Synergistic Interaction of 5-HT[sub.1B] and 5-HT[sub.2B] Receptors in Cytoplasmic Ca[sup.2+] Regulation in Human Umbilical Vein Endothelial Cells: Possible Involvement in Pathologies

• Published in: International journal of molecular sciences Vol. 24; no. 18
• Main Authors: Rybakova, Elena Yu, Avdonin, Piotr P, Trufanov, Sergei K, Goncharov, Nikolay V, Avdonin, Pavel V
• Format: Journal Article
• Language: English
• Published: MDPI AG 01.09.2023
• Subjects: Endothelium; Ethylenediaminetetraacetic acid; Mediation; Scientific equipment and supplies industry; United States; Germany
• ISSN: 1422-0067; 1422-0067
• DOI: 10.3390/ijms241813833

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