Monitoring of IP3 dynamics during Ca2+ oscillations in HSY human parotid cell line with FRET-based IP3 biosensors.
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 the present 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 demonstrated cell type-dependent differences in IP(3) dynamics; a non-fluctuating 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. 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+).
Tanimura
Akihiko
A
Department of Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan.
Morita
Takao
T
Nezu
Akihiro
A
Tojyo
Yosuke
Y
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002460:Cell Line; D002522:Chlorocebus aethiops; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D010306:Parotid Gland / Q000166:cytology / Q000378:metabolism*
D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium