Nitric oxide detection and visualization in biological systems. Applications of the FNOCT method

Area of Science:

• Biochemistry
• Cell Biology
• Analytical Chemistry

Background:

• Nitric oxide (NO) is a crucial signaling molecule involved in various physiological processes.
• Accurate measurement of NO release from cells and compounds is essential for understanding its biological roles.
• Existing methods for NO detection may lack sensitivity or spatial and temporal resolution.

Purpose of the Study:

• To develop and validate Fluorescent Nitric Oxide Cheletropic Traps (FNOCTs) for sensitive and high-resolution NO detection.
• To investigate NO release from specific cellular models and NO-donating agents.
• To demonstrate the capability of FNOCTs for monitoring NO dynamics at the single-cell level.

Main Methods:

• Application of FNOCTs with specific excitation and emission wavelengths for NO trapping.
• Utilizing FNOCT-4 to quantify extracellular NO release from activated macrophages.
• Employing membrane-permeating FNOCT-5 derivative for intracellular NO monitoring in single cells via fluorescence microscopy.

Main Results:

• FNOCTs demonstrated high sensitivity in detecting NO.
• Maximum extracellular NO release from LPS-activated macrophages was quantified at 37.5 nmol h(-1) (10(6) cells)(-1) at 11 hours post-activation.
• Intracellular NO production kinetics varied between endothelial cells (immediate) and macrophages (delayed, 4-8 hours).

Conclusions:

• FNOCTs provide a sensitive tool for monitoring NO release from single cells and NO-donating compounds.
• The method offers excellent temporal and spatial resolution for NO dynamics studies.
• FNOCTs are valuable for investigating NO signaling in complex biological systems.