Cell-based indicator to visualize picomolar dynamics of nitric oxide release from living cells.
We report a novel cell-based indicator that is able to visualize picomolar dynamics of nitric oxide release from living cells. Cells from a pig kidney-derived cell line (PK15) endogenously express soluble guanylate cyclase (sGC), which is a receptor protein for the selective recognition of NO. Binding of NO by sGC causes the amplified generation of guanosine 3',5'-cyclic monophosphate (cGMP). To make the PK15 cells into NO indicators, the cells are transfected with a plasmid vector encoding a fluorescent indicator for cGMP and fluorescence resonance energy transfer is recorded at 480 +/- 15 and 535 +/- 12.5 nm upon excitation of the cells at 440 +/- 10 nm. The cell-based indicator exhibits exceptional sensitivity (detection limit of 20 pM), selectivity, reversibility, and reproducibility. The outstanding sensitivity of the present indicator has led us to uncover an oscillatory release of picomolar concentrations of NO from hippocampal neurons. We present evidence that Ca2+ oscillations in hippocampal neurons underlie the oscillatory NO release from the neurons during neurotransmission. We also have succeeded in visualizing the extent of diffusing NO from single vascular endothelial cells. The present cell-based indicator provides a powerful tool to uncover picomolar dynamics of NO that regulates a wide range of cell functions in biological systems.
Sato
Moritoshi
M
Department of Chemistry, School of Science, The University of Tokyo, Hongo, Tokyo 113-0033, Japan.
Nakajima
Takahiro
T
Goto
Mariko
M
Umezawa
Yoshio
Y
D000818:Animals; D020013:Calcium Signaling / Q000502:physiology; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism; D042783:Endothelial Cells; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006162:Guanylate Cyclase / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D036103:Nanotechnology; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism* / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D009435:Synaptic Transmission / Q000502:physiology; D013997:Time Factors; D014162:Transfection / Q000379:methods
D005456:Fluorescent Dyes; D009569:Nitric Oxide; D006162:Guanylate Cyclase; D006152:Cyclic GMP