Objective The myogenic response sets the foundation for blood flow control. Recent findings suggest a role for G protein‐coupled receptors (GPCR) and signaling pathways tied to the generation of reactive oxygen species (ROS). In this regard, this study ascertained the impact of NADPH oxidase (Nox) on myogenic tone in rat cerebral resistance arteries. Methods The study employed real‐time qPCR (RT‐qPCR), pressure myography, and immunohistochemistry. Results Gq blockade abolished myogenic tone in rat cerebral arteries, linking GPCR to mechanosensation. Subsequent work revealed that general (TEMPOL) and mitochondrial specific (MitoTEMPO) ROS scavengers had little impact on myogenic tone, whereas apocynin, a broad spectrum Nox inhibitor, initiated transient dilation. RT‐qPCR revealed Nox1 and Nox2 mRNA expression in smooth muscle cells. Pressure myography defined Nox1 rather than Nox2 is facilitating myogenic tone. We rationalized that Nox1‐generated ROS was initiating this response by impairing the ability of the CaV3.2 channel to elicit negative feedback via BKCa. This hypothesis was confirmed in functional experiments. The proximity ligation assay further revealed that Nox1 and CaV3.2 colocalize within 40 nm of one another. Conclusions Our data highlight that vascular pressurization augments Nox1 activity and ensuing ROS production facilitates myogenic tone by limiting Ca2+ influx via CaV3.2.

Hydrogen peroxide at concentrations below cytotoxic ones causes an increase in the cytoplasmic calcium concentration in human umbilical vein endothelial cells as a result of calcium release from intracellular stores. Two-pore calcium channel blocker trans-NED19 partially suppresses the increase in the level of calcium ions in the cells in response to the addition of hydrogen peroxide. The staining of endothelial cells with the fluorescent stereoisomer cis-NED19 and LysoTracker confirmed the localization of two-pore calcium channels in lysosomes and endolysosomal vesicles.

Hydrogen peroxide, formed in the endothelium, acts as a factor contributing to the relaxation of blood vessels. The reason for this vasodilatory effect could be modulation by H2O2 of calcium metabolism, since mobilization of calcium ions in endothelial cells is a trigger of endothelium-dependent relaxation. The aim of this work was to investigate the influence of H2O2 on the effects of Ca2+-mobilizing agonists in human umbilical vein endothelial cells (HUVEC). We have found that H2O2 in concentration range 10-100 μM increases the rise of [Ca2+]i induced by 5-hydroxytryptamine (5-HT) and carbachol and does not affect the calcium signals of ATP, agonist of type 1 protease-activated receptor SFLLRN, histamine and bradykinin. Using specific agonists of 5-HT1B and 5-HT2B receptors CGS12066B and BW723C86, we have demonstrated that H2O2 potentiates the effects mediated by these types of 5-HT receptors. Potentiation of the effect of BW723C86 can be produced by the induction of endogenous oxidative stress in HUVEC. We have shown that the activation of 5-HT2B receptor by BW723C86 causes production of reactive oxygen species (ROS). Inhibitor of NADPH oxidases VAS2870 suppressed formation of ROS and partially inhibited [Ca2+]i rise induced by BW723C86. Thus, it can be assumed that vasorelaxation induced by endogenous H2O2 in endothelial cells partially occurs due to the potentiation of the agonist-induced calcium signaling.

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In this study, we investigated the effects of NAD(P)H oxidase (NOX) inhibitor VAS2870 (3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine) on the histamine-induced elevation of free cytoplasmic calcium concentration ([Ca2+]i) and the secretion of von Willebrand factor (vWF) in human umbilical vein endothelial cells (HUVECs) and on relaxation of rat aorta in response to histamine. At 10 μM concentration, VAS2870 suppressed the [Ca2+]i rise induced by histamine. Inhibition was not competitive, with IC50 3.64 and 3.22 μM at 1 and 100 μM concentrations of histamine, respectively. There was no inhibition of [Ca2+]i elevation by VAS2870 in HUVECs in response to the agonist of type 1 protease-activated receptor SFLLRN. VAS2870 attenuated histamine-induced secretion of vWF and did not inhibit basal secretion. VAS2870 did not change the degree of histamine-induced relaxation of rat aortic rings constricted by norepinephrine. We suggest that NOX inhibitors might be used as a tool for preventing thrombosis induced by histamine release from mast cells without affecting vasorelaxation.