Expression of ryanodine receptor type 3 and TRP channels in endothelial cells: comparison of in situ and cultured human endothelial cells.
OBJECTIVE
Ca(2+) mobilization plays an important role in endothelial function by stimulating Ca(2+)-dependent synthesis of vasodilating factors. In addition to inositol-1,4,5-trisphosphate (InsP(3)) mediated Ca(2+) mobilization, Ca(2+) release from ryanodine-sensitive pools and Ca(2+)-influx through TRP channels have been suggested to be important in endothelial Ca(2+)-signaling. However, the function and molecular identity of TRP channels and ryanodine receptors in human endothelium in situ are still elusive. We hypothesized that expression of ryanodine-receptors (RyR) and TRP channels differs between human endothelium in situ and in cultured cells.


METHODS
By combining single-cell RT-PCR and patch-clamp techniques, expression of RyR and TRP channels was determined in situ in endothelial cells of human mesenteric artery (HMAECs) obtained from patients undergoing bowel resection and in the endothelial 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, respectively. Expression of the RyR 1 and 2 was not detected in either HMAECs or EA.hy926. In patch-clamp experiments in HMAECs, applications of caffeine (0.5 mM) induced sustained hyperpolarization mediated by activation of Ca(2+)-activated K channels. In EA.hy926, caffeine-induced hyperpolarization was not detected. 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, Ca(2+)-store depletion activated non-selective cation currents leading to Ca(2+) entry.


CONCLUSIONS
Our findings suggest that, in addition to InsP(3) mediated Ca(2+) release, Ca(2+) release from ryanodine-sensitive stores mediated by RyR3 and Ca(2+) entry through TRP1 might represent important components of endothelial Ca(2+) signaling in situ and thereby of endothelial function in intact human blood vessels.
Köhler
R
R
Department of Endocrinology and Nephrology, Medical Center Benjamin Franklin, Freie Universität Berlin, Berlin, Germany. koe@zedat.fu-berlin.de
Brakemeier
S
S
Kühn
M
M
Degenhardt
C
C
Buhr
H
H
Pries
A
A
Hoyer
J
J
D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000032:analysis / Q000378:metabolism*; D002469:Cell Separation; D003106:Colon; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D008564:Membrane Potentials; D008638:Mesenteric Arteries; D018408:Patch-Clamp Techniques; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019837:Ryanodine Receptor Calcium Release Channel / Q000032:analysis*; D050052:TRPC Cation Channels
D015220:Calcium Channels; D019837:Ryanodine Receptor Calcium Release Channel; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D002118:Calcium