Allyl Isothiocianate Induces Ca2+ Signals and Nitric Oxide Release by Inducing Reactive Oxygen Species Production in the Human Cerebrovascular Endothelial Cell Line hCMEC/D3

Abstract

Nitric oxide (NO) represents a crucial mediator to regulate cerebral blood flow (CBF) in the human brain both under basal conditions and in response to somatosensory stimulation. An increase in intracellular Ca2+ concentrations ([Ca2+]i) stimulates the endothelial NO synthase to produce NO in human cerebrovascular endothelial cells. Therefore, targeting the endothelial ion channel machinery could represent a promising strategy to rescue endothelial NO signalling in traumatic brain injury and neurodegenerative disorders. Allyl isothiocyanate (AITC), a major active constituent of cruciferous vegetables, was found to increase CBF in non-human preclinical models, but it is still unknown whether it stimulates NO release in human brain capillary endothelial cells. In the present investigation, we showed that AITC evoked a Ca2+-dependent NO release in the human cerebrovascular endothelial cell line, hCMEC/D3. The Ca2+ response to AITC was shaped by both intra- and extracellular Ca2+ sources, although it was insensitive to the pharmacological blockade of transient receptor potential ankyrin 1, which is regarded to be among the main molecular targets of AITC. In accord, AITC failed to induce transmembrane currents or to elicit membrane hyperpolarization, although NS309, a selective opener of the small- and intermediate-conductance Ca2+-activated K+ channels, induced a significant membrane hyperpolarization. The AITC-evoked Ca2+ signal was triggered by the production of cytosolic, but not mitochondrial, reactive oxygen species (ROS), and was supported by store-operated Ca2+ entry (SOCE). Conversely, the Ca2+ response to AITC did not require Ca2+ mobilization from the endoplasmic reticulum, lysosomes or mitochondria. However, pharmacological manipulation revealed that AITC-dependent ROS generation inhibited plasma membrane Ca2+-ATPase (PMCA) activity, thereby attenuating Ca2+ removal across the plasma membrane and resulting in a sustained increase in [Ca2+]i. In accord, the AITC-evoked NO release was driven by ROS generation and required ROS-dependent inhibition of PMCA activity. These data suggest that AITC could be exploited to restore NO signalling and restore CBF in brain disorders that feature neurovascular dysfunction.

Cracking the Endothelial Calcium (Ca2+) Code: A Matter of Timing and Spacing

“…TRPA1 channels have also been detected in human cerebrovascular endothelial cells, where they are gated by 4-HNE but not by AITC. However, TRPA1-mediated Ca 2+ sparklets were not investigated in this study [119].…”

“…Studies on rodents have shown that SK Ca /IK Ca channel currents were detectable in freshly isolated mesenteric artery endothelial cells [101] and in cerebral [166] and pulmonary [103] resistance arteries. Conversely, they are absent in mouse capillary endothelial cells [167], although preliminary evidence suggests that they are present at the human BBB [119].…”

Two Signaling Modes Are Better than One: Flux-Independent Signaling by Ionotropic Glutamate Receptors Is Coming of Age

“…Mouse brain microcirculation has long been investigated to gain cellular and molecular mechanisms that regulate the functions subserved by the human BBB [51,220,240,241]. However, emerging evidence suggests that the blend of ion channels by which CECs detect neuronal activity can differ between mouse and human microvasculature [242][243][244]. Future work could assess whether NMDARs also signal in a flux-independent manner in mouse brain microvascular endothelial cells.…”

Mitochondrial dysfunction at the crossroad of cardiovascular diseases and cancer

“…Novel cyclosporine A analogues that lack immunosuppressant activity, such as Debio025 and NIM811, are currently under investigation. The efficacy of untargeted antioxidant strategies to attenuate ROS overproduction in mitochondria is also limited by their off-targeted effects on cytosolic ROS, which stimulate cellular survival and other primary functions at moderate levels [ 432 , 434 ]. It has been suggested to exploit the highly hyperpolarized ΔΨm to specifically deliver cationic antioxidant molecules into the mitochondrial matrix [ 435 ]; unfortunately, the ΔΨm is rather depolarized in many CVDs [ 432 ].…”