The mechanism of action of cannabidiol, one of the major constituents of cannabis, is not well understood but a noncompetitive interaction with mu opioid receptors has been suggested on the basis of saturation binding experiments. The aim of the present study was to examine whether cannabidiol is an allosteric modulator at this receptor, using kinetic binding studies, which are particularly sensitive for the measurement of allosteric interactions at G protein-coupled receptors. In addition, we studied whether such a mechanism also extends to the delta opioid receptor. For comparison, (-)-Delta9-tetrahydrocannabinol (THC; another major constituent of cannabis) and rimonabant (a cannabinoid CB1 receptor antagonist) were studied. In mu opioid receptor binding studies on rat cerebral cortex membrane homogenates, the agonist 3H-DAMGO bound to a homogeneous class of binding sites with a KD of 0.68+/-0.02 nM and a Bmax of 203+/-7 fmol/mg protein. The dissociation of 3H-DAMGO induced by naloxone 10 microM (half life time of 7+/-1 min) was accelerated by cannabidiol and THC (at 100 microM, each) by a factor of 12 and 2, respectively. The respective pEC50 values for a half-maximum elevation of the dissociation rate constant k(off) were 4.38 and 4.67; 3H-DAMGO dissociation was not affected by rimonabant 10 microM. In delta opioid receptor binding studies on rat cerebral cortex membrane homogenates, the antagonist 3H-naltrindole bound to a homogeneous class of binding sites with a KD of 0.24+/-0.02 nM and a Bmax of 352+/-22 fmol/mg protein. The dissociation of 3H-naltrindole induced by naltrindole 10 microM (half life time of 119+/-3 min) was accelerated by cannabidiol and THC (at 100 microM, each) by a factor of 2, each. The respective pEC50 values were 4.10 and 5.00; 3H-naltrindole dissociation was not affected by rimonabant 10 microM. The present study shows that cannabidiol is an allosteric modulator at mu and delta opioid receptors. This property is shared by THC but not by rimonabant.

1 We studied whether cannabinoid CB 1 receptor gene disruption (to yield CB 1 À/À mice) affects the electrically evoked tritium overflow from vas deferens and atrial pieces preincubated with [ 3 H]-noradrenaline (NA) ('noradrenaline release') and from cerebral cortex slices preincubated with [ 3 H]-choline ('acetylcholine release'). 2 NA release was higher by 37% in vas deferens from CB 1 À/À mice than in vas deferens from CB 1 þ / þ and CB 1 À/À mice in any preparation. 6 In conclusion, the increase in NA release associated with CB 1 receptor deficiency in the vas deferens, which cannot be ascribed to an alteration of tritium content of the preparations, suggests an endogenous tone at the CB 1 receptors of CB 1 þ / þ mice in this tissue. Furthermore, the effect of WIN 55,212-2 on NA release in the vas deferens and on cortical Ach release involves CB 1 receptors, whereas the involvement of non-CB 1 -non-CB 2 receptors can be excluded.

1 We determined the eects of nociceptin/orphanin FQ and the NOP receptor ligands acetyl-ArgTyr-Tyr-Arg-Ile-Lys-NH 2 (Ac-RYYRIK-NH 2 ) and naloxone benzoylhydrazone on transmitter release in vitro. 2 The electrically evoked tritium over¯ow from guinea-pig and mouse striatal slices and guinea-pig retinal discs preincubated with [ 3 H]-dopamine was inhibited by nociceptin/orphanin FQ (pEC 50 7.9, 7.6 and 8.6; E max 30, 50 and 55%). Ac-RYYRIK-NH 2 0.032 mM and naloxone benzoylhydrazone 5 mM antagonized the eect of nociceptin/orphanin FQ in striatal slices of the guinea-pig (apparent pA 2 9.1 and 6.8) and the mouse (apparent pA 2 9.2 and 7.5) and strongly attenuated the eect of nociceptin/orphanin FQ 0.1 mM in guinea-pig retinal discs. Ac-RYYRIK-NH 2 0.032 mM did not aect the evoked over¯ow by itself whereas naloxone benzoylhydrazone 5 mM inhibited it in each tissue. 3 The electrically evoked tritium over¯ow from mouse brain cortex slices preincubated with [ 3 H]-noradrenaline was inhibited by nociceptin/orphanin FQ (pEC 50 7.9, E max 85%), Ac-RYYRIK-NH 2 (pEC 50 8.3, E max 47%) but not aected by naloxone benzoylhydrazone 5 mM. Ac-RYYRIK-NH 2 and naloxone benzoylhydrazone showed apparent pA 2 values of 8.6 and 6.9. 4 In conclusion, the inhibitory eect of nociceptin/orphanin FQ on dopamine release in the striatum and retina and on noradrenaline release in the cerebral cortex is mediated via NOP receptors. Ac-RYYRIK-NH 2 behaves as an extremely potent NOP receptor antagonist in the striatum and retina and as a partial agonist in the cortex.

Different types of presynaptic inhibitory Gα(i/o) protein-coupled receptors usually do not act independently of each other but rather pre-activation of receptor X impairs the effect mediated via receptor Y. It is, however, unknown whether this interaction extends to the cannabinoid CB(1) receptor on cholinergic neurones and hence we studied whether its activation, pharmacological blockade, or genetic inactivation affects the function of other presynaptic inhibitory receptors. The electrically evoked acetylcholine or noradrenaline release was determined in superfused rodent tissues preincubated with (3)H-choline or (3)H-noradrenaline. The muscarinic M(2) receptor, Gα(i), and Gα(o) proteins were determined in hippocampal synaptosomes by Western blotting. Hippocampal anandamide and 2-arachidonoyl glycerol levels were determined by LC-MS/MS. The inhibitory effect of the muscarinic receptor agonist oxotremorine on acetylcholine release in hippocampal slices was increased by genetic CB(1) receptor ablation (mouse) and the CB(1) antagonist rimonabant (rat but not mouse) and decreased by a cannabinoid receptor agonist (mouse). In mouse tissues, CB(1) receptor ablation also increased the effect of a δ opioid receptor agonist on acetylcholine release in the hippocampus and the effect of oxotremorine on noradrenaline release in the vas deferens. CB(1) receptor ablation, to a very slight extent, increased Gα(o) protein levels without affecting either Gα(i) and M(2) receptor protein or the levels of anandamide and 2-arachidonoyl glycerol in the hippocampus. In conclusion, the CB(1) receptor shows an inhibitory interaction with the muscarinic and δ opioid receptor on cholinergic neurones in the rodent hippocampus and with the muscarinic receptor on noradrenergic neurones in the mouse vas deferens.

1 The cannabinoid CB 1 receptor inverse agonist/antagonist SR 141716 increases acetylcholine release in rodent hippocampus and improves memory in some experimental paradigms. Since drugs like SR 141716 may represent a novel class of cognition-enhancing drugs, we wanted to check whether the function of the CB 1 receptor is preserved during ageing. 2 Hippocampal and striatal slices from 2-to 3-and 24-to 28-month-old C57BL/6J mice were preincubated with [ 3 H]-choline or [ 3 H]-noradrenaline ([ 3 H]-NA) and superfused. 3 The cannabinoid receptor agonist WIN 55,212-2 inhibited, and SR 141716 facilitated, the electrically (3 Hz) evoked tritium overflow in hippocampal slices (preincubated with [ 3 H]-choline) from young and aged mice to the same extent. The evoked overflow per se was less by 33% in slices from aged animals. 4 WIN 55,212-2 and SR 141716 did not affect, but the muscarinic receptor agonist oxotremorine inhibited, the evoked (3 Hz) overflow in striatal slices (preincubated with [ 3 H]-choline) from young and aged mice to the same extent. The evoked overflow per se tended to be less in slices from aged animals. 5 The evoked (0.3 Hz) overflow in hippocampal slices (preincubated with [ 3 H]-NA) was not affected by WIN 55,212-2 and SR 141716, but was inhibited by histamine (via H 3 receptors) in slices from young mice and, to a somewhat less extent, in slices from aged mice. The evoked overflow per se did not differ between age groups. 6 In conclusion, the function of the CB 1 receptor involved in the tonic inhibition of hippocampal acetylcholine release is preserved in aged mice.