Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
, 141 (2), 253-62

Oleamide Is a Selective Endogenous Agonist of Rat and Human CB1 Cannabinoid Receptors

Affiliations
Comparative Study

Oleamide Is a Selective Endogenous Agonist of Rat and Human CB1 Cannabinoid Receptors

James D Leggett et al. Br J Pharmacol.

Abstract

1. The ability of the endogenous fatty acid amide, cis-oleamide (ODA), to bind to and activate cannabinoid CB(1) and CB(2) receptors was investigated. 2. ODA competitively inhibited binding of the nonselective cannabinoid agonist [(3)H]CP55,940 and the selective CB(1) antagonist [(3)H]SR141716A to rat whole-brain membranes with K(i) values of 1.14 microm (0.52-2.53 microm, Hill slope=0.80, n=6) and 2.63 microm (0.62-11.20 microm, Hill slope=0.92, n=4), respectively. AEA inhibited [(3)H]CP55,940 binding in rat whole-brain membranes with a K(i) of 428 nm (346-510 nm, Hill slope=-1.33, n=3). 3. ODA competitively inhibited [(3)H]CP55,940 binding in human CB(1) (hCB(1)) cell membranes with a K(i) value of 8.13 microm (4.97-13.32 microm, n=2). In human CB(2) transfected (hCB(2)) HEK-293T cell membranes, 100 microm ODA produced only a partial (42.5+/-7%) inhibition of [(3)H]CP55,940 binding. 4. ODA stimulated [(35)S]GTPgammaS binding in a concentration-dependent manner (EC(50)=1.64 microm (0.29-9.32 microm), R(2)=0.99, n=4-9), with maximal stimulation of 188+/-9% of basal at 100 microm. AEA stimulated [(35)S]GTPgammaS binding with an EC(50) of 10.43 microm (4.45-24.42 microm, R(2)=1.00, n=3, 195+/-4% of basal at 300 microm). Trans-oleamide (trans-ODA) failed to significantly stimulate [(35)S]GTPgammaS binding at concentrations up to 100 microm. 5. ODA (10 microm)-stimulated [(35)S]GTPgammaS binding was reversed by the selective CB(1) antagonist SR141716A (IC(50)=2.11 nm (0.32-13.77 nm), R(2)=1.00, n=6). 6. The anatomical distribution of ODA-stimulated [(35)S]GTPgammaS binding in rat brain sections was indistinguishable from that of HU210. Increases of similar magnitude were observed due to both agonists in the striatum, cortex, hippocampus and cerebellum. 7. ODA (10 microm) significantly inhibited forskolin-stimulated cyclic AMP (cAMP) accumulation in mouse neuroblastoma N1E 115 cells (P=0.02, n=11). ODA-mediated inhibition was completely reversed by 1 microm SR141716A (P<0.001, n=11) and was also reversed by pretreatment with 300 ng ml(-1) pertussis toxin (P<0.001, n=6). 8. These data demonstrate that ODA is a full cannabinoid CB(1) receptor agonist. Therefore, in addition to allosteric modulation of other receptors and possible entourage effects due to fatty acid amide hydrolase inhibition, the effects of ODA may be mediated directly via the CB(1) receptor.

Figures

Figure 1
Figure 1
(a) Inhibition of specific [3H]CP55,940 binding by cis-ODA (1 nM–100 μM), Ki=1.14 μm (0.52–2.53 μM, Hill slope=0.80, n=6). (b) Inhibition of specific [3H]SR141716A binding by ODA (0.1 μM–100 μM), Ki=2.63 μM (0.62–11.20 μM, Hill slope=0.92, n=4).
Figure 2
Figure 2
Inhibition of [3H]CP55,940 binding in hCB1 (transiently transfected HEK-293T) cell membranes by cis-ODA, Ki=8.13 μM (range 4.97–13.32 μM, n=2). The figure represents one of two experiments conducted in triplicate.
Figure 3
Figure 3
(a) ODA-stimulated [35S]GTPγS binding in rat brain membranes (EC50=1.64 μM, n=4–9, R2=0.99). (b) Inhibition of 10 μM ODA-stimulated [35S]GTPγS binding by SR141716A (IC50=2.11 nM, n=6, R2=1.00).
Figure 4
Figure 4
AEA-stimulated [35S]GTPγS binding in rat brain membranes (EC50=10.43 μM (4.45–24.42 μM), n=3, best fit curve: R2=0.98).
Figure 5
Figure 5
Antagonism of agonist-potentiated [35S]GTPγS binding to rat brain membranes and effects of stereo isomers of ODA. (a) Basal binding, (b) 1 μM HU210, (c) 0.1 μM HU210 plus 1 μM SR141716A, (d) 100 μM cis-ODA, (e) 10 μM cis-ODA plus 1 μM SR141716A, (f) 10 μM cis-ODA plus 10 μM LY320135, (g) 100 μM trans-ODA (significance levels: *P<0.01 compared to basal binding).
Figure 6
Figure 6
Regional increases in [35S]GTPγS (autoradiographic) binding after stimulation with HU210 or ODA (bars indicate s.e.m.) compared to [35S]GTPγS binding under basal conditions. Significance levels: *P<0.01 compared to basal.
Figure 7
Figure 7
Example autoradiograms showing (a) 14C calibration strip calibrated with disintegrations per minute (DPM) values from 275,000 DPM to 0 DPM (b) basal, (c) 1 μM HU210-treated slices, (d) 10 μM ODA-treated slices and (e) NSB. All examples are from the same experiment and show significantly enhanced [35S]GTPγS binding after treatment with HU210 or ODA.
Figure 8
Figure 8
Inhibition of forskolin (Forsk)-stimulated cAMP generation in mouse neuroblastoma N1E 115 cells by 1 μM HU210 and 10 μM ODA and attenuation of ODA-mediated inhibition by the selective CB1 receptor antagonist SR141716A (1 μM). ODA, HU210 and SR141716A had no effect alone. ++=P<0.01 compared to basal cAMP generation (n=11), *=P<0.05 compared to forskolin-stimulated cAMP generation (n=11).
Figure 9
Figure 9
Inhibition of forskolin (Forsk)-stimulated cAMP generation in mouse neuroblastoma N1E 115 cells by 10 μM ODA and blockade of ODA-mediated inhibition by pretreatment with 300 ng ml−1 pertussis toxin (PTX). Pretreatment with PTX significantly increased cAMP accumulation in Forsk only and ODA only treated cells compared to respective non-pretreated cells. *=P<0.05 compared to basal cAMP levels, **=P<0.001 compared to basal cAMP levels, +=P< 0.05 compared to Forsk-stimulated cAMP levels, $=P<0.05 compared to respective non-pretreated condition, $$=<0.001 compared to non-pretreated condition.

Comment in

Similar articles

See all similar articles

Cited by 32 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback