O-2050 facilitates noradrenaline release and increases the CB1 receptor inverse agonistic effect of rimonabant in the guinea pig hippocampus

Abstract

The cannabinoid CB1 receptors on the noradrenergic neurons in guinea pig hippocampal slices show an endogenous endocannabinoid tone. This conclusion is based on rimonabant, the facilitatory effect of which on noradrenaline release might be due to its inverse CB1 receptor agonism and/or the interruption of a tonic inhibition elicited by endocannabinoids. To examine the latter mechanism, a neutral antagonist would be suitable. Therefore, we studied whether O-2050 is a neutral CB1 receptor antagonist in the guinea pig hippocampus and whether it mimics the facilitatory effect of rimonabant. CB1 receptor affinity of O-2050 was quantified in cerebrocortical membranes, using (3)H-rimonabant binding. Its CB1 receptor potency and effect on (3)H-noradrenaline release were determined in superfused hippocampal slices. Its intrinsic activity at CB1 receptors was studied in hippocampal membranes, using (35)S-GTPγS binding. Endocannabinoid levels in hippocampus were determined by liquid chromatography-multiple reaction monitoring. O-2050 was about ten times less potent than rimonabant in its CB1 receptor affinity, potency and facilitatory effect on noradrenaline release. Although not affecting (35)S-GTPγS binding by itself, O-2050 shifted the concentration-response curve of a CB1 receptor agonist to the right but that of rimonabant to the left. Levels of anandamide and 2-arachidonoyl glycerol in guinea pig hippocampus closely resembled those in mouse hippocampus. In conclusion, our results with O-2050 confirm that the CB1 receptors on noradrenergic neurons of the guinea pig hippocampus show an endogenous tone. To differentiate between the two mechanisms leading to an endogenous tone, O-2050 is not superior to rimonabant since O-2050 may increase the inverse agonistic effect of endocannabinoids.

A pharmacological paradox: may a neutral antagonist shift an agonist concentration-response curve to the left?

“…In the study of Jergas et al (2014), the pK d of rimonabant, 7.8, was much higher than its pEC 50 , 5.0. To take this into consideration, the condition of cooperativity has to be assumed in addition to that of a receptor reserve (for quantification of the differences between pK d and pEC 50 see Limberger 1999 andSauermann 2005).…”

“…The pK d of O-2050, 6.8, was assessed convincingly (note that this pK d of 6.8 was very similar to the pA 2 of 7.0, Jergas et al 2014).…”

“…As it can be learned from the paper of Jergas et al By definition, a neutral competitive antagonist only changes the affinity of an agonist. Nevertheless, under the following assumptions, such a sole decrease in the agonist affinity can explain an increase in the potency of an inverse agonist, as observed by Jergas et al (2014), i.e., a shift to the left of its concentration-response curve. …”

“…As it can be learned from the paper of Jergas et al (2014), O-2050, assumingly a neutral CB 1 receptor antagonist, indeed shifts the concentration-response curve of the classical CB 1 receptor agonist WIN 55,212-2 to the right. The concentrationresponse curve of the inverse CB 1 receptor agonist rimonabant, however, is shifted to the left by O-2050.…”

“…Cooperativity means that two inverse agonists bind "non-independently" of each other to the CB 1 receptor dimer. We don't know explicitly from the data of Jergas et al (2014) whether this cooperativity is positive or negative or neutral with respect to the binding affinities at the first and the second receptor of the dimer, although negative cooperativity may occur more often (Franco et al 2007). We may only say that two inverse agonist molecules bind simultaneously to one CB 1 receptor dimer, and that binding of only one inverse agonist molecule is also sufficient to maximally reduce the constitutive 35 S-GTPγS binding.…”