On the Feasibility of Using an Acedane-Based Fluorescent Probe to Monitor Hydrogen Sulfide in Primary Neuronal Cultures

Hydrogen sulfide (H₂S), which, under physiological conditions, exists in cells mainly in the form of anion HS^–, is considered as a gaseous transmitter of inter- and intracellular signals along with nitrogen oxide and carbon monoxide. Analysis of the dynamics of H₂S content in living cells is impossible without creating sensitive and specific probes. Several acedan-based compounds have been synthesized in the group of K.H. Ahn (Singha et al., 2015. Anal. Chem. 87 (2), 1188–1195). In the presence of H₂S these probes attach to the sulfhydrilic group and form fluorescent carbocyclic compounds. The carbocyclic derivative of P3, compound csP3, was found to be optimal for fluorescence-microscopic studies in terms of spectral characteristics and response time to H₂S. In this work, we tested the suitability of csP3 to record H₂S changes in buffers mimicking the salt composition of the intracellular environment and in primary neuronal culture cells from rat cerebral cortex. It was found that reducing the polarity of the solution by adding dimethyl sulfoxide (30% by volume) caused a blue shift of the emission by ~10 nm and a twofold increase in fluorescence intensity. The csP3 fluorescence depends on the salt composition and increases in the presence of bicarbonate (NaHCO₃, 10 mM). Addition of P3 or csP3 to the neuronal culture caused a rapid increase in fluorescence, which was followed by a slow increase in fluorescence signal after 3–5 min. Glutamate (10 μM, in the presence of 10 μM glycine, 0 Mg²⁺) increased probe fluorescence, but only in those neurons in which delayed deregulation of calcium homeostasis did not occur. We conclude that the product of the reaction of P3 with H₂S is sensitive to a change in the salt composition of the intracellular medium and can be redistributed in cells between water and more hydrophobic environment. This means that an increase in P3 fluorescence in cells, especially after the addition of glutamate to neurons, does not necessarily indicate an increase in H₂S concentration. To confirm the feasibility of using P3 and structurally related probes as quantitative indicators of H₂S presence, additional studies of the properties of these compounds are needed.