The trace amine para-tyramine is structurally and functionally related to the amphetamines and the biogenic amine neurotransmitters. It is currently thought that the biological activities elicited by trace amines such as p-tyramine and the psychostimulant amphetamines are manifestations of their ability to inhibit the clearance of extracellular transmitter and/or stimulate the efflux of transmitter from intracellular stores. Here we report the discovery and pharmacological characterization of a rat G protein-coupled receptor that stimulates the production of cAMP when exposed to the trace amines p-tyramine, beta-phenethylamine, tryptamine, and octopamine. An extensive pharmacological survey revealed that psychostimulant and hallucinogenic amphetamines, numerous ergoline derivatives, adrenergic ligands, and 3-methylated metabolites of the catecholamine neurotransmitters are also good agonists at the rat trace amine receptor 1 (rTAR1). These results suggest that the trace amines and catecholamine metabolites may serve as the endogenous ligands of a novel intercellular signaling system found widely throughout the vertebrate brain and periphery. Furthermore, the discovery that amphetamines, including 3,4-methylenedioxymethamphetamine (MDMA; "ecstasy"), are potent rTAR1 agonists suggests that the effects of these widely used drugs may be mediated in part by this receptor as well as their previously characterized targets, the neurotransmitter transporter proteins.

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.

Angiogenesis, the formation of capillaries from pre-existing blood vessels, occurs as a result of various normal and pathological processes, including ovulation, wound healing, and ischemic disease. The angiogenic process requires endothelial cells to acquire an altered phenotype, modify cell-cell contacts, migrate, proliferate, and re-establish cell-cell contacts to form patent tubes or endothelial sheets. Finally, mechanisms that control microvessel regression balance pro-angiogenic processes to regulate the extent, magnitude, and duration of angiogenesis.It has become apparent that up-regulation of mechanisms that promote endothelial survival are essential during angiogenesis. This requirement was underscored by studies showing that inhibitors of ␣ v ␤ 3 integrin (but not ␤1 integrins) block angiogenesis by inducing apoptosis of migrating endothelial cells (1). While the precise mechanism by which blockade of ␣ v ␤ 3 induces apoptosis of endothelial cells is not clear, previous studies suggest that ␣ v ␤ 3 ligation modulates expression of apoptosis-regulatory genes (2, 3). For example, ␣ v ␤ 3 blockade caused activation of p53 and down-regulation of bcl-2 in endothelial cells isolated from CAM (2). In addition, previous studies implicate ␣ v ␤ 3 -mediated activation of NF-B as an important endothelial cell survival pathway (4). The transcription factor NF-B is a pleiotropic regulator of many genes involved in immune and inflammatory responses. The NF-B family of proteins consists of homo-or heterodimeric subunits of the Rel family. In unstimulated cells, NF-B is localized in the cytoplasm in complex with an inhibitory protein, IB (5). Upon stimulation, the inhibitory IB becomes phosphorylated, ubiquitinated, and subsequently degraded by the proteosome machinery (6). This allows NF-B to translocate to the nucleus, bind DNA, and transactivate transcription of specific genes. In endothelial cells, apoptosis induced by growth factor deprivation was blocked by osteopontin, an ␣ v ␤ 3 ligand (4). The protective effect of osteopontin required nuclear translocation of the transcription factor, NF-B, since overexpression of a nondegradable form of IB (super repressor) blocked the effect.Several recent studies have implicated NF-B as an important cell survival factor (4, 7-9). It has been hypothesized that NF-B-induced transcription of anti-apoptotic genes is responsible for its protective activity. In support of this, NF-B has been shown to up-regulate transcription of antiapoptotic molecules such as A1, A20, XIAP, TRAF2,. To determine whether these or unique factors might be responsible for the protective effects of NF-B in endothelial cells, we used suppressive subtraction hybridization to isolate endothelial genes that required active NF-B for induction by osteopontin. Here we report that one of the clones isolated in this screen was osteoprotegerin, a soluble member of the TNF 1 receptor superfamily and the recently discovered osteoclast differentiation inhibitory factor. We show that adding recombinant osteoprote...

We have previously shown that several thyronamines, decarboxylated and deiodinated metabolites of the thyroid hormone, potently activate an orphan G protein-coupled receptor in vitro (TAAR1) and induced hypothermia in vivo on a rapid time scale [Scanlan, T. S.; Suchland, K. L.; Hart, M. E.; Chiellini, G.; Huang, Y.; Kruzich, P. J.; Frascarelli, S.; Crossley, D. A.; Bunzow, J. R.; Ronca-Testoni, S.; Lin, E. T.; Hatton, D.; Zucchi, R.; Grandy, D. K. 3-Iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone. Nat. Med. 2004, 10 (6), 638-642]. Herein, we report the synthesis of these thyronamines. Additionally, a large number of thyroamine derivatives were synthesized in an effort to understand the molecular basis of TAAR1 activation and hypothermia induction. Several derivatives were found to potently activate both rTAAR1 and mTAAR1 in vitro (compounds 77, 85, 91, and 92). When administered to mice at a 50 mg/kg dose, these derivatives all induced significant hypothermia within 60 min and exhibited a hypothermic induction profile analogous to 3-iodothyronamine (1, T(1)AM) except 91, which proved to be more efficacious. On the basis of this result, a dose-dependent profile for 91 was generated and an ED(50) of 30 mumol/kg was calculated. Compound 91 proved to be more potent than T(1)AM for TAAR1 activation and exhibits increased potency and efficacy for hypothermia induction. These data further strengthen the pharmacological correlation linking TAAR1 activation by thyronamines and hypothermia induction in mice.

Background and Purpose-Mild hypothermia confers profound neuroprotection in ischemia. We recently discovered 2 natural derivatives of thyroxine, 3-iodothyronamine (T 1 AM) and thyronamine (T 0 AM), that when administered to rodents lower body temperature for several hours without induction of a compensatory homeostatic response. We tested whether T 1 AM-and T 0 AM-induced hypothermia protects against brain injury from experimental stroke. Methods-We tested T 1 AM and T 0 AM 1 hour after and 2 days before stroke in a mouse model of focal ischemia. To determine whether T 1 AM and T 0 AM require hypothermia to protect against stroke injury, the induction of hypothermia was prevented. Results-T 1 AM and T 0 AM administration reduced body temperature from 37°C to 31°C. Mice given T 1 AM or T 0 AM after the ischemic period had significantly smaller infarcts compared with controls. Mice preconditioned with T 1 AM before ischemia displayed significantly smaller infarcts compared with controls. Pre-and postischemia treatments required the induction of hypothermia. T 1 AM and T 0 AM treatment in vitro failed to confer neuroprotection against ischemia. Conclusions-T 1 AM and T 0 AM, are potent neuroprotectants in acute stroke and T 1 AM can be used as antecedent treatment to induce neuroprotection against subsequent ischemia. Hypothermia induced by T 1 AM and T 0 AM may underlie neuroprotection. T 1 AM and T 0 AM offer promise as treatments for brain injury.