Synergistic Interaction of 5-HT and 5-HT Receptors in Cytoplasmic Ca Regulation in Human Umbilical Vein Endothelial Cells: Possible Involvement in Pathologies.

The aim of this work was to explore the involvement of 5-HT and 5-HT receptors (5-HTR and 5-HTR) in the regulation of free cytoplasmic calcium concentration ([Ca]) in human umbilical vein endothelial cells (HUVEC). We have shown by quantitative PCR analysis, that 5-HTR and 5-HTR mRNAs levels are almost equal in HUVEC. Immunofluorescent staining demonstrated, that 5-HTR and 5-HTR are expressed both in plasma membrane and inside the cells. Intracellular 5-HTR are localized mainly in the nuclear region, whereas 5-HTR receptors are almost evenly distributed in HUVEC. 5-HT, 5-HTR agonist CGS12066B, or 5-HTR agonist BW723C86 added to HUVEC caused a slight increase in [Ca], which was much lower than that of histamine, ATP, or SFLLRN, an agonist of protease-activated receptors (PAR1). However, activation of 5-HTR with CGS12066B followed by activation of 5-HTR with BW723C86 manifested a synergism of response, since several-fold higher rise in [Ca] occurred. CGS12066B caused more than a 5-fold increase in [Ca] rise in HUVEC in response to 5-HT. This 5-HT induced [Ca] rise was abolished by 5-HTR antagonist RS127445, indicating that extracellular 5-HT acts through 5-HTR. Synergistic [Ca] rise in response to activation of 5-HT1BR and 5-HTR 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] measurements in single cells demonstrated that activation of 5-HTR alone by BW723C86 caused single asynchronous [Ca] oscillations in 19.8 ± 4.2% ( = 3) of HUVEC that occur with a long delay (66.1 ± 4.3 s, = 71). On the contrary, histamine causes a simultaneous and almost immediate increase in [Ca] in all the cells. Pre-activation of 5-HTR 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] oscillations in addition to single oscillations. In conclusion, to get a full rise of [Ca] in HUVEC in response to 5-HT, simultaneous activation of 5-HTR and 5-HTR is required. 5-HT causes an increase in [Ca] via 5-HTR while 5-HTR could be activated by the membrane-permeable agonist CGS12066B. We hypothesized that CGS12066B acts via intracellular 5-HTR inaccessible to extracellular 5-HT. Intracellular 5-HTR might be activated by 5-HT which could be accumulated in EC under certain pathological conditions.