5-HT2B receptor-mediated calcium release from ryanodine-sensitive intracellular stores in human pulmonary artery endothelial cells

1 We have characterized the 5-hydroxytryptamine (5-HT)-induced calcium signalling in endothelial cells from the human pulmonary artery. Using RT-PCR we show, that of all cloned G-protein coupled 5-HT receptors, these cells express only 5-HT1D beta, 5-HT2B and little 5-HT4 receptor mRNA. 2 In endothelial cells 5-HT inhibits the formation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) via 5-HT1D beta receptors but fails to activate phosphoinositide (PI) turnover. However, the latter pathway is strongly activated by histamine. 3 Despite the lack of detectable inositol phosphate (IF) formation in human pulmonary artery endothelial cells, 5-HT (pD(2) = 5.82 +/- 0.06, n = 6) or the selective 5-HT2 agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (pD(2) = 5.66 +/- 0.03, n = 7) elicited transient calcium signals comparable to those evoked by histamine (pD(2) = 6.44 +/- 0.01, n = 7). Since 5-HT2A and 5-HT2C receptor mRNAs are not detectable in pulmonary artery endothelial cells, activation of 5-HT2B receptors is responsible for the transient calcium release. The calcium transients are independent of the inhibition of adenylate cyclase, since DOI does not stimulate 5-HT1D beta receptors. 4 Both, the 5-HT- and histamine-stimulated calcium signals were also observed when the cells were placed in calcium-free medium. This indicates that 5-HT triggers calcium release from intracellular stores. 5 Heparin is an inhibitor of the IP3-activated calcium release channels on the endoplasmic reticulum. Intracellular infusion of heparin through patch pipettes in voltage clamp experiments failed to block 5-HT-induced calcium signals, whereas it abolished the histamine response. This supports the conclusion that the 5-HT-induced calcium release is independent of IP3 formation. 6 Unlike the histamine response, the 5-HT response was sensitive to micromolar concentrations of ryanodine and, to a lesser extent, ruthenium red. This implies that 5-HT2B receptors trigger calcium release from a ryanodine-sensitive calcium pool. 7 It has been postulated that cyclic ADP-libose (cADPR) is a soluble second messenger which activates ryanodine receptors. However, calcium signals similar to the 5-HT response could not be elicited by intracellular infusion with cADPR. Furthermore, the subsequent application of 5-HT or DOI elicited a calcium signal that was not affected by the above pretreatment. 8 We conclude that human 5-HT2B receptors stimulate calcium release from intracellular stores through a novel pathway, which involves activation of ryanodine receptors, and is independent of PI-hydrolysis and cADPR.