Divalent cation entry in cultured rat cerebellar granule cells measured using Mn2+ quench of fura 2 fluorescence.
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 microM 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 microM), but nitrendipine (2 microM), 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 alpha-amino-hydroxyisoxazole propionic acid (200 microM) and carbamyl choline (1 mM). Neither thapsigargin (10 microM) nor dantrolene (30 microM) 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+]i in the presence of normal [Ca2+]o but not in nominally Ca(2+)-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 Mn(2+)-impermeant channels.
Simpson
P B
PB
Department of Cell Physiology and Pharmacology, University of Leicester, UK.
Challiss
R A
RA
Nahorski
S R
SR
D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002412:Cations; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects*; D005453:Fluorescence; D016257:Fura-2; D008345:Manganese / Q000378:metabolism*; D016202:N-Methylaspartate / Q000494:pharmacology; D009568:Nitrendipine / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D015220:Calcium Channels; D002412:Cations; D013729:Terpenes; D008345:Manganese; D016202:N-Methylaspartate; D019284:Thapsigargin; D009568:Nitrendipine; D000252:Calcium-Transporting ATPases; D011188:Potassium; D002118:Calcium; D016257:Fura-2