Bastadin 12 and ryanodine reveal similarities between thapsigargin- and tetrabromobisphenol A-induced intracellular Ca(2+) 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 Ca(2+) release. Other studies have demonstrated that ryanodine is able to inhibit Ca(2+) efflux from skeletal sarcoplasmic reticulum (SR) membranes in response to the known Ca(2+) 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 Ca(2+) release have been observed in primary cultures of rat cerebellar granule cells (CGCs). Here, we used CGCs and the fluorescent intracellular Ca(2+) probe fluo-3 to test the following hypotheses: (1) TBBPA shares Ca(2+) 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 μM bastadin 12 alone induced an increase in the intracellular Ca(2+) level in CGCs, whereas 2.5 and 10 μM bastadin 12 had no effect on the basal Ca(2+) concentration. The thapsigargin-induced Ca(2+) release was partially reduced by co-administration of either 2.5 μM bastadin 12 or 200 μM ryanodine, and the release of Ca(2+) was nearly completely attenuated by these compounds when they were given together. TBBPA (5, 10 and 25 μM) administration caused a concentration-dependent increase in CGC Ca(2+) levels. Administration of 2.5 μM bastadin 12 with 200 μM ryanodine blocked the increase in intracellular Ca(2+) evoked by 10 μM 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 Ca(2+) release from the intracellular stores of cultured neurons, and our findings support the hypothesis that TBBPA and thapsigargin induce intracellular Ca(2+) release through a common mechanism.