Expression of Ryanodine Receptor 3 and Trp Channels in Endothelium of Human Mesenteric Artery: A Single-Cell Rt-Pcr and Patch-Clamp Analysis in Situ .

Abstract

P146 Ca 2+ mobilization plays an important role in endothelial function by stimulating Ca 2+ -dependent synthesis of vasodilating factors. In addition to InsP 3 - mediated Ca 2+ store depletion, Ca 2+ release from ryanodine-sensitive pools and Ca 2+ -influx through cation channels of the TRP-gene family have been suggested to be involved in endothelial Ca 2+ signaling. In cultured endothelial cells (EC) the function and expression of ryanodine-receptors (RyR) and TRP channels might differ substantially from those in native endothelium of human blood vessels. We, therefore, characterized expression and function of RyR and TRP channels in EC of small human mesenteric artery (MA) by use of single-cell RT-PCR and patch-clamp techniques in situ. MA were isolated from colon specimens of patients subjected to hemicolectomy. For single-cell RT-PCR and PC experiments single human mesenteric artery EC (HMAEC) were harvested directly from the luminal vessel with the patch pipette. Expression of the RyR subtype 3, but not of the RyR1 and RyR2 subtpyes was detected in 25% of HMAEC samples. Correspondingly, in PC experiments in HMAEC, application of caffeine (0.5 mM) induced Ca 2+ -release from ryanodine-sensitive stores and subsequently activation of Ca 2+ -activated K + channels leading to a sustained endothelial hyperpolarization from a resting potential of 28 ± 3 mV to -46 ± 4 mV. Single HMAEC expressed the TRP subtypes, TRP1 and TRP3, but not TRP 4 and 6. The TRP1 was the predominantly expressed TRP subtype as expression was detected in 16% of HMAEC. TRP3 expression was detected in only 3% of HMAEC. In single-channel and whole-cell patch clamp experiments in HMAEC, InsP 3 -mediated Ca 2+ -store depletion in response to bradykinin (100 nM) activated TRP related nonselective cation currents leading to Ca 2+ entry. In conclusion, 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 and thereby of endothelial function in intact human blood vessels.