Transient Receptor Potential Ankyrin 1 (TRPA1) mediates hydrogen sulfide-induced Ca2+ entry and nitric oxide production in human cerebrovascular endothelium.

Introduction: The gasotransmitter hydrogen sulfide (H 2S) modulates various brain functions, including neuron excitability, synaptic plasticity, and Ca2+ dynamics. Furthermore, H 2S may stimulate nitric oxide (NO) release from cerebrovascular endothelial cells, thereby regulating NOdependent endothelial functions, such as angiogenesis, vasorelaxation and cerebral blood flow (CBF). However, the signaling pathway by which H 2S induces NO release from cerebrovascular endothelial cells is still unclear. Methods: Herein, we exploited single-cell imaging of intracellular Ca2+, H 2S, and NO levels to assess how H 2S induces Ca2+-dependent NO release from the human cerebrovascular endothelial cell line, hCMEC/D3. Results: Administration of the H 2S donor, sodium hydrosulfide (NaHS), induced a dose-dependent increase in [Ca2+ ] i only in the presence of extracellular Ca2+ . NaHS-induced extracellular Ca2+ entry was mediated by the Ca2+-permeable TRPA1 channel, as shown by pharmacological and genetic manipulation of the TRPA1 protein. Furthermore, NaHSdependent TRPA1 activation led to NO release that was abolished by buffering the concomitant increase in [Ca2+ ] i and inhibiting eNOS. Furthermore, the endothelial agonist, adenosine trisphosphate (ATP), caused a biphasic increase in [Ca2+ ] i that was driven by cystathionine -lyase (CSE)-dependent H 2S production and by TRPA1 activation. Consistent with this, ATP-induced NO release was strongly reduced either by blocking CSE or by inhibiting TRPA1. Conclusions: These findings provide the first evidence that H 2S stimulates TRPA1 to induce NO production in human cerebrovascular endothelial cells. In addition, they show that this signaling pathway can be recruited by an endothelial agonist to modulate NO-dependent events at the human neurovascular unit.