Anandamide‐induced mobilization of cytosolic Ca²⁺ in endothelial cells

Abstract

1 Experiments were designed to determine whether anandamide a ects cytosolic Ca 2+ concentrations in endothelial cells and, if so, whether CB 1 cannabinoid receptors are involved. To this e ect, human umbilical vein-derived EA.hy926 endothelial cells were loaded with fura-2 to monitor changes in cytosolic Ca 2+ using conventional¯uorescence spectrometry methods. 2 Anandamide induced an increase in Ca 2+ in endothelial cells which, in contrast to histamine, developed slowly and was transient. Anandamide caused a concentration-dependent release of Ca 2+ from intracellular stores without triggering capacitative Ca 2+ entry, contrary to histamine or the endoplasmic reticulum Ca 2+ -ATPase inhibitor thapsigargin. 3 Anandamide pretreatment slightly reduced the mobilization of Ca 2+ from intracellular stores that was evoked by histamine. The mobilization of Ca 2+ from intracellular stores evoked by anandamide was impaired by 10 mM ca eine. 4 Anandamide and histamine each signi®cantly increased NO synthase activity in EA.hy926 cells, as determined by the enhanced conversion of L-The CB 1 cannabinoid receptor antagonist SR141716A (1 mM) only produced a marginal reduction of the mobilization of Ca 2+ produced by 5 mM anandamide. However, at 5 mM SR141716A elicited the release of Ca 2+ from intracellular stores. This concentration strongly impaired the mobilization of cytosolic Ca 2+ evoked by either anandamide, histamine or thapsigargin. 6 Pretreatment of the cells with either 200 mM phenylmethylsulphonyl¯uoride (to inhibit the conversion of anandamide into arachidonic acid) or 400 ng ml 71 pertussis toxin (to uncouple CB 1 cannabinoid receptors from G i/o proteins) had no signi®cant e ect on the mobilization of cytosolic Ca 2+ evoked by either anandamide, or histamine. 7 Taken together the results demonstrate that anandamide mobilizes Ca 2+ from a ca eine-sensitive intracellular Ca 2+ store that functionally overlaps in part with the internal stores mobilized by histamine. However, a classical CB 1 cannabinoid receptor-mediated and pertussis toxin-sensitive mechanism does not mediate this novel e ect of anandamide in endothelial cells. 8 The mobilization of cytosolic Ca 2+ in endothelial cells may account for the endotheliumdependent and NO-mediated vasodilator actions of anandamide. Due to its non-speci®c inhibition of Ca 2+ 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.

Cannabinoid-induced mesenteric vasodilation through an endothelial site distinct from CB1 or CB2 receptors

“…Therefore, these findings suggest that Abn-cbd causes NO-independent vasodilation in rat mesenteric arteries by triggering the release of an EDHF, such as K ϩ itself (39) or an epoxygenase product (40). Vascular endothelial cells contain Ca 2ϩ -activated K ϩ channels, which are opened by a rise in intracellular calcium (41), and anandamide has been shown to induce calcium transients in vascular endothelial cells by the activation of an SR141716A-sensitive mechanism (35,42). It has been proposed that EDHF itself may be an endocannabinoid acting on CB1-like receptors in vascular smooth muscle, based on the ability of SR141716A to inhibit EDHF-induced mesenteric vasodilation (30).…”

Anandamide-induced cell death in primary neuronal cultures: role of calpain and caspase pathways

“…Given that upregulation of intracellular Ca 2 þ is known to be crucial for calpain activation (especially for m-calpain) 43 and is a known action of anandamide, 15,44,45 we examined effects of anandamide on Ca 2 þ levels in cortical neuronal cells. We employed imaging analysis using the fluorescent indicator Fluo-3 for assessment of intracellular free calcium ion concentration.…”

“…14 Anandamide appears to regulate a number of crucial cellular events, including cytosolic Ca 2 þ levels and cell proliferation. [15][16][17] Endogenous cannabinoids, such as 2-arachidonoyl-glycerol (2-AG), are generally believed to play a neuroprotective role through actions at CB receptors, and anandamide has also been reported to provide neuroprotection through activation of CB receptors. 4,[18][19][20][21] However, in human neuroblastoma CHP100 and lymphoma U937 cell lines, anandamide induces apoptotic cell death through actions at VR1 receptors, leading to caspase activation.…”

Anandamide initiates Ca2+ signaling via CB2 receptor linked to phospholipase C in calf pulmonary endothelial cells

“…In human umbilical vein endothelial cells, anandamide and the CB1 receptor agonist HU210 have been found to upregulate iNOS (MacCarrone et al, 2000). In addition, in the human umbilical vein endothelial cell line EA.hy926, anandamide initiated the elevation of the intracellular Ca 2 þ concentration ([Ca 2 þ ] cyto ), which leads to the formation of nitric oxide, but the mechanisms by which anandamide initiates endothelial [Ca 2 þ ] sub signaling are still unknown (Mombouli et al, 1999) Nevertheless, although anandamide is obviously also able to activate vascular endothelial cells, the actual CB receptor isoform(s) in this type of cell has not been molecularly characterized and differentiation is entirely based on the use of CB1 or CB2 agonists/antagonists that are chemically very distinct from anandamide .…”