Effects of chronic ethanol administration on brain interstitial fluid levels of Methionine-enkephalin as measured by microdialysis in vivo.

The level of Met-enkephalin in the brain is inversely correlated with ethanol consumption and is controlled partially through efflux activity of peptide transport system-1 (PTS-1) at the blood-brain barrier (BBB). Prolonged alcohol drinking can perturb aspects of this system, including a loss of control of Met-enkephalin levels at the transcriptional and translational levels, and impaired release of Met-enkephalin from tissue sources. Met-enkephalin levels in whole brain homogenates often first paradoxically increase after a few days of ethanol drinking and then decrease with the development of physical dependence. Which of those various changes drives the others is unclear. To clarify these interactions, we here determined the levels of Met-enkephalin in striatal interstitial fluid (ISF) by microdialysis, striatal tissue homogenates, and serum after chronic ethanol treatment and alcohol withdrawal. Mice received ethanol (5%) in liquid diet for 7 days (ethanol-treated) and others withdrawn for a day following 7-day treatment (withdrawal). There was a significant (P<0.05) difference in the levels of Met-enkephalin in striatal microdialysate between the control (79.1+/-5.9 pg/ml) and ethanol-treated group (94.9+/-4.3 pg/ml), which was lost by withdrawing ethanol (83.9+/-3.8 pg/ml). In contrast, ethanol treatment did not affect Met-enkephalin levels in the striatal tissue. In the ethanol-treated group, there was a significant (P<0.05) reduction of the levels of Met-enkephalin in serum to 70.5% of control levels. This decrease was restored to the level of control by withdrawing ethanol. These reversible changes in ISF and serum are readily explained by the known changes in the efflux activity of PTS-1 at the BBB.

Preproenkephalin targeted antisenses cross the blood-brain barrier to reduce brain methionine enkephalin levels and increase voluntary ethanol drinking.

Antisense potentially can manipulate target gene expression in the brain if it can cross the blood-brain barrier (BBB). We designed three (10mer, 17mer, and 19mer) phosphorothioated antisenses (PS-ODNs) directed against the precursor molecule of methionine enkephalin (Met-Enk), an opiate peptide which suppresses voluntary ethanol drinking. We measured the ability of the antisenses to cross the BBB, accumulate in the brain and CSF, decrease levels of Met-Enk in brain and blood, and affect voluntary ethanol drinking. Each antisense readily crossed the BBB, with 0.07-0.16% of the i.v. dose accumulating per gram of brain. Capillary depletion and CSF sampling each confirmed that the antisenses entered the CNS. Gel electrophoresis of radioactivity recovered from brain and serum showed intact antisense and a higher molecular weight form likely representing antisense bound to protein, but no degradation products. Each antisense molecule and a cocktail of all three reduced Met-Enk levels in brain and serum. Met-Enk levels in the brain were reduced more rapidly and for a longer duration than Met-Enk levels in the serum, indicating a degree of selective targeting to the CNS. Additionally, administration of the cocktail was more effective in reducing Met-Enk levels than any of the individual antisenses. Each antisense increased voluntary ethanol drinking by about 20% and the cocktail increased it by about 80%. Taken together, these results used pharmacokinetic, immunochemical, and behavioral methods to show that PS-ODN antisenses that readily cross the BBB can decrease brain levels of Met-Enk and increase voluntary ethanol drinking.