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.

Second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) triggers Ca²⁺ release via two-pore channels (TPCs) localized in endolysosomal vesicles. The aim of the present work is to evaluate the role of TPCs in the action of norepinephrine (NE), angiotensin II (AngII), vasopressin (AVP), and 5-hydroxytriptamine (5-HT) on free cytoplasmic calcium concentration ([Ca²⁺]_i) in smooth muscle cells (SMCs) isolated from rat aorta and on aorta contraction. To address this issue, the NAADP structural analogue and inhibitor of TPCs, NED 19, was applied. We have demonstrated a high degree of colocalization of the fluorescent signals of cis-NED 19 and endolysosmal probe LysoTracker in SMCs. Both cis- or trans-NED 19 inhibited the rise of [Ca²⁺]_i in SMCs induced by 100 μM NE by 50–60%. IC₅₀ for cis- and trans-NED 19 were 2.7 and 8.9 μM, respectively. The inhibition by NED 19 stereoisomers of the effects of AngII, AVP, and 5-HT was much weaker. Both forms of NED 19 caused relaxation of aortic rings preconstricted by NE, with relative potency of cis-NED 19 several times higher than that of trans-NED 19. Inhibition by cis-NED 19 of NE-induced contraction was maintained after intensive washing and slowly reversed within an hour of incubation. Cis- and trans-NED 19 did not cause decrease in the force of aorta contraction in response to Ang II and AVP, and only slightly relaxed aorta preconstricted by 5-HT and by KCl. Suppression of TPC1 in SMCs with siRNA caused a 40% decrease in [Ca²⁺]_i in response to NE, whereas siRNA against TPC2 did not change NE calcium signaling. These data suggest that TPC1 is involved in the NE-stimulated [Ca²⁺]_i rise in SMCs. Inhibition of TPC1 activity by NED 19 could be the reason for partial inhibition of aortic rings contraction in response to NE.

The aim of this work was to explore the involvement of 5-HT_1B and 5-HT_2B receptors (5-HT_1BR and 5-HT_2BR) in the regulation of free cytoplasmic calcium concentration ([Ca²⁺]_i) in human umbilical vein endothelial cells (HUVEC). We have shown by quantitative PCR analysis, that 5-HT_1BR and 5-HT_2BR mRNAs levels are almost equal in HUVEC. Immunofluorescent staining demonstrated, that 5-HT_1BR and 5-HT_2BR are expressed both in plasma membrane and inside the cells. Intracellular 5-HT_1BR are localized mainly in the nuclear region, whereas 5-HT_2BR receptors are almost evenly distributed in HUVEC. 5-HT, 5-HT_1BR agonist CGS12066B, or 5-HT_2BR agonist BW723C86 added to HUVEC caused a slight increase in [Ca²⁺]_i, which was much lower than that of histamine, ATP, or SFLLRN, an agonist of protease-activated receptors (PAR1). However, activation of 5-HT_1BR with CGS12066B followed by activation of 5-HT_2BR with BW723C86 manifested a synergism of response, since several-fold higher rise in [Ca²⁺]_i occurred. CGS12066B caused more than a 5-fold increase in [Ca²⁺]_i rise in HUVEC in response to 5-HT. This 5-HT induced [Ca²⁺]_i rise was abolished by 5-HT_2BR antagonist RS127445, indicating that extracellular 5-HT acts through 5-HT_2BR. Synergistic [Ca²⁺]_i rise in response to activation of 5-HT1BR and 5-HT_2BR persisted in a calcium-free medium. It was suppressed by the phospholipase C inhibitor U73122 and was not inhibited by the ryanodine and NAADP receptors antagonists dantrolene and NED-19. [Ca²⁺]_i measurements in single cells demonstrated that activation of 5-HT_2BR alone by BW723C86 caused single asynchronous [Ca²⁺]_i oscillations in 19.8 ± 4.2% (n = 3) of HUVEC that occur with a long delay (66.1 ± 4.3 s, n = 71). On the contrary, histamine causes a simultaneous and almost immediate increase in [Ca²⁺]_i in all the cells. Pre-activation of 5-HT_1BR by CGS12066B led to a 3–4 fold increase in the number of HUVEC responding to BW723C86, to synchronization of their responses with a delay shortening, and to the bursts of [Ca²⁺]_i oscillations in addition to single oscillations. In conclusion, to get a full rise of [Ca²⁺]_i in HUVEC in response to 5-HT, simultaneous activation of 5-HT_1BR and 5-HT_2BR is required. 5-HT causes an increase in [Ca²⁺]_i via 5-HT_2BR while 5-HT_1BR could be activated by the membrane-permeable agonist CGS12066B. We hypothesized that CGS12066B acts via intracellular 5-HT_1BR inaccessible to extracellular 5-HT. Intracellular 5-HT_1BR might be activated by 5-HT which could be accumulated in EC under certain pathological conditions.