Anandamide‐induced mobilization of cytosolic Ca2+ in endothelial cells

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Abstract
1. Experiments were designed to determine whether anandamide affects cytosolic Ca2+ concentrations in endothelial cells and, if so, whether CB1 cannabinoid receptors are involved. To this effect, human umbilical vein-derived EA.hy926 endothelial cells were loaded with fura-2 to monitor changes in cytosolic Ca2+ using conventional fluorescence spectrometry methods. 2. Anandamide induced an increase in Ca2+ in endothelial cells which, in contrast 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 endoplasmic reticulum Ca2+ -ATPase inhibitor thapsigargin. 3. Anandamide pretreatment slightly reduced the mobilization of Ca2+ from intracellular stores that was evoked by histamine. The mobilization of Ca2+ from intracellular stores evoked by anandamide was impaired by 10 mM caffeine. 4. Anandamide and histamine each significantly increased NO synthase activity in EA.hy926 cells, as determined by the enhanced conversion of L-[3H]-arginine to L-[3H]-citruline. 5. The CB1 cannabinoid receptor antagonist SR141716A (1 microM) only produced a marginal reduction of the mobilization of Ca2+ produced by 5 microM anandamide. However, at 5 microM SR141716A elicited the release of Ca2+ from intracellular stores. This concentration strongly impaired the mobilization of cytosolic Ca2+ evoked by either anandamide, histamine or thapsigargin. 6. Pretreatment of the cells with either 200 microM phenylmethylsulphonyl fluoride (to inhibit the conversion of anandamide into arachidonic acid) or 400 ng ml(-1) pertussis toxin (to uncouple CB1 cannabinoid receptors from Gi/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+ from a caffeine-sensitive intracellular Ca2+ store that functionally overlaps in part with the internal stores mobilized by histamine. However, a classical CB1 cannabinoid receptor-mediated and pertussis toxin-sensitive mechanism does not mediate this novel effect of anandamide in endothelial cells. 8. The mobilization of cytosolic Ca2+ in endothelial cells may account for the 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 endogenous cannabinoids to endothelium-dependent control of vascular smooth muscle tone.