Synergistic Interaction of 5-HT_1B and 5-HT_2B Receptors in Cytoplasmic Ca²⁺ Regulation in Human Umbilical Vein...

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.