ABSTRACT
Transient receptor potential melastatin 2 (TRPM2) is a ligand-gated Ca2+-permeable nonselective cation channel. Whereas physiological stimuli, such as chemotactic agents, evoke controlled Ca2+ signals via TRPM2, pathophysiological stimuli such as reactive oxygen species and genotoxic stress result in prolonged TRPM2-mediated Ca2+ 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.
Subject(s)
Adenosine Diphosphate Ribose/analogs & derivatives , Clusterin/agonists , ADP-ribosyl Cyclase 1/chemistry , Adenosine Diphosphate Ribose/chemistry , Adenosine Diphosphate Ribose/pharmacology , Chromatography, High Pressure Liquid , Humans , Hydrogen Peroxide/chemistry , Jurkat Cells , Molecular Structure , Signal Transduction/drug effectsABSTRACT
RATIONALE: Tissue engineering may provide advanced in vitro models for drug testing and, in combination with recent induced pluripotent stem cell technology, disease modeling, but available techniques are unsuitable for higher throughput. OBJECTIVE: Here, we present a new miniaturized and automated method based on engineered heart tissue (EHT). METHODS AND RESULTS: Neonatal rat heart cells are mixed with fibrinogen/Matrigel plus thrombin and pipetted into rectangular casting molds in which two flexible silicone posts are positioned from above. Contractile activity is monitored video-optically by a camera and evaluated by a custom-made software program. Fibrin-based mini-EHTs (FBMEs) (150 microL, 600 000 cells) were transferred from molds to a standard 24-well plate two hours after casting. Over time FBMEs condensed from a 12x3x3 mm gel to a muscle strip of 8 mm length and, depending on conditions, 0.2 to 1.3 mm diameter. After 8 to 10 days, FBMEs started to rhythmically deflect the posts. Post properties and the extent of post deflection allowed calculation of rate, force (0.1 to 0.3 mN), and kinetics which was validated in organ baths experiments. FBMEs exhibited a well-developed, longitudinally aligned actinin-positive cardiac muscle network and lectin-positive vascular structures interspersed homogeneously throughout the construct. Analysis of a large series of FBME (n=192) revealed high yield and reproducibility and stability for weeks. Chromanol, quinidine, and erythromycin exerted concentration-dependent increases in relaxation time, doxorubicin decreases in contractile force. CONCLUSIONS: We developed a simple technique to construct large series of EHT and automatically evaluate contractile activity. The method shall be useful for drug screening and disease modeling.
