Endogenous opioid peptides modulate the effects of many drugs of abuse. Here we have analyzed the role of enkephalins and β-endorphin in animal models of ethanol preference, ethanol preference after stress exposure, ethanol withdrawal symptoms, and in a human case-control study with alcoholic patients. Our results indicate that β-endorphin modulates ethanol preference and stress-induced relapse in animals. In humans, polymorphisms in the β-endorphin encoding POMC gene are associated with alcoholism. Interestingly, we found sex-specific differences in animal and in human studies.
Our findings are entirely consistent with the previous demonstration that voluntary ethanol intake was decreased in MOP knockout animals (12
), i.e. in animals lacking the receptor with the highest affinity for β-endorphin. They also agree with the phenotype of DOP knockouts, which showed increased ethanol consumption. Together these data suggest that endorphin/MOP and enkephalin/DOP signalling modulates ethanol preference and consumption in mice. This is indeed one of the few examples showing a perfect match of receptor/ligand knockout phenotypes, thus lending further confidence to the relevance of these findings. Although enkephalins can also bind and activate MOP, it is unlikely that they contribute much to the MOP-mediated modulation of ethanol reward, because we and others found no difference between WT and ENK-KO mice in a two-bottle-choice and a conditioned place-preference paradigm (43
). Together these findings suggest that β-endorphin acting on MOP plays a critical role in ethanol reinforcement, whereas both opioid peptides seem to be involved in stress- induced relapse.
Our results do not agree with previous studies reporting on operant intravenous and oral self-administration of ethanol, as well as ethanol consumption in a two bottle-choice paradigm in β-endorphin deficient mice. We show here that mice lacking β-endorphin consumed less of an 8% ethanol solution than wildtype controls. In contrast Grisel and co workers found a higher ethanol preference in β-endorphin knockout mice compared to wildtype animals with an ethanol concentration of 7%, but not with 10% (44
). A study by Grahame and colleagues (45
) supported these findings, because they found no significant genotype effect in a free access paradigm with an ethanol concentration of 10%, but increased ethanol consumption in END-deficient mice in a scheduled access paradigm where EtOH was only available for 2 hours per day. They suggested that consumption of 10% alcohol during limited access is opioid dependent, because naltrexone treatment reduced drinking in wild type and in knockout mice (46
). In a paradigm for intravenous operant self-administration, END-deficient mice readily acquired self-administration behaviours, while wild-type mice did not. No genotype effects were observed when ethanol was delivered orally in the operant paradigm (45
). The reason for these disparate results is not clear. Because the genetic background of the animals was very similar in all studies (C57BL/6), procedural or environmental effects are most likely to account for these differences. One obvious environmental factor is the ambient stress level to which the animals are exposed. Environmental stressors include odorants, noise, vibrations and other factors that differ between animal facilities. Indeed, we found a significant genotype effect after stress exposure, which increased EtOH consumption in wild type, but not in END-KO mice. Endogenous opioids and their receptors are expressed in neuronal stress-response circuits including the paraventricular nucleus (PVN), the nucleus of the solitary tract and raphe nuclei (47
), which are also involved in the modulation drug reward (51
). It is well known that stress increases ethanol consumption in mice (36
) and contributes to ethanol-seeking behavior in humans (52
). Thus endogenous opioids may modulate ethanol consumption in stress conditions.
Because several previous studies have shown that female mice have a higher ethanol preference than males, we analyzed both sexes independently. We found that the difference in ethanol consumption between WT and END-KO mice was much larger in females than in males. Thus, the lack of β-endorphin affected female mice more strongly than males.
Important gender differences have also been observed for substance abuse disorders, including alcoholism, in humans (53
). These differences relate to the age of onset, motivation for drug use, co-morbidities, and numerous other factors. For example, in heavy drinkers adrenocorticotropic hormone and pituitary β-endorphin were significantly lower compared to controls, while plasma cortisol level were higher. These differences in hormone levels were more pronounced in female than in male subjects. It was therefore interesting to find that the POMC
two-marker haplotype (rs934778, rs3769671) was associated with alcoholism in females, but not in males. Because the female subgroup was relatively small, the effect size (odds ratio) of the polymorphism may have been overestimated. Nevertheless, we found the same association in the Swedish replication sample. In contrast, the association of markers rs2576581 and rs12545109 (PENK
) with the alcohol dependence phenotype in German females was not replicated in the Swedish cohort. Together our findings in humans and animals strongly support a contribution of β-endorphin to ethanol dependence in females. As previous studies in MOP receptor knockouts have also shown a stronger effect in females (12
), our finding further demonstrates a gender bias of the endogenous opioid system in alcohol dependence. Although alcohol consumption is higher in men compared to woman, this is probably culturally biased. Nevertheless, our results further contribute to the idea that the genetic regulation of drug reward and dependence differs between genders.
A recent study using a diverse cohort of European Americans from alcohol dependent families found no association between PENK
polymorphisms and alcoholism (29
). For PENK
, the study used different sets of markers. For POMC
, Xuei et al. also analyzed rs934778 (SNP of the haplotype linked with alcoholism in our sample), but did not find a significant association of this SNP with alcohol dependence. Nevertheless, this marker showed a significant association with a more severe subtype of alcoholism, which is characterized by co-morbidity with opioid dependence (29
). Unfortunately, Xuei et al did not differentiate between female and male gender in their sample, which encompassed predominantly males.
Taken together these data suggest, a contribution of endogenous opioid system to behaviours associated with alcoholism. β-endorphin seems to be important for ethanol preference, and enkephalin and β-endorphin modulate the effects of stress on ethanol consumption. A significant role of POMC in females was confirmed in our human association studies. Thus, the correlation of distinct alcohol related behaviours with the endogenous opioid peptides deserves further studies in humans.