Receptor- and calcium-dependent induced inositol 1,4,5-trisphosphate increases in PC12h cells as shown by fluorescence resonance energy transfer imaging

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The production and further metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] require several calcium-dependent enzymes, but little is known about subsequent calcium-dependent changes in cellular Ins(1,4,5)P3. To study the calcium dependence of muscarinic acetylcholine receptor-induced Ins(1,4,5)P3 increases in PC12h cells, we utilized an Ins(1,4,5)P3 imaging system based on fluorescence resonance energy transfer and using green fluorescent protein variants fused with the pleckstrin homology domain of phospholipase C-δ1. The intracellular calcium concentration, monitored by calcium imaging, was adjusted by thapsigargin pretreatment or alterations in extracellular calcium concentration, enabling rapid receptor-independent changes in calcium concentration via store-operated calcium influx. We found that Ins(1,4,5)P3 production was increased by a combination of receptor- and calcium-dependent components, rather than by calcium alone. The level of Ins(1,4,5)P3 induced by the receptor was found to be half that induced by the combined receptor and calcium components. Increases in calcium levels prior to receptor activation did not affect the subsequent receptor-induced Ins(1,4,5)P3 increase, indicating that calcium does not influence Ins(1,4,5)P3 production without receptor activation. Removal of both the receptor agonists and calcium rapidly restored calcium and Ins(1,4,5)P3 levels, whereas removal of calcium alone restored calcium to its basal concentration. Similar calcium-dependent increases in Ins(1,4,5)P3 were also observed in Chinese hamster ovary cells expressing m1 muscarinic acetylcholine receptor, indicating that the observed calcium dependence is common to Ins(1,4,5)P3 production. To our knowledge, our results are the first showing receptor- and calcium-dependent components within cellular Ins(1,4,5)P3.

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