Expression of ryanodine receptor type 3 and TRP channels in endothelial cells: comparison of in situ and cultured human endothelial cells

Objective: Ca2+ mobilization plays an important role in endothelial functio n by stimulating Ca2+-dependent synthesis of vasodilating factors. In addit ion to inositol-1,4,5-trisphosphate (InsP(3)) mediated Ca2+ mobilization. C a2+ release from ryanodine-sensitive pools and Ca2+ -influx through TRP cha nnels have been suggested to be important in endothelial Ca2+-signaling. Ho wever, the function and molecular identity of TRP channels and ryanodine re ceptors in human endothelium in situ are still elusive. We hypothesized tha t expression of ryanodine-receptors (RyR) and TRP channels differs between human endothelium in situ and in cultured cells. Methods: By combining sing le-cell RT-PCR and patch-clamp techniques, expression of RyR and TRP channe ls was determined in situ in endothelial cells of human mesenteric artery ( HMAECs) obtained from patients undergoing bowel resection and in the endoth elial cell line EA.hy926. Results: At the single cell level, expression of RyR 3 was detected in 25 and 5% of HMAECs and EA.hy926 samples, respectivel y. Expression of the RyR 1 and 2 was not detected in either HMAECs or EA.hy 926. In patch-clamp experiments in HMAECs, applications of caffeine (0.5 mM ) induced sustained hyperpolarization mediated by activation of Ca2+-activa ted K channels. In EA.hy926. caffeine-induced hyperpolarization was not det ected. Single HMAECs expressed the TRP genes, TRP1 and TRP3, but not TRP 4 and 6. The TRP1 was the predominantly expressed TRP gene in HMAECs in situ whereas TRP3 expression was rarely detected. EA.hy926 expressed only TRP1. In patch clamp experiments in HMAECs, Ca2+-store depletion activated non-se lective cation currents leading to Ca2+ entry. Conclusions: Our findings su ggest that, in addition to InsP(3) mediated Ca2+ release, Ca2+ release from ryanodine-sensitive stores mediated by RyR3 and Ca2+ entry through TRPI mi ght represent important components of endothelial Ca2+ signaling in situ an d thereby of endothelial function in intact human blood vessels. (C) 2001 E lsevier Science B.V. All rights reserved.