Imaging the nanomolar range of nitric oxide with an amplifier-coupled fluorescent indicator in living cells

Abstract

Since NO was found to be an endothelium-derived relaxing factor in 1987 (1, 2), it is now well established that NO is a ubiquitous messenger not only for vascular homeostasis but also for neurotransmission and immune systems (3). For the detection of NO, several methods have been devised so far. However, convincing methods are not currently available for visualizing NO dynamics in single living cells that have enough sensitivity (nM) and spatial resolution (sub-m). Bioimaging of NO has been reported based on chemiluminescence (4) and ESR (5); however, those methods have disadvantages for the functional analysis of NO, such as the use of cytotoxic H 2 O 2 or low spatial resolution. Electrochemical sensing using microelectrodes provides real-time detection of the nanomolar range of NO (6). However, available spatial information is limited to the electrode area and its positioning. Fluorescence methods are generally superior for bioimaging of molecular events in single living cells in terms of their high sensitivity, high spatial resolution (d 0 Х ͞2), and experimental feasibility (7). Several organic fluorescent indicators have already been developed for bioimaging of NO, such as diamino-fluoresceins (8), diaminorhodamines (9), diamino-boron dipyrromethenes (10), and diamino-cyanines (11). However, these organic dyes covalently react with NO ϩ but not with NO radical in the presence of dioxygen and are therefore not reversible sensors for NO. The sensitivity of the organic dyes in living cells has not been determined. The organic dyes easily accumulate in subcellular membranes and emit fluorescence signals there in an NO-independent manner. This membrane accumulation of the dyes substantially increases background signals and interferes with the detection of physiologic low concentration of NO in living cells. As to the concentration of cellular NO, the nanomolar range of NO is believed to be physiologically important for exerting its action. To overcome the limitations of previous fluorescent dyes, we have developed a genetically encoded fluorescent indicator for NO that reversibly detects NO with a high sensitivity (detection limit of 0.1 nM) and visualizes the nanomolar dynamics of NO in single living cells. Plasmid Construction. To construct cDNAs encoding soluble guanylate cyclase (sGC) ␣-CGY and sGC␤-CGY, each stop codon of sGC␣ and sGC␤ was deleted and a linker sequence, GGEQKLI-SEEDLLESR, was added to each C terminus of sGC␣ and sGC␤ by using PCR. Each cDNA encoding sGC␣ and sGC␤ was connected with cDNA encoding a fluorescent cGMP indicator, CGY. Each cDNA encoding sGC␣-CGY and sGC␤-CGY was subcloned at the NheI and NotI sites of a mammalian expression vector, pcDNA3.1(ϩ) (Invitrogen). Materials and MethodsCell Culture and Transfection. CHO-K1 cells were cultured in Ham's F-12 medium supplemented with 10% FCS and 1% penicillin͞ streptomycin at 37°C in 5% CO 2 . Vascular endothelial cells were cultured in Eagle's MEM supplemented with 20% FCS, 1% penicillin͞streptomycin, 1 mM sodium pyruvate, and 0.1 mM none...

Ginsenoside Re, a Main Phytosterol ofPanax ginseng, Activates Cardiac Potassium Channels via a Nongenomic Pathway of Sex Hormones

“…Because reliable methods to measure NO at nanomolar range have not been available until very recently (Sato et al, 2005), we did not directly assess NO release from cardiac myocytes induced by ginsenoside Re. However, our previous report that two different types of NO-trapper, carboxy-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and LNAC, abolished ginsenoside Re-induced I Ks activation (Bai et al, 2004) supports the idea that I Ks enhancement by ginsenoside Re is caused by NO.…”

Information processing deficits and nitric oxide signalling in the phencyclidine model of schizophrenia

“…Conceivably, synaptic NO levels are also increased but remain below the limit of detection using our method. To accurately monitor NO signalling at the synaptic level, other methods are likely needed (Sato et al 2005). Furthermore, the actual in vivo levels of NO have never been accurately determined, although many studies suggest that low nanomolar levels would be the likely physiological range ).…”

Sensing Inside the Living Cells

“…Because a single NO molecule leads to production of many cGMP molecules, this test system possesses a required very high sensitivity. The cGMP sensor is based on the change in relative intensity of two visible fl uorescent proteins that are coupled via FRET mechanism, which allows a wavelength-ratiometric response in microscopic images (Sato et al 2005 ). The attempts of direct NO sensing using functional fl uorescent dyes have also been reported (Pluth et al 2011 ).…”