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

Abstract

Hydrogen sulfide (H₂S), which under physiological conditions exists in cells mainly in the form of the HS^– anion, is considered as a gaseous transmitter of inter- and intracellular signals along with nitrogen monoxide and carbon monoxide. Analysis of the dynamics of H₂S content in living cells is impossible without the creation of sensitive and specific probes. The group of K.H. Ahn synthesized several acedan-based compounds, which in the presence of H₂S attached a sulfhydryl group, forming fluorescent carbocyclic compounds. According to the spectral characteristics and reaction rate with H₂S, the optimal substance was P3, which forms the carbocyclic compound csP3 with the same large Stokes shift as P3 (approx. 130 nm) and has a brighter fluorescence. In this work, we tested the suitability of csP3 for recording changes in H₂S in solutions simulating the minimum salt composition of the intracellular medium, as well as in cells of primary neuronal culture from the rat cerebral cortex. It was found that the fluorescence intensity of csP3, which was formed when Na₂S (H₂S donor, 100 and 300 µM) was added to the P3 solution, differed for solutions corresponding in salt composition to the extracellular medium and cytosol. In both cases, fluorescence increased in the presence of bicarbonate (NaHCO₃, 10 mM). A decrease in the polarity of solutions due to the addition of dimethyl sulfoxide (30% by volume) shifted the emission by approx. 10 nm to the shorter wavelength region and doubled the intensity. Glutamate (10 µM, in the presence of 10 µM of glycine, 0 Mg²⁺) increased the fluorescence of the probe, but only in those neurons in which delayed deregulation of calcium homeostasis did not occur. The addition of P3 or csP3 to the cell culture caused a rapid increase in the fluorescent signal, which was replaced by a slow signal growth after 3–5 min. It was concluded that the product of the reaction of P3 with H₂S was sensitive to changes in the salt composition of the intracellular medium and could be redistributed in cells between an aqueous and more hydrophobic environment. These circumstances made it difficult to interpret the growth of P3 fluorescence in cells as a quantitative indicator of the presence of H₂S and required additional studies of the properties of this and structurally related H₂S probes.