Soda Teresa, Brunetti Valentina, De Sarro Giovambattista, Biella Gerardo, Moccia Francesco, Berra-Romani Roberto, Scarpellino Giorgia
Department of Health Science, University Magna Graecia of Catanzaro, 88100 Catanzaro, Italy.
Department of Biology and Biotechnology, Laboratory of General Physiology, "L. Spallanzani", University of Pavia, 27100 Pavia, Italy.
Curr Neuropharmacol. 2025;23(9):1119-1133. doi: 10.2174/011570159X349872250124124612.
The gasotransmitter hydrogen sulfide (HS) modulates various brain functions, including neuron excitability, synaptic plasticity, and Ca dynamics. Furthermore, HS may stimulate nitric oxide (NO) release from cerebrovascular endothelial cells, thereby regulating NO-dependent endothelial functions, such as angiogenesis, vasorelaxation, and cerebral blood flow (CBF). However, the signaling pathway by which HS induces NO release from cerebrovascular endothelial cells is still unclear.
Herein, we exploited single-cell imaging of intracellular Ca, HS, and NO levels to assess how HS induces Ca-dependent NO release from the human cerebrovascular endothelial cell line, hCMEC/D3.
Administration of the HS donor, sodium hydrosulfide (NaHS), induced a dose-dependent increase in (Ca)i only in the presence of extracellular Ca. NaHS-induced extracellular Ca entry was mediated by the Ca-permeable TRPA1 channel, as shown by pharmacological and genetic manipulation of the TRPA1 protein. Furthermore, NaHS-dependent TRPA1 activation led to NO release that was abolished by buffering the concomitant increase in (Ca) and inhibiting eNOS. Furthermore, the endothelial agonist, adenosine trisphosphate (ATP), caused a long-lasting elevation in (Ca) that was driven by cystathionine γ-lyase (CSE)-dependent H2S production and by TRPA1 activation. Consistent with this, ATP-induced NO release was strongly reduced either by blocking CSE or by inhibiting TRPA1.
These findings demonstrate for the time that HS stimulates TRPA1 to induce NO production in human brain microvascular endothelial cells. Additionally, they show that this signaling pathway can be recruited by an endothelial agonist to modulate NO-dependent events at the human neurovascular unit.
气体信号分子硫化氢（HS）可调节多种脑功能，包括神经元兴奋性、突触可塑性和钙动力学。此外，HS可刺激脑血管内皮细胞释放一氧化氮（NO），从而调节依赖NO的内皮功能，如血管生成、血管舒张和脑血流量（CBF）。然而，HS诱导脑血管内皮细胞释放NO的信号通路仍不清楚。
在此，我们利用细胞内钙、HS和NO水平的单细胞成像来评估HS如何诱导人脑血管内皮细胞系hCMEC/D3中依赖钙的NO释放。
仅在细胞外钙存在的情况下，给予HS供体硫氢化钠（NaHS）可诱导细胞内钙（Ca）i呈剂量依赖性增加。如对TRPA1蛋白进行药理学和基因操作所示，NaHS诱导的细胞外钙内流由钙通透的TRPA1通道介导。此外，NaHS依赖的TRPA1激活导致NO释放，通过缓冲伴随的（Ca）增加和抑制内皮型一氧化氮合酶（eNOS）可消除这种释放。此外，内皮激动剂三磷酸腺苷（ATP）导致（Ca）的持久升高，这是由胱硫醚γ-裂解酶（CSE）依赖的H2S产生和TRPA1激活驱动的。与此一致，阻断CSE或抑制TRPA1均可显著降低ATP诱导的NO释放。
这些发现首次证明HS刺激TRPA1在人脑海微血管内皮细胞中诱导NO生成。此外，它们表明这种信号通路可被内皮激动剂募集，以调节人神经血管单元处依赖NO的事件。
