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Divalent Cation Entry in Cultured Rat Cerebellar Granule Cells Measured Using Mn 2+ Quench of Fura 2 Fluorescence
Author(s) -
Simpson P. B.,
Challiss R. A. J.,
Nahorski S. R.
Publication year - 1995
Publication title -
european journal of neuroscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.346
H-Index - 206
eISSN - 1460-9568
pISSN - 0953-816X
DOI - 10.1111/j.1460-9568.1995.tb01070.x
Subject(s) - nmda receptor , chemistry , biophysics , nitrendipine , dizocilpine , depolarization , thapsigargin , channel blocker , calcium , medicine , biochemistry , extracellular , receptor , biology , organic chemistry
In this study the rate of Mn 2+ quench of fura‐2 fluorescence evoked by glutamatergic and cholinergic agonists, depolarization and Ca 2+ store modulators was measured in cultured cerebellar granule cells, in order to study their effects on Ca 2+ entry in isolation from effects on Ca 2+ store release. The rate of fluorescence quench by 0.1 mM Mn 2+ was markedly increased by 25 mM K + ‐ evoked depolarization or by 200 μM N‐methyl‐D‐aspartate (NMDA), with a significantly greater increase occurring during the rapid‐onset peak phase compared to the plateau phase of the K + ‐ or NMDA‐evoked [Ca 2+ ] i response. The stimulatory effect of NMDA on Mn 2+ quench was abolished by dizocilpine (10 μM), but nitrendipine (2 μM), while decreasing the rate of basal quench, did not affect NMDA‐stimulated Mn 2+ entry. This suggests that nitrendipine may not act on NMDA channels in granule cells, at least under these conditions, and that voltage‐operated Ca 2+ channels are involved in control quench whereas the NMDA‐evoked quench is dependent on entry through the receptor channel. The t 1/2 of quench was unaffected by α‐amino‐hydroxyisoxazole propionic acid (200 μM) and carbamyl choline (1 mM). Neither thapsigargin (10 μM) nor dantrolene (30 μM) significantly affected the rate of quench under control or NMDA‐ or K + ‐stimulated conditions, which confirms that the previously reported inhibitory effects on [Ca 2+ ] i elevations evoked by these agents are due to actions on Ca 2+ stores. However, thapsigargin elevated [Ca 2+ Ii in the presence of normal [Ca 2+ ] i , but not in nominally Ca 2+ ‐free medium, indicating that it evokes Ca 2+ entry in cerebellar granule cells, probably subsequent to store depletion, which appears to be either too small to be detected by Mn 2+ quench or to occur via Mn 2+ ‐impermeant channels.