Cell-Based Indicator to Visualize Picomolar Dynamics of Nitric Oxide Release from Living Cells

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• 作者：Moritoshi Sato ; Takahiro Nakajima ; Mariko Goto ; Yoshio Umezawa
• 刊名：Analytical Chemistry
• 出版年：2006
• 出版时间：December 15, 2006
• 年：2006
• 卷：78
• 期：24
• 页码：8175 - 8182
• 全文大小：836K
• 年卷期：v.78,no.24(December 15, 2006)
• ISSN：1520-6882

文摘

We report a novel cell-based indicator that is able tovisualize picomolar dynamics of nitric oxide release fromliving cells. Cells from a pig kidney-derived cell line(PK15) endogenously express soluble guanylate cyclase(sGC), which is a receptor protein for the selectiverecognition of NO. Binding of NO by sGC causes theamplified generation of guanosine 3',5'-cyclic monophosphate (cGMP). To make the PK15 cells into NO indicators, the cells are transfected with a plasmid vectorencoding a fluorescent indicator for cGMP and fluorescence resonance energy transfer is recorded at 480 ± 15and 535 ± 12.5 nm upon excitation of the cells at 440 ±10 nm. The cell-based indicator exhibits exceptionalsensitivity (detection limit of 20 pM), selectivity, reversibility, and reproducibility. The outstanding sensitivity ofthe present indicator has led us to uncover an oscillatoryrelease of picomolar concentrations of NO from hippocampal neurons. We present evidence that Ca²⁺ oscillations in hippocampal neurons underlie the oscillatory NOrelease from the neurons during neurotransmission. Wealso have succeeded in visualizing the extent of diffusingNO from single vascular endothelial cells. The presentcell-based indicator provides a powerful tool to uncoverpicomolar dynamics of NO that regulates a wide range ofcell functions in biological systems.