Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Zoratti, C.
Endocannabinoid signalling in human endothelial cells
[ Dissertation ] Medical University of Graz; 2004. pp.
- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Graier Wolfgang
- Altmetrics:
- Abstract:
- Two cannabinoid (CB) receptors have been identified by molecular cloning: the CB1 receptor, which is expressed at high concentrations in the brain (Matsuda et al., 1990) but is also present in peripheral tissues (Shire et al., 1995), and the CB2 receptor, expressed by cells of the immune system (Munro et al., 1993) and endothelial cells (Zoratti et al.; 2003). Anandamide or arachidonoylethanolamide is one of the natural ligands for these cannabinoid receptors. Our experiments aimed to assess whether anandamide affects cytosolic Ca2+ concentrations in human endothelial cells and, if so, which cannabinoid or another putative receptor is involved. Cytosolic free Ca2+ü concentration was measured in single cells of the umbilical vein-derived endothelial cell line EA.hy 926 using fura-2/AM technique. Furthermore, the fluctuation of Ca2+ concentration in the endoplasmic reticulum (ER) and the mitochondria Ca2+ uptake were followed by vYC4-ER (Nagai et al., 2002) an improved cameleon targeting the ER, and the ratiometric- pericam-mt (Nagai et al., 2001). In the absence of extracellular Ca2+ anandamide yielded oscillatory increase of cytosolic free Ca2+ concentration with an EC50 of 7.3 (4.5-11.7) M. This effect was associated with a decrease of the ER Ca2+ concentration and could be blocked by the PLC inhibitor (U73122) and 2-APB an inhibitor of the IP3 receptors. The effect of anandamide on endothelial Ca2+ signalling was prevented by the CB1 receptor antagonist SR 141617A (Rinaldi-Carmona et al., 1994) but not by the CB2 antagonist SR144528 (Rinaldi-Carmona et al., 1998). These data were confirmed by molecular identification. Nevertheless, in the presence of extracellular Ca2+ anandamide failed to elevate the cytosolic Ca2+ concentration. The IC50 for Ca2+ to prevent the initiation of endothelial Ca2+ signal by anandamide was 7.7 (6.7-8.8) M. In the presence of Mn2+ (70M) that clusters integrins, anandamide achieved cytosolic Ca2+ elevation even in Ca2+containg solution. In contrast, clustering of integrins with fibronectin or its binding sequence RGD did not permit anandamide evoked Ca2+ signalling in the presence of extracellular Ca2+. The intefrin-cross-linking antibody LM609 (alfavbeta3) and the functional blocking antibodies B3A (alfa3) and JB1A (alfa1) completely prevented the Ca2+ response in response to anandamide in the absence of extracellular Ca2+. Moreover, evidence for the involvement of integrins was also provided in experiments in which basal RhoA activity, which is essential for integrin clustering, was inhibited by the ROCK inhibitor, Y 27632.