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Use of Fluorescence Resonance Energy Transfer-based Biosensors for the Quantitative Analysis of Inositol 1,4,5-Trisphosphate Dynamics in Calcium Oscillations*S⃞

By Akihiko Tanimura, Takao Morita, Akihiro Nezu, Akiko Shitara, Noboru Hashimoto and Yosuke Tojyo

Abstract

Inositol 1,4,5-trisphosphate (IP3) is an intracellular messenger that elicits a wide range of spatial and temporal Ca2+ signals, and this signaling versatility is exploited to regulate diverse cellular responses. In this study, we have developed a series of IP3 biosensors that exhibit strong pH stability and varying affinities for IP3, as well as a method for the quantitative measurement of cytosolic concentrations of IP3 ([IP3]i) in single living cells. We applied this method to elucidate IP3 dynamics during agonist-induced Ca2+ oscillations, and we demonstrated cell type-dependent differences in IP3 dynamics, a nonfluctuating rise in [IP3]i and repetitive IP3 spikes during Ca2+ oscillations in COS-7 cells and HSY-EA1 cells, respectively. The size of the IP3 spikes in HSY-EA1 cells varied from 10 to 100 nm, and the [IP3]i spike peak was preceded by a Ca2+ spike peak. These results suggest that repetitive IP3 spikes in HSY-EA1 cells are passive reflections of Ca2+ oscillations, and are unlikely to be essential for driving Ca2+ oscillations. In addition, the interspike periods of Ca2+ oscillations that occurred during the slow rise in [IP3]i were not shortened by the rise in [IP3]i, indicating that IP3-dependent and -independent mechanisms may regulate the frequency of Ca2+ oscillations. The novel method described herein as well as the quantitative information obtained by using this method should provide a valuable and sound basis for future studies on the spatial and temporal regulations of IP3 and Ca2+

Topics: Mechanisms of Signal Transduction
Publisher: American Society for Biochemistry and Molecular Biology
OAI identifier: oai:pubmedcentral.nih.gov:2659248
Provided by: PubMed Central
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