Adenosine 5′-diphosphoribose (ADPR) activates TRPM2, a Ca2+, Na+, and K+ permeable cation channel. Activation is induced by ADPR binding to the cytosolic C-terminal NudT9-homology domain. To generate the first structure–activity relationship, systematically modified ADPR analogues were designed, synthesized, and evaluated as antagonists using patch-clamp experiments in HEK293 cells overexpressing human TRPM2. Compounds with a purine C8 substituent show antagonist activity, and an 8-phenyl substitution (8-Ph-ADPR, 5) is very effective. Modification of the terminal ribose results in a weak antagonist, whereas its removal abolishes activity. An antagonist based upon a hybrid structure, 8-phenyl-2′-deoxy-ADPR (86, IC50 = 3 μM), is more potent than 8-Ph-ADPR (5). Initial bioisosteric replacement of the pyrophosphate linkage abolishes activity, but replacement of the pyrophosphate and the terminal ribose by a sulfamate-based group leads to a weak antagonist, a lead to more drug-like analogues. 8-Ph-ADPR (5) inhibits Ca2+ signalling and chemotaxis in human neutrophils, illustrating the potential for pharmacological intervention at TRPM2.
Transient receptor potential melastatin 2 (TRPM2) is a ligand-gated Ca-permeable nonselective cation channel. Whereas physiological stimuli, such as chemotactic agents, evoke controlled Ca signals via TRPM2, pathophysiological stimuli such as reactive oxygen species and genotoxic stress result in prolonged TRPM2-mediated Ca entry and, consequently, apoptosis. To date, adenosine 5'-diphosphoribose (ADPR) has been assumed to be the main agonist for TRPM2. Here we show that 2'-deoxy-ADPR was a significantly better TRPM2 agonist, inducing 10.4-fold higher whole-cell currents at saturation. Mechanistically, this increased activity was caused by a decreased rate of inactivation and higher average open probability. Using high-performance liquid chromatography (HPLC) and mass spectrometry, we detected endogenous 2'-deoxy-ADPR in Jurkat T lymphocytes. Consistently, cytosolic nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) and nicotinamide adenine dinucleotide (NAD)-glycohydrolase CD38 sequentially catalyzed the synthesis of 2'-deoxy-ADPR from nicotinamide mononucleotide (NMN) and 2'-deoxy-ATP in vitro. Thus, 2'-deoxy-ADPR is an endogenous TRPM2 superagonist that may act as a cell signaling molecule.
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Thyroid hormone (T3) is essential for normal development, differentiation and metabolic balance. We have performed DNA microarray experiments using hepatic RNA from hypothyroid and T3-treated hypothyroid rats in order to characterize T3-induced gene expression patterns after various time points (6, 24 and 48 h after the administration of the hormone). Sixty-two of 4608 different genes displayed a reproducible T3-response, and cluster analysis divided these differentially regulated genes into six expression patterns. Thirty-six genes were not significantly regulated within the first 24 h. Transient transfection experiments of eight late-induced gene promoters failed to detect a thyroid hormone response element within their regulatory elements, suggesting an indirect activation mechanism(s). In search for an intermediate factor of T3 action, we examined whether various rather ubiquitous transcription factors, peroxisome proliferator-activated receptors (PPARs) and coactivators of the PPAR coactivator 1 family (PGC-1) are regulated by T3. Only PPAR and PERC/PGC-1 exhibit a significant T3-response within the first 6 h after treatment, identifying these factors as candidate components for mediating the late-induced expression pattern. Regulation of early-induced genes within the first 6 h after administration of T3 on transcript levels correlates with altered protein levels after 24 and 48 h in vivo.
TRPM2 (transient receptor potential channel, subfamily melastatin, member 2) is a Ca2+-permeable non-selective cation channel activated by the binding of adenosine 5′-diphosphoribose (ADPR) to its cytoplasmic NUDT9H domain (NUDT9 homology domain). Activation of TRPM2 by ADPR downstream of oxidative stress has been implicated in the pathogenesis of many human diseases, rendering TRPM2 an attractive novel target for pharmacological intervention. However, the structural basis underlying this activation is largely unknown. Since ADP (adenosine 5′-diphosphate) alone did not activate or antagonize the channel, we used a chemical biology approach employing synthetic analogues to focus on the role of the ADPR terminal ribose. All novel ADPR derivatives modified in the terminal ribose, including that with the seemingly minor change of methylating the anomeric-OH, abolished agonist activity at TRPM2. Antagonist activity improved as the terminal substituent increasingly resembled the natural ribose, indicating that gating by ADPR might require specific interactions between hydroxyl groups of the terminal ribose and the NUDT9H domain. By mutating amino acid residues of the NUDT9H domain, predicted by modelling and docking to interact with the terminal ribose, we demonstrate that abrogating hydrogen bonding of the amino acids Arg1433 and Tyr1349 interferes with activation of the channel by ADPR. Taken together, using the complementary experimental approaches of chemical modification of the ligand and site-directed mutagenesis of TRPM2, we demonstrate that channel activation critically depends on hydrogen bonding of Arg1433 and Tyr1349 with the terminal ribose. Our findings allow for a more rational design of novel TRPM2 antagonists that may ultimately lead to compounds of therapeutic potential.
The effect of acute cold exposure on the concentration of glucagon in the blood was investigated in man and in intact and adrenalectomized rats. In man fasted overnight acute cold exposure, which caused a twofold increase in O2-consumption resulted in a rapid rise in plasma glucagon. The levels of insulin and blood glucose remained unaltered, while the concentration of serum free fatty acids and beta-hydroxybutyrate increased. In fasted intact rats acute cold exposure lead to similar effects. A close parallelism between the rise in plasma glucagon and the concentration of hepatic cycloAMP was observed. Adrenalectomy did not impair the cold induced rise in plasma glucagon and hepatic cycloAMP. It is concluded that acute cold exposure caused a rapid rise in the concentration of plasma glucagon leading to an increase in the concentration of hepatic cycloAMP, thus enhancing the rate of hepatic gluconeogenesis and ketogenesis. As these alterations were similar in the absence of glucocorticoids and medulla-derived catecholamines, it is suggested that glucagon may play a role in the metabolic adaptation to acute cold exposure.
TRPM4b is a Ca2+‐activated, voltage‐dependent monovalent cation channel that has been shown to act as a negative regulator of Ca2+ entry and to be involved in the generation of oscillations of Ca2+ influx in Jurkat T‐lymphocytes. Transient overexpression of TRPM4b as an enhanced green fluorescence fusion protein in human embryonic kidney (HEK) cells resulted in its localization in the plasma membrane, as demonstrated by confocal fluorescence microscopy. The functionality and plasma membrane localization of overexpressed TRPM4b was confirmed by induction of Ca2+‐dependent inward and outward currents in whole cell patch clamp recordings. HEK‐293 cells stably overexpressing TRPM4b showed higher ionomycin‐activated Ca2+ influx than wild‐type cells. In addition, analysis of the membrane potential using the potentiometric dye bis‐(1,3‐dibutylbarbituric acid)‐trimethine oxonol and by current clamp experiments in the perforated patch configuration revealed a faster initial depolarization after activation of Ca2+ entry with ionomycin. Furthermore, TRPM4b expression facilitated repolarization and thereby enhanced sustained Ca2+ influx. In conclusion, in cells with a small negative membrane potential, such as HEK‐293 cells, TRPM4b acts as a positive regulator of Ca2+ entry.
3,5-Di-iodo-L-thyronine (T2) is a naturally occurring metabolite of thyroxine (T4). Contrary to earlier findings, T2 has recently been shown to have rapid effects in rat liver and in mononuclear blood cells. In the experiments described here, T2 was tested to determine whether it has a TSH suppressive effect in rats in vivo and in rat pituitary fragments in vitro. In experiments over 2 weeks in rats in vivo, low doses of T2 (20-200 micrograms/100 g body weight per day) had no significant influence on body and organ weights, but significantly decreased TSh and T4 serum concentrations. At 200 micrograms/100 g per day, T2 suppressed TSH to 43% and T4 to 29% of control levels. At 1-15 micrograms/100 g per day, 3,5,3'-tri-iodo-L-thyronine (T3), used as a comparison to T2, had significant effects on TSH and T4 levels, and also on body weight. Fifteen micrograms T3/100 g per day decreased TSH to 44%, T4 to 25%, and body weight to 59% of control levels. In experiments over 3 months in rats in vivo, a low dose (25 micrograms/100 g per day) of T2 suppressed TSH to 60% and T4 to 57% of control levels and had no significant influence on other parameters. Conversely, 0.1 microgram/100 g per day T3 had significant effects on body and organ weights as well as pellet intake, but a less pronounced TSH suppressive effect: TSH concentrations were unchanged and T4 concentrations were down to 80% of control values.(ABSTRACT TRUNCATED AT 250 WORDS)
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