An enantiomeric study of salsolinol involvement in ethanol brain reinforcing effect, via the activation of the Gi pathway of µ-opioid receptors

Abstract

The reinforcing effect of ethanol is a major contributor to ethanol dependence. The formation of acetaldehyde, the first ethanol metabolite, is required for ethanol to be reinforcing, by activating dopaminergic neurons in the ventral tegmental area (VTA). Whether acetaldehyde can bind to a receptor to be reinforcing is unknown, but it can condense with dopamine to form racemic salsolinol [(R) and (S) SAL]. It has been proposed that SAL is the substance that exerts the reinforcing effect of ethanol consumption because SAL is a potent brain reinforcer when intracerebrally administered. Further, the levels of racemic SAL rise in the brain of rats after chronic ethanol administration. After an acute dose of ethanol, the levels of SAL in the brain reward system rise only if the metabolism of acetaldehyde is inhibited. The mechanism of SAL activity could be via µ-opioid receptors (µOR) since SAL displaces ligands from these receptors, but there is no evidence that SAL can directly activate µORs. This work aimed to determine whether SAL produced after an acute dose of ethanol activates the brain reward system of naïve rats via one of its enantiomers (either R or S) activation of µORs. Cell-based experiments showed that SAL activated µORs via Gi protein with no β-arrestin recruitment, and purified (S)-SAL was 50 times more potent than (R)-SAL. To support the experimental results we employed molecular dynamics simulations of (R) and (S)-SAL inside the binding pocket of the µOR. These simulations allowed us to explain that the enantiomeric specificity was caused by the interaction of the (S)-SAL chiral methyl group with the Y148 of the µOR, interaction that was not stable for (R)-SAL. The simulations showed that (S)-SAL, a small molecule compared to other opioid agonists, cannot interact with all the residues regarded as important for µOR activation, showing that (S)-SAL is a unique agonist of the receptor. Following, we determined whether (R) or (S)-SAL administration into the VTA of rats activate the brain reward system. The intra-VTA infusion of (R) and not of (S)-SAL induced a conditioned place preference in rats. The mechanism for this result may be different to µOR agonism since (S)-SAL did not induce an effect despite being a more potent agonist. Next, we intended to identify the reasons for the difficulty to detect SAL in the rat brain, despite being readily formed in vitro from acetaldehyde and dopamine at pH 7.4. The SAL activity at the dopamine transporter was explored using a cell-based assay, and we found that a millimolar SAL concentration was needed to be a ligand of the transporter, challenging the relevance of this uptake mechanism. To determine the effect of catechol-O-methyl transferase (COMT) in SAL elimination, SAL was injected intra-VTA of rats with or without the COMT inhibitor entacapone; then SAL levels were measured in VTA and substantia nigra homogenate supernatants. No differences were observed between the SAL levels measured from animals treated with, or without, entacapone. Finally, we aimed to detect and measure SAL in the brain of naïve rats administered with systemic ethanol. Rats were administered with ethanol, and brain homogenates assayed for SAL. No SAL was observed in either nucleus accumbens or VTA. When intra-VTA entacapone was administered concurrently with the ethanol administration, no SAL was observed either. In conclusion, SAL was found to be a µOR agonist, acting in vitro via (S)-SAL, activating Gi protein signaling. However, (S)-SAL was not active to induce a rat conditioned place preference after infused intra-VTA while (R)-SAL was able to. These results suggest that there is another target for (R)-SAL to be rewarding. We did not observe SAL in the brain of animals administered ethanol, even when a COMT inhibitor was infused intra-VTA. SAL may be rapidly eliminated, yet the results show that SAL is not a relevant ligand of the dopamine transporter and SAL metabolization by COMT experiment was inconclusive. To be reinforcing, SAL could reach proper levels in its receptor microenvironment, needing an accurate methodological approach to locally monitor its occurrence.