Anandamide-induced mobilization of cytosolic Ca2+ in endothelial cells

1 Experiments were designed to determine whether anandamide affects cytosol ic Ca2+ concentrations in endothelial cells and, if so, whether CB1 cannabi noid receptors are involved. To this effect, human umbilical vein-derived E A.hy926 endothelial cells were loaded with fura-2 to monitor changes in cyt osolic Ca2+ using conventional fluorescence spectrometry methods. 2 Anandamide induced an increase in Ca2+ in endothelial cells which, in con trast to histamine, developed slowly and was transient. Anandamide caused a concentration-dependent release of Ca2+ from intracellular stores without triggering capacitative Ca2+ entry, contrary to histamine or the endoplasmi c reticulum Ca2+-ATPase inhibitor thapsigargin. 3 Anandamide pretreatment slightly reduced the mobilization of Ca2+ from in tracellular stores that was evoked by histamine. The mobilization of Ca2+ f rom intracellular stores evoked by anandamide was impaired by 10 mM caffein e. 4 Anandamide and histamine each significantly increased NO synthase activit y in EA.hy926 cells, as determined by the enhanced conversion of L-[H-3]-ar ginine to L-[H-3]-citruline, 5 The CB1 cannabinoid receptor antagonist SR141716A (1 mu M) only produced a marginal reduction of the mobilization of Ca2+ produced by 5 mu M anandam ide. However, at 5 mu M SR141716A elicited the release of Ca2+ from intrace llular stores. This concentration strongly impaired the mobilization of cyt osolic Ca2+ evoked by either anandamide, histamine or thapsigargin. 6 Pretreatment of the cells with either 200 mu M phenylmethylsulphonyl fluo ride (to inhibit the conversion of anandamide into arachidonic acid) or 400 ng ml(-1) pertussis toxin (to uncouple CB1 cannabinoid receptors from G(i/ o) proteins) had no significant effect on the mobilization of cytosolic Ca2 + evoked by either anandamide, or histamine. 7 Taken together the results demonstrate that anandamide mobilizes Ca2+ fro m a caffeine-sensitive intracellular Ca2+ store that functionally overlaps in part with the internal stores mobilized by histamine. However, a classic al CB1 cannabinoid receptor-mediated and pertussis toxin-sensitive mechanis m does not mediate this novel effect of anandamide in endothelial cells. 8 The mobilization of cytosolic Ca2+ in endothelial cells may account for t he endothelium-dependent and NO-mediated vasodilator actions of anandamide. Due to its non-specific inhibition of Ca2+ signalling in endothelial cells , SR141716A may not be used to assess the physiological involvement of endo genous cannabinoids to endothelium-dependent control of vascular smooth mus cle tone.