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

ABSTRACT: Inositol 1,4,5-trisphosphate (IP(3)) is an intracellular messenger that elicits a wide range of spatial and temporal Ca(2+) signals, and this signaling versatility is exploited to regulate diverse cellular responses. In this study, we have developed a series of IP(3) biosensors that exhibit strong pH stability and varying affinities for IP(3), as well as a method for the quantitative measurement of cytosolic concentrations of IP(3) ([IP(3)](i)) in single living cells. We applied this method to elucidate IP(3) dynamics during agonist-induced Ca(2+) oscillations, and we demonstrated cell type-dependent differences in IP(3) dynamics, a nonfluctuating rise in [IP(3)](i) and repetitive IP(3) spikes during Ca(2+) oscillations in COS-7 cells and HSY-EA1 cells, respectively. The size of the IP(3) spikes in HSY-EA1 cells varied from 10 to 100 nm, and the [IP(3)](i) spike peak was preceded by a Ca(2+) spike peak. These results suggest that repetitive IP(3) spikes in HSY-EA1 cells are passive reflections of Ca(2+) oscillations, and are unlikely to be essential for driving Ca(2+) oscillations. In addition, the interspike periods of Ca(2+) oscillations that occurred during the slow rise in [IP(3)](i) were not shortened by the rise in [IP(3)](i), indicating that IP(3)-dependent and -independent mechanisms may regulate the frequency of Ca(2+) 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 IP(3) and Ca(2+).

SUBMITTER: Tanimura A 

PROVIDER: S-EPMC2659248 | biostudies-other | 2009 Mar

REPOSITORIES: biostudies-other