Rab5-dependent trafficking of the m4 muscarinic acetylcholine receptor to the plasma membrane, early endosomes, and multivesicular bodies.

The m4 subtype of muscarinic acetylcholine receptor regulates many physiological processes and is a novel therapeutic target for neurologic and psychiatric disorders. However, little is known about m4 regulation because of the lack of pharmacologically selective ligands. A crucial component of G protein-coupled receptor regulation is intracellular trafficking. We thus used subtype-specific antibodies and quantitative immunocytochemistry to characterize the intracellular trafficking of m4. We show that following carbachol stimulation, m4 co-localizes with transferrin, and the selective marker of early endosomes, EEA1. In addition, m4 intracellular localization depends on Rab5 activity. The dominant negative Rab5S34N inhibits m4 endocytosis initially following carbachol stimulation, and reduces the size of m4 containing vesicles. The constitutively active Rab5Q79L enhances m4 intracellular distribution, even in unstimulated cells. Rab5Q79L also produces strikingly enlarged vacuoles, which by electron microscopy contain internal vesicles, suggesting that they are multivesicular bodies. m4 localizes both to the perimeter and interior of these vacuoles. In contrast, transferrin localizes only to the vacuole perimeter, demonstrating divergence of m4 trafficking from the pathway followed by constitutively endocytosed transferrin. We thus suggest a novel model by which multivesicular bodies sort G protein-coupled receptors from a transferrin-positive recycling pathway to a nonrecycling, possibly degradative pathway.

Sensitization to daily morphine injections in rats with unilateral lesions of the substantia nigra.

Morphine indirectly enhances dopaminergic activity in the nigrostriatal system, and repeated administration of morphine progressively increases the locomotor activity of rats. We used the rotational behavior model to determine if daily morphine produces an increase in turning and produces cross-sensitization to d-amphetamine and cocaine. Rats with unilateral nigrostriatal lesions received daily injections of saline or morphine (10 mg/kg). Repeated morphine administration produced a progressive increase in turning over 13 days. Next, a morphine dose-response curve (1.0-30 mg/kg) was determined. Both the saline and morphine-treated groups showed dose-dependent increases in turning, but, the peak effect in the morphine group was higher than that in the saline group, indicating sensitization to morphine. The morphine-treated group did not show cross-sensitization to either d-amphetamine (0.1-3 mg/kg) or cocaine (1.0-30 mg/kg); in fact, it showed less cocaine-induced turning than the saline group. Seventy-one days after saline or morphine injections began, the morphine group was still significantly more sensitive to turning induced by 10 mg/kg morphine than the saline group was (200 vs. 750). Therefore, repeated daily injections of morphine produce a progressive sensitization to turning induced by morphine in the absence of cross-sensitization to turning induced by psychomotor stimulants.

Naltrexone and alcohol dependence. Role of subject compliance.

BACKGROUND: Two previous double-blind, placebo-controlled studies demonstrated that naltrexone (50 mg/d) reduces alcohol drinking in alcohol-dependent subjects. In both studies, treatment compliance was excellent. However, a robust treatment effect size for naltrexone relative to placebo has been shown for compliant subjects but not for subjects who missed research visits. The goal of this study was to determine the effectiveness of naltrexone in subjects who received psychosocial treatment in a more naturalistic setting with respect to the role of treatment attendance and medication compliance. METHODS: Ninety-seven alcohol-dependent subjects were randomly assigned to receive either naltrexone (n = 48) or matching placebo (n = 49) for 12 weeks. All subjects received individual counseling (twice per week for the first month followed by once per week). RESULTS: Overall, naltrexone showed only modest effects in reducing alcohol drinking for the 12 weeks of treatment. However, naltrexone treatment efficacy improved across a variety of outcome measures for subjects who completed treatment and were highly compliant with taking medication. CONCLUSIONS: Naltrexone is clinically effective relative to placebo in individuals who comply with the treatment protocol and take medication. The modest treatment effects in the entire sample suggest that the clinical efficacy of naltrexone could be improved by enhancing treatment compliance.

Naltrexone in the treatment of alcoholism: a clinical review.

The pooled results from our Veterans Affairs studies are presented for 99 men. The naltrexone-treated subjects reported a reduction in alcohol craving and drinking, as well as less euphoria when they ingested alcohol. Relapse rates were significantly lower for the naltrexone-treated subjects than they were for placebo-treated subjects. Together with the consistent results from other double-blind trials of naltrexone, we conclude that naltrexone is a safe and useful adjunct in the rehabilitation of alcohol-dependent patients. Although administration of naltrexone was shown to improve treatment outcome, subjects who attended all 12 research visits demonstrated larger treatment effects. These data suggest that the use of naltrexone as a pharmacological adjunct to psychosocial intervention is an effective treatment for alcohol dependence. The effectiveness of naltrexone may be improved by designing a treatment program that enhances compliance with the medication.

Medical management of alcohol dependence: clinical use and limitations of naltrexone treatment.

Historically, pharmacological and psychosocial treatments for alcohol dependence have demonstrated only modest effectiveness in reducing alcohol drinking. However, the recent US Food and Drug Administration approval of naltrexone for the treatment of alcohol dependence offers a new, safe and effective medication to reduce relapse following alcohol detoxification. This paper reviews the various psychosocial and pharmacological treatments currently available and the effectiveness of these treatments. This paper also reviews preclinical research which demonstrates the involvement of the opioid system in the reinforcing effects of alcohol. This research led to clinical trials on the use of the opioid antagonist, naltrexone, to reduce alcohol's pleasurable effects and enhance the effectiveness of psychosocial therapy. In two randomized clinical trials, naltrexone treatment reduced rates of alcohol relapse, number of drinking days and alcohol craving. The clinical efficacy of all pharmacological treatments for substance abuse are limited by compliance with taking the medication. Also, pharmacological treatment does not address the psychosocial complications which often result from chronic alcohol dependence. Therefore, the integration of medications such as naltrexone and psychosocial therapies may offer the best treatment. The further development and investigation of new pharmacological agents will enable matching of patient populations with specific treatments, offering more successful treatment outcomes.

Opiates and alcohol self-administration in animals.

During the past 25 years, numerous animal studies have demonstrated a relationship between alcohol consumption and opiates. Converging lines of evidence suggest that (1) alcohol consumption enhances opioid receptor activity and (2) conditions associated with relative deficiencies in opioid receptor activity stimulate increases in alcohol preference. This evidence leads to the hypothesis that alcohol drinking is reinforced, in part, by enhanced opioid receptor activity; thus, these effects should be blocked by opiate antagonists. In fact, the animal data are consistent with this prediction. Opiate antagonists reduce excessive alcohol intake without reducing the ingestion of other biologically important reinforcers.