Hydrogen Sulfide Activates cAMP Signaling Through Extracellular ATP in Periodontal Ligament Fibroblast

Objectives: Hydrogen sulfide (H₂S), a metabolic byproduct of anaerobic bacteria in periodontal pockets, is one of the major contributors of oral malodor. In fact, high concentrations of H₂S have been reported in periodontal pockets of patients. H₂S has recently been recognized as gaseous mediator with physiologically diverse functions, including modulation of inflammation. The human periodontal ligament fibroblast (PDLF) is capable of producing inflammatory cytokines in response to bacterial infection or environmental stresses. cAMP is an intracellular messenger involved in regulating inflammatory reaction. Extracellular-adenosine triphosphate (eATP) binds to membrane-bound purinergic receptors, providing danger signals to neighbouring cells in response to external stimuli. In this study, we hypothesized that H₂S activates cAMP signaling in PDLFs through eATP.
Methods: PDLFs were treated with H₂S donor and the amount of eATP was determined using bioluminescence assay. Cell viability was examined using the MTS assay. PDLFs were transfected with cAMP-response element (CRE)-reporter plasmid prior H₂S or ATP treatment, and cellular CRE-activation was examined by bioluminescent reporter assay. In addition to carbenoxolone that was used to block eATP release; suramin, a broad spectrum P2 purinergic receptors antagonist was also used to investigate the role of eATP in H₂S-mediated cAMP signaling in PDLFs.
Results: H₂S induced ATP release from PDLFs. Carbenoxolone abolished the effects of H₂S-mediated ATP release from PDLFs, suggesting the involvement of gap-junction hemichannel in this process. Either H₂S or ATP treatment significantly activated cAMP signaling in PDLFs. In addition, suramin substantially reduced H₂S-mediated CRE-activation.
Conclusions: eATP is an important extracellular messenger released under pathologic conditions. We have shown that H₂S induced ATP release in PDLFs through gap-junction hemichannel which in turn served as a signal to activate cAMP signaling. Thus H₂S may exert autocrine or paracrine effects in the periodontium through eATP, modulating genes under the control of cAMP signaling pathway.