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

Abstract

1 The endocannabinoid anandamide has been reported to affect neuronal cells, immune cells and smooth muscle cells via either CB1 or CB2 receptors. In endothelial cells, the receptors involved in activating signal transduction are still unclear, despite the fact that anandamide is produced in this cell type. 2 The present study was designed to explore in detail the effect of this endocannabinoid on Ca 2 þ signaling in single cells of a calf pulmonary endothelial cell line. 3 Anandamide initiated a transient Ca 2 þ elevation that was prevented by the CB2 receptor antagonist SR144528, but not by the CB1 antagonist SR141716A. These data were confirmed by molecular identification of the bovine CB2 receptor in these endothelial cells by partial sequencing. 4 The phospholipase C inhibitor 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5dione and the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenylborate prevented Ca 2 þ signaling in response to anandamide. 5 Using an improved cameleon probe targeted to the endoplasmic reticulum (ER), fura-2 and ratiometric-pericam, which is targeted to the mitochondria, anandamide was found to induce Ca 2 þ depletion of the ER accompanied by the activation of capacitative Ca 2 þ entry (CCE) and a transient elevation of mitochondrial Ca 2 þ . 6 These data demonstrate that anandamide stimulates the endothelial cells used in this study via CB2 receptor-mediated activation of phospholipase C, formation of inositol 1,4,5-trisphosphate, Ca 2 þ release from the ER and subsequent activation of CCE. Moreover, the cytosolic Ca 2 þ elevation was accompanied by a transient Ca 2 þ increase in the mitochondria. Thus, in addition to its actions on smooth muscle cells, anandamide also acts as a powerful stimulus for endothelial cells.

The Role of Mitochondria for Ca2+ Refilling of the Endoplasmic Reticulum

“…Cytoslic Ca 2ϩ Measurements-Changes in [Ca 2ϩ ] cyto were monitored using the Ca 2ϩ -sensitive dye fura-2 (24 (19,28). Notably, RP-mt was demonstrated to be a reliable Ca 2ϩ sensor when excited at 410 -440 nm, whereas the fluorescence of the permutated yellow fluorescent protein reflects mainly changes in the H ϩ concentration (30) when excited at 480 nm.…”

Aspects of endocannabinoid signaling in periimplantation biology

“…Both CB1 and CB2 are coupled with G proteins in the G i/o and G q families, and the activation of cannabinoid receptors has different biological effects that are cell-type dependent. Signals mediated by cannabinoid receptors include regulation of Ca 2+ channels [41][42][43][44][45], inhibition of adenylyl cyclase [4,46], activation of phopholipase C [47] and stimulation of mitogen-activated protein kinases (MAPKs) including ERK, JNK and p38 [44,48,49].…”

The endocannabinoid system in bull sperm and bovine oviductal epithelium: role of anandamide in sperm–oviduct interaction

“…CB2 bovine sequence has not yet been published. Zoratti et al (2003) used primers homologous to the human and mouse sequences for CB2 amplified the bovine CB2 receptor from bovine spleen, as this tissue is supposed to have a high expression level of this receptor. Alexa-Fluor555 goat antirabbit IgG and Alexa-Fluor594 donkey anti-mouse IgG were from Molecular Probes (Invitrogen), and URB597 (a selective FAAH inhibitor) was from Cayman Chemical Company (Migliore Laclaustra, Argentina).…”