Simultaneous imaging of phosphatidyl inositol metabolism and Ca  levels in PC12h cells

3 -induced conformational change to the open state. We also found that GFP-IP 3 R1 clusters colocalized with ERp44, a luminal protein of endoplasmic reticulum that inhibits its channel activity. This is the first example of ligandinduced clustering of a ligand-gated channel protein.Many extracellular stimuli are received by trimeric G protein-or tyrosine kinase-coupled receptors and relayed to signaling molecules including phospholipase C (PLC), 1 which leads to production of a second messenger, inositol 1,4,5-trisphosphate (IP 3 ) (1). IP 3 in turn activates an internal membrane receptor/Ca 2ϩ channel, the IP 3 receptor (IP 3 R), and induces Ca 2ϩ release from internal Ca 2ϩ stores (mostly endoplasmic reticulum (ER)). This IP 3 -Ca 2ϩ pathway is particularly important because it is located at the intersection of many signaling pathways and is therefore thought to play a central role in cellular signaling.Rather recently, evidence has been accumulating that the subcellular localization of IP 3 R is tightly controlled and changes according to circumstances (2-4). In addition, IP 3 R is known to form large clusters on the ER (5). Very importantly, theoretical modeling (6 -8) predicted that IP 3 R clustering affects both elementary events (e.g. Ca 2ϩ blips and puffs) and the formation of complex cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] C ) patterns (e.g. Ca 2ϩ oscillations and waves). Particularly Shuai and Jung (6) predicted that if IP 3 concentration is relatively low (i.e. physiological situation), there is an optimal extent of clustering of IP 3 receptor for effective elevation of cytosolic calcium concentration. When the clusters of IP 3 receptor would become very large, the whole calcium signals elicited by low IP 3 concentrations would be significantly reduced. Experimental verification of this hypothesis has not been performed mainly because the molecular mechanism of IP 3 R clustering remains totally unknown, and thus it is impossible to inhibit or artificially modulate it. Wilson et al. (5) suggested that IP 3 R clustering was triggered by increased [Ca 2ϩ ] C since not only Ca 2ϩ -mobilizing agonists but a Ca 2ϩ ionophore (ionomycin) and an inhibitor of sarco/ endoplasmic Ca 2ϩ -ATPase (thapsigargin) induced it. They also showed that it is independent of ER vesiculation (9). Later it was shown that two small GTP-binding proteins, Cdc42 and Rac, are involved in agonist-induced IP 3 R clustering but that was because they augmented Ca 2ϩ signals as a whole and not because they influenced intracellular trafficking (10). In short, the detailed molecular mechanism of IP 3 R clustering remains largely unknown, and it is important to know it to understand precise roles of IP 3 R clustering. In this study we simultaneously imaged the dynamics of enhanced green fluorescent protein (GFP)-tagged IP 3 R type 1 (GFP-IP 3 R1) and [Ca 2ϩ ] C by using fluorescent Ca 2ϩ indicators. Based on the results of the mutational and pharmacological analyses, we concluded that IP 3 R clustering is initiated by a conformational c...

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“…3C, upper plot). This phenomenon has been reported in other cell lines (20,21). Importantly the time course of the changes in [IP 3 ] (Fig.…”

supporting

confidence: 83%

Cluster Formation of Inositol 1,4,5-Trisphosphate Receptor Requires Its Transition to Open State

Tateishi

Hattori

Nakayama

et al. 2005

Journal of Biological Chemistry

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“…Both methods depend on the redistribution of fusion proteins from the plasma membrane to the cytosol. In our earlier study on PC12h cells, in which we used confocal microscopy, mAChR activation resulted in the production of a redistribution peak, which was significantly delayed relative to the increase in calcium levels [15]. By contrast, when we used FRET, we observed that calcium increase and Ins(1,4,5) P 3 production occurred within a similar time frame.…”

Section: Discussionmentioning

confidence: 70%

“…The expression vectors for CFP–PHD and YFP–PHD were constructed as described for the GFP fusion protein [15], using an expression vector containing the SRα promoter [31].…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2007

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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 .

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“…Previously, we showed that carbachol increased intracellular IP3 and calcium in this cell line (16). As PC12-derived cell lines express both Gq-and Gi-coupled mAChRs (17,18), which activate PI3K when expressed (19,20), carbachol presumably activates PLC and PI3K simultaneously.…”

Section: Introductionmentioning

confidence: 59%

Inhibitory Effect of the Phosphoinositide 3-Kinase Inhibitor LY294002 on Muscarinic Acetylcholine Receptor-Induced Calcium Entry in PC12h Cells

et al. 2007

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Abstract. Phosphoinositide-3 kinase (PI3K) and phospholipase C (PLC) utilize the same phosphoinositides as substrates to produce different signaling molecules. These enzymes are activated by a similar set of cell signaling mechanisms, i.e., tyrosine kinases and G proteins, and affect common cell functions, including proliferation, motility, and intracellular trafficking. Despite these similarities, the interplay between these enzymes is not well understood. To address this issue, the effects of the PI3K inhibitor LY294002 on carbachol-induced calcium increase in PC12h cells were examined. As carbachol stimulates both Gq-and Gi-coupled muscarinic acetylcholine receptors (mAChRs), PI3K and PLC are activated simultaneously in this protocol. LY294002 was found to reduce the carbachol-induced calcium increase, and the reduction was attributed to suppression of calcium entry. As LY294002 did not affect either carbachol-induced calcium release or calcium entry induced by calcium store depletion, this agent was found to suppress calcium entry directly activated by mAChRs. Although PI3K was supposed to compete for substrates with PLC, the PI3K inhibitor did not enhance PLC-dependent cellular responses. As LY294002 was still effective by treating cells after carbachol stimulation, it is likely that this agent blocks the calcium entry channels directly.

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Simultaneous imaging of phosphatidyl inositol metabolism and Ca levels in PC12h cells

Cited by 6 publications

References 21 publications

Cluster Formation of Inositol 1,4,5-Trisphosphate Receptor Requires Its Transition to Open State

Cluster Formation of Inositol 1,4,5-Trisphosphate Receptor Requires Its Transition to Open State

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

Inhibitory Effect of the Phosphoinositide 3-Kinase Inhibitor LY294002 on Muscarinic Acetylcholine Receptor-Induced Calcium Entry in PC12h Cells

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