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

Purpose 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. Methods Individual OHCs were isolated enzymatically from the guinea pig cochlea. Using H2S- and NO-sensitive dyes, 6-CdII and 4,5-diaminofluorescein diacetate respectively, we monitored the change in generation of H2S and NO in response to extracellular ATP in OHCs. Results We observed a gradual increase in 6-CdII 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-CdII fluorescence, which however was unaffected by cystathionine γ-lyase inhibitors, β-cyanoalanine or D-L-propargylglycine. L-NG-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. Conclusion 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.