As the activities of these kinase inhibitors directly on the KA receptor itself has not, to our knowledge, been determined, we can not rule out the possibility these kinases act directly on the KA receptor, or one of its domains, rather than act downstream of receptor activation. completely attenuated by the mixed CDK and MAP kinase inhibitor, olomoucine, in a concentration-dependent manner (50?C?600?M), and partially by roscovitine (1?C?100?M), a more selective CDK inihibitor. The p38 MAP kinase inhibitor, SB203580 (1?C?100?M), partially attenuated KA receptor-mediated apoptosis, as did the MAP kinase kinase inhibitors PD98509 (1?C?100?M) and U0126 (1?C?100?M). These findings provide new evidence for a complex network of interacting pathways involving CDK/MAPK that control apoptosis downstream of KA receptor activation in excitotoxic neuronal cell death. the N-methyl-D-aspartate (NMDA) receptors (Choi has been implicated in various apoptotic paradigms (Behrens dominant-negative mutants attenuate apoptosis in sympathetic neurones (Ham failed to cause apoptosis in hippocampal neurones, coincident with the reduction of c-phosphorylation (Yang models of seizure activity is prevented by ERK kinase inhibitors (Murray phosphorylation seems to be governed by p38 MAPK (Yamagishi (div). Cells were seeded at a cell density of 0.3106 cells cm?2 in 24-well NUNCTM plates (Denmark) precoated with poly-D-lysine (50?g?ml?1). Aphidicolin (2?g?ml?1) was added to the medium 18?C?24?h after plating to inhibit non-neuronal cell proliferation (Giardina (Cheung labelling of nuclear DNA fragments Apoptosis was analysed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin nick-end labelling (TUNEL) as previously described (Cheung test to compare individual treatments. Results Kainate neurotoxicity: preliminary observations KA exposure resulted in a reduction in cellular viability, with characteristics of apoptosis (see below), and was concentration-dependent (is responsible for the execution of apoptosis. As the activities of these kinase inhibitors directly on the KA receptor itself has not, to our knowledge, been determined, we can not rule out the possibility these kinases act directly on the Hydroxocobalamin (Vitamin B12a) KA receptor, or one of its domains, rather than act downstream of receptor activation. We however believe this is unlikely, as no neuroprotection is evident in the inactive isoform, iso-olomoucine. In conclusion, our results support Hydroxocobalamin (Vitamin B12a) the growing body of evidence relating to the cell MAP and routine kinases in excitotoxicity, and offer the first demo of the participation of MAPKs in KA receptor-mediated neuronal damage. Acknowledgments Supported with the National Health insurance and Medical Analysis Council (Australia), which P.M. Beart is normally a Senior Primary Analysis Fellow, and by grants or COCA1 loans in the Ramaciotti, Rebecca William and Cooper Buckland Foundations, and Perpetual Trustees. Abbreviations CDKcyclin-dependent kinaseCGCscerebellar granule cellsCNQX6-cyano-7-nitroquinoxaline-2,3-dionedivdays in vitro. Glu, glutamateKAkainateMAPkinase, mitogen-activated proteins kinaseMEKmitogen-activated proteins kinase kinaseMTT3-94,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromideTUNELterminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin Hydroxocobalamin (Vitamin B12a) nick-end labelling.
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