BASTADIN 12 AND RYANODINE REVEAL SIMILARITIES BETWEEN THAPSIGARGIN- AND TETRABROMOBISPHENOL A-INDUCED INTRACELLULAR Ca2+ RELEASE IN CULTURED CEREBELLAR GRANULE CELLS

Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant with recognized neuro- and cytotoxic properties that are presumably mediated by intracellular Ca2+ release. Other studies have demonstrated that ryanodine is able to inhibit Ca2+ efflux from skeletal sarcoplasmic reticulum (SR) membranes in response to the known Ca2+ releaser thapsigargin, provided that the macrocyclic brominated tyrosine derivative bastadin 5 is also present. Similar effects supporting the role of ryanodine receptors in thapsigargin-evoked Ca2+ release have been observed in primary cultures of rat cerebellar granule cells (CGCs). Here, we used CGCs and the fluorescent intracellular Ca2+ probe fluo-3 to test the following hypotheses: (1) TBBPA shares Ca2+ releasing properties with thapsigargin, and (2) synthetic bastadin 12 can replace bastadin 5 as a pharmacological tool to identify these similarities. The results demonstrated that either 200 nM thapsigargin or 30 mu M bastadin 12 alone induced an increase in the intracellular Ca2+ level in CGCs, whereas 2.5 and 10 mu M bastadin 12 had no effect on the basal Ca2+ concentration. The thapsigargin-induced Ca2+ release was partially reduced by co-administration of either 2.5 mu M bastadin 12 or 200 mu M ryanodine, and the release of Ca2+ was nearly completely attenuated by these compounds when they were given together. TBBPA (5, 10 and 25 mu M) administration caused a concentration-dependent increase in CGC Ca2+ levels. Administration of 2.5 mu M bastadin 12 with 200 mu M ryanodine blocked the increase in intracellular Ca2+ evoked by 10 mu M TBBPA, although these compounds were ineffective when applied separately. These results indicate that bastadin 12 may replace bastadin 5 when testing the ability of ryanodine to inhibit Ca2+ release from the intracellular stores of cultured neurons, and our findings support the hypothesis that TBBPA and thapsigargin induce intracellular Ca2+ release through a common mechanism.