Astrocytes in Culture Produce Anandamide and Other Acylethanolamides

Abstract

Anandamide (arachidonylethanolamide) is an endocannabinoid that belongs to the acylethanolamide lipid family. It is produced by neurons in a calcium-dependent manner and acts through cannabinoid CB1 receptors. Other members of the acylethanolamide lipid family are also produced by neurons and act through G-protein-coupled receptors: homo-gamma-linolenylethanolamide (HEA) and docosatetraenylethanolamide (DEA) act through CB1 receptors, palmitylethanolamide (PEA) acts through CB2-like receptors, and oleylethanolamide (OEA) acts through receptors that have not yet been cloned. Although it is clear that anandamide and other acylethanolamides play a major role in neuronal signaling, whether astrocytes also produce these lipids is unknown. We developed a chemical ionization gas chromatography/mass spectrometry method that allows femtomole detection and quantification of anandamide and other acylethanolamides. Using this method, we unambiguously detected and quantified anandamide, HEA, DEA, PEA, and OEA in mouse astrocytes in culture. Stimulation of mouse astrocytes with ionomycin, a calcium ionophore, enhanced the production of anandamide, HEA, and DEA, whereas PEA and OEA levels were unchanged. Endothelin-1, a peptide known to act on astrocytes, enhanced the production of anandamide, without affecting the levels of other acylethanolamides. These results show that astrocytes produce anandamide, HEA, and DEA in a calcium-dependent manner and that anandamide biosynthesis can be selectively stimulated under physiologically relevant conditions. The relative levels of acylethanolamides in astrocytes from rat and human were different from the relative levels of acylethanolamides in mouse astrocytes, indicating that the production of these lipids differs between species. Because astrocytes are known to express CB1 receptors and inactivate endocannabinoids, our finding strongly suggests the existence of a functional endocannabinoid signaling system in these cells.

Cannabinoids and neuroinflammation

“…In primary cultures of mouse microglia, 2-AG induces cell migration, and this is reversed by SR144528, cannabinol, and cannabidiol (Walter et al, 2003a). Perhaps under neuroinflammatory conditions, neurons or astrocytes produce endocannabinoids as a means of recruiting microglia (Walter et al, 2002;2003a;Walter & Stella, 2003b). Nitric oxide (NO) production by glial cells is also associated with immune-mediated cellular cytotoxicity and pathogenesis of MS and EAE (reviewed in Parkinson et al, 1997).…”

The endocannabinoid system in normal and pathological brain ageing

“…These data suggest that cannabinoids regulate glial activity primarily through CB2 receptors. Glial cells not only receive cannabinoid signals but can themselves produce cannabinoids such as anandamide [189], 2-AG [190] and palmitoylethanolamide [191], and also express the enzymes involved in the synthesis and degradation of endocannabinoids [192][193][194]. Because both neurons and glia express elements of the cannabinoid system, it was hypothesized that the cannabinoid system plays an important role in neuron-glia communication.…”

Nonpsychotropic Cannabinoid Receptors Regulate Microglial Cell Migration

“…Because microglial cells and BV-2 cells were routinely grown with FBS, cells were recovered in a defined cell culture medium ϳ18 hr before experiments. Specifically, we recovered them at 200,000 cells/ml MEM/Cellgro (Mediatech, Herndon, VA) (MEM supplemented with 10 mM HEPES, 5 mM NaHCO 3 , 100 U penicillin, 100 g/ml streptomycin, 2 mM L-glutamine, and 10% Cellgro) (Walter et al, 2002). Newborn mouse neopallia neurons in primary cultures were prepared in B27-supplemented Neurobasal as described by Stella and Piomelli (2001).…”

“…Newborn mouse neopallia microglial cells in primary cultures were prepared as described previously (Walter et al, 2002). BV-2 cells, a mouse microglial cell line, were grown in DMEM supplemented with FBS (3%), penicillin (100 U), and streptomycin (100 g/ml) and passaged every 3-4 d for a maximum of 30 passages.…”

“…In some experiments, we fixed the removed 9 ml of distant media by adding it to 9 ml of ice-cold methanol. Lipids from either cells plus adjacent media or distant media were then extracted with chloroform, and endocannabinoids were purified by HPLC and analyzed by chemical ionization gas chromatography/mass spectrometry (CI-GC/MS) as described previously (Stella and Piomelli, 2001;Walter et al, 2002).…”