Präkonditionierung des ASIC-vermittelten Ca 2+-Einstroms in Kortexneuronen der Maus durch Azidose

Cerebral ischemia is accompanied by tissue acidosis. Activation of acid-sensing ion-channels (ASICs) by protons leads to transient peaks in the neuronal Ca2+-concentration and finally neuronal death. The precise mechanism connecting activation of ASICs by acidosis to the Ca2+-peak in cortical neurons is unknown. Ischemic preconditioning and preconditioning via acidosis is known to have an neuroprotective effect against ischemic cell death. Some experiments suggest an effect of ischemic preconditioning on the expression pattern of ASICs. Here Fura-2-imaging was performed on primary cultured cortical neurons of WT- and ASIC1-/--mice in order to elucidate the cellular mechanism contributing to the acid induced peak of the Ca2+-concentration. The effects of preconditioning by different periods of acidosis on the acid induced Ca2+-peaks and on the expression pattern of the ASIC subunits 1a, 2a and 2b in cortical neurons were being anaylized by Fura-2-Imaging and real-time-PCR. Application of the L-type calcium-channel antagonist Nimodipine reduced the major part of the acid-induced Ca2+-peak in cortical neurons. Emptying the endoplasmatic reticulum with Thapsigargin had no effect on the acid-induced Ca2+-peak. In ASIC1-/--neurons a drop of pH did not provoke any change in neuronal Ca2+-concentration. Depending on the length of exposition preconditioning by acidosis reduced the amplitude of the acid-induced Ca2+-peaks in cortical neurons. The depolarisation-induced Ca2+-peaks in cortical neurons of WT-mice but not of ASIC-/--mice was beeing reduced by preconditioning by acidosis in the same manner. Real-time-PCR did not reveal any changes in the expression pattern of ASIC1a, -2a or -2b after preconditioning by acidosis. These results suggest that a large amount of acid-induced Ca2+-influx in neurons permeates via L-type calcium-channels activated scondary to ASIC-activation. Preconditioning by acidosis might reduce the acid-induced Ca2+-influx via L-type calcium channels. This effect might depend on ASIC1. These findings highlight the role of voltage-gated L-type calcium-channels in the context of ischemic neuronal death and preconditioning by acidosis. This might be further investigated in in-vivo-models of cerebral ischemia