Molekulare Wirkorte des Calcium-mobilisierenden Second Messengers cyclic ADP-ribose in T-Lymphocyten und Fibroblasten
Final Report Abstract
Cyclic ADP-ribose (cADPR) is a universal second messenger for Ca2+ mobilisation. Having obtained evidence for the role of cADPR in the process of sustained signal transduction and activation of human T cells, a prerequisite for an adequate immune response, we characterized in more detail chemical mimicks of cADPR, in particular a molecule that could easily cross biological membranes by addition of a lipophilic group, termed 8-bromo-N1-cyclic inosine diphosphoribose (8-Br-N1-clDPR). Findings from the present project can be summarized as follows: 8-Br-N1-clDPR turned out to be rock-solid in terms of metabolism. Thus, even during prolonged incubation of cells with 8-Br-N1-clDPR no metabolic degradation would influence the interpretation of biological or pharmacological data. Secondly, 8-Br-N1-clDPR was used to study the cADPR/Ca2+ signalling branch of T cells. Molecular targets of cADPR have been reported to be either the ryanodine receptor (RyR) localized to the endoplasmic reticulum or, more recently, the none-selective cation channel transient receptor potential - melastatin type 2 (TRPM2), localized in the plasma membrane. Clearly, 8-Br-N1-clDPR did not activate TRPM2 when overexpressed in HEK 293 cells. However, in a Ca2+ free/Ca2+ re-introduction protocol, 8-Br-N1-clDPR evoked Ca2+ release and Ca2+ entry indicating that the Ca2+ entry pathway activated here is the capacitative one. This, is further supported by its sensitivity to the ion channel blockers SKF 96365 and Gd3+ ions although these drugs are not very specific. In summary, 8-Br-N1-clDPR appears to be the first cADPR agonist affecting Ca2+ release and subsequent capacitative Ca2+ entry, without activating TRPM2.
Publications
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Kirchberger T, Wagner G, Xu J, Cordiglieri C, Wang P, Gasser A, Fliegert R, Bruhn S, Flügel A, Lund FE,Zhang LH, Potter BV, Guse AH. Cellular effects and metabolic stability of N1-cyclic inosine diphosphoribose and its derivatives. Br J Pharmacol. 2006 Oct;149(4):337-44.
