Divalent Cation Entry in Cultured Rat Cerebellar Granule Cells Measured Using Mn2+ Quench of Fura 2 Fluorescence |
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Authors: | P B Simpson R A J Challiss S R Nahorski |
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Institution: | Department of Cell Physiology and Pharmacology, University of Leicester, PO Box 138, Medical Sciences Building, University Road, Leicester, UK |
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Abstract: | In this study the rate of Mn2+ quench of fura-2 fluorescence evoked by glutamatergic and cholinergic agonists, depolarization and Ca2+ store modulators was measured in cultured cerebellar granule cells, in order to study their effects on Ca2+ entry in isolation from effects on Ca2+ store release. The rate of fluorescence quench by 0.1 mM Mn2+ 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 Ca2+]i response. The stimulatory effect of NMDA on Mn2+ quench was abolished by dizocilpine (10 μM), but nitrendipine (2 μM), while decreasing the rate of basal quench, did not affect NMDA-stimulated Mn2+ entry. This suggests that nitrendipine may not act on NMDA channels in granule cells, at least under these conditions, and that voltage-operated Ca2+ channels are involved in control quench whereas the NMDA-evoked quench is dependent on entry through the receptor channel. The t1/2 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 Ca2+]i elevations evoked by these agents are due to actions on Ca2+ stores. However, thapsigargin elevated Ca2+Ii in the presence of normal Ca2+]i, but not in nominally Ca2+-free medium, indicating that it evokes Ca2+ entry in cerebellar granule cells, probably subsequent to store depletion, which appears to be either too small to be detected by Mn2+ quench or to occur via Mn2+-impermeant channels. |
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Keywords: | N-methyI-D-aspartate Ca2+ entry nitrendipine thapsigargin Ca2+ release-activated Ca2+ entry |
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