Search

The aim of this work was to explore the involvement of 5-HT 1B and 5-HT 2B receptors (5-HT 1B R and 5-HT 2B R) in the regulation of free cytoplasmic calcium concentration ([Ca 2+ ] i ) in human umbilical vein endothelial cells (HUVEC). We have shown by quantitative PCR analysis, that 5-HT 1B R and 5-HT 2B R mRNAs levels are almost equal in HUVEC. Immunofluorescent staining demonstrated, that 5-HT 1B R and 5-HT 2B R are expressed both in plasma membrane and inside the cells. Intracellular 5-HT 1B R are localized mainly in the nuclear region, whereas 5-HT 2B R receptors are almost evenly distributed in HUVEC. 5-HT, 5-HT 1B R agonist CGS12066B, or 5-HT 2B R agonist BW723C86 added to HUVEC caused a slight increase in [Ca 2+ ] i , which was much lower than that of histamine, ATP, or SFLLRN, an agonist of protease-activated receptors (PAR1). However, activation of 5-HT 1B R with CGS12066B followed by activation of 5-HT 2B R with BW723C86 manifested a synergism of response, since several-fold higher rise in [Ca 2+ ] i occurred. CGS12066B caused more than a 5-fold increase in [Ca 2+ ] i rise in HUVEC in response to 5-HT. This 5-HT induced [Ca 2+ ] i rise was abolished by 5-HT 2B R antagonist RS127445, indicating that extracellular 5-HT acts through 5-HT 2B R. Synergistic [Ca 2+ ] i rise in response to activation of 5-HT1BR and 5-HT 2B R 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. [Ca 2+ ] i measurements in single cells demonstrated that activation of 5-HT 2B R alone by BW723C86 caused single asynchronous [Ca 2+ ] 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 [Ca 2+ ] i in all the cells. Pre-activation of 5-HT 1B R 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 [Ca 2+ ] i oscillations in addition to single oscillations. In conclusion, to get a full rise of [Ca 2+ ] i in HUVEC in response to 5-HT, simultaneous activation of 5-HT 1B R and 5-HT 2B R is required. 5-HT causes an increase in [Ca 2+ ] i via 5-HT 2B R while 5-HT 1B R could be activated by the membrane-permeable agonist CGS12066B. We hypothesized that CGS12066B acts via intracellular 5-HT 1B R inaccessible to extracellular 5-HT. Intracellular 5-HT 1B R might be activated by 5-HT which could be accumulated in EC under certain pathological conditions.

The new coronavirus infection (COVID-19) pandemic caused by SARS-CoV-2 has many times surpassed the epidemics caused by SARS-CoV and MERS-CoV. The reason for this was the presence of sites in the protein sequence of SARS-CoV-2 that provide interaction with a broader range of receptor proteins on the host cell surface. In this review, we consider both already known receptors common to SARS-CoV and SARS-CoV-2 and new receptors specific to SARS-CoV-2., Competing Interests: The authors declare that they have no conflict of interest., (© Pleiades Publishing, Ltd. 2023, ISSN 1990-7478, Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology, 2023, Vol. 17, No. 1, pp. 1–11. © Pleiades Publishing, Ltd., 2023.Russian Text © The Author(s), 2022, published in Biologicheskie Membrany, 2022, Vol. 39, No. 6, pp. 419–430.)

Here, we propose a new approach for quantitative estimation of von Willebrand factor (vWF) exposed on the surface of endothelial cells (ECs) using the ARC1779 aptamer that interacts with the vWF A1 domain. To visualize complex formation between vWF and the aptamer, the latter was conjugated with the Cy5 fluorescent label. Cultured human umbilical vein endothelial cells (HUVEC) were stained with the ARC1779-Cy5 conjugate and imaged with a fluorescence microscope. The images were analyzed with the CellProfiler software. vWF released from the Weibel-Palade bodies was observed as bright dot-like structures of round and irregular shape, the number of which increased several times after HUVEC exposure to histamine or thrombin. Staining with ARC1779-Cy5 also revealed long filamentous vWF structures on the surface of activated HUVEC. vWF secretion by ECs is activated by the second messengers cAMP and Ca2+. There is evidence that hydrogen peroxide also acts as a second messenger in ECs. In addition, exogenous H2O2 formed in leukocytes can enter ECs. The aim of our study was to determine the effect of H2O2 on the vWF exposure at the surface of HUVEC using the proposed method. It was shown that hydrogen peroxide at concentration 100 µM, which is lower than the cytotoxicity threshold of H2O2 for cultured HUVEC, increased several times the number of dot-like structures and total amount of vWF exposed on plasma membrane of HUVEC, which suggest that H2O2 acts as a mediator that activates exocytosis of Weibel-Palade bodies and vWF secretion in the vascular endothelium during inflammation and upon elevated generation of endogenous reactive oxygen species in ECs.

Second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) triggers Ca 2+ 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 ([Ca 2+ ] 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 [Ca 2+ ] i in SMCs induced by 100 μM NE by 50-60%. IC 50 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 [Ca 2+ ] 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 [Ca 2+ ] 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.

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 ([Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ] 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.