D-AP5 blocks the increase of intracellular free Ca~(2+) induced by glutamate in isolated cochlear IHCs

摘要

Objective To investigate the effect of D-AP5 (D-2-amino-5-phosphonopentanoate, a specific NMDA-antagonist) on the increase of intracellular free Ca~(2+) concentration ([Ca~(2+)]_i) induced by glutamate in isolated cochlear inner hair cells (IHCs), and to detect the autoreceptors of the IHC membrane. Methods When a laser scanning confocal microscope (LSCM) was used, the exogenous glutamate (Glu)-induced changes in [Ca~(2+) ]_i of isolated IHCs and OHCs of guinea pig cochlea were observed with fluo-3, a fluorescent probe for [Ca~(2+) ]_i. After D-AP5 or CNQX (6--cyano--7--nitroguinoxaline--2, 3--dione, a specific AMPA- antagonist) was administrated, the exogenous glutamate (Glu)-induced changes in [Ca~(2+)]_i of isolated IHCs were recorded. Results In the presence of a low concentration Glu (3.85 μmol/L), there was an increase of [Ca~(2+)]_i in IHCs, whereas there was no change in OHCs. When 50 μmol/L of D-AP5 was administrated in advance, Glu did not induce a corresponding increase in [Ca~(2+) ]_i in IHCs, and 50 μmol/L of CNQX did not completely block the increase of [Ca~(2+)]_i in IHCs. Conclusions These results suggest that the autoreceptors existing in the IHC membrane are mainly of NMDA type, while there are relatively few AMPA receptors. Exogenous Glu is capable of increasing [Ca~(2+)]_i in IHCs by acting on the NMDA autoreceptor of IHCs in a positive feedback manner.