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 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.

1 Atypical b-adrenoceptors resistant to propranolol, but blocked by bupranolol, increase contractile force and/or frequency of the heart in humans and rats. We compared the potencies of the enantiomers of bupranolol and examined the possible effects of seven bupranolol analogues including bevantolol (BEV) at this receptor in pithed and vagotomized rats. 2 CGP 12177, an agonist of the atypical b-adrenoceptor, increased heart rate dose-dependently. Its dose -response curve was shifted to the right by S-(À)-bupranolol 10 mmol kg À1 by a factor of 8.4, but not affected by the same dose of R-( þ )-bupranolol. 3 Desmethylbupranolol and compounds BK-21, BK-22, BK-23 and BK-25 also increased heart rate dose-dependently. The b 1 -adrenoceptor antagonist CGP 20712 given in combination with the b 2 -adrenoceptor antagonist ICI 118,551 (0.1 mmol kg À1 each) reduced the positive chronotropic action of the five bupranolol analogues without affecting that of CGP 12177. The potencies of the bupranolol analogues to increase heart rate were correlated (r ¼ 0.91, Po0.05) with their affinities for b 1 -adrenoceptor binding sites in rat brain cortex membranes labelled with [ 3 H]CGP 12177 (in the presence of ICI 118,551). 4 BK-26 and BEV, 10 mmol kg À1 each, had only minor effects on heart rate by themselves and did not antagonize the effect of CGP 12177. However, at 1 mmol kg À1 , they antagonized the increase in heart rate elicited by the b 1 -adrenoceptor agonist prenalterol. 5 In conclusion, bupranolol is a stereoselective antagonist at the atypical cardiostimulant b-adrenoceptor. The effects of the bupranolol analogues are related to the activation or blockade of b 1 -adrenoceptors, but not of atypical b-adrenoceptors.

We examined whether replacement of imidazole by a piperidine or pyrrolidine moiety will affect the potency and affinity of six H3-receptor antagonists. Potencies were determined in superfused mouse brain cortex slices preincubated with [3H]noradrenaline, in which the interaction of the antagonists with histamine with respect to its inhibitory effect on the electrically evoked tritium overflow was studied. Affinities were determined in mouse brain cortex membranes, using the radioligand [3H] N(alpha)-methylhistamine. The concentration-response curve of histamine for its effect on the evoked overflow from mouse brain cortex slices was shifted to the right by the 13 compounds under study. Replacement of the imidazole by a piperidine ring affected the p A2 value as follows: thioperamide, -2.7 log units; clobenpropit, -1.9; proxyfan, -1.3; FUB 138, -1.2. Potency hardly changed (< or =0.4 log units) when imidazole was replaced by piperidine in FUB 181 and by piperidine or pyrrolidine in FUB 153. Binding of [3H] N (alpha)-methylhistamine to mouse brain cortex membranes was inhibited monophasically by all compounds. The p K(i) values closely matched their p A2 values with three exceptions. The p K(i) values of proxyfan, FUB 138, and FUB 153 exceeded their respective p A(2) values by about 1 log unit. To reveal a potential partial agonism, the effect of the three drugs on (1) the electrically evoked tritium overflow and (2) [35S]GTPgammaS binding in mouse cortex preparations was determined. Proxyfan proved to be a partial agonist in both models (with intrinsic activities of 0.2 and 0.3, respectively) whereas FUB 138 and FUB 153 were devoid of agonistic effects. In conclusion, replacement of imidazole by piperidine or pyrrolidine affects the antagonist potencies of six H3-receptor antagonists in a very different manner. The piperidine analogue of FUB 181 (with a p A2 value as high as 7.7) may represent a lead for the development of non-imidazole H3-receptor antagonists. The discrepancy between the p K(i) and p A2 values may be accounted for by partial agonism in the case of proxyfan but can, at present, not be satisfactorily explained with respect to FUB 138 and FUB 153.

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