Involvement of cystathionine β-synthase in ATP-induced hydrogen sulfide production in cochlear outer hair cells

Endogenous hydrogen sulfide (H₂S) mediates several biological processes and is an important gaseous signal transmitter akin to carbon monoxide or nitric oxide (NO). The role of H₂S on auditory signaling pathway remains unknown. We have determined whether ATP can induce generation of H₂S in outer hair cells (OHCs) and examined whether H₂S can affect the ATP-stimulated generation of NO in OHCs.

Individual OHCs were isolated enzymatically from the guinea pig cochlea. Using H₂S- and NO-sensitive dyes, 6-Cd^II and 4,5-diaminofluorescein diacetate respectively, we monitored the change in generation of H₂S and NO in response to extracellular ATP in OHCs.

We observed a gradual increase in 6-Cd^II fluorescence induced by extracellular ATP, providing evidence for H₂S production in OHCs. This ATP-induced H₂S production was inhibited by suramin, an antagonist of the P2 receptor. ATP did not induce H₂S production in a Ca²⁺-free medium. This suggests that ATP-induced H₂S production was mediating by an influx of Ca²⁺ via activation of P2-purinergic receptors. The inhibitor of cystathionine β-synthase (CBS), amino-oxyacetic acid (AOAA), counteracted this increase in 6-Cd^II fluorescence, which however was unaffected by cystathionine γ-lyase inhibitors, β-cyanoalanine or D-L-propargylglycine. L-N^G-nitroarginine methyl ester, a non-selective inhibitor of NO synthase, also failed to inhibit ATP-induced H₂S production. In contrast, AOAA inhibited generation of NO in the presence of ATP in OHCs.

The current study suggests that ATP-induced H₂S production is mediating by an influx of Ca²⁺via activation of P2-purinergic receptors. These findings imply generation of H₂S by CBS in response to exposure to ATP in OHCs. Regulatory cross-talk among ATP, H₂S, and NO may also exist in the cellular signaling pathways in OHCs.