryptamine produced a dose-dependent negative inotropic effect as shown by reduced cardiac output (IC(50)=109 microM, 159 microM, and 242 microM, respectively). In the same preparation a similar effect was produced by thyronamine and 3-iodothyronamine, with IC(50)=94 microM and 27 microM, respectively. The negative inotropic effect of octopamine was confirmed in a papillary muscle preparation. All trace amines except tryptamine increased the heart rate, but this action could be attributed to their sympathomimetic properties, since it was abolished by propranolol. The negative inotropic effect of trace amines was significantly increased by the tyrosine kinase inhibitor genistein. Specific and saturable binding of [(3)H]-p-tyramine and [125I]-3-iodothyronamine was observed in ventricular tissue. While [3H]-p-tyramine was displaced by 3-iodothyronamine, [(125)I]-3-iodothyronamine was not displaced by p-tyramine. In conclusion, trace amines and thyronamines are negative inotropic agents. Their effect appears to be mediated by a subtype of trace amine-associated receptor which is characterized by the rank of potency: 3-iodothyronamine > thyronamine = octopamine = beta-phenylethylamine, while tryptamine and p-tyramine are significantly less active.

Protection of ischemic rat heart by dantrolene, an antagonist of the sarcoplasmic reticulum calcium release channel.

Abstract Cytosolic Ca2+ overload plays a major role in the development of irreversible injury during myocardial ischemia. Such overload is due at least in part to the release of Ca2+ from the sarcoplasmic reticulum. Therefore, we investigated whether dantrolene, a blocker of the sarcoplasmic reticulum Ca2+ release channel, may protect from ischemic injury. In binding experiments, we determined the effect of dantrolene on [3H]-ryanodine binding in rat cardiac tissue. In perfusion experiments, isolated rat hearts were perfused for 20 min in the working mode, in the presence of 0–45 μM dantrolene. The hearts were then subjected to 30 min of global ischemia and 120 min of retrograde reperfusion. Tissue injury was evaluated on the basis of triphenyltetrazolium chloride (TTC) staining and LDH release. The binding experiments showed that dantrolene displaced 4 nM [3H]-ryanodine with IC50 of 34 μM. In the perfusion experiments, tissue necrosis (i.e., TTC-negative tissue) averaged 28.3±1.6% of the ventricular mass under control conditions. Dantrolene was protective at micromolar concentrations: tissue necrosis decreased to 21.4±1.0% and 8.4±1.4% with 1 μM and 45 μM dantrolene, respectively (P < 0.05 and P < 0.01). Similar results were obtained with regard to LDH release. At low concentrations (up to 4 μM), dantrolene did not produce any significant hemodynamic effect, except for a slight increase in coronary flow, whereas at higher concentration a negative inotropic effect was apparent. In conclusion, dantrolene reduced ischemic injury even at concentrations that did not affect contractile performance. Modulation of sarcoplasmic reticulum Ca2+ release might represent a new cardioprotective strategy.

A3 adenosine receptor stimulation modulates sarcoplasmic reticulum Ca2+ release in rat heart

OBJECTIVE Stimulation of A3 adenosine receptors has been shown to protect cardiac myocytes from ischemic injury, but the mechanism of this action is unknown. We evaluated the effect of adenosine agonists and antagonists on the sarcoplasmic reticulum (SR) Ca(2+) channels. METHODS Isolated rat hearts were perfused with control buffer or different adenosine agonists and antagonists. Hearts were then homogenized and used to determine SR Ca(2+)-induced Ca(2+) release, assayed by quick filtration technique after loading with 45Ca(2+), and the binding of [3H]ryanodine, a specific ligand of the SR Ca(2+) release channel. In parallel experiments, hearts were challenged with 30 min of global ischemia and 120 min of reperfusion, and the extent of tissue necrosis was evaluated by triphenyltetrazolium chloride staining. RESULTS Perfusion with the A1>A3 agonist R-PIA and the A3>A1 agonist IB-MECA was associated with reduced [3H]ryanodine binding, due to reduced B(max) (by about 20%), whereas K(d) and Ca(2+)-dependence of the binding reaction were unaffected. These actions were abolished by the A3 antagonist MRS 1191, while they were not affected by A1 and A2 antagonists. The rate constant of SR Ca(2+) release decreased by 25-30% in hearts perfused with R-PIA or IB-MECA. Tissue necrosis was significantly reduced in the presence of R-PIA or IB-MECA. Protection was removed by MRS 1191, and it was not affected by A1 and A2 antagonists. Hearts were also protected by administration of dantrolene, a ryanodine receptor antagonist. In the presence of dantrolene, no further protection was provided by IB-MECA. CONCLUSION A3 adenosine receptor stimulation modulates the SR Ca(2+) channel. This action might account for the protective effect of adenosine.

NGFI-A expression in the rat brain after sleep deprivation

The effects of total sleep deprivation (SD) on the expression of the immediate-early gene NGFI-A were studied in the rat brain by in situ hybridization. Rats were manually sleep-deprived for 3, 6, 12 and 24 h starting at light onset (08:00 h) and for 12 h starting at dark onset (20:00 h). SD performed during the day induced a marked increase in NGFI-A mRNA levels with respect to sleep controls in many cerebrocortical areas and caudate-putamen, which was most evident after 6 h SD. A decrease was seen in hippocampus and thalamus, particularly after 12 h SD. Rats sleep-deprived for 12 h during the night showed an increase in NGFI-A expression in some cortical areas while rats sleep-deprived for 24 h showed few changes with respect to controls. The pattern of NGFI-A expression after forced wakefulness showed some differences from that observed after spontaneous wakefulness [M. Pompeiano, C. Cirelli and G. Tononi, Immediate early genes in spontaneous wakefulness and sleep: expression of c-fos and NGFI-A mRNA and protein, J. Sleep Res., 3 (1994) 80-96]. These observations are discussed with respect to the functional consequences of wakefulness in specific brain areas.