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

Abstract

Phosphatidylinositol hydrolysis and subsequent increases in intracellular calcium, activated by G-protein-coupled receptors or receptor tyrosine kinases, are important regulators of various cellular functions [1,2].The initial step in receptor-mediated phosphatidylinositol (PtdIns) metabolism involves the activation of phospholipase C (PLC), which in turn hydrolyzes PtdIns. When the substrate is phosphatidylinositol The production and further metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] require several calcium-dependent enzymes, but little is known about subsequent calcium-dependent changes in cellular Ins(1,4,5)P 3 . To study the calcium dependence of muscarinic acetylcholine receptor-induced Ins(1,4,5)P 3 increases in PC12h cells, we utilized an Ins(1,4,5)P 3 imaging system based on fluorescence resonance energy transfer and using green fluorescent protein variants fused with the pleckstrin homology domain of phospholipase C-d1. 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 storeoperated calcium influx. We found that Ins(1,4,5)P 3 production was increased by a combination of receptor-and calcium-dependent components, rather than by calcium alone. The level of Ins(1,4,5)P 3 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)P 3 increase, indicating that calcium does not influence Ins(1,4,5)P 3 production without receptor activation. Removal of both the receptor agonists and calcium rapidly restored calcium and Ins(1,4,5)P 3 levels, whereas removal of calcium alone restored calcium to its basal concentration. Similar calciumdependent increases in Ins(1,4,5)P 3 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)P 3 production. To our knowledge, our results are the first showing receptor-and calciumdependent components within cellular Ins(1,4,5)P 3 .

High Cell Density Upregulates Calcium Oscillation by Increasing Calcium Store Content via Basal Mitogen-Activated Protein Kinase Activity

“…In order to test the possibility that cell density affect calcium increase pattern by modulating inositol phospholipid metabolism, histamine-induced inositol phospholipid hydrolysis in HeLa cells were measured using a fluorescent resonance energy transfer (FRET) based imaging method [ 19 ]. Expression vectors containing fusion proteins of the pleckstrin homology domain, derived from rat PLCδ1, and cyan and yellow mutants of enhanced green fluorescent protein, were transfected into HeLa cells, and the interaction between these fusion proteins was determined by changes in FRET-based fluorescence ( Fig 7 ).…”

“…Expression vectors containing fusion proteins of the cyan and yellow variants of enhanced green fluorescent protein (EGFP) and the pleckstrin homology domain (PHD) derived from rat PLCδ1 were constructed and designated pCFP-PHD and pYFP-PHD, as described previously [ 19 ]. Histamine H1 receptor cDNA [ 20 ] was obtained by PCR amplification from bovine cDNA (Quick-Clone, BD bioscience, San Jose, CA) and used to construct an expression vector, pME-H1 using the SRα promoter [ 21 ].…”