We have synthesized iodinated resiniferatoxin bearing a 4-hydroxy-5-iodo-3-methoxyphenylacetate ester (I-RTX) and have characterized its activity on rat and human TRPV1 (VR1) receptors, as well as in behavioral assays of nociception. In whole cell patch-clamp recordings from transfected cells the functional activity of I-RTX was determined. Currents activated by capsaicin exhibited characteristic outward rectification and were antagonized by capsazepine and I-RTX. On rat TRPV1 the affinity of I-RTX was 800-fold higher than that of capsazepine (IC 50 ϭ 0.7 and 562 nM, respectively) and 10-fold higher on rat versus human receptors (IC 50 ϭ 0.7 and 5.4 nM, respectively). The same difference was observed when comparing the inhibition of [ 3 H]RTX binding to rat and human TRPV1 membranes for both RTX and I-RTX. Additional pharmacological differences were revealed using protons as the stimulus. Under these conditions capsazepine only partly blocked currents through rat TRPV1 receptors (by 70 to 80% block), yet was a full antagonist on human receptors. In contrast, I-RTX completely blocked proton-induced currents in both species and that activated by noxious heat. I-RTX also blocked capsaicin-induced firing of C-fibers in a rat in vitro skin-nerve assay. Despite this activity and the high affinity of I-RTX for rat TRPV1, only capsazepine proved to be an effective antagonist of capsaicin-induced paw flinching in rats. Thus, although I-RTX has limited utility for in vivo behavioral studies it is a high-affinity TRPV1 receptor antagonist that will be useful to characterize the functional properties of cloned and native vanilloid receptor subtypes in vitro.The transient receptor potential TRPV1 vanilloid receptor (also known as VR1; see revised TRP channel nomenclature Montell et al., 2002) gates a nonselective cation channel that is expressed by sensory neurons and that can be activated by protons, heat, and capsaicin, the pungent ingredient of chili peppers (Caterina et al., 1997;Tominaga et al., 1998). Ligands acting at the TRPV1 vanilloid receptor subtype have the potential therapeutic utility to treat thermal hyperalgesia-related pain and some inflammatory conditions (for review, see Szallasi and Blumberg, 1999;Caterina and Julius, 2001). One of the first antagonists described for the capsaicin receptor was capsazepine (Bevan et al., 1992). This ligand has been used widely to explore the functional significance of TRPV1 receptors in pain. However it has relatively low micromolar affinity for TRPV1 receptors and because it also blocks voltage-gated calcium channels, this has made interpretation of functional data with this compound less straightforward (Docherty et al., 1997).To date, one of the highest affinity ligands reported for the TRPV1 receptor is the natural plant product resiniferatoxin (RTX), which was first isolated from Euphorbia resinifera (Szallasi and Blumberg, 1989). Interestingly, it has been shown recently that iodination of this agonist RTX confers antagonist-like properties to the ligand wit...

The LXR nuclear receptors are intracellular sensors of cholesterol excess and are activated by various oxysterols. LXRs have been shown to regulate multiple genes of lipid metabolism, including ABCA1 (formerly known as ABC1). ABCA1 is a lipid pump that effluxes cholesterol and phospholipid out of cells. ABCA1 deficiency causes extremely low high density lipoprotein (HDL) levels, demonstrating the importance of ABCA1 in the formation of HDL. The present work shows that the acetyl-podocarpic dimer (APD) is a potent, selective agonist for both LXR␣ (NR1H3) and LXR␤ (NR1H2). In transient transactivation assays, APD was ϳ1000-fold more potent, and yielded ϳ6-fold greater maximal stimulation, than the widely used LXR agonist 22-(R)-hydroxycholesterol. APD induced ABCA1 mRNA levels, and increased efflux of both cholesterol and phospholipid, from multiple cell types. Gas chromatographymass spectrometry measurements demonstrated that APD stimulated efflux of endogenous cholesterol, eliminating any possible artifacts of cholesterol labeling. For both mRNA induction and stimulation of cholesterol efflux, APD was found to be more effective than was cholesterol loading. Taken together, these data show that APD is a more effective LXR agonist than endogenous oxysterols. LXR agonists may therefore be useful for the prevention and treatment of atherosclerosis, especially in the context of low HDL levels.

We recently described the identification of a non-peptidyl fungal metabolite (L-783,281, compound 1), which induced activation of human insulin receptor (IR) tyrosine kinase and mediated insulin-like effects in cells, as well as decreased blood glucose levels in murine models of Type 2 diabetes (Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M. T., Pelaez, F., Ruby, C., Kendall, R. L., Mao, X., Griffin, P., Calaycay, J., Zierath, J. R., Heck, J. V., Smith, R. G. & Moller, D. E. (1999) Science 284, 974 -977). Here we report the characterization of an active analog (compound 2) with enhanced IR kinase activation potency and selectivity over related receptors (insulin-like growth factor I receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor). The IR activators stimulated tyrosine kinase activity of partially purified native IR and recombinant IR tyrosine kinase domain. Administration of the IR activators to mice was associated with increased IR tyrosine kinase activity in liver. In vivo oral treatment with compound 2 resulted in significant glucose lowering in several rodent models of diabetes. In db/db mice, oral administration of compound 2 elicited significant correction of hyperglycemia. In a streptozotocin-induced diabetic mouse model, compound 2 potentiated the glucose-lowering effect of insulin. In normal rats, compound 2 improved oral glucose tolerance with significant reduction in insulin release following glucose challenge. A structurally related inactive analog (compound 3) was not effective on insulin receptor activation or glucose lowering in db/db mice. Thus, small molecule IR activators exert insulin mimetic and sensitizing effects in cells and in animal models of diabetes. These results have implications for the future development of new therapies for diabetes mellitus.Insulin elicits a diverse array of biological responses by binding to its specific receptor (1). The insulin receptor (IR) 1 is a heterotetrameric protein consisting of two extracellular ␣ subunits and two transmembrane ␤ subunits. The binding of the ligand to the ␣ subunit of IR not only concentrates insulin at its site of action, but also induces conformational changes in the receptor, which in turn stimulates the tyrosine kinase activity intrinsic to the ␤ subunit of the IR. Extensive studies have indicated that the ability of the receptor to autophosphorylate and phosphorylate intracellular substrates is essential for its mediation of the complex cellular responses of insulin (2-5).Insulin receptors trans-phosphorylate several immediate substrates (on Tyr residues), including insulin receptor substrate (IRS) proteins 1-4, Shc, and Gab 1, each of which provide specific docking sites for other signaling proteins containing Src homology 2 domains (6). These events lead to the activation of downstream signaling molecules, including phosphatidylinositol 3-kinase (PI 3-kinase). Numerous studies have adduced that PI 3-kinase is required for the metabolic effec...

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Peroxisome proliferator-activated receptors (PPARs) are a class of ligand modulated transcription factors with a prominent role in the regulation of metabolic processes. This report is intended to provide a limited introduction to the PPAR field, sketched with reference to one early series of PPAR ligands.