Endocannabinoids and the Control Of Behavior and Cardiovascular Function

National Institute On Alcohol Abuse And Alcoholism

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Abstract

In my FY20 annual report I summarized our findings, published in 2019 in Cell Metabolism, that the peripherally restricted CB1 receptor antagonist JD5037 was equally effective with the brain-penetrant CB1 antagonist rimonabant in markedly reducing total alcohol intake as well as ethanol preference in wild-type but not in CB1R-/- C57BL6 mice, using a 'two bottle, free choice' paradigm as well as a 'drinking in the dark' paradigm. Furthermore, peripheral CB1 blockade significantly reduced plasma levels of the biologically active octanoylated ghrelin, with no change in the level of its precursor, desacyl ghrelin, suggesting that the ghrelin acylation process, catalyzed by the enzyme GOAT, may be a CB1R target. In additional experiments we found that alcohol preference and intake are lower in ghrelin knockout and ghrelin receptor1 (GHSR-1) knockout mice than in their wild-type littermate controls. We also found that afferent vagal (sensory) denervation of the stomach increased alcohol drinking and drinking preference and abolished the efficacy of peripheral CB1R blockade as well as GHSR-1 blockade to reduce drinking. These findings are compatible with a scheme whereby alcohol drinking increases ghrelin acylation through CB1 receptor activation, and the released acylghrelin activates ghrelin receptors (GHSR1) on vagal sensory terminals to promote ethanol drinking behavior. As for the cellular mechanism involved in the selective decrease in acyl-ghrelin production by peripheral CB1 blockade, we found that in a ghrelin-producing stomach cell line JD5037 suppressed octanoyl-ghrelin but not desacyl-ghrelin production by reducing the levels of octanoyl-carnitine generated from palmityl-carnitine due to an increase in fatty acid oxidation. While depletion of the GOAT substrate octanoyl carnitine by JD5037 is a plausible mechanism for the reduced drinking preference observed in vivo, CB1 receptors on ghrelin-producing stomach cells may not be its only targets. Specifically, CB1 receptors are known to be present on the peripheral terminals of vagal afferent neurons, and their blockade may affect vagal afferent signaling in a way that would inhibit the drive to drink. As a first step to explore this possibility, we have generated conditional CB1R knockout mice with selective genetic deletion of Cnr1 (the gene encoding CB1R) in specific subsets of afferent neurons of the nodose ganglion. This was achieved by crossing CB1R floxed mice with avilin-cre or Trpv1-cre mice, advilin and Trpv1 being markers of distinct subsets of vagal afferent neurons. These animals and their wild-type littermates are currently being tested in different paradigms of voluntary alcohol drinking. In separate experiments, we are testing the effects on alcohol drinking and preference of our hybrid CB1R/iNOS inhibitor MRI-1867, in comparison with the effects of single target CB1R inhibitors. The purpose of these experiments is to test whether the additional targeting of iNOS and the resulting anti-inflammatory effect may contribute to the reduction in alcohol drinking, based on published evidence of M1 macrophage infiltration in the stomach of mice tested in models of binge drinking. By using central vs systemic administration of the CB1R antagonists we also tested whether or not CB1R in the CNS are dispensable for the effects of these compounds on alcohol drinking behavior. We now report that the globally active CB1R antagonist rimonabant and the peripherally restricted CB1R antagonist JD5037 both inhibit voluntary alcohol intake upon systemic but not after intracerebroventricular administration, the latter in doses that elicited anxiogenic behavior and blocked CB1R agonist-induced hypothermia and catalepsy. The orally administered hybrid peripheral CB1R/iNOS inhibitor S-MRI-1867 was also effective in reducing alcohol intake, while its CB1R inactive/iNOS active R enantiomer was not. The two MRI-1867 enantiomers were equally effective in inhibiting an alcohol-induced increase in portal blood endotoxin concentration, a marker of increased gut permeability. We conclude that (i) activation of peripheral CB1R plays a dominant role in promoting alcohol intake, and (ii) the iNOS inhibitory function of MRI-1867 helps mitigating the alcohol-induced increase in endotoxemia. This work has just been published in a special issue of Molecules in honor of the 90th birthday of Professor Raphael Mechoulam.