Compartmentalization of endocannabinoids into lipid rafts in a microglial cell line devoid of caveolin-1

Abstract

Background and purpose: N-acyl ethanolamines (NAEs) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids and along with related lipids are synthesized on demand from membrane phospholipids. Here, we have studied the compartmentalization of NAEs and 2-AG into lipid raft fractions isolated from the caveolin-1-lacking microglial cell line BV-2, following vehicle or cannabidiol (CBD) treatment. Results were compared with those from the caveolin-1-positive F-11 cell line.

Experimental approach: BV-2 cells were incubated with CBD or vehicle. Cells were fractionated using a detergent-free continuous OptiPrep density gradient. Lipids in fractions were quantified using HPLC/MS/MS. Proteins were measured using Western blot.

Key results: BV-2 cells were devoid of caveolin-1. Lipid rafts were isolated from BV-2 cells as confirmed by co-localization with flotillin-1 and sphingomyelin. Small amounts of cannabinoid CB(1) receptors were found in lipid raft fractions. After incubation with CBD, levels and distribution in lipid rafts of 2-AG, N-arachidonoyl ethanolamine (AEA), and N-oleoyl ethanolamine (OEA) were not changed. Conversely, the levels of the saturated N-stearoyl ethanolamine (SEA) and N-palmitoyl ethanolamine (PEA) were elevated in lipid raft fractions. In whole cells with growth medium, CBD treatment increased AEA and OEA time-dependently, while levels of 2-AG, PEA and SEA did not change.

Conclusions and implications: Whereas levels of 2-AG were not affected by CBD treatment, the distribution and levels of NAEs showed significant changes. Among the NAEs, the degree of acyl chain saturation predicted the compartmentalization after CBD treatment suggesting a shift in cell signalling activity.

Linked articles: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Figure 1

Distribution of membrane raft markers across BV-2 fractions. (A) [18:0]-sphingomyelin is highest in fraction 8 (significantly different from fractions 1–5, and 11–16, P < 0.05, n= 3; one-way anova, Fisher's LSD post hoc). Data are presented as the total quantity in picomoles (pmol) recovered from each fraction. Error bars represent standard error of the mean. (B) Western blot showing the distribution of flotillin-1, LAMP-1, and β-actin. The membrane raft marker flotillin-1 is concentrated in fractions 6–10. β-actin is localized mostly to fractions 1–7, with small amounts in the lighter fractions 8–9. LAMP-1, a lysosomal marker is spread through the lipid rafts and lighter fractions 6–13. (C) Comparison of [18:0]-sphingomyelin distribution between BV-2 (left y-axis) and F-11 (right y-axis) cells. Data for F-11 cells (from Rimmerman et al. 2008) was re-plotted in picomoles. (D) Western blot showing that BV-2 cells are devoid of the protein caveolin-1, CHO cells serve as a positive control.

Figure 2

Distribution of endocannabinoids and CB receptors across BV-2 fractions. (A) 2-AG (in pmol) is highest in interface fraction 6, and significantly different from fractions 1–5 and 10–16 (P < 0.05, n= 3, one-way anova, Fisher's LSD post hoc). (B) Comparison of 2-AG distribution between BV-2 (left y-axis) and F-11 (right y-axis) cells. Data for F-11 cells was re-plotted from Rimmerman et al. (2008). (C) AEA is found in non-lipid raft fraction 5, 6 and lipid raft fraction 9. (D) CB₂ receptors and the vanilloid receptor TRPV2 are localized to non-lipid raft fractions 1–5 and interface fraction 6. CB₁ receptors are present mostly in non-lipid raft fractions 1–5 and 6. Small amounts are present in lipid raft fractions. (E) Western blot analysis of fractions obtained using a second fractionation method confirmed the compartmentalization of β-actin, TRPV2 and most of CB₁ receptors in the non-lipid raft fractions. Flotillin-1, LAMP-1 and part of the CB₁ receptors are present in lipid raft fractions. Caveolin-1 is not found in any of the fractions. These results are consistent with the OptiPrep fractionation method.

Figure 3

Distribution of the NAEs, OEA, PEA and SEA, across BV-2 fractions. (A) OEA levels are highest in interface fraction 6 which is significantly different from fractions 1–5 and 10–16 (P < 0.05, n= 3, one-way anova, Fisher's LSD post hoc). (B) PEA levels are highest in interface fraction 6 which is significantly different from fractions 1–5 and 10–16 (P < 0.05, n= 3, one-way anova, Fisher's LSD post hoc).(C) SEA is highest in interface fraction 6 which is significantly different from fractions 1–4 and 10–16 (P < 0.05, n= 3, one-way anova, Fisher's LSD post hoc).

Figure 4

Distribution of membrane raft markers and CBD across BV-2 fractions following CBD treatment. (A) [18:0]-sphingomyelin is highest in fraction 9 (significantly different from fractions 1–6, and 12–16, P < 0.05, n= 3; one-way anova, Fisher's LSD post hoc). (B) CBD is highest in fractions 6 and 7 (CBD levels found in fraction 6 are significantly different from fractions 1–5 and 8–16; P < 0.02; n= 3, one-way anova, Fisher's LSD post hoc). (C) Lipid raft marker flotillin-1 is concentrated in fractions 6–11. β-actin is localized mostly to fractions 1–6. LAMP-1 is spread through lipid rafts and the lighter fractions 6–14. CB₂ receptors and the vanilloid receptor TRPV2 are localized to non-membrane raft fractions 1–5, and interface fraction 6. CB₁ receptors are compartmentalized mainly to non-lipid raft fractions with small amounts appearing in lipid raft fractions.

Figure 5

Distribution of the endocannabinoids AEA and 2-AG across BV-2 fractions following CBD treatment. (A) 2-AG levels and distribution are not affected by CBD treatment. (B) Following CBD treatment, AEA was found in the heavier fraction 5, interface fraction 6, and lipid raft fractions (7 and 9).

Figure 6

Distribution of the NAEs, PEA, SEA, and OEA across BV-2 fractions following CBD treatment. (A) OEA levels are highest in fraction 7 in the CBD-treated cells and are significantly different from fractions 1–5 and 10–16 (P < 0.05; n= 3, one-way anova, Fisher's LSD post hoc. The levels of OEA are not significantly increased in CBD-treated cells compared with vehicle-treated cells. (B) In CBD-treated cells, PEA peaked in fraction 7 and was increased in fractions 6–10 (fraction 7 is significantly different from fractions 1–5 and 11–16; P < 0.02; n= 3, one-way anova, Fisher's LSD post hoc). Lipid raft fractions 7–10 in CBD-treated cells have significantly increased levels of PEA when compared with vehicle-treated cells (#, fraction 7, P < 0.03; fraction 8, P < 0.06; fraction 9, P < 0.03; fraction 10, P < 0.05; n= 3, one-way anova, Fisher's LSD post hoc). (C) SEA levels are highest in fraction 7 in the CBD-treated cells and are significantly different from fractions 1–5 and 11–16, P < 0.01; n= 3, one-way anova, Fisher's LSD post hoc). Lipid raft fractions (7–10) in the CBD-treated cells have significantly increased levels of SEA (fraction 7, P < 0.005; fraction 8, P < 0.02; fraction 9, P < 0.05; fraction 10, P < 0.02; n= 3, one-way anova, Fisher's LSD post hoc).

Figure 7

2-AG and NAE levels in whole cells + media, following CBD treatment. (A) 2-AG levels in whole cells + growth medium are not significantly different between CBD-treated and vehicle-treated cells. (B) AEA levels are increased in whole cells + growth medium from CBD-treated cells at 60 min and 210 min, #, P < 0.001; n= 3, at CBD 210 min, n= 2, Bonferroni post hoc. (C) OEA levels are increased in whole cells + growth medium from CBD-treated cells compared with control at 60 min #, P < 0.02; at 210 min, *P < 0.001; n= 3 (at CBD 210 min, n= 2), one-way anova, Bonferroni post hoc. (D) PEA levels in whole cells + growth medium are not significantly different between CBD-treated and vehicle-treated cells. (E) SEA levels in whole cells + growth medium are not significantly different between CBD-treated and vehicle-treated cells.